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Neurochemical studies of the pathogenesis of four central nervous system disorders : Parkinson's disease,… Yong, Voon Wee 1986

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NEUROCHEMICAL STUDIES OF THE PATHOGENESIS OF FOUR CENTRAL NERVOUS SYSTEM DISORDERS: P A R K I N S O N ' S D I S E A S E , D I A L Y S I S ENCEPHALOPATHY,  HUNTINGTON'S  CHOREA,  AND HALLERVORDEN-SPATZ SYNDROME  By  VOON WEE YONG B.  S c . (Hons),  The U n i v e r s i t y  of Manchester,  A T H E S I S SUBMITTED IN PARTIAL F U L F I L L M E N T  1981  OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE S T U D I E S Department o f Pharmacology Faculty  We a c c e p t t h i s  and T h e r a p e u t i c s  of Medicine  t h e s i s as c o n f o r m i n g t o  the required  standard  TttE UNIVERSITY OF B R I T I S H COLUMBIA May 1 9 8 6 © Voon Wee Y o n g , 1986  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree t h a t for  that  Library  s h a l l make  for reference  and  study.  I  f o r extensive copying of  h i s or  be  her  copying or  f i n a n c i a l gain  shall  g r a n t e d by  publication  not  be  of  further this  Columbia  thesis  head o f  this  my  i t is thesis  a l l o w e d w i t h o u t my  of  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  the  representatives.  permission.  Department  University  the  s c h o l a r l y p u r p o s e s may  understood  the  the  I agree that  permission by  f u l f i l m e n t of  advanced degree at  Columbia,  department or for  in partial  written  -  ii  -  ABSTRACT  My  thesis  central  nervous  standing with  of  relied  pathogenesis  or  dead  are  on  firstly,  t o ..one o r  and s e c o n d l y ,  chapters,  the  use  that  with  PD  model  of  with  neurotoxins  ture  second c h a p t e r  neuronal  result  neurotoxin  studies  of  injected  were  repeatedly  control  subjects,  exposed  in  results  from both  PD  that  either  of  such  loss  with  serum  types  of  i n t h e s e r u m of  larger  than  molecule 10000  unclear.  to  particular  to the  Several  Animal  understudies  experiments  an or  study or  experiments  via  up  to  reactive  chap-  time  of  radicals,,  may be  chorea  or  that (HC)  useful  exogenous  experiment  ultrafiUrate  CSF  from  identity  This  may  origin. in  which  from  HC  patients  are suggestive HC.  the  premabe  for  or  the of  Two  main  rats  were  patients  or were  controls.  The  t h e p r e s e n c e of  putative  putative  a  explants  neurotoxin  t o serum p r o t e i n s , of  the  i n the degradation  i n which r a t s t r i a t a l  with  the  free  as a - t o c o p h e r o l  failure  i r r e v e r s i b l y bound The  exposed  the hypothesis  in vivo  serum  patients  conclusions of t h i s  Huntington's  endogenous  serum  daltons.  a  i n PD.  tested  in  performed:  culture  e i t h e r be a s m a l l  presently  occurs  may be  may a c t  thesis  a n d , an i n v i t r o  tissue  neurotoxin  cule  that  (PD).  performed.  Two m a j o r  compounds  this  with  i s devoted  disease  a g e n e t i c a l l y - d e t e r m i n e d enzymatic  of  circulating types  death  in  chapter  were  PD.  slowing the progression of neuronal The  dealing  N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine  of  antioxidant  each  Parkinson's  patients  more  that  The f i r s t  of  patients  t o c r e a t e an a n i m a l  death  in  into.4  system d i s o r d e r .  heavily  (MPTP)  divided  the  living  ter  is  or,  a  may  a mole-  neurotoxin  is  In  the  third  normalities died with  that  levels  in  is  the  attempts  ties  of  in  The  in  the  fourth  autopsied  Hallervorden-Spatz  2  SD  above  syndrome  those  dioxygenase, reduced that of  in  the the  of  decreased  serve  to  activity  chelate  iron,  radicals  that  damage in  even  who  the  the  HSS.  died  we  of  DE  or  not  failed  injected  we  of  Alum-  matter  rats  to with  employed  hemi-  administration  globus  local  with  a  contents  globus  other  combination neuronal  patients.  neurochemical  patient,  dioxygenase.  for  of  who  a deficiency  gray  though  for  patients.  the  patients  because  lithium  cysteine  of b o t h  The  observed  in  the  converts  accounting  ate  changes  On  cortical  ab-  in  aluminum.  one  disulfide  cysteine  of H S S .  case  was  of DE  brains  chronic  In  finding  of  in c l a r i f y i n g whether  we s e a r c h e d  l o c a l l y in  pallidus  morphological  the  the  patients  2  (HSS).  pallidus  of  in  neurochemical  brains  principally  and/or  of  that  the1globus free  DE,  was  controls.  c y s t e i n e accumulates  frontal  e x c r e t i o n of  mixed  enzyme  globus  in  for  brains  unsuccessful  chapter,  brain  of g l u t a t h i o n e - c y s t e i n e  the  content  nephrectomy,  the  A major  in  factor  latter  examined  autopsied  autopsied  were  aluminum  the  of  high  decrease the renal  Finally,  in  we  (DE).  regions  causative  5/6  to  present  experiments  hydroxide.  nephrectomy,  thesis,  encephalopathy  elevation  aluminum  be  this  abnormally  Animal  aluminum  of  several  were  patients.  produce  might  dialysis  GABA c o n t e n t s inum  chapter  hand, to  rare of  cystine  activity  pallidus  in  HSS  Accumulated increase  in  cysteine  of  the  a  cause  result  cysteine content  and c y s t e i n e may to  was  hypothesis as  iron  and  elevated  sulfinate,  We p r o p o s e  membranes  disease,  p a l l i d u s were  cysteine  of i r o n  abnormali-  the  may in  genertypical  -  iv  -  TABLE OF CONTENTS  Page  Abstract... Table of contents L i s t of Tables L i s t of Figures L i s t of Appendices Acknowledgements Dedication  i i iv vi x xi xii xi i i  CHAPTER 1 :  P A R K I N S O N ' S DISEASE  . I. II. III.  Abstract Introduction....... O u t l i n e o f e x p e r i m e n t s and m e t h o d s . R e s u l t s and D i s c u s s i o n i . S e a r c h f o r an a n i m a l model o f P a r k i n s o n ' s d i s e a s e i i . S t u d i e s o f t h e n e u r o t o x i c e f f e c t s o f MPTP i n C57 b l a c k m i c e . . . i i i . P o s s i b l e m e c h a n i s m o f MPTP t o x i c i t y i v . Experiments using b u t y l a t e d hydroxyanisole, glutathione monoethyl e s t e r , b u t h i o n i n e s u l f o x i m i n e , and d i e t h y l m a l e a t e . . v . Experiments t h a t seek t o i d e n t i f y t h e n a t u r a l neurotoxin(s) • r e s p o n s i b l e f o r most c a s e s o f i d i o p a t h i c P a r k i n s o n ' s d i s e a s e . . v i . S m o k i n g and P a r k i n s o n ' s d i s e a s e v i i . S t a t u s o f MAO-B i n r e l a t i o n t o P a r k i n s o n ' s d i s e a s e v i i i . G l u t a t h i o n e c o n t e n t and g l u t a t h i o n e t r a n s f e r a s e a c t i v i t y i n the autopsied brains of Parkinson's disease patients IV. C o n c l u s i o n s V. R e f e r e n c e s  CHAPTER 2 :  I.  II. III. IV.  V. VI.  Abstract Introduction i . Perspectives i i . Pathogenesis o f neuronal Outline of experiments In v i v o s t u d i e s Tissue culture s t u d i e s . . . i . Methods i i . R e s u l t s and D i s c u s s i o n Conclusions References  i 4 14 17 25 32 41 50 59 64 67 75 76  HUNTINGTON'S CHOREA 85  death  i n Huntington's  chorea  87 92 101 103 107 108 112 125 126  - V -  CHAPTER 3 : D I A L Y S I S ENCEPHALOPATHY Abstract I. Introduction i . Perspectives i i . Etiology of d i a l y s i s encephalopathy: aluminum I I . Outline of experiments I I I . Human s t u d i e s i . Method i i . R e s u l t s and D i s c u s s i o n IV. Animal experiments i . Method i i . R e s u l t s and D i s c u s s i o n V. C o n c l u s i o n s . . . . . . VI. References  •  Abstract.. Introduction Outline of experiments Results Discussion Conclusions References  133 134  The p o s s i b l e r o l e  CHAPTER 4 : HALLERVQRDEN-SPATZ  I. II. III. IV. V. • VI.  Page  of 137 -142 143 143  •  149 151 158 160  SYNDROME 165 166 169 171 177 182 183  -  vi  L I S T OF CHAPTER 1 :  -  TABLES  PARKINSON'S  DISEASE  Table • 1.  2.  Page  T y r o s i n e h y d r o x y l a s e (TH) a c t i v i t y and c o n t e n t s o f d o p a m i n e and i t s m e t a b o l i t e s i n r a t s t r i a t u m a f t e r c h r o n i c L-DOPA and c a r b i d o p a treatment  19  Effects rats  20  of  BSO on t o t a l  and l i v e r  T y r o s i n e h y d r o x y l a s e a c t i v i t y and c o n t e n t s o f d o p a m i n e m e t a b o l i t e s i n r a t s t r i a t u m a f t e r MMT t r e a t m e n t  7.  8.  9.  10.  11.  12.  13.  14.  15.  Effects of parameters  15 of  after  brain  4.  S t r i a t a l parameters of ( 8 mg/kg) f o r 10 d a y s  brain  of  Manganese  6.  of r a t  contents  3.  5.  contents  glutathione  of  MMT t r e a t m e n t . . . . .  non-scorbutic guinea pigs  21 and  its 22  injected with  MPTP 24  d a i l y i n j e c t i o n s o f MPTP (8 m g / k g ) on n e u r o c h e m i c a l s c o r b u t i c guinea pigs  Time c o u r s e o f n e u r o c h e m i c a l changes a f t e r an i n j e c t i o n o f 4 0 mg/kg MPTP  in s t r i a t u m of  C57  black  25  mice 28  C o m p a r i s o n o f d i f f e r e n t d o s a g e s c h e d u l e s o f MPTP on t h e s t r i a t a l c o n t e n t s o f d o p a m i n e and i t s m e t a b o l i t e s , i n m i c e s a c r i f i c e d 1 month a f t e r t h e l a s t i n j e c t i o n  29  C o m p a r i s o n o f d i f f e r e n t d o s a g e s c h e d u l e s o f MPTP on t h e s t r i a t a l c o n t e n t s o f d o p a m i n e and i t s m e t a b o l i t e s , i n m i c e s a c r i f i c e d 24 h a f t e r the l a s t i n j e c t i o n  30  S t r i a t a l neurochemical parameters of mice s a c r i f i c e d t i m e s a f t e r a s i n g l e i n j e c t i o n o f 40 mg/kg MPTP  31  C o n t e n t s o f d o p a m i n e and i t s m e t a b o l i t e s t r e a t e d w i t h MPTP and p r o t e c t i v e a g e n t s E f f e c t s o f MPTP i n j e c t i o n s on t o t a l and b r a i n s t e m o f m i c e  i n the  glutathione  at  various  s t r i a t u m of  mice 34  content  of  striatum 35  T o t a l g l u t a t h i o n e c o n t e n t s o f b r a i n s t e m and s t r i a t u m o f m i c e i n j e c t e d w i t h 4 0 mg/kg MPTP, w i t h o r w i t h o u t antioxidant pretreatment E f f e c t s o f L-DOPA and c a r b i d o p a t r e a t m e n t on s t r i a t a l d o p a m i n e and i t s m e t a b o l i t e s o f t h e mouse  contents  S t r i a t a l c o n t e n t s o f d o p a m i n e and i t s m e t a b o l i t e s i n m i c e g i v e n mg/kg MPTP, w i t h o r w i t h o u t L-DOPA and c a r b i d o p a p r e t r e a t m e n t .  37 of 38 20 38  -  16.  17.  18.  20.  21.  22.  23.  24.  25.  26.  27.  E f f e c t s o f BHA on t o t a l t r a n s f e r a s e a c t i v i t y of Effects  of  BHA  pretreatment  on t h e n e u r o c h e m i c a l ...  C o n t e n t o f amino a c i d s 10 mmol/kg g l u t a t h i o n e  2 40  effects  of  MPTP  in 44  E f f e c t s o f BSO p r e t r e a t m e n t on t h e n e u r o c h e m i c a l mg/kg MPTP i n C57 b l a c k m i c e . . . L i v e r and b r a i n s t e m t o t a l w i t h DEM  43  ..  i n l i v e r o f mouse s a c r i f i c e d 2 h monoethyl e s t e r  glutathione  contents  after 47  effects  of  20 48  of  mice  injected 49  C o n t e n t s o f d o p a m i n e and i t s m e t a b o l i t e s i n s t r i a t u m o f m i c e t r e a t e d w i t h DEM, DEM p l u s 20 mg/kg MPTP, o r 20 mg/kg MPTP o n l y  50  E f f e c t s o f MPTP a n a l o g s on s t r i a t a l m e t a b o l i t e s o f C57 b l a c k m i c e  54  contents  of  dopamine  E f f e c t s o f p a r a q u a t and r e d u c e d p a r a q u a t on s t r i a t a l d o p a m i n e and i t s m e t a b o l i t e s o f C57 b l a c k m i c e  and  its  contents  of 57  E f f e c t s o f p r e t r e a t m e n t w i t h t h i o c y a n a t e o r h y d r a z i n e on MPTPi n d u c e d d e p l e t i o n o f s t r i a t a l d o p a m i n e and i t s m e t a b o l i t e s i n m i c e .  61  F a i l u r e of hydrazine s t r i a t u m of mice  61  to  i n h i b i t MAO-B a c t i v i t y  B activity  28.  Inhibition  l i v e r monoamine  29.  Monoamine o x i d a s e disease patients  B activity  Total glutathione human b r a i n s  contents  31.  i n mice given  g l u t a t h i o n e c o n t e n t and g l u t a t h i o n e l i v e r and b r a i n s t e m o f t h e mouse  Monoamine o x i d a s e non-smokers  30.  -  S t r i a t a l c o n t e n t s o f d o p a m i n e and i t s m e t a b o l i t e s d i f f e r e n t d o s a g e r e g i m e n s o f MPP +  mice 19.  vii  of  rat  in p l a t e l e t s of  in l i v e r  and  human s m o k e r s  and 63  oxidase  B activity  in brain of c o n t r o l s  by human u r i n e  and  65  Parkinson's 66  in various  Glutathione transferase a c t i v i t y s u b s t a n t i a n i g r a o f PD p a t i e n t s  regions  of  rapidly  frozen 69  i n the f r o n t a l  cortex  and 74  - v i i i  CHAPTER 2 :  -  HUNTINGTON'S  CHOREA  Table  Page  1.  Striatal  in rats  i n j e c t e d w i t h whole  serum  2.  GABA c o n t e n t and CAT a c t i v i t y c o n c e n t r a t e d serum f i l t r a t e s  in striatum of rats  injected  3.  4. 5.  6.  7.  8.  GABA c o n t e n t  105 with 105  C h a r a c t e r i s t i c s o f HC p a t i e n t s culture experiments  whose s e r a w e r e u s e d i n t i s s u e 108  C h a r a c t e r i s t i c s of t i s s u e c u l t u r e experiments  109  I n d i v i d u a l v a l u e s o f GAD a c t i v i t y and GABA c o n t e n t o f r a t s t r i a t a l e x p l a n t s e x p o s e d t o 3 0 % w h o l e . s e r u m o r 15 % d e p r o t e i n i z e d s e r u m o f , Experiment 8 E f f e c t s o f 1 5 % w h o l e s e r u m f r o m HC p a t i e n t s s t r i a t a l explants E f f e c t s o f 15 % d e p r o t e i n i z e d of r a t s t r i a t a l explants  of rat 119  s e r u m f r o m HC p a t i e n t s  on GAD  activity 119  E f f e c t s o f 15 % C S F f r o m HC p a t i e n t s explants  CHAPTER  on GAD a c t i v i t y  113  on GAD a c t i v i t y  of r a t  striatal 120  3 : DIALYSIS  ENCEPHALOPATHY  Table 1.  Regional patients  Page GABA c o n t e n t s  of autopsied brain  i n DE a n d c o n t r o l  ; 144  2.  Pyridoxal  phosphate  content  3.  Contents  4.  Choline acetyltransferase  o f a u t o p s i e d b r a i n o f DE p a t i e n t s  o f monoamine m e t a b o l i t e s activity  i n a u t o p s i e d b r a i n o f DE p a t i e n t s in cerebral cortex  146  o f DE  patients  147  5.  Treatments  6.  Neurochemical  7.  Evidence A1(0H)3  8.  146  given  to aluminum-injected  parameters  animals  i n Group A r a t s  for c i r c u l a t o r y absorption of  after  149 chronic Al exposure.  153  subcutaneously-injected  E f f e c t s o f A l ( 0 H ) 3 o r s a l i n e i n j e c t i o n s s t a r t e d 24 h a f t e r nephrectomy i n L i C l - p r e t r e a t e d r a t s  154 5/6 156  -  CHAPTER 4 :  ix  -  HALLERVORDEN-SPATZ  SYNDROME  Table 1.  Page  B r a i n c y s t i n e and g l u t a t h i o n e - c y s t e i n e m i x e d d i s u l f i d e i n H a l l e r v o r d e n - S p a t z syndrome  172  2.  B r a i n GABA c o n t e n t  173  3.  Choline acetyl transferase a c t i v i t y  4.  Dopamine  i n Hal l e r v o r d e n - S p a t z  and m e t a b o l i t e s  syndrome  in Hallervorden-Spatz  i n t h e s t r i a t u m o f HSS C a s e 1  syndrome..  176 176  -  X -  L I S T OF CHAPTER 1 :  FIGURES  P A R K I N S O N ' S DISEASE  Figure 1.  2.  Page  Changes r e l a t i v e t o c o n t r o l s o f t o t a l g l u t a t h i o n e c o n t e n t o f l i v e r s o f C57 b l a c k o r C D l S w i s s a l b i n o m i c e , a t v a r i o u s t i m e i n t e r v a l s a f t e r an i n j e c t i o n o f 10 mmol/kg g l u t a t h i o n e m o n o e t h y l e s t e r . . . . . . .  45  Structural  52  formulae  o f MPTP,  CHAPTER 2 : 1.  2.  3.  Chemical acid  structures  2.  and t h e i r  HUNTINGTON'S  of glutamic  acid,  analogs  CHOREA  kainic acid,  and q u i n o l i n i c 96  GAD a c t i v i t i e s o f e x p l a n t s e x p o s e d t o 30% w h o l e s e r u m f r o m HC p a t i e n t s f o r 1 and 3 w e e k s , e x p r e s s e d as % o f t h e i r r e s p e c t i v e controls  116  GAD a c t i v i t i e s o f e x p l a n t s e x p o s e d t o v a r i o u s f o r m s o f HC serum f o r 1 w e e k , e x p r e s s e d a s °l° o f t h e i r r e s p e c t i v e c o n t r o l s  CHAPTER 4 : 1.  MPP+,  HALLERVORDEN-SPATZ  Enzyme a c t i v i t y o f c y s t e i n e d i o x y g e n a s e 3 regions of autopsied brain of control %change i n c y s t e i n e d i o x y g e n a s e increasing brain i r o n content  122  SYNDROME  i n t i s s u e homogenates from , a d u l t s a n d HSS p a t i e n t s . . . .  enzyme a c t i v i t y  as a f u n c t i o n  174  of 175  -  L I S T OF  xi  -  APPENDICES  Appendix P e r r y , T. L . , Y o n g , V . W., I t o , M . , F o u l k s , J . G . , W a l l , R. A . , G o d i n , D. V . , C l a v i e r , R. M. N i g r o s t r i a t a l dopaminergic neurons remain undamaged in rats given high doses of L-DOPA and c a r b i d o p a c h r o n i c a l l y . J . Neurochem. 4 3 : 9 9 0 - 9 9 3 , 1984. Yong, V. W., Perry, T. L., Godolphin, W. J . , Jones, K. A., Clavier, R. M., Ito, M., Foulks, J. G. Chronic organic manganese a d m i n i s t r a t i o n i n t h e r a t d o e s n o t damage d o p a m i n e r g i c n i g r o s t r i a t a l neurons. N e u r o t o x i c o l o g y 7 : 1 9 - 2 4 , 1986. P e r r y , T. L., Yong, V. W., Ito, M., Jones, K., W a l l , R. A., Foulks, J. G., Wright, J . M., Kish, S. J . l-Methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) d o e s n o t d e s t r o y n i g r o s t r i a t a l neurons i n the s c o r b u t i c guinea p i g . L i f e S c i . , 36:1233-1238, 1985. P e r r y , T. L., Yong, V. W . , J o n e s , K . , W a l l , R. A . , C l a v i e r , R. M., Foulks, J. G., W r i g h t , J . M. E f f e c t s of N-Methyl-4-phenyl1,2,3,6-tetrahydropyridine, and its metabolite, N - m e t h y l - 4 - p h e n y l - p y r i d i n i u m i o n , on d o p a m i n e r g i c n i g r o s t r i a t a l neurons i n the mouse. Neuroscience Letters 58:321-326, 1985. P e r r y , T. L . , Y o n g , V . W . , C l a v i e r , R. M . , J o n e s , K . , W r i g h t , J. M., Foulks, J. G., Wall, R. A . P a r t i a l p r o t e c t i o n from the dopaminergic neurotoxicity of N-Methyl-4-phenyl-l,2,3,6t e t r a h y d r o p y r i d i n e by 4 d i f f e r e n t a n t i o x i d a n t s in the mouse. Neuroscience L e t t e r s 60:109-114, 1985. Yong, V. W., Perry, T. L., Krisman, A. A. Depletion of glutathione in brainstem of mice caused by N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine is prevented by antioxidant pretreatment. Neuroscience L e t t e r s 6 3 : 5 6 - 6 0 , 1986. Y o n g , V. W . , Perry, Parkinson's disease.  T. J.  L. Monoamine o x i d a s e B , s m o k i n g , N e u r o l . S c i . 7 2 : 2 6 5 - 2 7 2 , 1986.  P e r r y , T. L . , Yong, V. W . , Hansen, S . , F o u l k s , J . G . , Kish, J. Is a c i r c u l a t i n g n e u r o t o x i n i n v o l v e d i n the p a t h o g e n e s i s H u n t i n g t o n ' s c h o r e a ? . J . N e u r o l . S c i . 6 7 : 3 5 1 - 3 5 8 , 1985.  and  S. of  P e r r y , T. L . , Yong, V. W., K i s h , S. J . , Ito, M., Foulks, J. G., G o d o l p h i n , W. J . , S w e e n e y , V . P . Neurochemical a b n o r m a l i t i e s in brains of renal failure patients treated by repeated hemodialysis. J . Neurochem. 4 5 : 1 0 4 3 - 1 0 4 8 , 1985. P e r r y , T. L . , N o r m a n , M. G . , Y o n g , V . W . , W h i t i n g , S . , C r i c h t o n , J. U., Hansen, S., Kish, S. J. Hallervorden-Spatz disease: C y s t e i n e a c c u m u l a t i o n and c y s t e i n e d i o x y g e n a s e d e f i c i e n c y i n t h e globus p a l l i d u s . Ann. N e u r o l . 1 8 : 4 8 2 - 4 8 9 , 1985.  - x i i  -  AKNOWLEDGEMENTS  I w i s h t o thank encouragement example I  throughout  of s c i e n t i f i c  am g r a t e f u l  and K a r e n J o n e s , I  Wall,  comments  questions  Medical the  appreciative Dr.  study  Wright  Dr.  f o r h i s g u i d a n c e and  and f o r p r o v i d i n g  Seung  should  a  superb  Council  of  of  I  Dr.  David. Godin,  valuable  Kim deserves  that  to  made  and  constructive  special  possible  Dr.  mention  the  in  for vitro  my w i f e ,  Fiona,  for  her  challenging  thesis. grants  Canada,  Canada.  financial  Last but not l e a s t ,  U.  Hansen  technical assistance.  Foulks,  their  Shirley  2.  go  by  in research,  skilled  James for  equipment  supported  Society  their  Dr.  in editing this  was  Research  acknowledge  of  James  and  thanks  and h e l p  Huntington  work,  who g e n e r o u s l y h e l p e d me w i t h  and  Perry,  and c o l l e a g u e s  described i n Chapter  special  This  my f r i e n d s  expertise  experiments  Thomas L .  the course of t h i s  and c r i t i c i s m s .  providing  Dr.  d e d i c a t i o n and e x c e l l e n c e .  to  am d e e p l y  Richard  A  my s u p e r v i s o r ,  to  Dr.  Thomas  the Parkinson To t h e s e  L.  Foundation  granting  Perry  from  the  of Canada,  and  agencies,  I  gratefully  contributions.  am e x t r e m e l y  C a n a d a f o r i t s award o f a p r e d o c t o r a l  grateful  to the Huntington  s c h o l a r s h i p t o me.  Society  of  -  xiii  -  DEDICATION  This  thesis  is dedicated  t o my w i f e ,  Fiona,  and o u r  families.  - 1 CHAPTER 1 .  PARKINSON'S DISEASE  ABSTRACT  Speculations  as  to  abound.  A prevailing  exposure  to  port  this  for  one  or  monkeys  understanding  In  C57  produced  a  substantia (90%)  of  toxicity any or  one  contents  of  of  scavenger, of  tathione that  This  the  be  might  be  could  the  in was  be or  the  from  important  requires  of  by  by  of  in  in  the  However,  property  the  of  free  of  an  of  the  mice  The with  ascorbate in  the  injection  endogenous  results  MPJP  reduction  radicals  after  decreased.  of  striatum.  e-carotene,  the  of  free  radi-  Furthermore,  this  of  mice  suggested  detoxification prior  by  metabolites.  compacta  pretreatment  h  meperhumans,  4 0 mg/kg  zona  pretreatment  These  sup-  conversion  long-lasting  of  stem of  by  of  PD.  the a  a  in  neurotoxic  of  Twenty-four brain  One  neurons  o-tocopherol,  significantly  MPTP.  in  involvement  prevented  synthesis  the  metabolites  e-carotene.  critically  the  injection  bodies  (PD)  a result  neurotoxins.  but  of  prevented  toxicity.  content  be f o r m e d  its  antioxidants,  MPTP  a-tocopherol  might  and  as  f o r m one o r more t o x i c  accompanied  partially  glutathione,  glutathione  was  in  neurotoxic,  cell  disease  N-methyl-4-phenyl-l,2,3,6-  the pathogenesis  neuronal  arises  nigrostriatal  subcutaneous  suggesting of  that  formed  B (MAO-B) t o  dopamine  N-acetylcysteine, mechanism  i s not  c l u e s as t o  different  MPTP,  observation  a single  could  four  antioxidants  cals  MPTP  disorder  m o l e c u l a r mechanism(s)  33% o f (SN).  4 0 mg/kg o f  loss  mice,  nigra  of  molecular  cal  the  of  itself  oxidase  lead to  loss  the  the  Parkinson's  environmental  dopaminergic  MPTP  black  is  or  damage  of  MPTP m i g h t w e l l  that  a contaminant  t h e enzyme monoamine An  is  idiopathic  (MPTP),  can  and m i c e .  of  endogenous  contention  analog,  etiology  hypothesis  more  t e t r a h y d r o p y r i dine idine  the  treatment  of of  with that free mice  the gluradiwith  - 2  diethylmaleate,  a  content  did  by  70%,  subsequently  commonly various ic  not  MPTP c a n p r o d u c e  occurring  agent  analogs of on  compounds  tried,  brain  dopaminergic  of  be  tested  it  idiopathic  PD.  involved  i n C57  in  black  paraquat,  glutathione  dopaminergic  lesion,  nigrostriatal  included  total  discrepancy remains  cases  might  stem  striatal  a parkinsonian  that  which  the  This  most  MPTP w e r e  the  depleted  potentiate  responsible for  effects  that  a d m i n i s t e r e d MPTP.  Although neurotoxin  compound  -  c e r t a i n l y not To  the  identify  a  pathogenesis  mice f o r  possible  However,  decreased  of  unexplained. is  system.  toxicity  more  of  PD,  neurotox-  none  striatal  the  of  the  contents  of  dopamine. Several  epidemiological  quently  among  tective  effect  for  causing  precursor  cigarette of  PD  smokers  smoking  resemble  to  neurotoxic  inhibits  this  enzyme,  might  be  explained.  heavy  cigarette  compared  to  cigarette Using the  another  pretreated  ette zine  than  in  among  first  Using  the  have  enough  Although to  by  as  a mean  the  the  occurs  The  less  fre-  appparent  pro-  compound(s)  responsible  of  an  inactive  and  if  cigarette  mechanism  of  cigarette  source  MAO-B,  of  interesting, for  PD  conversion  MAO-B,  decrease  account  If  requiring  protective  platelets  that  non-smokers.  unexplained.  metabolite(s) then  reported  of  25% o f this  MAO-B  decrease  protection  that  we  smoking !  smoking  found  that  activity  when  might is  not  be  afforded  by  smoking.  development  tine)  MPTP  smokers  large  have  remains  non-smokers.  functionally  studies  known  mice to  smokers,  of  approach PD  with  to  that  discern  appears  one  of  3  accumulate  in  the  and t h e n  pretreatment  gave  be  compounds  mechanism  conferred  fluids  i n j e c t i o n of  reduced  by  (hydrazine,  physiological  them an  substantially  to  the  the  MPTP.  toxicity  of  protection  cigarette  against  smoking,  we  thiocyanate  and  and  of  cigar-  that  hydra-  tissues  We f o u n d of  MPTP,  nico-  suggesting  - 3that  the lower  incidence of  presence of hydrazine Measurements frontal found  cortex) when  neurotoxic  development  chapter  of  patients to  to  when  the report  compared  continued  such  with  It  thereby  by  patients showed  to  in the autopsied brains  (SN and  PD.  appears  No  significant  unlikely  that  changes an  were  overactive  an i n a c t i v e  predisposing certain  PD p a t i e n t s  death.  as a - t o c o p h e r o l  sion of neuronal  in  death  The or  al.  significant  precursor  individuals  to  the  and c o n t r o l  (1982)  that  PD.  Of  reduction  We s u g g e s t  the  subjects,  glutathione 6 regions in  total  this  content  measured,  glutathione  possibilities  that  i n t h e SN o f PD b r a i n s may be t h e r e s u l t  the  detoxification  might  results  3-carotene  i n PD.  et  who d i e w i t h  controls.  consumption  of  Perry  of glutathione  and t h a t  the time  chronic  by t h e i n c l u s i o n o f more PD p a t i e n t s  t h e SN i n PD b r a i n s  radicals,  to  PD.  decreased content its  who d i e d  metabolite(s),  updates  content  be due i n p a r t  causes e x c e s s i v e conversion of  was d e c r e a s e d i n t h e SN o f only  might  bodies.  controls.  in brain  of  Finally,  in their  MAO-B a c t i v i t y w e r e made  compared  MAO-B a c t i v i t y to  of  PD among s m o k e r s  well also might  of  potentially  be e x p o s e d suggest  to  that  be u s e f u l  in  of  neurotoxic  such n e u r o t o x i n s  antioxidant slowing  the  the  up  compounds progres-  -  I)  Idiopathic Parkinson. and  Parkinson's  a disturbance  have  dence  people  than  90^ of  (Bianchine The  i n persons cases,  fundamental  dopaminergic  of  nucleus of  in  a  locus  lesser  brain.  the  might  and p u t a m e n ) only  Losses  rigidity,  1982);  about  however,  after  the  may  200 o f  the  hearing-1  (Barbeau  tremor,  patients  affects  rate  by James  inci-  i n 40 has  1984).  In  age o f  55  have  (1982).  changes  are the only  which  compacta  of  more years  when  degeneration. of from  substantia  decrease  pathway,  (Ehringer  originates the  i s a marked  in  other  the striatum  et  al.  approximately  neuromelanin-  nigra  i n dopamine  1960;  80 % o f  (SN).  A  content  in  (comprised  motor  changes  nigrostriatal  been  been  succinctly  described  summarised  T h e s i g n i f i c a n c e o f some death  1982). important  of  specific  f o r the loss  Perhaps ones  nuclei  n u c l e u s o f t h e vagus  have  neurons,  the abnormalities f o r the c l i n i c a l  as  (1982) is  the  and  uncertain.  might  of dopaminergic  neurons  parkinsonian  Marsden  but  1973).  1982).  the  o f t h e changes  the  although  such  (Forno  for  by  of  Hornykiewicz  neuromelanin-containing  t o compensate  (Marsden  well-defined:  of perikarya are also c o n s i s t e n t l y observed,  n o t b e due t o  striatum  pathway  onset  PD a p p e a r s  the n i g r o s t r i a t a l  extent,  These  functional  its  pathway,  degeneration  neurochemical  Hornykiewicz Many  of  c o e r u l e u s and t h e d o r s a l  Many  (Kurtzke  i n 1817  as 30 % o f  PD  a prevalence  has  the pars  PD a p p e a r  h a v e been l o s t . to  disease  nigrostriatal  neurons  terminus  Symptoms  a s many  t h e a g e o f 65 y e a r s  pathology  consequence o f t h i s  caudate  that  above  the  bradykinesia,  1985).  population  age such  described  1985).  containing  the  (Bianchine  i n the general  been r e p o r t e d  include  In a d d i t i o n ,  dementia  increases with  (PD) was f i r s t  symptoms  posture.  an a c c o m p a n y i n g  100,000  the  of  INTRODUCTION  disease  Characteristic  4 -  merely  influence  be upon  of the n i g r o s t r i a t a l  expression  of  the d i s -  -.5  ease.  Evidence  covered which  neurotoxin,  selectively  dopamine produce 1979;  destroys  application  brains  (Bianchine  other  (these  include  inhibiting  because i n t h e normal  activity.  brain  Thus,  i n PD,  the s t r i a t a l  a c t i v i t y of cholinergic The  benefits carbidopa  often  dramatically,  is  or  not maintained  cross  agonists  studies  Of  dopamine  of  patients  the  blood-brain  (L-3,4-dihydroxyphenylalanine) i s metabolized o f L-DOPA w i t h  choice  via  other such  latter  nigrostriatal  from  benserazide) with  et a l .  for  PD  deficiency  a  as  peripheral  (Bianchine  1985).  mechanisms  bromocriptine)  or  of  useful  drugs  is  by  of the striatum are  (Bartholini  results  the  is beneficial.  dopaminergic  class  pathway  outside  et a l . 1975).  in a relative  over-  neurons.  resulting  (plus  of  The  dopamine  of  MPTP c a n  (Davies  treatment  the c h o l i n e r g i c interneurons  i n h i b i t e d by t h e d o p a m i n e r g i c  loss  neurochemical  not r e a d i l y  o f L-DOPA  PD, a c t  dopaminergic  cholinergic  doses,  s u c h as c a r b i d o p a o r b e n s e r a z i d e  treat  dis-  (MPTP),  resultant  the demonstration  combined: a d m i n i s t r a t i o n  to  direct  extensive  L-DOPA  now t h e t r e a t m e n t  used  a  a n d monkeys  the r a t i o n a l  does  precursor  inhibitor is  drugs  a recently  In s u f f i c i e n t  that  9 5 % o f an .'.oral- d o s e  DOPA d e c a r b o x y l a s e  The  is  has l e d to  immediate  1985),  combination  the rather  Dopamine . i t s e l f  its  by  et a l . 1983).  PD p a t i e n t s  in the striatum  S i n c e about  This  of  with  o f PD i n humans  of  but  supplied  neurons  in the striatum.  important  barrier,  brain  nigrostriatal  a l l t h e symptoms  the d i s e a s e .  is  N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine  Burns  deficiency  does.  contention  Langston et a l . 1983;  autopsied  with  this  and i t s m e t a b o l i t e s nearly  An !.on  supporting  -  the  are remarkable.  a restoration  indefinitely.  introduction  After  of  90%of  of m o b i l i t y . about  therapy  3 years  patients  However, of  with  this  treatment,  L-DOPA respond, response former  -  disabilities ment,  most  (Shaw  et  the  begin patients  worse  than  Curtis  before  continues  Marsden  et  unabated  PD.  loss  can be f o u n d ,  o f symptoms  cell  death  debated 1984) tory Kondo  might  free  the  i s that  fact,  after  L-DOPA  clinical  if of  the e t i o l o g y  radical  aging  of  level  some  several  therapy  of  years of  i n nature  treat-  disability show  of  that  treatment  nigrostriatal  (Ludin of  of  studies  the loss  effects  the cause  et  a l . 1976;  t h e drugs  presently  and do n o t c u r e  on t h e e t i o l o g y  of  the disease  or slowed  if  of  the  idiopath-  the n i g r o s t r i a t a l  may b e p o s s i b l e  In a f f l i c t e d  have r e l e v a n c e  that  patients,  the molecular  f o r the disease.  neuronal  by  devising  the  progres-  basis  of  nigral  with More  akineto-rigid  study  Kurland  the  genetic  recently,  subtype  reported  2 familial  toxicity,  discussion. o f PD h a s b e e n  a positive  a familial  in-  neurotoxins.  i n 1903 ( c i t e d  having  viral  in  Duvoisin  family  frequency  his-  o f 16%.  t h e m u l t i f a c t o r i a l - t h r e s h o l d model  component  transmitted  and m e r i t  b y Gowers  PD f o l l o w s  dopamine  to the pathogenesis  reported  (1958)  heredity,  and e n v i r o n m e n t a l  thesis  of heredity  PD a b o u n d :  system,  mechanisms,  123 p a t i e n t s  (1972) c o n c l u d e d t h a t  idiopathic  for this  An e a r l y  15% o f  of  the nervous  scavenging  f o r many y e a r s .  environment.  In  Clearly,  strategies.  possible contribution  noted  with  be h a l t e d  accelerated  susceptibilty,  an  even  prevention  as t o  These h y p o t h e s e s The  treatment.  6 years  c o u l d be d i s c e r n e d .  Hypotheses  adequate  a l . 1984). patients  After  pretreatment  PD a r e s y m p t o m a t i c  pharmacological  sion  infection,  their  much r e s e a r c h h a s c e n t e r e d  The e x p e c t a t i o n  appropriate  patients.  disorder.  In r e c e n t y e a r s , ic  of  to  of  Thus,  i n the treatment  underlying  et  L-DOPA  a l . 1977).  i n many  returned  of the majority  neurons  used  have  a l . 1980;  condition  is  t o reemerge  6 -  being  subgroups  greater  than  o f PD h a v e  as an a u t o s o m a l  that  been  of  of the  reported:  recessive t r a i t ,  and a  -  subtype of  with  family  other  prominent  members  authors  that  present  study  with  have  not  with  'multisystem  (Duvoisin  and  only  19  1  opment  most c a s e s o f  Between  the  lethargica.  years  Many  postencephalitic patients  of  over  90% l o s s  the  the  than  ism this  PD  of the  neuronal  differences (for  example,  autopsied  that  Marttila  12  However,  when  other  disorders  PD  (for  example,  excluded  against  twins.  a  from  case  had  (Ward  the  significant  Among 43  disease  al.  1920  SN,  monozygo-  definite et  factor  and  the  idiopathic  of  idiopathic tangles) of  (1977,  developed Harris  (8  PD,  al.  1983).  in the  devel-  PD  1978)  et  dopamine  those  of  them  had  brains,  idiopathic supported  1937).  severe  results et  was  neuronal  et  even al.  In more  1973).  parkinsonism  postencephalitic  this  his-  1973).  i n c l u s i o n s which  1973;  disorder  PD.  indicated  al.  depletion  al.  These  a positive  (Bernheimer  et  called  idiopathic  showed  postencephalitic  Lewy body  encephalitis  condition al.  (Bernheimer  (Bernheimer  the  of  of  a  neurochemical  disorder  between  epidemic  and 1 9 2 6 )  content  and  instead  an  1928;  patients  the  exist  was  resembling  years  loss  some f o r m s et  1982).  important  later  features  dopamine  the  there  (Zeigler  striatal  of  prevalence  al.  were  index  the  be an  individuals  of  neurofibrillary  suggests  1926,  of  95% o f  showed  a virus.  and  compacta  some  idiopathic about  1916  pars  that  the  high  PD.  clinical  of  of  a  to  argument  study  for  u n l i k e l y to  examination  degree  Although  in  thus  e n c e p h a l i t i s between  in  severe  concordant  parkinsonism  displayed  loss  fact,  was  tremor)  strongest  which  et  symptoms  essential  in  and  subgroups  and  a recent  pairs  affected  Neuropathological tory  is  signs  the  from  (Barbeau  familial  benign  twin  pair  inheritance of  comes  dizygotic  such  Perhaps  inheritance,  tremor  similar  and  1982).  monozygotic  Genetic  found  atrophies'  -  dominant  essential  rather  genetic contribution tic  tremor,  7  might  are  contention  found  parkinson-  Forno be  and  1982),  caused by  by  report-  -  ing that pathic  viral  PD who w e r e  between of  1916  viral  PD.  antibodies  attempted  to  of  viral  of  a viral  system.  In normal  compacta  of  and of  again  normally  declines that  reveal become  t h e c a s e (Ward  the  d e c l i n i n g motor  PD,  was t a k e n  the  or  decade  various not  a slow  unsuccessful  types  they  the h i s t o l o g i c a l  pigmented age.  et - a l .  have  findings  virus,  (Calne be  advancing  identical  concordance  et a l . 1983). function  Thus,  (1983)  of  have twins  and  et a l .  attributed  e t a l . (1983)  controls  reaction  put f o r t h  (McGeer  time  as i t  et a l .  with  age,  Studies  and speed  trends  of  a r e more  t o b e an  exagger-  2 arguments  against  age a t t h e same However,  t h e f a i l u r e o f L-DOPA  aging  patients  1966).  a g e , but both PD a p p e a r s  of  falls  (Hornykiewicz  should  i n the pars  the case  f o r the d i s e a s e .  Secondly, normal  neurons  in the striatum  i n PD  1981).  et.;al.  In  o f age-matched  sharper  reasoned,  by C a l n e  with  dopamine  slower with  s h o u l d show a h i g h  not  of  t h e same r e l a t i o n s h i p :  Calne  they  of  i s much  i n PD ( E v a r t s  Firstly,  t h e number  exceeds  decline  form o f a g i n g .  and t h u s  been  or  idio-  PD i s due t o a c c e l e r a t e d a g i n g o f t h e n e r v o u s  the content  function  pronounced  this.  have  o f whether  a conventional have  in  m o s t c a s e s o f i d i o p a t h i c PD c a n n o t  i s that  reduction  this  neural  ated  should  do n o t show  them  individuals,  Similarly,  movement  from  t h e SN o r d i n a r i l y  PD, t h i s  1977).  regardless  by e i t h e r  that  lethargica  with  infection.  Another hypothesis  with  encephalitis  PD p a t i e n t s  isolations  i n the sera of p a t i e n t s  a l l individuals  sera,  infection  I t would appear  found  to  However,  in their  the brains  been  exposed  and 1 9 2 6 .  characteristic  1983).  not  antibodies  Also,  have  8 -  does  as a p h a r m a c o l o g i c a l  for  rate,  t h i s was  to  correct  patients  separation  with  of a g -  i n g and P D . The striatal  possible tract,  toxicity is  of  the basis  dopamine, of  yet  the neurotransmitter another  hypothesis.  of In  the vivo  nigroand vn  -  vitro, and  dopamine  can  induce  1983). cals  is cytotoxic DNA  The m e c h a n i s m o f produced  during  enzyme monoamine Sinet  et  al.  hydrogen (u^)*  hydroxyl  1978;  Halliwell  the  Slater  Indeed,  waste product  that  is  dopamine, pathway 1976;  Graham  neurons via  when  from  et  studies  neurological  increased  Cohen  injected of  into free  manganese  disorder  led  of  free  Support  this  al.  toxicity  which (Thoenen  semiquinones 1975).  which,  groups or  into  clinical  man,  symptoms  1980;  subsequent t o be a  the  hypothesis destroys et  line  suggest  et a l .  comes  from  nigrostriatal  a l . 1973),  may a c t  semiquinone,  et of  of  oxidative  (Cohen  -al.  support  can produce  and  to  metabolism  radicals  (Cohen  Another in  of b i o -  al.  and  i n c l u d i n g 6-hydroxydopamine  dopamine et  several  dopamine  for  al.  (Graham 1 9 7 9 ) .  of r e a c t i v e  animals  et  an  membranes,  et  integrity  as  oxygen  i n t h e SN a r e t h o u g h t  6-hydroxydopamine,  radicals,  with  has  cell  Deneke  o f dopamine  shunting  t h e SN o f  Sachs  membrane  such  (McCord  of  the  e t a l . 1978;  a variety  compartmentalisation,  1982).  resembles  a l . 1973; of  dopamine  transport,  the neurotoxin  chemically  Heikkila  of  1973;  pigments  excessive formation  1979;  the formation  which  of  phospholipids  via  Possessing  reactivity  can d i s r u p t  (Tappel  c a n be l o s s  of  in  great  radi-  singlet  (Op,  al.  free  species  semiquinones.  radicals  peroxidation  toxicity  resulting  that  these  the neuromelanin  a disorder  and thus  evidence  them  d e r i v e d from the o x i d a t i o n  potential  PD  lipid  on  polyunsaturated  The r e s u l t  death.  The  the  anion  to  et  or  Graham  1984),  1978),  (Moldeus  (autoxidation,  Graham  dopamine  (Wick  to r e a c t i v e  (Tse e t a l . 1976;  1981;  cells  fibroblasts  metabolism  and  confers  cancer  attributed  superoxide  a l . 1985),  of  1982).  al.  (HO" ) ,  which  especially  process  et  human  has been  dopamine  (H2O2),  et  in  oxidative of  radical  and m u r i n e  breakage  Maker  electron,  molecules,  cell  oxidase)  1980;  human  toxicity  the  peroxide  unpaired  by  strand  to  9 -  a  1974; comes  permanent  neuropathological  and  -  neurochemical Bernheimer  10  characteristics similar  et  al.  1973)..  -  to  Donaldson  idiopathic  et  al.  PD  (Mena  et  al.  (1980)  have  reported  considerably  more  by  1967;  that  the  2+ autoxidation  of  dopamine  was  enhanced  Mn  ions  than 2+  2+ by  other  biologically-important  2 + 2 + Ni  ,  mine  ,  and Mg  et  al.  dopamine,  an  reactive with  free  defence  natural tase vate  of  mechanisms al.  antioxidants radical  et  of  Jakoby  and  which  the  and  al.  1980;to  al.  the  Cu  of  free  the  (Brannan  Another  oxidised  to  Sinet  al.  et  ,  Zn  ,  dopa-  radicals  :  (Tappel  body  et  al.  0,f  and  its  disulfide  1980).  In  GSH  inacti-  the  an  important GSH  (Habig  with et  a  al.  mechanisms,  also  1977;  glu-  reaction,  enzymatic  al.  peroxi-  the  conjugates  probably  dismu-  during  also  semiquinone  et  Such  reduced  GSSG,  this  is  these  Hafeman  which  with  of  equipped  species.  1981)  reaction  a-tocopherol  be  superoxide  by  to  source  glutathione  system which  1965;  would  these  enzyme,  addition  and  potential  enzymes  rate-limiting.  In  a  the  i n c l u d i n g dopamine  ascorbate  in vivo  is  scavenge  include  transferase  1980).  as  or  peroxides  be  electrophiles,  quenchers  and  catalase  becomes  glutathione  such  of  that  detoxify  other  believed  et  likely  respectively.  H2O2  are  generation  is  exist,  1969)  H2O2  GSH  variety  1974;  as  autoxidation  neurotransmitter,  can  et  of  endogenous  that  (Deneke  component wide  such  1980).  mechanisms  (GSH),  process  increased  it  detoxifies  tathione  manganese-enhanced  then  and  levels  This  radicals,  defence  (McCord  dase,  .  associated with  HO- ( D o n a l d s o n If  cations  2+  Ca was  divalent  may  Seregi  act  as  et  al.  1978). In have the in  accordance with  shown SN i s  that  in  the  the  above,  normal  s i g n i f i c a n t l y lower  patients  with  PD,  the  GSH  it  is  significant  autopsied than  in  content  human  other of  brain,  brain  the  that  SN  the  regions is  Perry,et  al.  content analysed,  significantly  of  (1982) GSH  and  in that  decreased  -  from t h a t to  put  of  forth  results  a regional we  the most  observation  that  GSH  the  loss  deficiency  shall  t h e work  support  see  reported  popular  More  meperidine in  analog,  humans  Jenner  et  the  to  al.  et  1984),  such  PD  is  due  years before neurons  onset  in the  pars  dopamine  content,  begin  be m a n i f e s t e d .  to  nigrostriatal  symptoms w o u l d nervous In matter  system, summary, of  but  of  normal  not  to  the  With  In  the  other  the  to  the  the  in  et  al.  a l . 1983; al.  to  etiology  of  observations  the  initial  chronic  man  (Mena  Wright et  These  (1983)  results  is  and  in  threshold  et  al.  1984a; sug-  neurotoxic damage  to  suggested many  a significant  loss  decreases for  would  in  striatal  symptoms  associated  loss  be  of  reached insult  to furand  to  the  grace'.  i d i o p a t h i c PD that  neur-  reports  have  an e n v i r o n m e n t a l  ' c o u p de  a  neurotoxins  necessary the  al. of  al.  a selective  environmental  then  et  synthesis  1983;  is  man-  nigrostriatal  1984a).  and L a n g s t o n  PD  neurotoxins.  of  the  of  i r r e v e r s i b l e neuronal  after  the  to  Langston  may c o n t a i n  aging,  words,  vulnerable  etiology  in  dopaminergic  that  critical  PD  was  ability  SN and p a r a l l e l  extent  (1982) in  environmental  formed  more  This  aging provides  The  lead  al.  neurons  region  symptoms  et  or  normal  the  from  et  Calne  one  compacta of  therefore,  speculation.  can  symptoms*  neurons,  appear.  (Burns  environment  to  to  Langston  Indeed,  exposure,  came  (Heikkila  exposure  pathway. to  the  the  as  exposure  to. damage  monkeys  this  observation  hypothesis  1979;  et  thesis.  contaminant  found  and m i c e  that  nigrostriatal  a  al.  species of  this  parkinsonian  MPTP,  p o s s i b i l i t y that  substance(s)  ther  induce  Perry  nigrostriatal  rendering  in this  current  prompted  of  later,  hypothesis  h a s been  (Davies  several  that  this  recently,  1984),  gest  for  intoxication  1967).  of  This  -  t h e d i s e a s e o c c u r s as a r e s u l t o f  ganese  the  As  o f much o f  Earlier,  ons  controls.  hypothesis  injury.  Perhaps that  the  from  oxidative point  normal  11  MPTP  can  remains induce  presently a  a  selective  - 12 -  lesion of  of  the  PD have  sibly PD. or  nigrostriatal  lent  strong  endogenous)  or  an  is  in  MPTP  1984;  to  is  oxidase  not  al.  clorgyline, Heikkila of  been  shown  rats  (Heikkila  prenyl et  al.  to  dol  et in  al.  the and  of  the  symptoms  to  amount in  be  posof  of  GSH  affording  described  and f o r  MPTP  (or  pathogenesis  the  PD,  of  addition  (Javitch al.  to  1985)  of  that  et  al.  1985)  MAO-B  requires  more  this  research  et  al.  ion  (MPP+)  or  in  reas-  done  has  been  al.  is  when to  by  1985a),  amfolenic  and  13098  (Pileblad  not  MPTP  al.  the  MAO-A  1985),  charged  which  the  1985).  In  the  latter  In  contrast  after  injections  deprenyl  uptake  al.  et  1985)  of  sub-  by  de-  Bradbury MPP+  to  administration.  blockers  (Sundstrom et  this,  of  experi-  prevented to  has  brain  embryonic  be  et  terminal  rat  to  ani-  (Markey  The  into  al.  of  of  1985).  acid  et  positively  injected  reported  dopamine  enzyme  expl ants  al.  neurotoxicity  but  of  the et  the  (Markey  1984b).  intracerebroventricular  inhibitors,  GBR  al.  MAO-B,  added et  et  et  by  Pretreatment  toxicity  (Castagnoli  (Cohen  MPP +  1984b).  the  neurons  when  metabolites  and d e p r e n y l ,  Langston by  conversion  toxic  prevents  oxidation  (Mytilineou  found  or  dopaminergic  neurotoxicity  (1985)  one  tissue culture  pretreatment  (Mayer e t  of  important  model  highlights  but  1984b,  m i c e p r o d u c e d a more e x t e n s i v e In  an a n i m a l  Langston  al.  after  destroy  nigra  the  in  experiments  s u c h as p a r g y l i n e  N-methyl-4-phenylpyridinium  ment,  the  all  environmental  important  critically  of  the  completely et  MPTP,  to  1984b;  inhibitor  stantia  Some  that  mechanisms,  be  neurotoxic,  MAO-B i n h i b i t o r s  metabolite  are  radical  of  nearly  hypothesis  might  some  mals w i t h  1984;  free  create  B (MAO-B)  H e i k k i l a et  al_.  the  produce  order.  itself  monoamine  via  MPTP t o  and  factors  systems  introduction  others  to  susceptibility.  thesis utilise  on,  act  scavenging  resistance this  support  neurotoxic  MPTP c o u l d w e l l other  pathway  al. can  s u c h as 1985),  mazincocaine  completely  or  -  partially active found  prevent  uptake to  the  system  be a p o o r  toxicity for  MPTP  substrate  synaptosomal  preparations  is  at  taken  may  be  up  the  converted  concentrations  same  of  recent  increase atum.  the  Both  atropine bitor  to  cell  publication  dopamine  et  gested  that  extensive  to  degeneration  of  neurons.  uptake  However,  system  of  rat  MPP+  on  dopamine  itself.  This  suggests  (possibly which  in  then  the  of  MPTP  cells  which  gains  access  into  is  striatal  other  glial  an  hand,  that  MPTP  have  high  the  dopa-  destruction occurs.  by  r e s p o n s e s were  MAO-B o r  possibility  1985b).  Hadjiconstantinou  a c e t y l c h o l i n e and d e c r e a s e t h e  of  the  al.  MPP+,  et  al.  dopamine  b l o c k e d by p r e t r e a t m e n t  and t r i h e x y p h e n i d y l .  either  the  (Javitch  MAO-B)  suggesting  dopaminergic  the  as  -  MPTP,  into  extraneuronally  m i n e r g i c n e u r o n s where A  of  for  rate  13  of  Since atropine the  activation  dopamine of  dopaminergic  with  the  mouse  to  these  receptors  nigrostriatal  of  MPTP  to  stri-  anticholinergics  thought  system,  cholinergic  showed  contents  was n o t  uptake  (1985)  be an  inhi-  authors  sug-  might  neurons  after  MPTP  toxicity  contribute  MPTP  admini-  stration. To and  date,,  therefore,  uncertainties  metabolite(s)  of  exist  MPTP.  the as  molecular to  the  mechanism o f precise  identity  of  the  is  unknown  neurotoxic  -  II)  The  following  their  respective  dealt  with:  OUTLINE OF  experiments, sections  of  search for  an a n i m a l  ii.  The  neurotoxic  effects  iii.  The  p o s s i b l e mechanism(s)  Attempts whether  v.  vi.  to the  for  for  most c a s e s o f  The  possible cause(s)  The  i n the  status  activity  would  much  appendices reader In  are  general,  Animals were then Those  the  were  of  to  of  autopsied  protective  performed.  content  and  such  that  in  They  to  determine  conditions,  might  effect  activity  be  responsible  of  smoking  against  in  tissues  might  the  content  already  repeat  the  methodologies  the  following  end  be  of  used  been  this  and  glutathione  transferase  published in  or  detail  thesis  to  is  in  here.  which  press,  it  A series  the  of  interested  details.  procedures  by  a  patients.  has  to  be  PD.  b r a i n s of. PD  experimental  to  MAO-B  work  sacrificed  areas  were  discussed  PD.  glutathione  cervical  d i s s e c t e d according to  brain  be  neurotoxicity,  neurotoxin(s)  the  will  mice,  glutathione  increased  total  this  for  black  MPTP i s a l t e r e d u n d e r  idiopathic  attached  can r e f e r  which  PD.  MPTP  total  natural  that  in the  be r e d u n d a n t  of  of  DISCUSSION,  MPTP i n C57  development  of  of  of  METHODS  PD.  likelihood  The  Since  the  of  of  of  search  factor viii.  toxicity  AND  model  brain  The  development vii.  change  rationale  RESULTS  The  -  EXPERIMENTS AND  the  i.  iv.  14  the for  were  common t o m o s t  dislocation.  instructions the  various  of  experiments:  Relevant Glowinski  neurochemical  brain et  al.  regions (1966).  analyses  were  o  weighed,  and  histological  then  kept  frozen  examination  were  at  -70  C prior  immersed  in  to  analysis. buffered  Brain 10%  stems  formalin  for upon  -  15  -  excision. Analyses  of s t r i a t a l  lic  acid  its  metabolite  in  (HVA)  aH.,  Life  natant  5-hydroxyindoleacetic  electrochemical  injected  chromatographic  into  column  reference  0. 1  M NaH^PO^  per  liter.  was 1 m l / m i n .  1985).  (ODS  HPLC  was a t  External to  system,  60  mg  pH 3 . 6 ± 0 . 0 1 . standards  compensate  the amplitude  were for  were  included  and  a  amperometric  carried  out  C (Perry  e_t  100  mg  injected after  sodium of  respect  octyl  a  t o an  solution  of  sulfate phase  second to  fourth  in sensitivity  due t o  o f t h e compounds  t h e peak. d e f 1 e c t i o n s  with  the mobile  every  decreases  contents  detector  aqueous  rate  super-  reverse-phase  V with  was an  and  and s e r o t o n -  i n Appendix  which  The f l o w  The b r a i n  of  (5-HIAA),  phase  slight  noradrenaline  (MHPG),  was s e t a t + 0 . 7  Na 2 EDTA  homovanil-  25 y l o f e a c h s t r i a t a l  and an  The m o b i l e  e l e c t r o d e and c o l u m n c h a n g e s . from  (DOPAC),  acid  In b r i e f ,  Hypersil)  electrode.  sample o f s t r i a t u m  culated  acid  as d e s c r i b e d  The p o t e n t i a l  containing  This  detection  the  g l a s s y carbon e l e c t r o d e . Ag/AgCl  and i t s m e t a b o l i t e s  3-methoxy-4-hydroxy-phenylethyleneglycol  S c i . 36:1233-1238,  was  o f dopamine  and 3 , 4 - d i h y d r o x y p h e n y l a c e t i c  and i t s m e t a b o l i t e  by HPLC w i t h  contents  they  were  produced  on  cal-  chroma-  tograms. Measurements for  the  Waymire  of  synthesis et - a l .  tyrosine  hydroxylase  of  dopamine)  (1971),  i n which  01.  Total  glutathione  content  t r y  technique of Tietze  et a l . (1968,  of  and o f  brain  and  f o r mouse  the  method  of  livers Tietze  rat  (1969)  a  in  either  by t h e  acid  analyser  The l a t t e r  allowed  monoethyl multiple  the  method  using the for  the  pro-  specimens  experiment,  experiment.  analyses,  of  spectrophotome-  sulfoximine ester  enzyme  2-mercaptoethan-  was u s e d o n l y  the buthionine  glutathione  of  replaced the  o r on an amino  1981).  (the r a t e - l i m i t i n g  modification  was d e t e r m i n e d  brain  i n the  by  dithiothreitol  (1969),  cedure of P e r r y human  were  activity  amino  While acid  -  analyser was  technique  used  for  gave  the  as s t a n d a r d .  For  histological  were  then  stained  compacta  c u t from  of  peduncular nerve. form  with  was a v e r a g e d  will  Cresyl  bodies  and i i ) of  were  about  counted  methods  activities)  be r e f e r r e d  (for  will  stated,  Student's  of  stems  at  -  per day. protein  serial  in  fixed  content,  at 2 l e v e l s : the l e v e l  of  sections  number i)  of  length, the 2  be  assays  all  t-test  of  described  respective  of  bovine  serum  20 vim i n  thick-  These  the l e v e l of  sections  sections,  of  stained  the  zona inter-  cranial  and  fusicount  blind.  and g l u t a t h i o n e  RESULTS  were  the  and t h e c e l l  performed  MAO-B  in  the t h i r d  moderately  in  (1961)  AND  trans-  DISCUSSION,  or  appendices.  statistical  (P =  at  Lowry  perikarya  emergence  These a n a l y s e s were example,  of  of  using  in formalin.  and t h e  each  The method  cyrostat  20 um i n  either  to in t h e i r  otherwise  two-tailed  result  violet,  f o r each mouse.  other  Unless the  the brain  nucleus,  i n shape,  ferase  analyses,  t h e SN was c o u n t e d  Cell  All  1  determination  albumin  ness  only  16  0.05).  analyses  were  performed  using  -  III)  Ill.i)  reduced  glutathione  b r a i n s when oxidants, in  the  Perry  SN  was  compared t o et  makes  this  PD,  nigral  then  acid  have  subsequent  onset  content  disease, the  to  and  potential  development  of  first  replenishing)  al.  1973;  In  might  in  the  was  stop  slow are  be  toxicity  Schultz  quite  a key  of  as  of  that  of in  the  of  The  GSH  result  development  neuronal  loss  the  s i n c e the  and brain  with  the  (assuming  after  the  that  symptoms  disease. drugs  of  ascorbic  can e l e v a t e  neurons  of  or  afflicted  even  progression  PD  reactive  depletion  that  of  of  PD.  already  importance,  of  a-tocopherol  Compounds  loss  autopsied  injury.  nigrostriatal  continues  the  SN o f  scavenger  factor  such  t h e mean c o n t e n t  the  oxidative  further  the  loss  model . t h a t  By  the  of  nigral  resembles the and  manipulating  such a m o d e l ,  used  to  disease  These  presently  fail.  an a n i m a l  required.  in  patients  factor(s)  strategies  i n PD m i g h t  Before dopamine  might  p o s s i b i l i t y that PD,  that  hypothesis  drugs  neurochemical, pharmacological,  is  compound  drugs  is  a contributing  useful.  PD e v e n t u a l l y the  the  prevention  neurotoxic  treatment  test  is  the  be  therefore  used to t r e a t  tics,  also  found  and c o n s e q u e n t symptoms  antioxidant  in  GSH  p a r k i n s o n i a n symptoms.  antioxidant  appear),  logical,  of  might  exposure  To  value  Since  susceptible  loss  with  Parkinson's  (1982)  advanced  region  deficiency  treatment  might  GSH  GSH  al.  of  s i g n i f i c a n t l y lower  (1982)  c o u l d be m a r k e d n e u r o n a l If  et  controls.  al.  DISCUSSION  an a n i m a l model  Perry  (GSH)  -  RESULTS AND  Search for  As d e s c r i b e d e a r l i e r ,  17  perhaps  nigral  etiological  GSH  contributes  diseasein behavioral  GSH  content  importance  of  to  the  neuropathocharacteris-  (depleting this  or  endogenous  determined. of  MPTP  widely  1982).  was to  However,  discovered,  produce we  the  animal  decided  neurotoxin  models  not  to  of use  PD this  6-hydroxy(Thoenen  et  neurotoxin  -  because does  of  the f o l l o w i n g  not penetrate  1982).  effects  pathway  of  minergic  et  free  (Uretsky  less  e t a l . 1970;  nuclei)  dopamine  and  possible  carbidopa  brain,  et  that  to  if  produce  dopaminergic  a l . 1981; rats  nigrostriatal  by s p e c i f i c a l l y  or not the p r i o r Table oral  dose  without to  1  that  L-DOPA  are 993,  from  described 1984).  treatment  failed  et  behavioral in detail  these  of  greater  (that i s ,  are d i f f i c u l t  120  to  react-  e t a l . 1978;  Sinet  days  amounts  a  Normal  a l . , J.  it  L-DOPA i n the  In  addition,  GSH  synthesis  to  test  whether  o f L-DOPA.  maximally-tolerated  and c a r b i d o p a ,  examinations. et  of  content  the t o x i c i t y with  1984),  the rate-limiting  was u s e d  neurochemical  (Perry  Graham  blocks  synthetase,  in rats.  and h i s t o l o g i c a l  of  dopamine  which  0.94 g/kg/day)  tract  generation  be d e s t r o y e d .  et a l . 1979b),  A  dopa-  containing  injections  fed large  might  to produce  i n Appendix  to the  Indeed, those  a l . 1983;  a compound  for  dopaminergic  degen-  To a c h i e v e  Graham  elevation  neurons  (approximately  BSO p r e t r e a t m e n t ,  the n i g r o s t r i a t a l  obtained  1978;  chronically  (Griffith  produce  animals.  d e p l e t i o n o f GSH c o u l d p o t e n t i a t e  shows  of  However,  inhibiting y-glutamyl-cysteine  enzyme f o r GSH s y n t h e s i s  than  intracerebral  Wick  (BSO),  Intracerebroven-  tract.  damage  it  Schultz  in addition  et a l . 1972).  Moldeus  were  neurons  v i a the metabolic  a sustained  DL-buthionine-S,R,-sulfoximine  to  are necessary. i n small  a l . 1973;  to perform,  nigrostriatal  local  r a d i c a l s (Tse e t a l . 1976; Maker  et  peripherally,  the b r a i n .  simple  Sachs  may b e n e u r o t o x i c  Injected  (Thoenen  into  susceptible  6-hydroxydopamine,  brain  a l . 1980;  seemed  directly  are r e l a t i v e l y  p e r f o r m r e l i a b l y and a c c u r a t e l y  ive  barrier  the dopaminergic  appear  s e l e c t i v i t y with  Since  i t s use.  on a l l c a t e c h o l a m i n e - c o n t a i n i n g  neurons  specific  be made  which  interest,  noradrenaline  into  must  injections,  erative  with  the blood-brain  Injections  tricular  problems  18 -  with  evidence of results  or  damage  were  also  The methods  used  Neurochem.  43:990-  -  Table  1.  Tyrosine  hydroxylase  19 -  (TH) a c t i v i t y  and i t s m e t a b o l i t e s  and c o n t e n t s  in rat striatum  c h r o n i c L-DOPA and c a r b i d o p a Drug t r e a t m e n t of animals  TH activity , , , . . x (nmol/h/mg p r o t e i n )  DOPAC  ± 0.11 (15)  15.0 ± 0.5 (15)  L-DOPA,  carbidopa  2.63 ± 0.14 (19)  16.4 ± 0.3 (19)  2.85 ± 0.08 (10)  16.7 ± 0 . 6 (10)  2.70 ± 0.10 (15)  16.5 ± 0.6 (15)  V a l u e s a r e mean ± S E M , w i t h number o f t h e means d i f f e r s i g n i f i c a n t l y .  Table  2 shows t h a t  although  d i d cause a s u b s t a n t i a l  in  the brain  the l i v e r ,  Thus,  levels  in the BSO-pretreated  istration  to  inability  o f BSO t o d e c r e a s e  2  are pooled  BSO  produce  data  administration  ed a t e a c h p o i n t  from  a  were  rats,  brain  1.79 ± 0.11 (8)  0.85 ± 0.05 (8)  in brackets.  They were  d i d not d i f f e r  those  of  at 2 , pooled  lesion  controls.  4,  7,  admin-  may r e f l e c t  The r e s u l t s  for  content  o f L-DOPA a n d c a r b i d o p a  GSH c o n t e n t .  had s t a r t e d .  glutathione  from  nigrostriatal  sacrificed  None  2 0 mM BSO t o r a t s  in total  not d i f f e r e n t  animals  of s a c r i f i c e  0.82 ± 0.05 (8)  of oral  decrease  the f a i l u r e  dopaminergic  1.96 ± 0.26 (8)  of animals  administration  up t o 14 weeks  HVA  ( u3q /a q w e t w e 3i q h t ) ^ '  2.77  controls  after  Dopamine  carbidopa,  Untreated  dopamine  treatment  L-DOPA, BSO  BSO  of  shown  the  in  Table  10 o r 14 weeks  after  because the values  obtain-  s i g n i f i c a n t l y f r o m one a n o t h e r .  -  Table  2.  Effects  of  of  20  -  BSO on t o t a l  brain  glutathione  and l i v e r  of  contents  rats  Control  BSO  Whole b r a i n  2.2  ± 0.1 (10)  Liver  2.8 ± 0.7  2.1  ± 0.1 (10)  0.3 ±  (5) V a l u e s a r e u m o l / g w e t w e i g h t , mean ± m a l s a n a l y s e d shown i n p a r e n t h e s e s . . c a n t l y from c o n t r o l s : < 0.01.  Why  was  Possibly, However, imine,  BSO  this  compound  BSO  was r e p o r t e d  to  regularly  brain.  BSO,  methylene  with  group,  technique  stration  to  of  repeatedly  dopamine  et  al.  followed  to  high  doses  which  a  to  sulfox-  (Griffith  gain  conferred more  barrier.  prothionine  i n mice  able  brain  concentrations  produce  has  of  access  by  the  et  al.  into  the  additional  readily.  Unfortunately, after  in  the  rat  so t h a t  the  i s s u e cannot  only  11  permanent  animal  reported  model  to  injections  was  administration of  an  of  brain  PD,  methylcyclopentadienyl  been  experiment  reminiscent by  was  blood-brain  group,  convulsions  solubility  available  the  agent?  admini-  be  resolved  were  injected  time.  attempt  manganese  a methylene  the  of a n i signifi-  glutathione-depleting  penetrate  compound  entered  d r u g was n o t  This  to  elicit  lipid  BSO  SEM, w i t h t h e number Values that d i f f e r  brain  lacks  latter  i n mice a f t e r  1982).  disturbance be  determine  a compound  of  chronic  with  the  greater  at t h i s  another  that  s h o u l d have  this  conclusively  (MMT),  that  a  unable  of  implying  In  was  as  a congener  1979a),  a  ineffective  0.1J  (6)  of  decrease over  additional  produces  schizophrenia; neurological  an the  a 21  rats  manganese the day  period  phase  pyschosis  disorder  striatal  interest  initial  with  tricarbonyl  (Gianutsos  because of  then  symptoms  content  in  man,  pyschiatric subsides, and  to  neuro-  -  pathological  and n e u r o c h e m i c a l  PD ( B e r n h e i m e r For  et a l . 1973;  21  findings  design, refer  icology  7:19-24,  Table  organic  manganese  manganese c o n t e n t last  of  treated of  75  3 shows  administration  (24  was e l e v a t e d more  injections  given  over  Presumably,  to  those  this  to Appendix that  seen  in  idiopathic  than 2 - f o l d .  content  was due t o  B (Yong e t a l . , N e u r o t o x -  immediately  injections  a 5-month  a n i m a l s h a d a mean manganese  controls.  similar  Mena e t a l . 1 9 6 7 ) .  the experimental 1986).  -  over  48  However,  period, only  after  the period  days),  of  s l i g h t l y higher  rapid  clearing  cerebellar  1 month  the brain  of  after  Manganese  Animals  contents of r a t brain after  Cumulative MMT d o s e  . .Og/kg) . .  1055  3605  dry wt)  than  the  % increase over corresponding c o n t r o l s  168  ± 0.15  2.88 ±  0.11§  18  V a l u e s a r e mean ± S E M , w i t h number o f a n i m a l s shown i n p a r e n theses. B r a i n r e g i o n used f o r a n a l y s i s o f Groups 1 and 3 r a t s was t h e c e r e b e l l u m , whj^le t h a t f o r G r o u p s 2 and 4 a n i m a l s was o c c i p i t a l c o r t e x . P < 0.001; § P < 0 . 0 5 .  those  manganese  MMT t r e a t m e n t  ± 0.03  4.59 ± 0.18  2.44  Group 2 : Controls, 75 i n j e c t i o n s , k i l l e d 1 mo a f t e r l a s t i n j e c t i o n (10) Group 4 : MMT, 75 i n j e c t i o n s , k i l l e d 1 mo a f t e r l a s t i n j e c t i o n (11)  U g / g 1.71  Group 1 : Controls, 24 i n j e c t i o n s , k i l l e d 24 h a f t e r l a s t i n j e c t i o n (8) Group 3 : MMT, 24 i n j e c t i o n s , k i l l e d 24 h a f t e r l a s t i n j e c t i o n (13)  Manganese content  the  manganese-  compound f r o m t h e b r a i n .  Table 3 .  of  . .  -  The  neurochemical  no r e d u c t i o n ity,  in  either  latter  striatal  immediately  group  which might drug  results,  of  animals  have  Thus,  was n o t  t o x i c to  was  chronic  in  in  the  order  SN,  to  assess  large  Tyrosine  hydroxylase  Tyrosine  (8) (13) (10) (11)  in rat  amounts of  striatum after  11.2 11.4 8.5 8.2  ± 0.4 ± 0.4 ± 0.4 ± 0.3  activ-  any  were  organic  The  changes  reversible  upon also  manganese  rats.  MMT  of  treatment HVA  DOPAC  (yg/9  0.1 0.1 0.2 0.2  of  a c t i v i t y and c o n t e n t s  protein)  2.5 ± 2.5 ± 2.7 ± 2.8 ±  were  was  ended.  histologically,  Dopamine  hydroxylase  had  whether  exposure  there  hydroxylase  injections  observed  of  indicate that  tyrosine  MMT  p e r i o d of  administration  (nmpl/h/mg 1 3 2 4  the the  d o p a m i n e and i t s m e t a b o l i t e s  Group Group Group Group  in  4,  dopaminergic n i g r o s t r i a t a l neurons  Table 4.  Animals  or  after  used  Perikarya  in Table  content  month  occurred during  withdrawal.  normal.  1  -  tabulated  dopamine or  22  wet  weight)  1.4 ± 0.1 1 . 2 ± O.f 3.3 ± 0.1 3.2 ± 0.1  0.9 0.8 1.2 1.2  ± = ± ±  0.0 0.0 0.0 0.0  V a l u e s a r e mean ± S E M , w i t h number o f a n i m a l s i n b r a c k e t s . SEM l e s s t h a n 0 . 0 5 i s shown as 0 . 0 . The e x p e r i m e n t a l c h a r a c t e r i s t i c s o f t h e v a r i o u s g r o u p s a r e t h e same a s t h o s e shown on T a b l e 3 . . Values t h a t d i f f e r s i g n i f i c a n t l y from t h e i r r e spective controls: § P < 0.05.  Possible chronic  reasons f o r  administration  colleagues  and  of  rats  our  treated  technique  neurotoxicity i n o r g a n i c Mn  I  of  failure  to  MMT  have  been  that  the  of  suggest was o f  of  the  the  manganese  to o x i d i s e to  has Mn  rather  could been  not  a model  discussed  elevated  organic  measurement  produce  in  manganese than  the  of  (Graham 1 9 8 4 ;  to  in  Appendix content  inorganic  differentiate  suggested  PD  the  B. in  by  Here,  my  the  form,  between  rat  brains although  them.  reside  in  the  ability  Donaldson  et  al.  1982).  The of  -23  The  latter  might  generating potential ity  dopamine to  generate  As  an  the  with  an  humans  of  inorganic  neurons  et  in  that  before  1983;  MPTP  handling, (like  it  treated dant, of  with  might  MPTP.  (Perry  et  Animals  treated increase  of  and  to  guinea  diet.  after  not  reactions,  that  have  have  the  inorganic  would  be  the  capabil-  manganese.  interesting  of  produce  dopaminergic next  al•  to  L-DOPA/carbidopa  degeneration  al.  s e r i e s of  that  1984a),  produce  1984).  A  dopamine  al.  1983).  of  the  efforts  to  Since  in  to  a model  of  PD  synthesize  in  the  species  more  susceptible  their  rate  of  lack  are  of  in  own  to  was  pigs  of  1983; the  re-  chronically  cost  animal  and e a s e  p i g s made  of  scorbutic  ascorbate)  the in  a model  (Kolata  ascorbic acid,  described  Early  non-primate of  guinea  that  protocols  terms  their  of  nigrostri-  produce  guinea  a smaller,  hope was  The  toxic  possible exception  content  neurons  experiments.  MPTP was  particularly in  cannot  10% o f  nigrostriatal  had been g e n e r a l l y u n s u c c e s s f u l  S c i . 36:1233-1238,  about  control  only  et  experimental  lost  et  et  pigs  this  to the  many a d v a n t a g e s ,  Life  might  it  might  demonstrated  striatal  render  bate-deficient  was  Sahgal  The  al.  toxic  rodents  MPTP.  The  radicals  administration  compound  (Heikkila  I attempted  primates,  manganese  experiments,  prompted  (Chiueh  model w o u l d o f f e r  free  transfer  rats.  primates  p o r t e d -50% r e d u c t i o n treated with  one-electron  other  simultaneous  MPTP was  i n mice  al.  above  manganese  PD i n s m a l l l a b o r a t o r y Chiueh  and  Organic  the that  pathway  and l o w e r  by  r a d i c a l s as- h a s been s u g g e s t e d f o r  possibility  t h e MPTP s a g a , atal  free  reports  dopamine  neurons.  extension  nigrostriatal The  oxidise  semiquinones  damage  to  test  then  -  and  then  an  antioxi-  neurotoxic  effects  detail  in  Appendix  on  the  C  1985). initial weight  scorbutic  animals.  a rescue  ascorbate  body loss  Their  weight was  weights  injection  (20  while  similar began  for to  mg/animal)  the  ascorMPTP-  stabilize was  or  given.  -  Clearly,  t h e animals were  the a s c o r b a t e - f r e e Both show for  normal  and s c o r b u t i c  about 4 h a f t e r  or  not,  signs  each  neurochemical  scorbutic  severely  scorbutic during  their  p e r i o d on  diet.  any n e u r o l o g i c a l  The  rendered  24 -  animals  other  injected  than  with  sedation  8 mg/kg  MPTP  and h y p o t o n i a ,  failed  which  results  are not  Striatal  in Tables  highly  parameters  injected with Parameter  Tyrosine hydroxylase Dopamine DOPAC HVA Noradrenaline 5-HIAA  lasted  injection. 5 and 6  susceptible  to  indicate the  that  guinea  neurotoxic  Control  of n o n - s c o r b u t i c  MPTP (8 m g / k g )  f o r 10  MPTP (killed 2 h after last injection)  guinea  pigs  days MPTP ( k i l l e d 30 d a f t e r last injection)  (8)  (4)  (9)  7.5 ± 0 . 3  6.7 ± 0 . 3  6.6 ± 0.3  4.8 ± 0.6 3.4 ± 0.3 3.0 ± 0.2 0.08 ± 0.02 0.21 ± 0.02  6.1 ± 0.6 1.5 ± 0.3* 2.4 ± 0.5 0.07 ± 0 . 0 0 0.36 ± 0.10  5.3 ± 0.6 2.9 ± 0.3 3.0 ± 0.3 0.07 ± 0.02 0.27 ± 0.04  T y r o s i n e h y d r o x y l a s e a c t i v i t y i s e x p r e s s e d i n nmol/h/mg p r o t e i n , w h i l e c o n t e n t s o f n e u r o t r a n s m i t t e r s and m e t a b o l i t e s a r e i n y g / g wet w e i g h t . A l l v a l u e s a r e mean ± S E M , w i t h number of animals i n d i c a t e d in parentheses. SEM l e s s t h a n 0 . 0 0 5 a r e shown as 0 . 0 . Significantly different from c o n t r o l s : + P < 0.01.  pigs,  effects  MPTP.  Table 5.  to  of  -  Table 6.  Effects  o f 15 d a i l y  neurochemical Parameter  25 -  i n j e c t i o n s o f MPTP (8 mg/kg) on  parameters  of s c o r b u t i c guinea  A n i m a l s k i l l e d 24 h a f t e r MPTP i n j e c t i o n s  ... CAT Tyrosine hydroxylase Dopamine DOPAC HVA Noradrenaline 5-HIAA  A n i m a l s k i l l e d 60 d a f t e r MPTP i n j e c t i o n s  MPTP  Control  MPTP  Control  (8)  (8)  pigs  .  (15)  .  .  11.2 ± 0 . 5 5.5 ± 0.1  6.3 ± 0.2  4.9  ± 0.2*  9.0 ± 0.4 1.4 ± 0.1 1.9 ± 0.2 0.28 ± 0.03 0.11 ± 0 . 0 2  7.6 ± 0.6§ 1.3 ± 0.1 2.1 ± 0.3 0.19 ± 0.02§ 0.13 ± 0.02  (15)  .  11.0 ± 0 . 5 4.3 ± 0.1§  6.1 ± 0.7 3.4 ± 0.1 4.2 ± 0.3 0.14 ± 0.03 0.17 ± 0.02  5.0 ± 0.3 2.6 ± 0.1§ 2.9 ± 0.2§ 0.17 ± 0 . 0 2 0.13 ± 0.02  C h o i i n e a c e t y l t r a n s f e r a s e (CAT) a c t i v i t y ( n m o l / 1 0 min/mg protein) was m e a s u r e d i n t h e f r o n t a l c o r t e x w h i l e a l l o t h e r a n a l y s e s w e r e of the s t r i a t u m . T y r o s i n e h y d r o x y l a s e a c t i v i t y i s i n nmol/h/mg protein. Neurotransmitters and m e t a b o l i t e s a r e e x p r e s s e d i n y g / g wet w e i g h t . V a l u e s a r e mean ± SEM w i t h t h e number o f a n i m a l s shown i n p a r e n t h e s e s . Values that d i f f e r from c o n t r o l s k i l l e d at t h e same t i m e : *P < 0.001; § P < 0 . 0 5 .  The ple)  to  could of  apparent  r e s i s t a n c e of  the neurotoxic  be t h a t  the metabolic  MPTP a r e d i f f e r e n t  appear  effects  of  pigs  MPTP  pathway(s)  i n guinea  t o be a s u i t a b l e s m a l l  111. i i ) S t u d i e s  guinea  pigs.  animal  remains or  the report  susceptible  to  the  this  of  mice  strain  effects  of  MPTP  by H e i k k i l a  on t h e  dopaminergic  of  These  for  exam-  unexplained.  It  l o c a l i s a t i o n and p e r s i s t e n c e the guinea  p i g does  not  parkinsonism.  effects  o f MPTP  mice  et a l .  effects  pursued.  presently  f o r MPTP-induced  of the neurotoxic  neurotoxic were  the  to primates,  In a n y e v e n t ,  i n C57 b l a c k Following  (relative  (1984a) MPTP,  began  that  C57 b l a c k  various with  nigrostriatal  mice  experiments  an e x a m i n a t i o n  neurons  of  these  were using  of  the  mice.  -  Experiments  were  designed  a single  1)  depletion  loss  i n t h e zona compacta o f t h e S N , 3)  of  c a u s e d by d i f f e r e n t  MPTP f o r t h e d e p l e t i o n  ous  (sc)  given  experimental  are described  1985).  dosage  the time of  MPTP,  2)  the degree  regimens  striatal  course  of  A l l administrations  striatal  the extent  of  of  striatal  MPTP,  dopamine  of  and 4)  content  dopa-  neuronal  dopamine  the  de-  selectivity  as o p p o s e d  o f MPTP w e r e b y t h e  to  other  subcutane-  route.  The above  injection  of  neurotransmitter. amines.  -  assess:  mine  pletion  after  to  26 -  To  assess  protocols  used  i n Appendix the  for  the  D (Perry  selectivity  of  et  h and 1 month  and  i t s 2 metabolites  metabolite  after  5-HIAA,  and  of  and HVA,  of  MPTP,  and t h e n  the i n j e c t i o n .  DOPAC  a l . Neurosci;  action  a s i n g l e s c i n j e c t i o n o f 40 mg/kg,  2.5  determination  In  were  addition  striatal  noradrenaline  1,  Lett.  C57  2,  to  black  mice  of  dopamine  serotonin  its  metabolite  of motor  impairment  were  a t 30 m i n ,  contents of  and 3  12:321—326,  sacrificed  contents  and  of  and i t s  MHPG,  were  measured. Behavioral 20 m i n u t e s (though  after  moved  extended  h after  indicated  when  hind  At  most  were  mice  tremors. often  Body  seen  3 o r 4 days  extended  hind  still posture  this  after  3 0 mg/kg MPTP c a u s e d v e r y  absent.  displaying  they  animals appeared  behavioral  24  and  occurred  could  marked  resembling  Thereafter,  little  tremors  h  difficulty between  e v e n when after  the  hypokinesia  gradually normal.  when  2 and  disturbed. injection, and  often  that  recovered,  Sedation,  in  observed  posture  Injections  as  sedation  be r e a d i l y  the kyphotic  change.  as e a r l y  consisted of  tremors,  impairment  time,  was  was h u n c h e d ,  r e c e i v i n g MPTP,  These  i n i n i t i a t i n g movements  limbs  sedated,  i n PD p a t i e n t s .  slight  Maximal  and m i c e h a d d i s t i n c t d i f f i c u l t y of  MPTP.  disturbed),  limbs.  the i n j e c t i o n .  Retraction  signs  an i n j e c t i o n o f 4 0 mg/kg  animals  retracting 2.5  observations  is  and b y  o f 20 o r observed,  -  lasted of  for  only 0.5  2 a n i m a l s so Our  the  1  h.  60 mg/kg  suggest  dose  that  results  changes At  in  30  and  60  dopamine  However,  in  was  at  but  SN t o  139 ± 11 Why (90%)  and  MPTP?  function  of  the lase,  partial  after  of  to  2  loss The  mg/kg  animals.  MPTP  is  10%  of  About  of  months  at 4 . 5  6 control ± SEM)  a single  neurochemical 40  was  C57  black  the  at  severeproduced  striatum.  MPTP  injection,  nigrostriatal  striatum,  increased  as  the  DOPAC  striatal  mice  in  MPTP.  and  almost  dopaminergic the  mg/kg  injection,  after  of in  was  it  in  of  metabolites  recovery  ratio,  of  increased,  MPTP  h,  MPTP  released  course  the  its  4.5  normal  (mean  disparity of  as  24  h  judged and  by  at  one  months.  mice  showed  perikarya  i n j e c t i o n of  probable  enzyme been  between  neurons  many d o p a m i n e r g i c  have  40  per  the  unit  40 mg/kg  pars  compacta  area.  Those  of  MPTP c o n t a i n e d  of 7  only  (P<0.001). such  rate-limiting might  molar  towards  and  for  dopamine  examinations  there  40 mg/kg  of  injection  and b y 24  dopamine  of  1 month  the  lethal  modestly  after  40 mg/kg  evidence  perikarya is  Thus,  time  was  h  reduced,  live  c o n t a i n 206 ± 5  mice k i l l e d  was r a p i d l y  most  a single  2.5  to  had r e t u r n e d  Histological the  By  of  of  of  the  content  allowed  turnover  injection  show  after  dopamine  HVA)/dopamine  +  7  mice  one m o n t h .  mice  sc  survival  Table  of  decrements  The  t h e . (DOPAC month,  in  striatum  clear  neurons.  however,  such a d o s e .  c o n t e n t was m a r k e d l y  long-lasting  there  given  minutes,  r e d u c e d as  MPTP,  single  with  comparably r e d u c e d .  content  ly  the  a  compatible  animals generally die a f t e r The  -  treated.  results  highest  to  27  for  (33%)  reason  is  neurons, the  chronically  the at  of  month  after  1  that but  magnitude  the  MPTP  d i d not  synthesis inhibited  of by  dopamine  injection  injection  kill  them.  dopamine, MPTP.  an  reduction  impaired For  the  instance,  tyrosine  This  of  appears  hydroxylikely  -  since  Hirata  lase  activity  the  et a l . in  (1985)  tissue  have  slices  28 -  shown of  the rat  i n h i b i t e d neurons would r e c o v e r ,  Table 7. of  Time  striatum.  tyrosine  Given  time,  hydroxyhowever,  as b o r n e o u t b y t h e d o p a m i n e c o n t e n t  30 min  (26)  60 min  2.5 h  (9)  (5)  (9)  11.8 ± 0.4 0.96 ± 0.04 1.22 ± 0.07 0.16 ± 0.01  13.8 ± 0 . 6 § 1 4 . 1 ± 1.1 0.42 ± 0.04 0.55 ± 0.09 1.32 ± 0 . 0 6 . 1.22 ± 0 . 0 6 . 0.11 ± 0.01 0.11 ± 0.01  24 h .(5) Dopamine DOPAC HVA (DOPAC + HVA)/ Dopamine  inhibits  at  an i n j e c t i o n o f 4 0 mg/kg MPTP  Controls  Dopamine DOPAC HVA (DOPAC + HVA)/ Dopamine  MPTP  course of neurochemical changes i n s t r i a t u m  C57 b l a c k m i c e a f t e r  .  that  1  6.4 ± 0.5 0.46 ± 0.05 2.11 ± 0.07 0.35 ± 0.04  month  4.5  . .(141.  .  1.5 ± 0.4 „ 0.20 ± 0.03 0 . 6 3 ± O.IO" 0.54 ± 0 . 0 8 '  months (8).  4.4 ± 0.34 0.74 0.24 ±  1.1 ± 0 . 1 0.22 0.04 0.39 0.02" 0.52 ± 0.06  1.1 0.06 0.10' 0.02  V a l u e s shown (mean ± SEM) a r e i n p g / g w e t w e i g h t , e x c e p t t h a t (DOPAC + H V A ) / D o p a m i n e r a t i o s a r e m o l a r . M i c e were sacrif i c e d at the time i n t e r v a l s s p e c i f i e d a f t e r a s i n g l e i n j e c t i o n o f 4 0 mg/kg o f MPTP. The number o f a n i m a l s a r e shown i n ^parentheses. Values that d i f f e r s i g n i f i c a n t l y from c o n t r o l s : P < 0.001; § P < 0 . 0 5 .  4.5  months  than that Table in  animals  mg/kg. last  after at 1  t h e s i n g l e 4 0 mg/kg  given  the s t r i a t a l  either  groups  injection.  (Table  7),  which  was h i g h e r  month.  8 compares  Both  injection  of  The  contents  a s i n g l e 4 0 mg/kg a n i m a l s were  results  show  of  injection,  sacrificed that  a  dopamine or  a month  single  and i t s 5  injections of  after  insult  metabolites  the s i n g l e  resulting  from  20 or a  -  large than  dose  of  multiple  MPTP  is  injections  Table  9,  which  still  significantly  lowering  These  achieved  only  reached. ic  At  Table  that  of  less  results a  lower  contents  suggest critical  striatal  of  dopaminergic  a  This  from  at  24  than  of  parameters Dopamine DOPAC HVA  1 x 40  mg/kg  of  0.1 0.05 0.06  (13  the a  by  last  injection,  injection  such  of  neurotoxic  MPTP  (and  detoxifying  that  thus  agents  data  x 30 m g / k g )  single  the  neurons  the  effect  of  dosage s c h e d u l e s of  dopamine  and i t s the  MPTP on  5 x 20  mg/kg  4.6 ± 0.39 ± 0.83 ±  neurotoxpresent  injection of  significance 0.4 0.03 0.07  P < P < P <  0.001 0.01 0.001  V a l u e s a r e e x p r e s s e d i n u g / g w e t w e i g h t , mean ± SEM, with number of mice analysed shown in parentheses. Student's t w o - t a i l e d t - t e s t was u s e d t o d e t e r m i n e l e v e l o f s i g n i f i c a n c e . C o n t r o l v a l u e s , i n u g / g w e t w e i g h t (mean ± S E M ) , are: dopamine, 11.8 ± 0 . 4 ; DOPAC, 0 . 9 6 ± 0 . 0 4 ; HVA, 1 . 2 2 ± 0 . 0 7 .  40  is  the  Level  in  species  metabolites, last  was  is  the  may be  of  in  injury  toxicity.  (10)  (10) 1.1 ± 0.21 ± 0.39 ±  supported  after  that  i n mice s a c r i f i c e d 1-month a f t e r Striatal  h  of  nigrostriatal  390 mg/kg  threshold  MPTP),  different  contents  is  dose o f  concentrations  formed  Comparison  to  concentration  s u f f i c i e n t to reduce the  8.  -  doses.  effective,  sub-threshold are  damaging  a cumulative  dopamine  when  species that  amounts  show  striatal  mg/kg.  more  29  in  - 30 -  Table  9.  Comparison striatal  of  different  contents  of  dosage s c h e d u l e s of  dopamine  and i t s  i n m i c e s a c r i f i c e d 24 h a f t e r Striatal  1 x 40  parameters  to Table 8 f o r  Table  10  to  shows  the  and  those  noradrenaline of  the 1 h or different  the from  ous1 amines. affected.  MAO  amines.  These  inhibitor  1 month  mark,  controls. At  1  month,  of At  contents,  controls.  a c t e d as a weak  Not  selectivity  neurotransmitter  injection Level  mg/kg  (Fritz only  only  30  al.  1985;  dopaminergic  acutely, the  MPTP  dopamine  ns ns  MPTP on  have  0.05  P<  ns.  after  dopamine,  might et  of  minutes  as w i t h  changes  Thus,  significant:  action  of  significance  do) 2.4 ± 0.2 0 . 2 3 ± 0.02 0.48 ± 0.03  legend.  the  metabolites,  last  x 30  .  1.5 ± 0.4 0.20 ± 0.03 0.63 ± 0.10  Refer  tonin  13  (5)  Dopamine DOPAC HVA  various  mg/kg  the  MPTP on  striatal 40  were  been  Singer  to et  al.  were  remains  of  sero-  relative  MPTP's  non-selectively system  MPTP,  increased  due  parameters  mg/kg  content  having  1985).  At  statistically affects  vari-  selectively  -  Table 10.  Striatal  at v a r i o u s  times  31  neurochemical after  -  parameters  of mice  sacrificed  a s i n g l e i n j e c t i o n o f 4 0 mg/kg  MPTP  MPTP Controls  11.8  Dopamine  •' 30 m i n  ± 0.4 (26)  2.5 h 6.4  13.8 ± 0 . 6 § (9)  DOPAC  0.96 ± 0.04 (26)  0.42 ± (9)  HVA  1.22 ± 0.07 (26)  1.32 ± 0.06  Serotonin  0.37 ± 0.03 (21)  0.55 ± (9)  5-HIAA  0.24 ± 0.04 (26)  Noradrenaline  0.26 ± 0.04  0.08 ± 0.01 (10)  ± 0.5* (5)  1.1  ± 0.1* (14)  0.46 ± 0 . 0 5 * (5)  0.22 ± 0 . 0 4 * (14)  2.11  0.39 ± 0 . 0 2 * (14)  (9)  (7) MHPG  0.04*  1 mo  ± 0.07* (5)  0.72 ± 0 . 0 7 * (5)  0.33 ± 0.04 (14)  0.25 ± 0.02 (9)  0.19 ± 0 . 0 3 (5)  0.22  ± 0.03 (13)  0.49 ± 0.06 + (4)  0.34 ± 0.06 (5)  0.17  ± 0.03 (8)  0.09 ± 0.02 (5)  0.05 ± 0.00 (5)  0.07 ± 0.02 (8)  0.09§  V a l u e s (mean ± SEM) a r e i n y g / g w e t w e i g h t , w i t h t h e number o f a n i m a l s a n a l y s e d shown i n p a r e n t h e s e s . SEM l e s s t h a n 0 . 0 0 5 a r e g i v e n as 0 . 0 0 . Values t h a t d i f f e r s i g n i f i c a n t l y from c o n t r o l s : P < 0.001;+ P < 0.01; § P < 0 . 0 5 .  In  summary,  single gic  lasting  seen Thus,  effects  of  i n j e c t i o n o f 4 0 mg/kg  neurons  not  the  in  of the  of  striatal  serotonin first  several  a s i n g l e 4 0 mg/kg  chemical magnitude  and of  or  of  C57  of  noradrenaline. days,  dose  of  but MPTP  black  a loss  t h e SN.  contents  neuropathological the  in  MPTP p r o d u c e s  i n the zona compacta  reduction  those  MPTP  about  appears  to  is  as  follows:  33% of  not  impairment persist  produce of less  human  by a  than  is  long-  that  but  readily  indefinitely.  i n mice PD,  a  dopaminer-  and i t s m e t a b o l i t e s ,  Behavioral does  are  i s accompanied  dopamine  this  changes  of  This  characteristics  neuropathological  mice  the  neuro-  although seen  in  the the  - 32 -  human c o n d i t i o n .  111. i i i ) A question minergic olites  that  has  reactive  MPTP, free  such  as  radicals  experiment  have  60:109-114,  could been  toxicity  whether is  1985).  example,  dopamine  antioxidants  teine)  saline  or  the  third  injection  of  once  day  to  MPTP.  metabolites.  performed  prevent  In  brief,  exerted  the in  all The  11  MPTP-injected pared to  shows  control  than those o f  mice not  Since  radicals, toxicity  the  or  the  antioxidants  involvement  appeared  likely.  of  of  the  t h e s e were  4  1  with or  differof  one  this Lett. of  administered month of  4  N-acetylcyssc  after  the  dopamine  and  blind  contents 4  were  in  MPTP  a  still  latter  protected  common in  the  reduced  antioxidants,  property  when  in.  com-  s i g n i f i c a n t l y higher  a simultaneous a n t i o x i d a n t .  partially  share the  MPTP  performed  dopamine  a n y one  MPTP,  were  injected  contents  were  This  saline.  striatal  with  one o f  ascorbate  for  formation  Neurosci.  mg/kg  by  neurotoxicity.  al.  sacrificed  analysed  whose  et  were  examinations  given  mice  or  (MPP+),  Details  Mice  m i c e g i v e n MPTP w i t h o u t  antioxidant-pretreated  ion  MPTP.  40  although  mice p r e t r e a t e d  were  e-carotene,  s t r i a t u m was  that  metab-  any  with  groups.  Histological  of  (Perry  mice  dopa-  d i r e c t l y by  MPTP  5 days,  mice p r e t r e a t e d w i t h a - t o c o p h e r o l Table  black  E  to  MPTP m e t a b o l i t e s ) .  whether  neurotoxicity  C57  for  assess  Appendix  damage  semiquinones,  presence of  to  (o-tocopherol, daily  the  N-methyl-4-phenylpyridinium  described  different  MPTP.  is  MPTP  increased in the  were f i r s t  antioxidants  its  MPTP  d e a l s w i t h the p o s s i b l e m o l e c u l a r mechanism o f  Experiments  on  unanswered  c a u s e d by  the  (for  f r o m d o p a m i n e m i g h t be  ent  remained  n i g r o s t r i a t a l neurons  of  section  P o s s i b l e mechanism o f  from of  molecular  the  Thus,  the  toxicity  of  detoxifying  free  mechanism of  MPTP  -  Histological  examinations  indicated  W h i l e 7 m i c e a d m i n i s t e r e d 4 0 mg/kg (mean ± S E M ) ,  those  ± 2 perikarya  (P<0.001).  karya gic al  per u n i t  neuronal  area.  cell  ity  came f r o m  our  studies stem.  an e n d o g e n o u s  Lett.  free  time  Tietze  (1969)  in  obtained.  No c h a n g e s  striatum.  In  initial  the  scavenger, radicals.  an  glutathione stem  area  had 215  showed 206 ± 5 of  injection,  free  in total  Appendix of  this  of  contents  peri-  dopaminer-  this  neuron-  a  i n MPTP  F  as  (Yong e t a l .  MPTP,  measured  Table  12  might  C57  black  at. mice  b y t h e method gives  response  i n c r e a s e a t 1 h was f o l l o w e d b y a s i g n i f i c a n t r e d u c t i o n  be  Neurosci.  Briefly,  glutathione  biphasic  con-  glutathione  content  experiment.  were  toxic-  glutathione  inasmuch  4 0 mg/kg  i n the t o t a l  however,  radicals  glutathione  and s t r i a t u m .  observed  stem,  of  total  injection  brain  per u n i t  was a 3 3 % l o s s  h e r e was t h a t  after  were  result.  and o - t o c o p h e r o l  no MPTP  changes  details  brain  impressive  perikarya  a s i n g l e MPTP  provides  Total the  there  MPTP-induced  via free  intervals  MPTP  given  involvement  The r a t i o n a l e  1986)  sacrificed.  of  more  by a - t o c o p h e r o l .  the  radical  MPTP w o r k s  63_:56-60,  various were  if  for  both  animals  after  prevented  even  MPTP had 139 ± 11  whereas  1 month  evidence  tent of the brain  reduced,  Control  bodies  an  injected with  Therefore,  l o s s was c o m p l e t e l y Additional  is  ( n = 7)  33 -  the  content  of  results of  the  was  seen.  An  in  total  Table  11.  Contents  of  dopamine and i t s m e t a b o l i t e s  o f m i c e t r e a t e d w i t h MPTP and p r o t e c t i v e  Dopamine  Controls  MPTP alone  MPTP + a-tocopherol  (26)  (14)  (12)  MPTP + B-carotene  i n the  striatum  agents MPTP + ascorbate  MPTP + N-acetylc y s t e i ne  (7)  (4)  (7)  11.8  ±  0.4*  1.1  ±  0.1  4.5  ±  0.4*  3.5  ±  0.4*  3.9 ±  0.8*  4.4  ±  0.6*  DOPAC  1.0  ±  0.0*  0.2  ±  0.0  0.4  ±  0.0  +  0.3  ±  0.0  0.6 ±  0.2  +  0.7  ±  0.2+  HVA  1.2  ±  0.1*  0.4  ±  0.0  0.8  ±  0.0*  0.6  ±  0.0*  0.8 ±  0.1*  0.8  ±  0.1*  V a l u e s (mean ± SEM) a r e e x p r e s s e d i n y g / g w e t w e i g h t , w i t h SEM l e s s t h a n 0.05 l i s t e d a s 0.0. Number o f a n i m a l s a n a l y s e d a r e shown i n b r a c k e t s . E x c e p t f o r c o n t r o l s , a l l m i c e w e r e g i v e n a s i n g l e s c i n j e c t i o n o f 40 mg/kg M P T P , and k i l l e d 1 mo a f t e r . P r o t e c t i v e agents were i n j e c t e d sc d a i l y f o r 5 d a y s , s t a r t i n g 48 h b e f o r e MPTP. D a i l y doses were: a-tocopherol, 2.35 g/kg; e - c a r o t e n e , 100 m g / k g ; L-ascorbate, 100 m g / k g ; and N - a c e t y l c y s t e i n e 500 m g / k g . V a l u e s t h a t d i f f e r s i g n i f i c a n t l y f r o m MPTP a l o n e : *P  < 0.001;  +  P <  0.01.  -  Table  12.  Effects  content  of  35  -  o f .MPTP i n j e c t i o n s on t o t a l s t r i a t u m and b r a i n s t e m o f  Controls  glutathione mice  MPTP 30 m i n  1 h  2.5  h  24 h  24h ( a f t e r 3rd injection)  Striatum 1.08  1.01  ± 0.07 (19)  0.93 ± 0.12 (10)  ± 0.04 (22)  1.02  1.01  ± 0.06 (10)  1.16  ± 0.05 (14)  1.06  Brain  stem  ± 0.06§ (16)  1.12  ± 0.05 (15)  1.01  ± 0.03 (18)  1.10  ± 0.07 (14)  0.75  ± 0 . 0 5 * 0.76 (18)  ± 0.04 (15)  ± 0.04* (15)  T o t a l g l u t a t h i o n e . v a l u e s (mean ± SEM) a r e e x p r e s s e d i n y m o l / g w e t w e i g h t , w i t h t h e number o f a n i m a l s shown i n p a r e n t h e s e s . Mice were g i v e n a s i n g l e s c i n j e c t i o n o f 40 mg/kg o f MPTP and k i l l e d a t t h e time i n t e r v a l s i n d i c a t e d , except f o r animals in the l a s t column, w h i c h w e r e k i l l e d 24 h a f t e r t h e l a s t o f 3 d a i l y MPTP ( 4 0 m g / k g ) injections. Values that d i f f e r s i g n i f i c a n t l y from c o n t r o l s : P < 0.001; § P < 0.05.  glutathione in  mice which  were k i l l e d Because out  content  this  therefore  had  at been  24 h a f t e r of  area  the  changes  SN,  in  of  and n o t  the  change i n t h i s  in Table  12  for  size  study  reduction  injections  of  was  not  more  40 mg/kg  pronounced  MPTP  and  of  a much  the  SN,  free  of  larger  I  was  unable  to  contaminating t i s s u e mass,  reliably  dissect  structures.  the  brain  stem,  w h i c h d i s s e c t i o n was e a s y and r e p r o d u c i b l e .  surrounding  in  the  brain  non-nigral  s t r u c t u r e would stem.  which  injection.  brain  observed  brain  latter  3 daily  the t h i r d  and f o r  glutathione  This  given  frozen  to  i n c l u d e d the SN,  h.  minute  from  forced  24  probably  stem were tissues, have  occurring  then  the  been g r e a t e r  only  actual than  I  was which  If  the  in  the  magnitude that  shown  -36  One of  plausible  MPTP-induced  synthesis followed  and by  free hence  were  after  formed  neuronal  tection on  the in  MPTP  the  the  MPTP-induced  mined.  Animals  dants.  40  thione An  content  of  brain 3  was  t h e mice were  as  an  the it  that  brain  is  suggests of  the  property,  glutathione  in  follows.  stem.  This  in  the  glutathione  that  The  increase in  consumed  the  presence  glutathione  rise  is  process  content  free  nigrostriatal  and  then  of  de-  did  not  radicals,  tract,  MPTP,  the  stem  daily  and e - c a r o t e n e effects  total  if  not  any,  in  injections  injected  of  immediately Appendix  F  i n c l u d i n g the doses of 13  show  that  the  these  data  these  exogenous  the  is  that  pretreatment content  either  after  the  by each of sharing  of  the had  MPTP-derived  MPTP was a t t e n u a t e d  (Table  loss  these  third  them deter-  antioxi-  injection. details  of  used. of  these same  total  free  gluta-  antioxidants. free  substituted  11).  pro-  with  were  further  antioxidants  MPTP-induced  by  of  the  provides  antioxidants  detoxification  the p r o c e s s , the t o x i c i t y of  of  had e a c h o f f e r e d  glutathione  i n t h e b r a i n s t e m was p r e v e n t e d of  as  initial  striatal  sacrificed.  in Table  interpretation  aided,  of  design,  in  a-tocopherol  given  MPTP  to  is  striatum.  toxicity  were  mg/kg  results  scavenging  in the  changes  the experimental The  bodies  results  lead  content  injection  cell  the  the  depletion,  antioxidants  against  24 h l a t e r  its  observation  terminals  Since  of  The  of  r a d i c a l s may  glutathione  toxification. change  intepretation  -  radicalfor,  radicals.  or In  -  Table  13.  Total  glutathione  37 -  contents  o f b r a i n stem and s t r i a t u m  o f m i c e i n j e c t e d w i t h 4 0 mg/kg MPTP, w i t h antioxidant Controls  without  pretreatment  No p r e t r e a t m e n t  (Saline)  or  s-carotene  a-tocopherol  and MPTP  and MPTP  (MPTP-alone)  Brainstem  1.01 ± 0 . 0 4 * (22)  0.74 ± 0.05 (18)  1.07 ± 0 . 0 6 * (10)  0 . 9 8 ± 0.04+ (10)  Striatum  1.08 ± 0.07 (19)  1.01 ± 0.03 (18)  1.04 ± 0.05 (10)  1.08 ± 0.04 (10)  V a l u e s a r e i n y m o l / g w e t w e i g h t , mean ± S E M , w i t h number o f s a m p l e s a n a l y s e d shown i n b r a c k e t s . D a i l y doses o f a - t o c o p h e r o l a n d s - c a r o t e n e w e r e 2 . 3 5 g / k g a n d 100 mg/kg r e s p e c t i v e ly. Values that d i f f e r s i g n i f i c a n t l y from MPTP-alone a n i m a l s :  * P < 0.001; t p < 0 . 0 1 . If then  free  radicals  is their  are involved  identity?  Castagnoli  c a l s d e r i v e d from dopamine might mice  either  days.  Saline  particulars be  a normal  unusually  turnover Despite shown  diet  o r 2 0 mg/kg  of  i n Table  however,  be i n v o l v e d .  and a g g r e s s i v e ,  the toxicity  1 5 , where  and c a r b i d o p a Appendix which  F  the dopaminergic  t h e L-DOPA  o f MPTP i s i n d e p e n d e n t  and c a r b i d o p a  of free radical  diet. formation  mice  (Table  that  given  the to  dopaminergic  i t c a n be s e e n  were n o t  for 3  gives  caused  increased  diet  radi-  I f e d C57 b l a c k  in the striatum  f e d a normal  what  free  was n o t p o t e n t i a t e d .  different  neurotoxicity  that  MPTP  animals  those  treatment,  ratio]  this,  toxicity,  of  the s t r i a t a of MPTP-treated from  sc.  substantially  molar  MPTP  suggested  L-DOPA  injected  The L-DOPA  of  To t e s t  containing  MPTP was t h e n  [(DOPAC + H V A ) / d o p a m i n e this,  e t a l . (1985)  or a d i e t  the experiment. active  i n t h e mechanism  14).  This  is  parameters  in  statistically  Apparently, from  the  dopamine.  -  Table striatal  14.  Effects  contents  38 -  o f L-DOPA and c a r b i d o p a t r e a t m e n t  o f dopamine  and i t s m e t a b o l i t e s  diet)  1st  (26) Dopamine DOPAC HVA (HVA + DOPAC)/ dopamine  :  11.8 0.96 1.22 0.16  i n t h e mouse  L-DOPA and c a r b i d o p a  Controls (normal  on  2nd  group3  11.1 4.92 6.68 1.00  0.4 0.04 0.07 0.01  ± ± ± ±  groupk (10)  (13) ± ± ± ±  diet  0.7 0.73* 0.29* 0.13*  12.0 0.80 1.26 0.15  ± ± ± ±  0.8 0.07 0.07 0.01  V a l u e s f o r dopamine and i t s m e t a b o l i t e s a r e e x p r e s s e d i n u g / g wet w e i g h t , while (DOPAC + H V A ) / d o p a m i n e v a l u e s are molar ratios. A l l v a l u e s a r e mean ± S E M , w i t h number o f s a m p l e s shown i n b r a c k e t s . a M i c e s a c r i f i c e d i m m e d i a t e l y a f t e r 3 d a y s o f t h e L-DOPA and c a r b i d o p a d i e t , a t t h e t i m e when o t h e r s i m i larly-treated mice were given 2 0 mg/kg MPTP (Table 15). ^ M i c e s a c r i f i c e d 3 weeks a f t e r c e s s a t i o n o f 11 d a y s o f t h e s p e c i a l d i e t , when m i c e g i v e n 2 0 mg/kg MPTP p l u s L-DOPA and c a r b i d o p a d i e t , were s a c r i f i c e d ( T a b l e 1 5 ) . Values that diff e r s i g n i f i c a n t l y from c o n t r o l s : *P < 0.001.  Table  15.  Striatal  mice g i v e n  contents 20 mg/kg  o f dopamine  MPTP, w i t h  and c a r b i d o p a Normal  Dopamine DOPAC HVA  and i t s m e t a b o l i t e s  or without  in  L-DOPA  pretreatment  diet  L-DOPA + c a r b i d o p a  Controls  MPTP  and MPTP  (26)  (9)  (9)  11.8 ± 0.4* 0.96 ± 0.04 1.22 ± 0 . 0 7 §  6.9 ± 0.4 0.59 ± 0.04 1.02 ± 0.06  7.0 ± 0.8 0.58 ± 0.05 0.98 ± 0.07  V a l u e s a r e mean ± S E M , u g / g w e t w e i g h t . Number o f a n i m a l s a n a l y s e d a r e shown i n p a r e n t h e s e s . Values that d i f f e r signif i c a n t l y f r o m 2 0 mg/kg MPTP m i c e on n o r m a l d i e t : P< 0 . 0 0 1 ; § P< 0 . 0 5 .  -  To  determine  of  its  of  MPP+.  after body  parent  whether  compound,  Behavioral  an  injection  posture  was  up k y p h o t i c  its  to  In  3  mg/kg  animals,  MPP +  was  was  detail  trated  the  seen  the  (within  before  death.  In  Thus,  This  MPP+  is  sc As  with  prevents highly  from  15  of  doses minutes  and  the  MPTP.  Doses  group  as  sedated  differed  animals  one  various  early  4 0 mg/kg  with  (which  neurotoxic ' effects  appeared  injection. h),  death.  mice  h after  2.5  the  follows.  the  24  for  injected  out').  at  pargyline  their  hunched-  However,  20 mg/kg  or  all high-  usually exhibiting  of  mice  the  given  40  toxicity  toxic,  even  mg/kg  of  more  a  MPTP)  so  than  MPTP. MPP+  14.5  content  nmol/g the  nmol/g.  in  as  MPP+,  with  while  10  were  2 h after  just  compound  nmol/g,  brain in  prevent  mice were  ('stretched is  -  responsible  black  mg/kg  a l l mice r a p i d l y  parent  61.2  normal  pretreatment  failed  10  that  seizure  was  observations of  posture  generalized MPP+,  C57  altered  animals appeared er k i l l e d  MPP+  39  wet  the  method  0.  blood-brain  In  value  for  Hence,  barrier  pooled  weight.  calculated  The  Appendix  of  whole the  of  when  and  in  of  MPP+  injected  was  striatum  MPP +  analyses  brain  1  h  the  blood  100  content  was  trapped  content  is  subcutaneously,  preferentially  after  in  the  described MPP +  accumulated  penein  the  striatum. Although mg/kg is  MPP +  did not  shown  by  confirmed (mean  ±  suffer the  this SEM)  penetrated any  damage  neurochemical by  indicating  dopaminergic  had 236 ± 3 p e r i k a r y a .  ments,  MPP +  proved  not  t o be more l e t h a l  to  brain,  data that  in  than  MPTP.  Table  while  Thus,  neurotoxic  mice  given  dopaminergic  perikarya  (n = 5)  was  the  to  per  the  mice  area,  (n = 6) the  conditions  nigrostriatal  of  neurons.  Histological  control  under  injections  nigrostriatal  16.  unit  10  This  examinations had  206  MPP+-treated of  neurons,  10  these  ±  5  mice  experi-  although  it  -  Table  16.  Striatal  40  contents of  d o p a m i n e and i t s  in mice g i v e n 2 d i f f e r e n t Controls  -  dosage regimens of  (26)  .  11.8 ± 0.96 ± 1.22 ±  MPP +  1 x 4 0 mg/kg MPP  Dopamine DOPAC HVA  metabolites  0.4 0.04 0.07  10 x 10  .  (5) 10.9 ± 0.6 0.82 ± 0.09 1.20 ± 0.04  experiment;  has  2  possible  the neurotoxic metabolite of in  the  into  SN  or  striatum  below t h o s e  since  the was  61.2  than  the  MPP +  (1984) piece  10 of  due  results show  already  nmol/g content  minutes  to  utilized  nigrostriatal discussed  damage.  This  were  this  weight)  the  lack  at  the  dosage  multiple  suggests  that  10  of  of  was  of  by  mg/kg  MPP+  site  schedules  4 0 mg/kg  order  of  a  threshold  region)  were  possible,  of  100  mg/kg  magnitude  lower  Markey  et  MPTP.  Another  action  8  neurotoxic  to  lower  al.  might  comes  Tables  i n j e c t i o n s of  not  taken  neurotoxicity  MPTP.  more  is  is  appears  injection  found  with  MPP+  latter  latter  an  an  injected  concentrations  than  was  wet  that  i n j e c t i o n of  after  MPP+  MPP+ a c h i e v e d  that  in this The  ± 0.6 ± 0.03§ ± 0.04  Either  (assuming  terminals  hour  nmol/g  mice  neurons  above,  an  weight.  different  a s i n g l e MPTP  injection  .  with that  the c o n c e n t r a t i o n s of  produce  suggests  insufficient  that  to  (870  after  or,  the  content  wet  evidence which  that  minergic  MPP+  interpretations.  by n e u r o n a l  required  striatal  MPP+  be  after  n i g r o s t r i a t a l neurons  still  MPTP;  (7)  11.8 0.73 1.20  V a l u e s a r e e x p r e s s e d i n y g / g w e t w e i g h t , mean ± SEM, number o f samples a n a l y s e d i n p a r e n t h e s e s . Values d i f f e r s i g n i f i c a n t l y from c o n t r o l s : § P < 0 . 0 5 .  This  mg/kg  MPP+  +  from and  9  dopa-  doses.  concentration  As must  be r e a c h e d b e f o r e damage c a n be a c h e i v e d . MPP+  is  widely  believed  to  be  the  neurotoxic  metabolite  of  MPTP.  -  MPP+ al.  is  reportedly  1985),  and,  toxic  when  to  41  nigral  -  explants  injected centrally  to  in  rats  tissue  (Heikkila  might  t h i s be r e c o n c i l e d w i t h  the  involvement  city,  as my r e s u l t s  One  p o s s i b i l i t y is that  derived plore  from  the  atal  MPP+,  perhaps  using  Heikkila  et  al.  toxicity  of  MPP+  pretreatment  the  1985)  have  or.not  deprenyl  in  these r e a c t i v e  hydroxyanisole  elevating  effect  brain)  sulfoximine  monoethyl  (Ip  biosynthesis,  an  increase in  et  be  toxicity  of  brain  total  The  the  et  shown  mice  compounds  glutathione  1985; the  deprenyl  to  determine  of  to  have  effect  on  1985).  to  the  an  et  The  to  of  brain  liver  MPTP.  was  not  compound, (but  not  with  buthionine  in  glutathione  enzyme al.  increase  glutathione-  Another  treated  an a t t e m p t  levels.  such a  free  detoxifica-  that  increase  rate-limiting (Puri  in the  neurotoxicity  previously  in  MPTP may i n v o l v e  suggest  the  al.  glutathione  diethyl-maleate  important  reduce  Y-glutamylcysteine synthetase these  and  observations  been in  is ex-  Since by  interest  to  al.  prevented  be o f  toxi-  nigrostri-  neurotoxic.  hydroxyanisole,  Jaeschke  content  important  (Cohen  been r e p o r t e d  has  be  others  to  How  radical(s)  to  would  tissues.  inhibitor  d e s c r i b e d below u t i l i z e d ed  has  free  e_t  properties.  might  1984;  ester,  glutathione (BSO),  (BHA)  peripheral  determined  total  These  1985).  damage  be c r i t i c a l l y  contents  Butylated  in  to  species.  normal. glutathione  glutathione  it  the mechanism o f  appears  the  would  ester,-buthionine-sulfoximine,  Glutathione  previously  1985),  has a n t i o x i d a n t  As d i s c u s s e d e a r l i e r ,  of  al.  al.  (Cohen  r a d i c a l s i n MPTP  reportedly  Experiments, using b u t y l a t e d  monoethyl  radicals.  MPP +  et  prevent  which  c u l t u r e : was  free  It  might  under  reported  in, tissue  of  radical.  antioxidants  conditions  (Mytilineou* et  Ill.iv)  tion  an •MPP  p o s s i b i l i t y that  neurons  whether  suggest?  culture  1983).  Experiments  produce  objective  was  a sustainto  deter-  - 42 -  mine  whether  another liver  MPTP  group (but  of  not  The  mg/kg  The a  given  total  BSO  (BHA  + MPTP)  received  MPTP  second group o f mice of  5 days, This  total  liver Tietze  content  tathione after  stem.  still  in Table  last  Total  17,  and o f of  content  evident  1562 o f  5  BHA  is  a very  thione  had  120  lipid  daily  111. v i i i .  in  in  prior 2)  glutathione  addition,  could  of  accentuate  of  to  the its  last  brain  is  content  human  5 days  was  The  the  injec-  BHA the  effects  activity  analysed  elevation  both  for last  of  BHA  in  the  using  was  the  measured  and  r e s u l t s would  as  total  i n mouse  liver  parameters  activity  to  was this  the  is  manipulation  be  24  h  remained glu-  liver  were  content  was  controls).  likely that  glu-  total  173 % o f  suggest  potent  will  in  glutathione  activity  resistant  of  i n c r e a s e s i n both  (total  barrier,  PD,  BHA  after  activity  activity  However,  r e q u i r i n g a very  idiopathic  or  content  injection  rather  5th  500  s i n g l e MPTP i n j e c t i o n .  document  transferase  the  the  mice. of  same d a i l y d o s e o f  a substantial  stem.  blood-brain  black  111.viii.  transferase  compound,  the  C57  injections  transferase  transferase  the b r a i n  after  3, to  of  8 daily  transferase  injections.  glutathione  groups  of  2,  glutathione  glutathione  the  1,  served  produced  the  2  glutathione  glutathione  soluble  change of  relevance  BHA  h after  penetrated  content  nificant have  and  controls,  If  depletion  the  (Table  r e c e i v e d the  mice  and  while  unchanged from c o n t r o l s tathione  if  immediately  sacrificed  content  (1969),  content  the  In  a month a f t e r  alone)  of  in  a total  using the procedure described in Section As shown  conditions.  determine  injected  second group  brain  protocol  (BHA  and w e r e  glutathione  and t h e  to  such  subcutaneously  These a n i m a l s were s a c r i f i c e d  total  under  glutathione  injected  w i t h 4 0 mg/kg  injection. on  was  group  BHA,  tion.  m i c e was  toxic  MPTP.  (Sigma)  first  still  brain)  the t o x i c i t y of BHA  was  since  the  it  gluta-  with  a  stimulus.  This1  discussed  in  sigmight  Section  -  Table glutathione  17.  Effects  43 -  o f BHA on t o t a l  transferase  activity  glutathione  of l i v e r  and b r a i n GSH  Total glutathione contents ( y m o l / g wet w e i g h t ) Control  BHA  Liver  8.8 ± 0.46 (10)  Brainstem  1.06  s t e m o f t h e mouse  transferase (ymol/min/mg  activity protein) BHA  Control  ± 0.73* (10)  1.84  ± 0.09 (17)  4.84  ± 0.20 (10)  1.04 ± 0.05 (5)  0.16  ± 0.00 (4)  0.17  ± 0.01 (5)  17.6  ± 0.04 (5)  c o n t e n t and  V a l u e s a r e mean ± SEM, w i t h number o f s a m p l e s a n a l y s e d shown in brackets. BHA m i c e w e r e s a c r i f i c e d 24 h a f t e r t h e l a s t o f 5 d a i l y i n j e c t i o n s o f 500 mg/kg. Values that d i f f e r signific a n t l y from t h e i r respective controls: P < 0.001. SEM l e s s t h a n 0 . 0 0 5 a r e shown as 0 . 0 0 .  In  Table  metabolites from to  18,  HPLC a n a l y s i s  showed  the t o x i c i t y  produce  transferase crease  an  that of  i n both  pretreatment  MPTP.  elevation  activity  18.  in  with  total  stem.  to protect  o f dopamine  BHA d i d n o t o f f e r  glutathione  in the l i v e r  of  content  The s u b s t a n t i a l  only,  but not  mice a g a i n s t  o f BHA p r e t r e a t m e n t effects  contents  may be a r e f l e c t i o n  the brain  parameters  Effects  This of  an i n s u f f i c i e n t c o n d i t i o n  Table  of the s t r i a t a l  protection  the f a i l u r e and o f  of  and l o n g - t e r m  i n the b r a i n ,  the neurotoxicity  1.43 ± 0.19 0.17 ± 0 . 0 2 0.43 ± 0.03  o f MPTP.  BHA and MPTP (6) 1.51  ± 0.22  0.14 ± 0 . 0 1 0.48  ± 0.02  V a l u e s a r e mean ± S E M , i n u g / g w e t w e i g h t . Number o f s a m p l e s in parentheses. None o f t h e means d i f f e r s i g n i f i c a n t l y .  in-  appeared  on t h e n e u r o c h e m i c a l  (22)  BHA  glutathione  o f MPTP i n m i c e MPTP-alone  Dopamine DOPAC HVA  any  and i t s  - 44 -  The  next  ester,  s e r i e s of  w h i c h was s y n t h e s i z e d  confirm that as  experiments  reported  mmol/kg  of  it  could  by  Puri  the  et  the  killed  at  same t i m e  using  the  total  glutathione  Thereafter, decreased, et  al.  content  of  a return  to  a l b i n o mice mice. liver  from  the  of  content  were  of  content the  at  2 h after  levels  (Swiss-Webster), only  was o b s e r v e d a t  a  small 15  at  Anderson  at  1  injected  and  1969). noted liver do  not  (2  A at  and 3 0 m i n u t e s  in  total  small  10  30  in  unchanged,  Since  total  the  on CD1  glutathione  of  or Puri  glutathione  same d o s e ,  these  in  minutes.  r e p l i c a t e those  fold)  1).  obtained  increase  and  remained  was r e p e a t e d  (figure  liver,  sc with  results  15  a s i n g l e i n j e c t i o n of  increase  the  30 m i n u t e s ,  i n d i c a t e s the  increase  experiment  of  To  were  8 h post-injection. the  content  (1985).  s a l i n e - i n j e c t e d mice a l s o  the  results  al.  1.5  was  of  15  et  monoethyl  1,  (Tietze  liver  glutathione  mice were  Control  r e p o r t e d , a marked  liver  black  Figure  the  Thus,  use o f  glutathione  sacrificed  analysis  glutathione  control  Again,  C57  intervals.  controls.  who  total  injection.  method  the  procedure of  (1983),  and  single  total  (1983)  al.  compound,  Tietze  the  indeed e l e v a t e  and 2 h a f t e r the  by  involved  with  authors Swiss  content  used  albino of  the  - 45 -  140  —  c  CU +->  120  c o u  CO o C iO -t-> •r- C  -ET o  •«-) (J  100  (TJ  4-> 4_=J O  o>»«  to <3 n j  i—  O -o 4-> Ol  80  </> C (0  • r - QJ i.  10 CL CU X c n  c  ro  co  60  JC  o  40  1  1 \~ 15 min  30 min  + 1 h  1.5  H h  2 h  Time p e r i o d s a f t e r i n j e c t i o n of glutathione monoethylester Figure 1. Changes r e l a t i v e t o c o n t r o l s o f t o t a l glutat h i o n e c o n t e n t o f l i v e r s o f C57 b l a c k o r CD1 S w i s s a l b i n o m i c e , a t v a r i o u s t i m e i n t e r v a l s a f t e r an i n j e c t i o n o f 10 mmol/kg g l u t a t h i o n e m o n o e t h y l e s t e r . % mean + ( o r - ) SEM are shown. Each p o i n t i s t h e average of 3 t o 5 m i c e i n j e c t e d and a n a l y s e d . Open t r i a n g l e s are of C57 black m i c e , w h i l e c l o s e d t r i a n g l e s a r e o f CD1 S w i s s a l b i n o m i c e .  -  There  is  one  and t h e method mice the  with  in  for  content  Thus,  was  et  al.  increase  my  have  been  10  l i v e r of  mmol/kg  injection.  of  This  so  19  glycine) and  lists  and o f  its  liver  its  However,  dioxygenase  pathway  ine  and  ine,  taurine.  and  thione  content  technique. increased, Since acid  glycine  the  (Schneider The  in  was  table  the  unchanged,  probably latter  content  is The  was  because not  an  mouse,  ester  normal,  and  its  homeostatic  in  total  gluta-  et  a C57  and  al.  (1968,  b l a c k mouse  sacrificed  rapid  1981)  was  injected  2 h after  c a t a b o l i s m of  the  glutathione  increase. glutathione (Y-glutamylcysteinyl-  amino  acids  in  the  2  mouse  the  net  detected  in  of  cysteine  metabolism,  et  al.  of  1983),  the  mouse  confirming  amino listed  are  2  of  given  results  to  could  in  Table  19  in  the  not  not  These  are  hypotaur-  taurine,  ester.  hypotaur-  Total  using  glutamic  be  are  cysteine  of  indicate mouse  and the  a-ketoglutarate  it  Cysteine  via  obtained  glutathione,  acid,  initially  the  livers.  samples,  shown.  increased contents  transamination  increased  Perry  and o f  its  product  results  glutathione  total  shows  liver  rate-limiting  content  increase  by  of  products  A n o t h e r breakdown  analysis.  thione  2  procedure of  contents  component  any  caused  liver  glutathione  their  normal.  document  d i s u l f i d e , c y s t i n e , were  tabulated.  to  that  employed,  pretreated  the  the  initially  assess whether  c o u l d e x p l a i n my i n a b i l i t y t o Table  were  I  content  because and  where  that  authors  glutathione  inhibited  monoethyl  to  protocol  occurred  effective  a control  done  total  enzymes  glutathione  was  These  was  acid a n a l y s i s using the  p e r f o r m e d on t h e  used.  in  c o n t e n t was r a p i d l y r e t u r n e d  Amino  the  experiment  degradative  might  between  probably  synthesis In  -  (1983)  experiment  subnormal. and  difference  the  glutathione  mechanisms  with  Puri  their  biosynthetic  thione  that  BSO.  ester  enzyme  important  46  the  acid, had  given  the  Tietze was  not  occurred.  detected that  gluta-  by  liver  amino gluta-  ester,  but  -  47  -  g l u t a t h i o n e was t h e n r a p i d l y h y d r o l y s e d  Table  19.  Content  2 h after Animal  Total glutathione  the  15.05 18.02  0.58 4.03  1.60 1.03  0.99 1.18  values  a r e i n y m o l / g wet  group  of  injection  of  10  after  a sc  mice  died  analyses mice  of  was  the  of  this  for  of  black  of  C57  pretreated  black  a month  mine  and  its  from  those  of  after  metabolites mice  fed  with  and  However,  compound a g a i n s t t h e group  liver, 40  to  t o x i c i t y of  mice were MPTP  the  MPTP  in  these  ordinary  the  the  mg/kg  one  BSO  the  were  water  ester.  or  9  of  histological  pathway rate  suggest  (20mM)  in  i n j e c t e d s c on t h e  injection,  tap  not  (sc),  of  seen a  these in  the  protective  MPTP.  fed  animals  MPTP  monoethyl  lethality  does  a sustain-  and p r e s u m a b l y o f  neurochemical  group  2.16 4.18  produce  dopaminergic n i g r o s t r i a t a l  possible. ester  ester  glutathione  injection, the  the  mice  mmol/kg  7 d a y s , w i t h 20 mg/kg o f  sacrificed  (Table  not  10  of  Glycine  weight.  monoethyl  content  C57  MPTP  integrity  monoethyl  Another water  the  therefore  glutathione role  after  ester Glutamine  brain, hour  sacrificed  Glutamic acid  glutathione  a  monoethyl  acids.  Hypotaurine  ed i n c r e a s e i n t o t a l injected  l i v e r o f mouse  glutathione  f a i l u r e of. g l u t a t h i o n e  I  amino  Taurine  All  Despite  i t s component  amino a c i d s i n  10 mmol/kg  8.44 7.97  Control Ester  of  into  and  striatal not  their  fourth  day.  contents  significantly  injected  drinking  with  20  When  of  dopa-  different  mg/kg  MPTP  20).  To s u m m a r i s e  the  experiments  of  total  glutathione  i n the  is  more  susceptible  to  brain  change,  above, is but  the  results  difficult in  the  to  case  suggest alter. of  that That  the in  glutathione  the  content liver  monoethyl  -  ester, tion  an i n i t i a l  of  total  ficient  increase  glutathione  is  Table 20.  Effects effects  returned the  20 mg/kg  transferase  of  reacts  8.4 0.44 1.03  et  al.  1981).  s p a c e d 24  sacrificed 1 h after  second  injection,  total  glutathione  for  (1969).  As shown  ly  analysis  90% d e p l e t i o n  brain  stem.  In  in Table of  the  ing  few  injection  and of  Behavioral  long-lasting, DEM.  Seven  loss  and t h e  by  content  of  that  in  body  weight  mice  at  died.  of  We  of  mice were  the  produced the  days  therefore  of  glu-  inject-  stem of  was were  Tietze  approximate-  liver  to  to  mice.  group  method  observed  7  One  and b r a i n  sedation  least  black  presence  allowed  was  C57  attempt  the  both  were  consisting for  DEM  0.5 0.02 0.06  an  DEM/kg.  liver  content  mice  occurring 10  in  ml  in of  groups  0.8  of  changes  of  brain  2 i n j e c t i o n s of  glutathione  rapid  of  ± ± ±  number of the means  (Sigma)  and  Two  h apart,  the  second group  second i n j e c t i o n ,  obvious  21,  total  the  days.  of  (DEM)  the of  glutathione  ed s c w i t h 2 i n j e c t i o n s ,  obtained  with None  liver  with  suf-  the neurochemical  0.6 0.03 0.05  contents  (Plummer  the  a  (5)  an e l e c t r o p h i l e t h a t  tathione  be  (10)  diethylmaleate  is  to  BSO and 20 mg/kg MPTP  used  glutathione  appear  altera-  20 mg/kg MPTP  More  total  prior  black mice  y g / g wet w e i g h t , in parentheses.  deplete  not  The  MPTP.  on  V a l u e s a r e mean ± SEM, s a m p l e s a n a l y s e d shown differ significantly.  we  normal.  does  of  MPTP i n C57  7.3 ± 0.50 ± 1.04 ±  recently,  to  1 iver  BSO p r e t r e a t m e n t  Dopamine DOPAC HVA  DEM  of  -  the n e u r o t o x i c i t y  of  of  rapidly  content  c o n d i t i o n to modify  48  and  survive over  and after  the  tremors the  elected  to  the after ensuwere  second treat  -  mice w i t h  only  1  i n j e c t i o n of  MPTP c o u l d be p o t e n t i a t e d  Table  21.  Liver  DEM,  after  49  to  DEM  -  assess whether  and b r a i n s t e m t o t a l  glutathione  the  toxicity  of  contents  of  mice  DEM DEM  Control Two  B r a i n stem  not  pretreatment.  injected with  Liver  or  9.05 ± 0.54 (14)  1.63  1.14  0.22  ± 0.03 (12)  One  injections  3.13  ± 0.32' (10) ±  injection ±  0.19  (5) 0.32 ± 0.03'  0.02'  (10)  (5)  V a l u e s a r e mean ± SEM, ymol/g wet w e i g h t . DEM m i c e w e r e s a c r i f i c e d 1 h a f t e r 1 o r 2 i n j e c t i o n s o f 0 . 8 ml D E M / k g . Values that differ s i g n i f i c a n t l y from controls:*P< 0.001. T o t a l g l u t a t h i o n e c o n t e n t s o f b o t h t h e l i v e r and b r a i n s t e m o f DEM m i c e g i v e n 1 injection, differ s i g n i f i c a n t l y from t h o s e o f DEM m i c e i n j e c t e d w i t h 2 d o s e s o f DEM (P < 0.01, Student's t - t e s t ) .  Four  groups  i n j e c t i o n of glutathione As  shown  of  total  of  C57  content  in Table  i n the 21,  glutathione  analysis  (Table 22). 1  h later  fourth  of  by an  group t h a t  DEM-pretreated  liver  these  content  used.  animals of  group  of  the  i n j e c t i o n of  DEM,  20 mg/kg  h later  and b r a i n  group  for  received  analysis of  22,  (about stem.  and was s a c r i f i c e d and  its  MPTP.  These  MPTP, was n o t  0.8  total  (1969).  depletions  a month  ml  in  of  afterwards  the  striatum  DEM/kg,  followed  killed  striatal  an  A second group  animals,  MPTP, w e r e  t h e mean  70%)  metabolites  administered  20 mg/kg o f  in Table  mice given  1  first  substantial  liver  m i c e was  received only As shown  had  dopamine  of  The  and b r a i n s t e m b y t h e T i e t z e method  same d o s e o f  contents  A third  MPTP i n j e c t i o n . in  mice were  0 . 8 ml DEM/kg and was s a c i f i c e d  animals r e c e i v e d the for  black  together  with  1 mo a f t e r  content  of  significantly different  a the  dopamine from  that  -  of  mice that  were  given  s l i g h t l y elevated  Table  22.  only  MPTP.  i n the former  Contents  mice t r e a t e d with Animals  However,  and i t s m e t a b o l i t e s  DEM p l u s 2 0 mg/kg MPTP, Dopamine  DEM o n l y  (13)  contents  11.2  of  DOPAC  and HVA  were  group.  o f dopamine  DEM,  50 -  in striatum  o r 2 0 mg/kg MPTP  DOPAC  ± 0.7*  of only  HVA  0.58 ± 0.03§  1.08 ± 0 . 0 6 +  20 mg/kg MPTP ( 1 8 )  7.3 ± 0.3  0.49 ± 0.02  0.88 ± 0.03  DEM p l u s 20 mg/kg MPTP ( 1 0 )  8.4 ± 0.5  0.61 ± 0 . 0 2 *  0.99 ± 0.05s  V a l u e s a r e mean ± S E M , u g / g w e t w e i g h t , w i t h t h e number o f s a m p l e s shown i n p a r e n t h e s e s . DEM p l u s 20 mg/kg MPTP m i c e w e r e g i v e n an i n j e c t i o n o f 0 . 8 ml DEM/kg 1 h b e f o r e an i n j e c t i o n o f 20 mg/kg MPTP. A l l a n i m a l s w e r e s a c r i f i c e d 1 month a f t e r t h e i n j e c t i o n o f MPTP o r ^ D E M . Values that d i f f e r sig§ n i f i c a n t l y f r o m 20 mg/kg MPTP: P < 0.001; P < 0.05; + P < 0.01.  Thus, content It  is  III.iii toxic  despite  a substantial  in DEM-pretreated difficult  to  species  explained  that  mice,  reconcile  which suggest  a role  might  noted  i s that  These  the  brain  toxicity present  from  of  stem  MPTP  results  for glutathione  be f o r m e d  Experiments  that  responsible formost  sis  the  of  total  glutathione  was n o t  potentiated.  with  those  of  i n the d e t o x i f i c a t i o n  MPTP.  This  discrepancy  Section  of  neuro-  cannot  be  presently.  Ill.v)  As  depletion  toxins  seek  have  the natural  neurotoxin(s)  cases of i d i o p a t h i c P a r k i n s o n ' s  i n the i n t r o d u c t o r y PD i s t h e r e s u l t  to i d e n t i f y  section,  of exposure  y e t t o be i d e n t i f i e d .  the c u r r e n t l y  disease  prevailing  t o one o r more e n v i r o n m e n t a l Although  MPTP p r o d u c e s  hypothetoxins.  a parkinson-  -  ian  syndrome,  idiopathic MPTP,  it  PD.  other  meperidine  and  its  analog  to  (or  might  perhaps  incidence of this  with  monly  found  in  verted  by  paraquat, duced  a  I  a  diol  base  MPTP  have  of  strategies PD.  For  pounds  (Testa  from  these  from  to  create  of  MPTP  into  seeds  analog  of  sources  of  exposed. toxin  avoid  of  a  If  were  an  iden-  or  detoxify  it  it  occurred  in  if  the  might  diet  cases  synthesis  been  to  of  bears  a model or  the a  a  MPP+,  of  analog  betel  structural  by of  decrease  the  injecting  C57  which  (which  in  nut  complete  PD  some  cinnamaldehyde  an MPTP  without  that  moieties; compound  condensation et  al.  from et  chemical of  the  example,  might  vivo);  palm;  of  al.  the  (Barker  similarity  all  The to  be  con-  arecoline,  a  ring  resemblance  structure;  to  MPP+;  re-  bears  2  et  al.  1981)  compounds  with  which  is  formed  neuroactive  by  indole-  N-methyl-l,2,3,4-tetrahydro-6,7-isoquinoline-  condensation  1985).  com-  2-methyl-l,2,3,4-tetrahydro-B-carbol-  carbonyl  1985);  are  N,N-dimethylcin-  pyridine  N-methyl-l,2,3,4-tetrahydroisoquinol ine,  in vivo  Injections  during  people  most  environmental  parkinsonism-causing  included  formation  endogenous  (Col 1 ins  formed  their  known  for  [ 4 , 4 ' - b i s ( l - m e t h y l - l , 2 , 3 , 6 - t e t r a h y d r o p y r i d i n e ) ] which  trace  and  no  few  devise  analogs These  herbicide  intracellular amines  to  attempted  N-methyl-tetrahydropyridine ine,  are  responsible  a contaminant  simply excluding  alkaloid  paraquat  as  occurrence  chemical  an  there  agent  disease.  Schiff  namylamine,  the  nature.  cholinomimetic  the  relatively  possible  reason,  mice  -  p o s s i b l y endogenous)  then  black  be  because  which  be  the  to  formation  prevent  foods,  For  is  than  it  certain  unlikely  This  environmental tified,  is  51  of  phenyl e t h y l amines  structural  MPTP  and  compounds  MPP+,  t h e a n a l o g s w e r e by t h e s c  formulae are route.  of  which  with these  illustrated  might  carbonyl compounds in  Figure  be comand 2.  - 52 -  I  VI  i!  Ill  VII  Figure 2. Structural pounds a r e : I) II) III) IV) V) VI) VII) VIII) IX) X)  VIII formulae  of  MPTP,  IX MPP+,  and t h e i r  X analogs.  The  N - m e t h y l - 4 - p h e n y l - l , 2 , 3 , 6 - t e t r a h y d r o p y r i d i n e (MPTP), N,N-dimethylcinnamylamine, Cinnamaldehyde, Arecoline, 2-methyl-l,2,3,4-tetrahydro-6-carboline, N-methyl-l,2,3,4-tetrahydro-6,7-isoquinolinediol, N - m e t h y l - l , 2 , 3 , 4 - t e t r a h y d r o i s o q u i n o l i n e (MTIQ), N-methyl-4-phenylpyridinium ion (MPP+), Paraquat, Reduced p a r a q u a t [ 4 , 4 ' - b i s ( l - m e t h y l - l , 2 , 3 , 6 - t e t r a h y d r o p y r i d i n e ) ] .  com-  -  Cinnamaldehyde day  period  this  was  The  last  in  (Fisher),  a total  injection  9  were  of  each  animal  last  i n j e c t i o n , when During  the  course  observed  highest  received  when  dose  parkinsonian-1ike These  were  sometimes  of  this  of  for  dose.  the  striatal  s l i g h t l y higher  bromide  (Sigma)  from 5 to 200 each was  also  was  necessary  last HVA  i n j e c t i o n of  report  that  10 the  of  et  24  last  (Table  or  each  during  was  and  did  period  29 d a i l y  as t h e dose  mice were from  salt). per  was  The  mouse  the  injected,  of  injection  any  show  were  Convulsions  not  HVA  remain-  reduction  levels  of  intense  14  arecoline  increased  last was  injected with  manifestations  were  6  i n j e c t i o n s were  1360  mg/kg.  atropine  mg/kg.  This  atropine  cover  muscarinic  tremors.  Nine  days  Striatal  from c o n t r o l s  (Table 23).  In  prevented  i s possible that  the if  to  which  excessive  view  Prior  of  (Sigma),  sacrificed.  pretreatment  hydro-  progressively  mice were  it  when  the 4  i n j e c t i o n s of  Dosage  to  a l . 1985),  effects  changes,  although  the  kyphosis  injection. the  dose  longer.  However,  tremor  reached.  adverse  injections)  30 and  h after  any  no  lower.  DOPAC,  was  cumulative  survive  behavioral  2  differ  or  injection  0.9%NaCl.  and  not  a  23).  given  salivation  900 mg/kg total  over  40 mg/kg  sacrificed 1  akinesia,  died  given  d o s e was  injections,  h after  dopamine  increased  anticholinergic  (Hadjiconstantinou  The  daily  obvious  the  a r e c o l i n e the  d i d not  dose.  9  mice  cumulative  prevent  excessive  contents  6 of  (expressed  progressively  of  mg/kg  last  least  in  a dose  they might  700  at  i n j e c t i o n the  to  that  was  starting  M i c e were  c o n s i s t i n g of  m i c e were  total  until  g/kg.  the  h after  dissolved  The  arecoline  including  of  200 mg/kg  mg/kg.  for  than c o n t r o l s  Another group  The  latter  was  Despite  contents  10% e t h a n o l ,  cinnamaldehyde  dosage  elicited.  highest  12  the  symptoms  ing mice s a c r i f i c e d 1 in  was  mg/kg  persisted  this  appeared  the  (900  seen.  it  in  20 i n j e c t i o n s .  each  injections  -  dissolved  increased at  that  were  of  53  activity, after  dopamine of  the  toxicity  a r e c o l i n e had  the and  recent  of an  MPTP  -  Table of  23.  Effects  dopamine  of  -  MPTP a n a l o g s on s t r i a t a l  and i t s m e t a b o l i t e s  Analog  of  C57  Dopamine  S a l i n e c o n t r o l s (44) C i n n a m a l d e h y d e (4) A r e c o l i n e (8) N,N-dimethylcinnamylamine (7) N-methyl-1,2,3,4tetrahydro-6,7i s o q u i n o l i n e d i o l (10) N-methyl-1,2,3,4tetrahydroi s o q u i n o l i n e (9) 2-methyl-l,2,3,4tetrahydro-3c a r b o l i n e (6)  T  54  contents  black  mice  DOPAC  11.3 11.6 11.8 11.2  ± ± ± ±  0.4 1.2 0.9 1 . 3  0.95 0.84 0.66 0.80  ± ± ± ±  13.1  ± 0.6  0.84  14.3  ± 0.3  13.8  ± 0.8§  HVA  0.04 0.32. 0.04 0.11  1.27 1.94 1.04 1.41  ± ± ± ±  0.05 0.54+ 0.08 0.18  ± 0.03  1.31  ±  0.07  0.76  ± 0.01§  1.24  ±  0.03  0.84  ± 0.07  1.32  ±  0.07  *  V a l u e s a r e mean ± SEM, i n y g / g w e t w e i g h t , w i t h t h e number o f m i c e shown i n p a r e n t h e s e s . Dosages a r e d e s c r i b e d i n t h e t e x t . Values t h a t d i f f e r s i g n i f i c a n t l y from c o n t r o l s : "P < 0 . 0 0 1 ; P < 0.01; 5 P < 0.05.  MPTP-like tection our  mechanism in  action  my e x p e r i m e n t  attempts  described  of  to  by  u n l i k e l y to  might  protect  mice  Hadjiconstantinou have n e u r o t o x i c  daily for  cumulative toxicity  dose when  200 m g / k g ,  a  per the  7  in  mouse  was  jection,  m i c e began  to  ors then  developed,  but  tion,  the  was  particular  constant  have  from  MPTP  et  al.  was  or  motion  as  Animals 120  around  continued  disappeared  40  in  conditions  to  Appendix  cage. At  E)  200 m g / k g . no  minutes  move.  However,  arecoline  signs  However,  Five  to  the  pro-  is  system.  showed  mg/kg.  their  atropine  Thus,  described  observed.  move c o n t i n o u s l y animals  using  failed.  i n c r e a s i n g from  80  the  manifestation.  atropine,  (1985),  890 mg/kg.  syndrome  the  with  its  on t h e n i g r o s t r i a t a l  doses  40,  neurotoxicity,  prevented  (synthesized  days,  dose  similar  effects  N,N-dimethylcinnamylamine injected  and  Total of  drug  at  150  or  after  the  in-  Ataxia 1.5  was  h  and  trem-  post-injec-  and was r e p l a c e d b y r e p e t i t i v e  chewing  -  on w o o d - s h a v i n g s  while  standing  animals  normal  again.  tion),  appeared striatal  dopaminergic  55 -  i n one s p o t . At  4  sacrifice  parameters  h after  (10 days  were  the i n j e c t i o n ,  after  unchanged  the  the l a s t  injec-  control  values  from  (Table 2 3 ) . N-methyl-l,2,3,4-tetrahydro-6,7-isoquinolinediol in 0 . 9 % NaCl, of  4 0 mg/kg  ficed and,  was a d m i n i s t e r e d (expressed  20 days  after  a s shown  a s t h e HC 1  the l a s t  the  from  brain,  presence  an a n a l o g  hydroisoquinoline  group were  injection.  of  2  lacks  groups  hinder  the  ( M T I Q ) , was t e s t e d  N-methyl i s o q u i n o l inium  was d i s s o l v e d i n w a t e r ,  dried, ated  a small to  dryness  through  of  HC1  black  daily  mice.  (dosage  160 m g / k g ,  injections  The d o s a g e  tion  of  This  lasted  giving  for  was o b s e r v e d ,  N-methyl-1,2,3,4of  this  effects  (obtained  as  of  the  (but  and i s o p r o -  i n 70% y i e l d ) . ,  a period  of  3  were  oxygen.  The e t h e r  added.  MTIQ  form-  s o l u t i o n was  salt  was  evapor-  the hydrochloride  administered  lower h,  sc to  progressively  salt).  cumulative not of  in mice.  salt  of  NMR s p e c t r o m e t e r .  MTIQ w e r e  HC1  ether.  of  into  N-methyl-l,2,3,4-tetra-  to exclude  Identity  tetra-  compound  methyl i o d i d e  iodide  with  was i n c r e a s e d  a total  160 mg/kg  sacri-  and i t s m e t a b o l i t e s  and t h e h y d r o c h l o r i d e  and r e c r y s t a l l i z e d .  expressed  in  groups,  the solution  added,  MTIQ was c o n f i r m e d u s i n g a p r o t o n Eight  behavior  dopamine  isoquinoline with  a n d was e x t r a c t e d  amount  and a n i m a l s . w e r e  and 4 moles o f e x c e s s s o d i u m b o r o h y d r i d e  g a s was b u b b l e d layer  injections  for possible neurotoxic  panol.  a s an o i l y  5 daily  passage  the hydroxyl  by r e a c t i n g  ed  of mice.  given,  of striatal  MTIQ was s y n t h e s i z e d  Nitrogen  dissolved  No a b n o r m a l  hydroxy!  might  that  The p r o d u c t ,  (Sigma),  controls.  hydro-6,7-isoquinolinediol the  salt)  i n Table 2 3 , contents  remained unchanged Since  t o another  HC1  dose  The of  last  5  which  mice  After  became  animals  40 t o  C57  mg/kg  were  each  rapidly  appeared  of  160  injections  1040 mg/kg.  doses),  after  from  a group  of  injec-  sedated.  normal.  A  -  single  mouse  injected with  and d i e d w i t h i n 15 compatible with  trols  of  dopamine  and  that  system of  Another  sacrificed  the  MTIQ  of  tallized  from  solution  through  mice  The  injected  (Table  starting  ies  precipitate The  last  which  of  was  compound, was  did  highest  injection. than  to  dose  Striatal  those  explain,  of  con-  they  effects  on  days  with  and  80 mg/kg,  injection,  4  in  the  excess methyl  nonenigro-  was  treated  formed  was  identity  any  iodide  a  The  in  and  was boil-  recrys-  water-methanol  with  an  filtered, as  resonance.  cause  in  2-methylMTHBC  washed,  dissolved  its  excess  washed,  MHTBC was first  of and  confirmed  injection  of  behavioral  change.  Subse-  sedation,  ataxia,  tremors  which  produced  These  lasted for  striatal  0.9% NaCl..  filtered,  bubbled,  not  convulsions.  treatment  used.  mg/kg  seizures  2 h.  When s a c r i f i c e d  dopaminergic parameters  were  1  normal  23).  Another were  be t h e  last  for  dissolved  c o l o r l e s s product  40 m g / k g ,  the  daily  and n u c l e a r m a g n e t i c  and o c c a s i o n a l c l o n i c after  the  difficult  (Sigma) w i t h  from water-methanol,  i n j e c t i o n s were  month  to  neurodegenerative  (MTHBC)  nitrogen  The  by mass s p e c t r o m e t r y  quent  are  was  yellow  which  sodium b o r o h y d r i d e .  of  generalised  s i g n i f i c a n t l y higher  no  isopropanol.  recrystallized  appeared  after  had  by r e a c t i n g n o r h a r m a n  isopropanol.  MHTBC was  a month  results  l,2,3,4-tetrahydro-3-carbolihe  ing  MTIQ d e v e l o p e d  mice.  group  synthesized  of  160 mg/kg  DOPAC w e r e  Although  indicate  striatal  Thus,  -  survival.  (Table 23).  theless  2 0 0 mg/kg  minutes.  MTIQ a n i m a l s w e r e contents  56  with  (doses  The 6.3 are  first  was  of  paraquat  utilized  mg/kg, expressed  then  8.5  here  i n b e h a v i o r were o b s e r v e d ,  5  as  in  0 . 9 % NaCl.  daily  injections  mg/kg,  and  the  free  Three of  finally  base).  the mice l o s t weight  dosage  the 3  schedules  diiodide  injections  Although rapidly  no  and 7  of  salt, 12.7  abnormalitof  10  mice  -  died  within  24 h a f t e r  the l a s t  f i c e d a t t h i s 24 h m a r k , the  striatum,  Table  although  24.  Effects  contents  57  -  injection.  and t h e y  The r e m a i n i n g  had n o r m a l  contents  mice were  o f dopamine  sacri-  a n d HVA i n  DOPAC l e v e l s w e r e s l i g h t l y d e c r e a s e d ( T a b l e 2 4 ) .  of paraquat  o f dopamine  and r e d u c e d p a r a q u a t  and i t s m e t a b o l i t e s  Cumulative  on s t r i a t a l  o f C57 b l a c k  Dopamine  mice  DOPAC  doses  (ug/g wet  HVA  weight)  (mg/kg)  Controls  (44)  11.3  ± 0.4  0.95 ± 0.05  1.27  Paraquat  (3)  Paraquat  Paraquat  5 2 . 9 , over 5 injections  13.1  ± 0.7  1.03 ± 0.12  0.89 ± 0.14  (9).  12.7, single injection  12.5 ± 0.7  0.72 ± 0.04§  1.20  (7)  4 3 . 5 , over 3 injections  14.5 ± 0 . 4 *  0.76 ± 0.04  1.30 ± 0.07  Reduced (10) paraquat  3 4 1 . 6 , over 6 injections  12.7  0.80 ± 0.03  1.28 ± 0.06  ± 0.7  ± 0.05  ±0.06  V a l u e s f o r d o p a m i n e a n d i t s m e t a b o l i t e s a r e mean ± SEM. The number o f a n i m a l s a n a l y s e d i s shown i n b r a c k e t s . Cumulative doses f o r p a r a q u a t and r e d u c e d p a r a q u a t a r e e x p r e s s e d as t h e free base. Values that d i f f e r s i g n i f i c a n t l y from c o n t r o l s : P < 0.001; § P < 0 . 0 5 .  The tion  of  month. deficits  second dosage 12.7  mg/kg  No m o r t a l i t y in striatal  schedule of paraquat  (expressed  as t h e f r e e  was e n c o u n t e r e d . contents  of  diiodide base),  Table  dopamine  employed  a single  injec-  a n d s a c r i f i c e was a f t e r  24 shows  that  1  t h e s e m i c e h a d no  and i t s m e t a b o l i t e s  when  compared  -  to  58  normals. The  third  dichloride. and  were  another died  the  schedule of  Injections  spaced  soon  3  after  died.  days 10  the  last  mg/kg  apart.  The  latter  10  third  paraquat. water,  treatment  This  then  mice given  injection.  contents  oxygen, formed  excess as  an  was d r i e d , reduced  oily  a small  paraquat  product  layer  extracted  of  HC1  was to  dihydrochloride  C57  black  diHCl,  in  (doses  expressed  behavioral last  salt  as  the  of  free  abnormalities striatal  6  7.3,  1  the  salt  of  the  after  reduced  to.  paraquat  ether  was  in  exclude  solution  dihydrochloride  reduced paraquat  10  paraquat  Reduced The  of  24).  solution  added.  3  in  dose  month  recrystallized.  of  injections  29.1,  58.1,  Throughout  evident.  contents  and  daily  14.5,  base).  were  of  design,  dissolving  ether.  and  same  increased (Table  were  with  because  the  normal  base)  salt  of  Identity  of  confirmed  by  spectrometry.  received  s u c c e s s i v e doses  injection, (Table  mice  were  through  added,  dryness  present  first  borohydride  n u c l e a r m a g n e t i c r e s o n a n c e and mass Ten  HVA  of  paraquat  free  necessary  dihydrochloride  bubbled  was  evaporated  the  by  and  amount  was  as t h e  sodium  was  of  as t h e  injections  c o n t e n t was the  injections  (expressed  3 daily  DOPAC and  was  3  timing  synthesized  gas  of  each  Despite  received  was  nitrogen  amounts  of  dopamine  group  compound  while  involved  14.5  of  Striatal  paraquat  were o f  l a s t i n j e c t i o n , although The  the  dosage  experiment,  animals  mal  -  When  the  of 116.3  and  and  its  1  paraquat  116.3  experimental  sacrificed  dopamine  reduced  month  metabolites  mg/kg  period,  no  after  the  were  nor-  tested  were  24). +  In  summary,  therefore,  ineffective  in  damaging  the  mice.  is  in spite  of  ing  the  This  course  of  the  various  analogs  dopaminergic some  injections  of  MPTP  nigrostriatal  interesting drugs  of  such  behavioral as  or  MPP  pathway changes  cinnamaldehyde  of  C57  black  observed and  dur-  N,N-di-  -  methylcinnamylamine. does not  mean  neurons doses,  that  primates  and  but  in mice,  much  et  al.  ible  toxicity  ates.  This  Barbeau  et  several  (cited  between  regions  (Barbeau  et  al.  brain  of  obtain  incidence  paraquat bicide  s h o u l d be is  in  paraquat  in  doses  paraquat  which might  use.  In  compatible  of  Ill.vi)  shown  interesting  Clearly,  more  thoroughly  with  more r e a d i l y  g i c n i g r o s t r i a t a l neurons  An  MPTP.  my  the  in  several  epidemiological  cigarette  smokers  than  al_.  1971;  Baumann  protective put  forth.  sure of  et  al.  mechanism o f Perry  et  carbon monoxide  1980;  (1982) in  the an  same that  low  1984a;  the  poss-  in  prim-  paraquat.  correlation  pesticide  use  in  investigators paraquat  pipiens,  could  although  this  possible neurotoxicity  in  primates,  survival,  well  the  brain,  as  did  not  of  since this  dosage  the  to  tested  of  showing  as  al.  excellent  the  the  very  herbicide  level  Rana  et  various  and P a r k i n s o n ' s  disease  PD  that  research  studies  non-smokers  smoking  al.  to  at  as  be  mice  her-  schedules  reduced damage  of  form  of  dopaminer-  mouse.  by  among  answer  in  nigrostriatal  experiments,  enter  Smoking  observation  (Langston  addition,  frogs,  to  primates  should  the  results in  to  found  and  In  concentrations  investigated  common  have  reported  r e d u c t i o n was more m a r k e d w i t h  they  regard  PD  Canada.  have  dopamine  of  toxic  equivocal  with  changes  neurotoxic  required  man,  1985a)  be  is  less in  parkinsonian  not  are  a  important  Quebec,  1985)  doses  Lewin,  the  produce  itself  MPTP-analogs  in  -  might  MPTP  higher  especially  al.  (r = 0.967)  man.  To  these  to  compounds  1984a).  of is  failure  these  of  Heikkila  reduce  The  59  brain  to  is  occur  (Nefzger  Godwin-Austen remains  unknown,  suggested cells  of  that  al.  al.  cigarette  has  frequently 1968;  1982).  several the  disease  less  et  et  the  smokers  among  Kessler Although  theories  increased  been  have  partial  might  et the  been pres-  provide  a  -  reducing from  oxidant  pounds a  environment  in  damage.  smoke  molecule,  the neurotoxin It  is  possible  protective  effect  more o f  the  cules.  To  of  3 compounds  of  cigarette  ^1_. 1 9 7 7 ) .  Neurol.  one  of  these  MPTP  was  The  in  nicotine that,  if  the all  for  the  MPTP-induced  pretreatment  of  died  anything,  or  striatum  within nicotine  5  the  days  detoxify to  ascertain  MPTP.  if  fluids The  mice were  before  an  one  tissues tested  Voght  pretreated  and  et  G ( Y o n g e_t  injection  sacrificed  mole-  compounds  brief,  or  any  and  1974;  the  one  MPTP-like  Appendix  of the  40  with mg/kg  striatum  metabolites. 25.  While to  thiocyanate  was  26).  effect  Mice the  an  to of  an  hydrazine  pretreated MPTP  i n t e n s i f i e d the neurotoxic  comparable  MPTP  inhibitor  of  for  cannot  10  of  injection  MAO-B  injection effects  very  nigrostriatal  protection,  prior  not  following  offered  dopaminergic  considerable  protective  (Table  that  in  damage  it  is  first  described  pretreatment  since  The  al.  were  offered  antioxidant  MAO-B i n h i b i t i o n  liver  mice  Table  explain  et  days  assumes  molecule.  (Liu  In  and i t s  in  of  also  to  can  and h y d r a z i n e  later,  MPP+),  hypothesis  physiological  are  (or  com-  hypotheses  model  toxicity  1986).  dopamine  mechanism  the  several  displayed  with  The  of  in  experiment  month  from  found  to  this  smoke  MPTP  neurons  pyridine  i s an M P T P - l i k e  other  MPTP mouse  accumulate  72^265-272,  are  to  vity  of  One  hydrazine  attributed  the  used  3  many  This  smoking.  tobacco  can p r e v e n t  contents  11).  we  compounds  neurons,  (Table  in  to  Sci.  protection  that  present  of  neurons.  cigarette  sodium t h i o c y a n a t e ,  results  slight  by  at  the  uptake  least  afforded  given.  the  nigrostriatal  formulate  this,  3  analysed for  inhibit  nigrostriatal  that  to  Details  J.  postulated  i d i o p a t h i c PD  smokers  al.  protect  responsible for  known  were n i c o t i n e ,  -  partially  (1985)  might  into  compounds test  could  Snyder  cigarette  pyridine  that  which  60  days  be  actiwith  suggesting MPTP.  -  Table  25.  Effects  of  61  pretreatment  on M P T P - i n d u c e d  -  with  d e p l e t i o n of  striatal  and i t s m e t a b o l i t e s  Dopamine DOPAC HVA  thiocyanate  i n mice  MPTP  Thiocyanate  (39)  (23)  (8)  ±0.4 ± 0.05* ± 0.05*  hydrazine  dopamine  Controls  11.3 0.97 1.22  or  1.3 ±0.2 0.21 ± 0.03 0.43 ± 0.03  Hydrazine (16)  2.7 ± 0.7+ 0.31 ± 0.06 0.62 ± 0 . 0 9 +  5.7 ±0.5 0.51 ± 0 . 0 7 * 0.89 ± 0.04*  V a l u e s a r e mean ± S E M , i n y g / g w e t w e i g h t , w i t h number of samples i n d i c a t e d i n p a r e n t h e s e s . Values that d i f f e r signif i c a n t l y f r o m m i c e i n j e c t e d w i t h MPTP a l o n e : P < 0.001; + P <0.01.  Table 26.  Failure  activity  of  in l i v e r  hydrazine  to  i n h i b i t MAO-B  and s t r i a t u m o f  mice  Controls  Hydrazine  Liver  65.1 ± 3.4 (10)  67.1  ± (7)  5.0  Striatum  2 8 . 0 ± 2.1 (15)  32.7  ± (7)  1.6  V a l u e s a r e mean ± SEM, n m o l e s / h / m g p r o t e i n , w i t h number of samples indicated in parentheses. Hydrazine-treated mice were s a c r i f i c e d 1 h a f t e r t h e l a s t o f 8 d a i l y i n j e c t i o n s o f 11 m g / k g . None o f t h e means a r e s t a t i s t i c a l l y s i g n i f i c a n t .  Thus,  if  an M P T P - l i k e then the  cigarette  Of  neurotoxin  responsible for  m o l e c u l e whose  apparent  compound.  venting  the  toxicity  protective  course,  smoke w h i c h  it might  the t o x i c i t y of  effect  is  of  possible  be more  MPTP.  can  most  cases of  idiopathic  s i m i l a r l y be r e d u c e d by  smoking might that  potent  there than  other  hydrazine  in  is  hydrazine,  be a t t r i b u t a b l e are  PD  to  substances partially  this in pre-  -62  The the  second h y p o t h e s i s  observation  (Markey  that  that  requires  conversion  inhibits  this  neurotoxic effect  if  Heikkila et  the  agent(s)  enzyme s o t h a t  species  is  evaluate  compared w i t h  of  they  contain  or  possibility,  (who e a c h  only  G.  the  MAO-B a c t i v i t y  in platelets  brain  tissues  Table  27 shows  cantly  lower  in  results  of  Oreland  Although enough of  to account  1980).  in  et  excess  for  al  most  et  cases  the  It  is  idiopathic  PD  cigarette  an i n a c t i v e  on  neurotoxic  1984b).  of  and i f  then  the  more  smoking  precursor(s)  appparent  of  to a  protective  activity  (1981)  than who  reduction  the protection  80% b e f o r e  that  chosen the  of  as t h e s o u r c e o f only  readily  persons.  et a l .  in  accessible  a  might  afforded  i n the  This  much not  not  human  be  seen  In  the  population.  functionally  a r e MAO i n h i b i t o r s must is  signifi-  confirms  smaller  by s m o k i n g .  improvement  or  1981).  non-smokers.  studied  have MAO-B,  Whether  its activity  of  a d a y ) was  o f MAO-B i n p l a t e l e t s was  (25 )  clinical  activity  20 c i g a r e t t e s  in living  (Sandler  MAO-B  t h e enzyme m e a s u r e m e n t s  and a r e  studies  smokers  platelet  than  i s a r e l i a b l e index  t h e mean  al.  the  Details  B isozyme  antidepressants of  Langston  of  P l a t e l e t s were  i s uncertain  cigarette  interesting,  depression,  MAO-B  that  smoked  non-smokers.  s o u r c e o f t h e enzyme f o r r o u t i n e  or other  become  neurotoxic,  reduced,  smoking r e l i e s  by MAO-B t o  for  the conversion  second  been d e s c r i b e d i n A p p e n d i x since  a l . 1984b;  role of  be e x p l a i n e d .  smokers  that  conversion  t o become  prevented  this  cigarette  the p r o t e c t i v e  responsible  b y MAO-B  o f smoking might  To heavy  MPTP r e q u i r e s  e t a l . 1984;  possible  regarding  -  the  large  treatment  inhibit (Youdim  platelet et  al.  -  Table 27.  Monoamine  oxidase  63 -  B activity  in platelets  of  human s m o k e r s and n o n - s m o k e r s Subjects  Monoamine  Mean age  (nmol/h/mg  (years) Cigarette (32)  smokers  Non-smokers (41)  Values  are  mean  parentheses.  I  B activity  protein)  37 ± 2  4.08  ±  34 ± 2  5.46  ± 0.24  SEM,  with  number  of  subjects  0.26*  shown  in  *P < 0.001.  What c o m p o n e n t this,  ±  oxidase  of  measured  rat  cribed  i n Appendix  G,  smoke.  These were  cigarette liver  smoke  MAO-B  inhibits  activity,  i n the presence  cotinine  of  ( t h e major  MAO-B ? To  using  one o f  an  attempt  in  vitro  4 constituents  metabolite  of  or  hydrazine.  totally  ineffective  lowered  platelet  might  resulting  of  nicotine),  it as  in  in  cigarette  at  inhibitors  of  the  10  thiocyanate,  to  synthesis  of  10  M,  were  the cause of  appears  in vivo,  cigarette  —3  Although  smokers  i s certainly possible that  cigarette  in the l i v e r ,  converted  this  tested  i n i n h i b i t i n g MAO-B a c t i v i t y .  possible explanation  ingredient  perhaps be  act  compounds,;  des-  not  to  be d u e  one o f t h e s e  molecules  of  the  com-  MAO-B,  i n d e c r e a s e d a c t i v i t y o f t h e enzyme.  A third an  4  MAO-B a c t i v i t y  to t h e s e compounds, pounds  All  answer  system  —fi  cyanide,  to  into  hypothesis  smoke  which  of  the  protective  induces  destroy  the  metabolite  h a v e y e t been  performed.  by  of  production  the neurotoxin  the active  role  MAO-B.  smoking  of  precursor No  other before  experiments  is  that  enzymes, it to  can test  -  III.vii) An  important  toxic, If  but  the  such  Status finding  requires  The  in  human al.  ating  the  activity  might  result  amount  of  and  control  the  subjects  urinary  MAO-B  employed, supplied  clusion  reduced t h i s form of is  MAO-B.  of  was  shows  PD  for  a  The  subjects, and  or  and  72:265-272,  description  in  there  of  the  neuro-  PD  requires  MAO-B  activity  the d i s e a s e . h a s . been  al.  demon-  1982;  Bhatta-  responsible for  it  f a c i l i t a t e conversion  of  in  PD.  urine  also was  to  regul-  produce  the  were  not  metabolites.  a failure  thus  1986)  of  et  were  then  platelet  in  inhibitor  resulting  not  toxic  idiopathic  Glover  inhibitor  is  To of  test PD  obtained  this,  patients from  a correlation  some  between  MAO-B  activity.  gives  details  of  PD  controls,  subjects,  and  Appendix the  G  method who  platelets. that  inhibition  the  PD  patients  the  difference  rat  than being  liver for  MAO-B b y  control  treated  but  one o f  the  while  few  of  controls  might  of  have  between  the  the  various  drugs  accounted control  PD  for  were  and PD  signifi-  However,  exdrugs  were r e c e i v i n g  taking  their  their  was  anticholinergic  patients  and  urine  subjects.  with  All  presence  patients  of  patients  currently  difference.  drug treatment,  PD  1980;  MAO  Platelets  a s c e r t a i n whether  as  more  of  disease  itself  variations  inhibitor(s)  measured.  Sci.  or  then  species,  potency  MPTP  one  endogenous al.  MAO  that  i n the development  et  neurotoxic  endogenous  to  well  possible that  urine  to  u r i n e s and  of  factor  Parkinson's  cases  same e n z y m e ,  MAO-B i n human  J.-Neurol.  greater  to  most  an e n d o g e n o u s  inhibitory  as  T a b l e 28 cantly  of  subjects  al.  MAO-B for  to  is  i n c r e a s e d MAO-B a c t i v i t y ,  control  (Yong e t  by  (Glover  If  precursors  the  research  an u n i d e n t i f i e d  1982).  in  the  urine  charya et  inactive  by  be a c r u c i a l  presence of  strated  i n MPTP  responsible  a transformation  -  MAO-B i n r e l a t i o n  conversion  neiirotoxin(s)  i n t i s s u e s might  of  64  patients  any d r u g s .  metabolites  greater is  some  in  inhibition unlikely  to  It the of be  - 65 -  due t o d i f f e r i n g subjects (r  showed  = 0.26,  of  a  mean a g e s ,  no c o r r e l a t i o n  linear  decreased  patients, amounts  urinary  the urinary  in  control  MAO-B  This  arguement  expected  MAO-B  suggests  assumes  that  frontal The  measurements  cortex)  experimental  jects  from  platelet  MAO-B  Also,  the former, regulator  inhibition  there  poor  MAO-B whose  MAO-B  evidence  by  the  to s y n t h e s i z e  the  control  of  was no  inhibitor(s)  had t h e y f a i l e d  and p l a t e l e t  for the  adequate  correlation  activity identity  PD  be-  (r = 0.13) is  unknown,  o f MAO-B a c t i v i t y o f t i s s u e s .  MAO-B a c t i v i t y  i s an a c c u r a t e  reflect-  t i s s u e s s u c h as t h e b r a i n .  patients  details,  as w e l l  Inhibition  of  regions  was a l s o  b y human  (SN a n d  performed.  t h e PD and c o n t r o l  are d e s c r i b e d i n Appendix  of r a t l i v e r  Mean age subjects  brain  subjects  as d e s c r i p t i o n s o f  B activity  providing urine  in autopsied  and c o n t r o l  b r a i n s were u s e d ,  oxidase  any c a s e ,  the  o f MAO-B a c t i v i t y  whose p o s t - m o r t e m  Subjects  that  PD  Table 28.  In  inhibitor.  endogenous  i o n o f MAO-B a c t i v i t y o f o t h e r Direct  of  inhibitor(s)  m i g h t n o t be an i m p o r t a n t  age a n d u r i n a r y  analysis).  h a v e been  subjects  between  excretion  an e n d o g e n o u s  tween 21  regression  as m i g h t of  since linear regression analysis  G.  monoamine urine % Enzyme i n h i b i t i o n by u r i n e  Control subjects (49)  47 ± 3  20.9 ±  1.3  A l l Parkinson's disease patients (31)  62 ± 2 *  34.5 ± 4 . 0 *  Parkinson's disease patients not r e c e i v i n g a n t i c h o l i n e r g i c drugs (21)  63 ± 2 *  27.7  ± 3.4§  Mean ± SEM a r e shown f o r age i n y e a r s , and f o r urinary MAO-B i n h i b i t i o n i n . Number o f subjects analysed is shown in parentheses. Values that differ significantly from t h a t of c o n t r o l s u b j e c t s : *P < 0.001, P < 0.05.  sub-  -  In  Table  29,  it  c a n be s e e n  increased in the f r o n t a l MAO-B  activity  1980),  a n d t h e somewhat  due t o t h e i r In brain  increases  greater  summary,  of  patients. causes  the  MAO-B  in  appears  was n o t  significantly  brain  regions  i n t h e SN o f  (Fowler  t h e PD p a t i e n t s  described  that  conversion  of  showed  brains  of  of  urinary  PD  patients.  MAO-B  an o v e r a c t i v e an  no s i g n i f i c a n t  Monoamine  controls  inactive  oxidase B a c t i v i t y  and P a r k i n s o n ' s  Subjects  disease  There  inhibitor(s)  MAO-B a c t i v i t y precursor  in brain  to  a  of  patients  Mean age  MAO-B  (years)  (nmol/h/mg  activity protein)  Frontal-cortex C o n t r o l s (20) Parkinson's disease  78 ± 2 * Substantia  Controls (11) Parkinson's disease  24.7 ± 1.8 25.2 ± 1.4  63 ± 2 (11)  (7)  et  al_.  may b e  increase  i n PD.  Table 29.  control.  death.  excretion  unlikely  activity  most  activity  i n the autopsied  excessive  metabolite(s)  age  the experiments  a decreased It  with  higher  that  o r SN o f PD b r a i n s when c o m p a r e d t o  mean age a t  MAO-B a c t i v i t y  evidence  cortex  66 -  nigra 41.2 ± 5.2 52.9 ± 8.1  60 ± 2 79 ± 3 *  V a l u e s a r e i n mean ± S E M , w i t h t h e analysed in parentheses. Values that l y from c o n t r o l s : P < 0.001.  number differ  of subjects significant-  in in  in  was no living tissues  neurotoxic  - 67 -  111. v i i i )  Glutathione  in This that  has  glutathione SN  (6)  updates  from  PD  as o f  to  study  inclusion  of  reduced  the mixed  thione  content  of  tectable frozen  in  rapidly  in  parkinsonian et  al.  GSH  the  a more  during  et  with  al.  (1982)  PD.  Since  brains.  (GSSG)  Perry  since  et  GSH,  Part  forms  A  small  then, of  al.  of  there  this  sec-  this  were  (1968,  1981).  2xGSSG,  compound  death,  mainly  is  and  from  glutathione,  GS-SCy,  and  representation  Upon  GS-SCy a c c u m u l a t e s ,  study.  and c y s t e i n e ,  sum o f  biopsies.  Perry  (1982). '  accurate  life  of  activity  patients  patients  that  and o x i d i s e d  as  disease  SN o f  procedure of  taken  frozen  immediately,  used  Perry  (GSH)  enables  present  was  transferase  observation  in the  d i s u l f i d e of  was  GS-SCy  content  the  more  of  an amino a c i d a n a l y s e r b y t h e glutathione  by  brains  the o b s e r v a t i o n  Measurements as w e l l  initiated  Parkinson's  c o n t e n t was r e d u c e d  been o p p o r t u n i t y  tion  and g l u t a t h i o n e  the a u t o p s i e d b r a i n s of  r e s e a r c h was  sample of  content  of  total  not  The gluta-  normally the  de-  brain  autolysis  on  Total  GS-SCy.  unless  the  done  of  is  gluta-  thione. Brains  used f o r  consisting and o f (DE),  of  persons  patients  d a t a were f r o m died  without  Huntington's  olivopontocerebellar atrophy  glutathione interval  have  In  8  h  been al.  did  mean ± SEM,  none o f  metabolism.  of  (Perry et brains  who  dying with  schizophrenia.  would  control  not P<  or  All  match  the  0.001).  of For  so e x t e n s i v e With  any  (OPCA),  the  PD  to  brains  However,  (HC),  is  there  intervals,  the  age  (controls  linear  =  subjects  neurological  disease,  lateral  a n y known had  a  regression  sclerosis,  or  abnormality  of  of  h a v e become  subjects 57  encephalopathy  death-to-freezing  autolysis  would of  group of  dialysis  studied  quantitation  respect  of  amyotrophic  brains  longer  that  signs  chorea  these disorders  less.  1981).  a heterogeneous  at  ± 2 years, analysis  glutathione unreliable  death, PD in  = the  control 75  ±  3,  control  -  group r  showed  =0.02;  no  frontal  tical  biopsied  total  glutathione  ate  cortex,  specimens in  representation  biopsied  a small  pathological  Again,  no  (r =  was  glutathione the other cal  and f o r  b i o p s i e s of  normal  The  cerebral  age  of  was  found  age  (SN,  we a n a l y s e d 19  cor-  a more  These  surgical  surgery  cortex  subjects between  excision.  gives  life.  underwent  with  after  course  during  who  The  this,  immediately  abscess.  the a  total  large  glutathione  group  s i g n i f i c a n t l y reduced content  was  somewhat  5 brain regions  significance  death-to-freezing patients  frozen  present  brain  correlation  confirm  content  age  removal  order  ranged and  accur-  specimens  required  in  The  of  a  sacrifice  to  reach  from  16  total  of  the  to  64.  glutathione  0.36).  patients,  content  or  glutathione To  subjects  lesion.  T a b l e 30 p r e s e n t s PD  content  apparently  deeper  content  were  from  tumour  area of  linear  that  -  total  r = 0.05).  the  were  brain  of  promptly-frozen  of  material  deep-seated of  correlation  68  (3.9  intervals  ± 0.3  thione  reduction  nigral  glutathione  in  and  4.0  the  SN  by  in  the  lower  in  PD  of  0.5 PD  of  SN  of  PD  patients  t-test,  almost  identical  at  d e f i c i e n c y t h a t was p r e s e n t  than  for  during  the  mean  ±  in  Although  controls  for  reach  statisti-  Since  the  control SEM),  probably life.  for  glutathione  patients.  d i d not  autopsy  regions  Total  P = 0.05).  h respectively, patients  6 brain  subjects.  these reductions  2-tailed  were ±  control  only  examined,  (analysed  of  contents  the  mean  and  PD  gluta-  represents  a  -  Table 30.  Total  glutathione  69  contents  rapidly frozen Brain  region  Frontal  cortex  Cerebellar  Caudate  cortex  nucleus  Putamen  Substantia  in various regions  human  nigra  Parkinson's Disease  1.79  ± 0.07 (53)  1.54  ± 0.12 (15)  1.67  ± 0.07 (53)  1.44  ± 0.12 (12)  1.98  ± 0.08 (52)  1.66  ± 0.18 (8)  2.23  ± 0.09 (56)  1.85  ± 0.21 (10)  2.13  ± 0.11 (30)  1.72  ± 0.19 (10)  1.61  ± 0.07 (53)  1.16  ± 0.11+ (15)  V a l u e s a r e mean ± SEM, u m o l / g w e t w e i g h t , w i t h number o f j e c t s shown i n p a r e n t h e s e s . + P < 0 . 0 1 , as compared t o t r o l mean f o r t h e same b r a i n r e g i o n .  Animal ester, is  BHA)  rather  tent  of  are at is  a  have  SN  the  total  manipulation.  Why t h e n  The  atrophy  since g l i a , there  which  i s no  the c e r e b e l l a r cortex  in  of  PD  this  replaces  glutathione OPCA.  brain lost  (BSO,  is  the  total  neurons,  deficiency of  in  to  is  region.  L a r g e numbers  content  compared first  subcon-  glutathione  glutathione  least 4 possible interpretations. of  lower, in  thesis  when  reflection  much  that  this  brains  addition,  areas  described in  indicated  r e s i s t a n t to the  unlikely In  experiments  of  brains  Controls  cortex  Occipital  -  of  the  brain  glutathione controls ?  that  the  However,  lower this  also  contain  the  striatum  neurons  monoethyl  are  conThere  amount  apppears  glutathione. i n HC  l o s t from  or  in  these  in these 2 d i s o r d e r s .  The  second p o s s i b i l i t y i s  that  the  reduced  total  glutathione  content  in  -  the  SN o f  PD p a t i e n t s  is a result  patients  we s t u d i e d  of  (we have no c l e a r r e c o r d s  death  selves al.  it  an a t t e m p t appears a  to produce  terminus  decrease of  been e x p o s e d  because  that  drug  being  that  the lower these  total  levels  of  the reduction  treated  was n o t due t o L-DCPA t r e a t m e n t ,  patients  who w e r e  now f e w and f a r A third  never  p o s s i b i l i t y i s that  i s due t o  thione.  For  which must not et  an  brain  A  fourth  content ments  them  activity  of  The  pathway,  time them-  Tse e t represent  possibility  similarly  show  striatum,  would  Obviously, content  being  surely  have  o n e means  of  i n t h e SN o f  PD  would  be t o s t u d y  drug.  However,  such  deficiency  of  Also,  failure  o n e , inasmuch  of  areas argues a g a i n s t  indicate  autopsied  SN o f  patients  are  y-glutamylcysteine  inherited of PD,  as s i m p l e  which  free  significant  in  might  explain  relies  radicals  might  certain  individuals of  If  individuals mechanism  t o PD  does (Ward  glutathione  in  defect.  lower from  the  predisposition  depletion  on r e s u l t s  gluta-  be r e d u c e d .  the genetic  genetic  the  of  synthetase,  might  then  t h e SN i n PD  breakdown  a g e n e r a l i s e d enzymatic  PD p a t i e n t s that  accelerated  i n the s u s c e p t i b i l i t y of  the lack  possibility  or  of glutathione,  t o t h e development  complex  i n t h e SN o f  which  synthesis  enzymatic  t o be a f a c t o r  a l . 1983).  other  the  inherent  be a v e r y  this  should  content.  the glutathione  enzyme f o r t h e : s y n t h e s i s  predisposes  appear  decreased  example,  rate-limiting were  with  the  o r L-DOPA  this  the  between.  patients  this  treated  up t o  However,  glutathione  patients  a l l of  of glutathione  or dopamine.  total  L-DOPA  PD p a t i e n t s  nigrostriatal  L-DOPA  or  (Graham e t a l . 1 9 7 8 ;  radicals. of  Most  dopamine  levels  glutathione  of  with  Since  radicals  the striatum  of  treatment.  of t h i s ) .  the dopaminergic  to^high  of  free  b y t h e SN t o d e t o x i f y  untenable  confirming  probably  is conceivable  significant  the  were  can a u t o x i d i s e  1976),  70 -  total  glutathione  our animal  be i n v o l v e d  experi-  i n the molecular  -  mechanism  of  MPTP  important  in  the  striatal  then  ification,  a  appears would  the  of  the  these  et  occurs  a  its  to  the  restoration thus  Calne et  briefly  al.  in  1985)  many y e a r s  brain  the  areas  from  at  detoxresult.  might  death?  levels  that  of  be  the  This  probably Calne  who s u g g e s t e d t h a t  before  free  area.  glutathione  differs  nigro-  forms  be  neurotoxin(s) of  of  process of  would  in t h i s  critically  that  the  other  be  cause  neurotoxin  a neurotoxin to  to  the  levels,  opposed  hypothesis  1983;  is  If  glutathione  be e x p o s e d  This  appears  radicals.  in  SN a s  otherwise,  al.  neurotoxin  decrease the  still  occurred.  (Calne  of  l o c a l i s a t i o n of  patient  glutathione  i d i o p a t h i c ,PD  consequent  likely since,  Langston to  the  have  in  deficiency in  a r e f l e c t i o n of Would  loss  -  that  e x c e s s i v e consumption  with  localised  and  detoxification  neuronal  radicals,  The  toxicity,  71  appearance  and  exposure of  symp-  the  puta-  toms. What i s tive  the r e l e v a n c e of  neurotoxin?  clinical  trial  deprenyl  (an  patients  at  an M P T P - l i k e and  if  achieve. might cate to  MAO-B the  early  to be  it  which is  destroys  this  is  benefit, indeed  neurotoxin  potential  then these p o t e n t i a l  that  treatment  we  If  the  my r e s u l t s  years  or  not  possibly  On  before  to  the  drugs  content from  of  a  multi-centre  symptoms PD  via  then slow  to  a-tocopherol  of  idiopathic  speculate,  none o f  task.  that  progression  neurons  can r a i s e  a difficult many  the  exposure  reported  candidates  that  although  occurred  as  of  whether  disease.  continous  agents  1985b)  slow  the  t h e d i s e a s e , an e f f e c t  duration  determine  could  of  promising  Similarly,  that  stage  neurotoxin  a l s o be o f  the  inhibitor)  and  (Lewin  was b e i n g p l a n n e d t o  would  g r e s s i o n of  time  A recent review  exposure  deprenyl  the  is  free  in  PD  caused  by  radicals,  a-tocopherol or  or  stop  the  or pro-  c u r r e n t l y used can brain  glutathione  animal  experiments  the  other  hand,  the  appearance  s t r a t e g i e s a r e u n l i k e l y t o be o f  if of  indi-  exposure symptoms,  benefit.  -  Assuming brains and  the decreased  was due t o  GSH,  the  enzymatic ty  that  is  an  in nature,  the  peroxides  that  and i f  glutathione by  with  GSSG ( D e n e k e e t a l . 1 9 8 0 ; ciently rate,  reduced  back  the overall  action of these Another  are  et  could of  s o , what  i s t h e enzyme  the  system,  GSH.  In  nature  the  the  the  the  its  glutathione  is unlikely  or  it  other  oxidised  the l a t t e r  reductase  Is  possibili-  H,^  becomes  However,  PD  derivatives)  ? One  inactivates GSH  SN o f  reaction.  involved  process,  content  (or  of  which  et a l . 1980).  glutathione  enzyme  GSH w i t h  a l . 1980).  transformed  is  GSH b y  detoxifying  lowered  a neurotoxin  in  is  system.  effi-  At  t o be a l t e r e d  to  any  by t h e  enzymes.  which conjugates Jakoby  total  content  remains  Sinet  to  glutathione  between  peroxidase  reacting  -  total  interaction  question  72  In  to  involves  a wide v a r i e t y the process  by a p r o c e s s back  system  that  GSH.  It  of e l e c t r o p h i l e s  of  produces  derivatives seemed  content  of  the  which  levels  cannot  possible total  transferase,  (Habig e t a l . 1974;  detoxification,  therefore  be r e s p o n s i b l e f o r t h e r e d u c e d  glutathione  that  of  GSH  be  readily  this  enzyme  glutathione  i n t h e SN  PD p a t i e n t s . We m e a s u r e d  tex  and SN o f  sis  of  died  PD.  patients Brains  without  patients  the activity  encephalopathy  died  that  with  (DE).  It  because  insufficient  assay.  The OPCA o r DE  thione  metabolism.  assays  matched  glutathione  who had d i e d w i t h  any e v i d e n c e  who  of  served  as  of neurological  patients  PD b r a i n s  were  disease;  olivopontocerebellar was n e c e s s a r y  include  brains  brains  respect  used  mainly  were of for  from  however,  atrophies  h a d no e v i d e n c e  control with  to  i n the f r o n t a l  a pathologically-confirmed  controls  non-neurological  The  transferase  cor-  diagno-  subjects some w e r e  who from  (OPCA)  or  dialysis  among  the  controls  these  available  for  any a b n o r m a l i t y glutathione  to d e a t h - t o - f r e e z i n g  the  enzyme  of  gluta-  transferase  intervals  of  the  -  brain,  as w e l l  age m a t c h e d  (Table  Analysis Spearman  as t h e  of  et  al.  (1984)  homogenised  were  centrifuged for  of  storage  in at  of  50  transferase  t h e method  volumes  10,000  analysis.  (w/v)  g for  Into  a  of  15  100  ul  was  added.  1.0  ml.  was  seconds. blank 9.6  average  change  as  done  in  conjugate  duplicate  m i n e d by t h e method o f  and  cortex  of  controls.  The  be  their  due  frontal  to  cortex  a c t i v i t y with 0.74  for  SN).  PD  SN o f  t h e age o f  final  room  t h e y were  not  340  nm  a blind  using  Brain The  volume  5  mM  buffer of  15  seconds  was  col-  GSH, at  1  pH  mM 6.5,  the mixture and  its  for  corrected  calculated of  content  was  progress  seconds  Analysis Protein  samples  then  absorptivity  was  of  homogenates  supernatants  a molar  fashion.  for  value and  90 a of ex-  each  sample  was  deter-  (1951).  in Table 31.  mean  the  every  protein.  in  modification  (1974).  temperature  absorbance/15 and  al.  the  phosphate  formed/min/mg  s l i g h t l y higher  and  at  by  containing  activity  patients  greater  and  transferase  Lowry  The r e s u l t s a r e shown frontal  However,  M sucrose.  cuvet  at  present),  glutathione  nmol  in  et  0.25  The  c a r r i e d out  (when no t i s s u e was mM~*cm-*,  done  mM s o d i u m  spectrophotometrically  The  pressed was  supernatant  r e a c t i o n was  followed  was Habig  reaction 100  The  -70°C.  minutes,  and  brain  at  of  l-chloro-2,4-dinitrobenzene of  -  31).  glutathione  were  lected  duration  73  did  Mean enzyme a c t i v i t i e s i n t h e  not  activity age  controls subject  at  differ in  at  both  death.  revealed death  significantly regions  Linear  of  from PD  regression  SN  and  those  of  brains  analyses  a s i g n i f i c a n t increase in (r  = 0.81  for  frontal  could of  enzyme  cortex,  and  -  Table 31.  Glutathione  cortex  transferase  Mean age a t (years)  Substantia  Values trols:  Th-us,  (mean ± SEM) P < 0.05; *P  the  results  interaction(s)  leading  patients.  oxidants, 1980),  PD  frontal  patients  GSH  death  i n the  transferase  (nmol/min/mg  .  activity protein)  60 ± 3 77 ± 2 *  183.1 ± 202.3 ±  10.6 13.5  63 ± 4 . 77 ± 2-  204.2 ± 221.2 ±  12.9 15.7  nigra  Controls (16) Parkinson's disease (12)  PD  activity  cortex  C o n t r o l s (23) Parkinson's disease (17)  of  -  and s u b s t a n t i a n i g r a o f  Subjects  Frontal  74  akin  that differ < 0.001.  presently to  the  A persistent to  c a n n o t be  the  direct  excluded.  significantly  available  decreased  do  total  non-enzymatic r e a c t i o n of  not  from  clarify  glutathione interaction  a-tocopherol  of  with  con-  the  nature  content  in  of the  glutathione oxidants  the SN  with  (Witting  - 75 -  IV)  Several ter.  animal  How h a v e  pathic  PD?  a n d human  these  aided  My r e s u l t s  CONCLUSIONS  experiments  have  been  i n the understanding  suggest  the following  described  in this  of the pathogenesis important  points  chap-  of  idio-  about  idio-  p a t h i c PD: 1.  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A s s o c .  s t u d i e s on p e r s o n s who h a v e had e p i d e m i c e n c e p h a l i t i s , 9 1 : 1 3 8 - 1 4 0 , 1928.  -85  Chapter  2 :  -  HUNTINGTON'S CHOREA  ABSTRACT  We t e s t e d in  the hypothesis  Huntington's  enzymatic  chorea  failure  (HC)  in the  endogenous  or exogenous  vivo  i n which r a t s  study  filtrate which  from  In  or  patients,  might  origin. were  study,  content  there  of  GABA  neurotoxin  contributes  a  molecule  that  examine  acid some  a small  further  than met  10000 with  f r o m HC p a t i e n t s .  were  with and  in tissue  was a s m a l l in  rats  of  either  performed:  an i n  serum or serum an  in  culture  vitro to  ultra-  study  serum  in  or  but s i g n i f i c a n t reduction  treated  with  bound  greater  the in vivo molecule daltons. equivocal  the  to  serum  than 10,000 explants  consistently  of  The r e s u l t s  showed  synthetic the  tightly  bound  CSF  Experiments results,  P o s s i b l e reasons  as  to did  o f HC,  i n the from  treated  that  vf  i t must or  HC with  a  cir-  either  a large  be  com-  daltons. t o 30% whole  deficiency  of  enzyme  GABA.  for  neurotoxin  that to  serum  in rats  proteins,  exposed  putative  experiments,  whole  suggested  to the pathogenesis  striatal  characteristics  be e i t h e r  exposed  tightly  decarboxylase,  as w i t h  larger  is  study,  patients  indicate,  studies  subjects,  HC p a t i e n t s .  a molecular weight  In t h e i n v i t r o  glutamic  of  occurs  genetically-determined  GABA d e f i c i e n c y was o b s e r v e d  culating  HC  a  that  a c i r c u l a t i n g neurotoxin  Two t y p e s  were  from  adult-onset  of  of  death  controls.  b u t no s t r i a t a l  pound w i t h  neuronal  be t h e r e s u l t  control  serum u l t r a f i l t r a t e  small  premature  injected repeatedly  or  explants  the i n v i v o striatal  the  degradation  patients  rat striatal  from p a t i e n t s  mean  HC  that  the  in  proteins,  delineate experiments  these that  of  Attempts  to  culture  material or  from  activity  tissue  the detrimental  plasma  serum  a  may  molecule  possibilities utilized  f o r the d i s c r e p a n c i e s are d i s c u s s e d .  CSF  - 86  The r e s u l t s o b t a i n e d vide s u g g e s t i v e but i n t h e serum of  not  from both  the  -  in vivo  conclusive evidence for  patients with  HC.  and the  in v i t r o  experiments  presence of  a  pro-  neurotoxin  -  I)  relentlessly autosomal  chorea  dominant  the  American  majority  30 and  50  age o f  15  of  In  the  mother.  the  symptoms. common  HC  the  signs  other  dystonia  Thus,  there  dysarthria  chorea  is  in  the of  minimal  or  addition  observed  in  both  the  to  the  the  Bird  found  the by  be  of  body  about  by  a  inherited  in  in  a an  gene  has  In  the  1983).  100,000.  between  the  In  ages  of  symptoms  before  'juvenile  chorea'  or  Remarkably,  in  1980). father  more  the  is  al. 10  the  that  disease,  causative  et  called  1973;  to  The  occurs  inherited  (Went  than  twice  causative  sex-related  et as  gene  the  al.. many  from  modifying  an  factor  disorder. a combination the  adult-onset become  features  of  face,  fingers  patients.  affected.  emerge,  physical  and  in  patients  mental  and t o e s  As  Late  and  the  the  are  are  disease  course  of  troubled  by  swallowing.  onset  absent  a l s o have m u s c l e s p a s m s , In  course  is  is  manifest  e s p e c i a l l y of  parkinsonian  juvenile  HC  transmitted  appears  that  (Gusella  cases  (1983)  onset of  in  4  subgroup  studied  movements  and d i f f i c u l t y  contrast,  disorder  symptoms  al.  is  characterized  parts  and  et  al.  they  is  early  et  cases  of  clinical  (Byers  Huntington's  penetrance.  10% o f  disease  Myers  Choreiform  progresses,  In  HC  onset  About  i n f l u e n c e s t h e age o f Clinically,  HC,  of  contrast,  as  incidence of  distinct  cases,  late-onset  affected that  variant  juvenile  1984).  a  complete  the  1984).  and f o r m  known  r e s i d e on chromosome  patients,  (Martin  also  with  population,  of  the Westphal most  to  Perspectives  neurodegenerative  fashion  r e c e n t l y been shown North  (HC),  progressive  -  INTRODUCTION  I.i) Huntington's  87  (Byers  tremor, motor  juvenile  cases t y p i c a l l y e x h i b i t et  al.  1973;  disturbance of  disability, and  Spokes  1981).  eye movements,  mental  adult-onset  muscle r i g i d i t y ,  and  and  emotional  varieties  of  HC.  They  and may  epilepsy.  problems A  are  gradual  -  *  ,  intellectual ture  of  the  ficulty et  during  the  1978).  are f r e q u e n t l y  from  intervenes) tional  symptoms  of  symptoms  capacity rating  scale  for  13  for In  (see  a  patients large  below),  Patients  nucleus,  Bruyn  sults  putamen,  in  striatum, 8.5  pm  these  et  al.  neuronal  putamen, a  there  in  diameter)  small  neurons  in  appearance,  have  a regular  fea-  increasing  dif-  organizational  of  their  tasks  some  point  committing  suicide  and s u c h  patients  psychotic,  as  a  HC  may  studied  many a r e a s  cortex. being  showing  to  reach  or  recurved  1.8  the  the  death  a  func-  ranging  from  zero for  bed-  conventional  drug  to  functional  age o f  of  the  units  35 y e a r s  most  collectively of  the  loss  of  large 80%  per  tended  endings  of  degeneration  lateral small  in  the  called  neurons  the  to  appendages  (which 1981).  Type are  II  sumis  caudate nucleus  striatum,  ventricles.  (Lange of  the  caudate  neurons  techniques,  and  b r a i n has been  marked  Dendrites  staining  HC  widespread  Deterioration  enlargement  Golgi  of  devised  units  abilities  rather  consistently  compared  by  before  allots  of  accidental  has  receiving  loss  Although  70  It  duration  1981).  disproportionate as  (1981)  patients  a mean  The  s u i c i d e or  retaining, f u l l  of  are  structures  (unless Shoulson  appeared  (1979).  downhill.  patients.  of  status  and c e r e b r a l  is  HG  (Shoulson  characteristic  striatum,  risk  frankly  years.  group  losses  these  the  out  is  s e r i o u s l y depressed at  the  death  still  whose symptoms  The n e u r o p a t h o l o g i c a l  observed,  patients  carrying  inexorably  he o b s e r v e d  more r a p i d l y  by  and  to  15  marized  and  become  is  about  deteriorate  and  become  is  treatment  and  disease,  patients  of  usually  ones.  year.  problems  dementia,  m i s d i a g n o s e d as s c h i z o p h r e n i c s .  onset  a maximum o f ridden  the  10% o f  progression  disease  full-blown  Many p a t i e n t s  course of About  to  -  Memory d e t e r i o r a t e s ,  arithmetical  al.  high.  The  progressing  disease.  doing  (Caine  is  decline,  88  In  rethe  average Loss  of  neurons  of  markedly  (Graveland  altered et  al.  -  1985).  In t h e s u b s t a n t i a  fined  to  the  more  nigrostriatal nigra  ventral  tract,  pathway, 1984).  It  i n the s t r i a t u m ,  major  input  al.  1983).  general, atrophy  to  than t h a t  HC p a t i e n t s .  i n many b r a i n a r e a s include  nigra.  t h e s i s e s GABA,  striatum,  cortex, with  juvenile  HC  have  deficiency  been of  nucleus,  i s decreased  loss  of s t r i a t a l  of  and s t r i a t o n i g r a l  choline  acetylcholine. 1974).  This  Substance  P  in  contents  nigra  also  suggesting  evidence  finding  occurs  (Kanazawa  of the c h o l i n e r i n HC  et  disease.  greater  In  degree  of  e t a l . 1951).  acid  (GABA)  e t a l . 1975;  globus  pallidus  (GAD),  nucleus,  of  is  present  Perry  1982a).  and  substantia  t h e enzyme w h i c h s y n -  putamen,  et a l . 1975).  the afore-mentioned as w e l l  (Clark  It  and g l o b u s  pal-  is likely  that  areas  as G A B A e r g i c  represent  neurons  a  in the  pathways. i n HC b r a i n s  acetyl transferase  and s u b s t a n t i a  Urquhart  interneurons,  (Shoulson  in the autopsied brains  y-aminobutyric  Urquhart  a  et  glutamatergic  Alzheimer's  (Campbell  reported  i n the caudate  most  may h a v e  cases  putamen,  t h e GABA and GAD d e f i c i e n c i e s o f  activity  in  acid decarboxylase  and 1 9 7 4 ;  Another neurochemical  seen  (Cross  atrophied  t o be n o r m a l  patients  e t a l . 1973;  of glutamic  GABAergic  is  in adult-onset  (Perry  provides  i s reported what  is  substantia  i n HC  the  both  striatopal1idal  intact  to  changes  ( B i r d e t a l . 1973  relatively  which  dopaminergic  compacta of the  projection  the caudate  Activity  The  the c o r t i c o s t r i a t a l  observed  A marked  1980).  not  of  Many n e u r o c h e m i c a l  lidus  is  is usually, con-  or  contrasts  the b r a i n s  (Bird  neurons  whether  the cerebral This  of  i n the pars  The n u c l e u s b a s a l i s o f M e y n e r t ,  gic  These  reticulata  originates  i s unclear  another  the degeneration  pars  which  and t e r m i n a t e s  al • 1983).  nigra,  89 -  about  (CAT), 50% of  are reduced et  subnormal  a l . 1977;  in  i s a decrease the  enzyme  patients the Emson  concentrations  that  (Bird  striatum,  in the s t r i a t a l  et  synthesises al.  globus  1973;  pallidus  et  a l . 1980).  There  is  of  methionine-enkephalin  -  in  the s t r i a t o p a l 1 i d a l  the o t h e r  hand,  and s t r i a t o n i g r a l  the content  t h e s t r i a t u m and g l o b u s In  general,  changes cortex major  that  treat or  symptoms  haloperidol,  choreic  effects  nigrostriatal excess  due  (Shoulson  therapies  antipsychotic  loss  useful,  Of  1986).  since they  al.  a l . 1980).  On  i s increased in  et a l . 1983).  neurons  extend  from  and n e u r o c h e m i c a l  within  the  the  cerebral  striatum,  bear  of  use a n t i d e p r e s s a n t  movements  drugs  striatonigral  drugs,  such  (Shoulson  probably  t r a n s m i s s i o n which  antipsychotic  derive  as  the  from  GABAergic  drugs  to  thioridazine  1984).  The  anti-  a reduction  may be i n a s t a t e  this  loss  i n HC  contention  is  derived  to: a l a r g e  65 u n t r e a t e d  Thus, treat  attempts  been  made  of  t h e HC  amino 1968).  acid  to to  kindred  HC p a t i e n t s  the  of  of  relative  inhibitory  influence  correct  does  Attempts  studied,  drug  drugs  (Shoulson from  near  t h e mean  are  efficacy  Additional  experience  Maracaibo rate  drug-treated  therapies  no  1981).  the  Lake  have  of  in  HC  Vene-  decline  patients  palliative  of  ob-  ( Y o u n g e_t in  nature,  symptoms.  provide  brain.  living  of North American  conventional  only  and a n t i d e p r e s s a n t  neuronal  s e r v e d was s i m i l a r t o t h a t  this  of  HC p a t i e n t s  choreiform  the progressive  belonging  areas  (Aronin  and a n t i p s y c h o t i c  of  patients  have  that  of  reduce  supporting  In  somatostatin  neuropathological  populations  to  evidence  al.  et  i n HC.  depression,  the  of  (Emson  1984).  slowing  zuela.:  pattern  and t h o s e  dopaminergic to  Although in  drug of  the neuropeptide  certain  of pathology  Conventional  pathways  p a l l i d u s o f HC b r a i n s  and t h e s t r i a t u m , brunt  of  the o v e r a l l  suggests  90 -  more  effective  the d e f i c i e n c y  Administration  not r e a d i l y have  been  cross made  to  therapeutic of  of  GABA  GABA  content  itself  the b l o o d - b r a i n slow  the  management,  is  observed  efforts in  ineffective  barrier  enzymatic  many since  (Roberts  et  degradation  of  - 91  GABA w i t h et  al.  inhibitors  1980)  failed  to  agonists  s u c h as m u s c i m o l  azid to  et  al.  1975)  results  (INH), this  potent  INH  in the  clinical mental  itself  is  of  INH,  3  (Perry  et-  only  INH  trial  blind)  by  markedly  that were  in  6  in  1  their  a large  time of  is  this  tion atric  of  scale  needed sort  to  might  acid  (Perry  approach GABA  has  receptor  imidazole-4-acetic  produced  no  9  functional  patients  by  acid  improvements  in  showed  i n the  role  of  answer  the  question  who trial  that  4  of  an  later  of  from  In  a  11  results  are  therapy  of  patients  with  INH  in  the  as  to  whether  or  slow  agent  with  treatment  the  or  of  potential  for  improved  HC.  HC.  and  as  Clear-  period  chronic  neurological  (non-  equivocal  long  not  Nega-  agent  were  the  a  al.  double-blind  same  over  in and  et  therapy.  Thus,  extending  non-blind  (Perry  patients  a  who  double-blind  a  is  brain  improvement  INH  the  of  patient  conducted of  but  HC  investigators  benefited  trial  the  Another  same  favorably  Elevation  1985).  a  isoni-  is hydrazine,  significant  However, the  clarify  HC.  showed  another  prevent  respond  which  brain al.  agent,  inhibitor,  1981).  et  (1982),  placebo-controlled  in  al.  (Perry  McLean  INH  may  one o f  autopsied  capacity.  can  et  patients  by  of  patients  GABA-transaminase  INH  although  b r a i n GABA c o n t e n t  deterioration  a  1979a).  al..- (1983)  to the b e n e f i c i a l e f f e c t s ly,  this  symptoms.  or  but  some HC  the  trial  obtained  et  5  of  patients;  Stober  1978)  but  brain GABA-elevating  (Perry  in  al.  1982b)  results  al.  many m e t a b o l i t e s ,  of  crossover  tive  not  with  placebo-controlled showed  1984),  or mental  another  inhibitor  treatment  symptoms  al.  tried,  because  demonstrated  under  trial  et  been  with  liver, into  recently  while  also  mention,  GABA-transaminase  GABA was died  have  et  physical  (Shoulson  treatment  deserve  metabolized  in  s u c h as a m i n o o x y a c e t i c  functions.  of  drug.  (Scigliano  improvement  or c o g n i t i v e  The  GABA t r a n s a m i n a s e  y-vinyl-GABA  produce  (Shoulson motor  or  of  -  A  of  trial eleva-  psychi-  treatment  of  -  HC,  baclofen, will  be d i s c u s s e d i n  Thus,  much  although  cal  changes  ent  time.  i n HC,  management  risk  of  I.i i)  Pathogenesis  death  which of  death.  The  destroys of  HC, The  in the all  third  these  dealt with  the  Menkes e t  of  of  study, be  al.  or  neuropathological  remains do n o t  or  of  the  most  at  the  pres-  progressive  the  onset  a better  occurs  neurochemi-  slow the  Improvement o f  requires  which  and  unsatisfactory halt  prevention  therapeutic  of  symptoms  understanding markedly  HC  HC  in a l l  in  of  in the  the  stri-  the  cell  mutant  Three  main  The  predisposing presence  gene  for  second  cells  of  to  an  involves premature  a neurotoxin  p r i n c i p a l l y with  HC  hypotheses  gene may c o d e f o r  membranes.  the  chorea  which  the  etiology  be  involved  discussion.  i s b a s e d on  patients.  Since  brain  were  cells.  c h a r a c t e r i s t i c s of that  membrane  used Early  skin  cultured  defect  may  publications  abnormalities  tissues  these  cell  a s e r i e s of  membrane  reported  how  the mutant  deals  generalized  in  is  neurons.  thereby  chapter  merit  of that  postulates  found  expressed  (1973)  suggests  this  in Huntington's  be a n s w e r e d  mechanism  a  death  populations  peripheral  growth  to  i s present  that  have  tissues  might  section.  disease.  neuronal  first  Because  pathogenesis  for  the  needs  hypothesis  suggestion  to  the  therapies  of  possibilities  claiming  cesible  The  repair  neurons.  peripheral  gene  DNA  about  death  certain  p r o t e i n which  defective  next  disease,  neuronal  h a v e been a d v a n c e d .  a  the  patients  HC,  the  -  cortex.  A key q u e s t i o n  abnormal  with  for  the  cerebral  the  available  patients  at  pathogenesis  causes  of  c h a r a c t e r i s t i c of  of  individuals  atum and  treatment  Currently  deterioration  i s known  92  in  cells with  cells are the  accounts  fibroblasts fibroblasts  in the obtained  relatively hope in  that  the  in tissue from  1970s, from inacthe  HC  literature culture.  HC p a t i e n t s  had  -  a  reduced  growth  potential  d e r i v e d from normal  replicative  life  and c o - w o r k e r s that  of  firmed  span  to  several  groups ( K i r k  On t h e o t h e r  a  hand,  meters data  could  coded  samples,  (1979)  and c o n t r o l  al.  by  computer.  detected  (1981)  for  t h e most  to  from  the  growth  be s i m i l a r studies  were s m a l l  their  that  those were  Among t h e s e type of l o c a l  can i n f l u e n c e a r e t h e medium anesthetic  Goetz e t a l . 1979).  detect  any  between  cultured  et a l . (1979),  Eleven  of  the  differences  resulting the 3  Meanwhile,  time  between  para-  between  measured. this  in  i n d i v i d u a l s , and  results.  analyses  study,  difference,  using  blind-  cultured  fibro-  and c o n t r o l s .  of  of  HC  controls.  either  not  differential used,  Early  growth  of  and s i z e  in  reports blind,  in the report  the s i t e  to obtain  fibroblasts  performed  (two p a t i e n t s  used  by  parameters  original  characteristics to  the  from  corroborated  convincing  significant  medium  c l a i m and c o n -  'at-risk'  no s t a t i s t i c a l d i f f e r e n c e s  in blind studies  Many f a c t o r s  No  Menkes  later  Cassiman 22  longer  did  density,  discriminant  any o f  repeated  and f o u n d  samples.  o f 22 c o n t r o l s ,  perhaps  culture  that  fibroblasts  to  population  claimed  HC s k i n  was  unable  those  thereafter,  e t a l . 1978;  were  to  and h a d a  original  that  This  Leonardi  o r i n maximum  d e r i v e d f r o m HC p a t i e n t s  Thus,  came  time  provided  be  et  appear  Gray e t a l .  analysed  Goetz  blasts  e t a l . 1977;  Shortly  their  (1975)  density.  were . t e s t e d : and s t a t i s t i c a l  were  groups  al.  et  compared  density  a different  of Goetz  blind-coded study  25 HC p a t i e n t s ,  using  when  e t a l . (1975)  confluent  retracted  o f HC p a t i e n t s  a thoroughly  Goetz  experiment,  cell  span  fibroblasts.  a l . 1977),  greater  in generation  fibroblasts  In c o n t r a s t ,  control  et  the observations grow  either  than  previous  indeed  life  grew t o a g r e a t e r  (Barkley  their  and d e c r e a s e d  controls*  c u l t u r e d HC f i b r o b l a s t s  93 -  the skin biopsy,  of or  of Kirk  cells of  tissue a  culture  difference  sample  numbers  et a l . 1977).  in tissue  the skin  culture.  biopsy,  and t h e method  the used  -  to  replenish  Beverstock  the  culture  defects  in erythrocytes  E r y t h r o c y t e membrane electron  subjects by  at  least  al_. 1 9 8 2 ;  spin  et  (ESR)  a l . 1977;  have  also  fibroblasts  from  HC  et  tors  (Lakowicz  1979),  after  have  1981).  This  al.  1981;  been r e p o r t e d to  summary,  control  could  reported from  t o have  those  this  of  h a s been  a l . 1981;  by  dif-  control disputed  Beverstock  Claims  been  have  challenged protein  gene  in  et  al.  either  been  by o t h e r s . peripheral  (Moronha  does  i n the c e l l  i n HC a f f e c t s  a membrane differed  defect  from  a fluorescent  et  not  probe  support  retracted any r a t e ,  tissues  consistently  a l . 1979).  investigafrom  would  by t h e i r  help  and m a r k e d l y  only  to  capping observa-  structural  t i s s u e s f r o m HC  original  the detection  HC  1985).  generalized  of p e r i p h e r a l  not  This  (Beverstock  a  in  (Pette-  Lymphocytes  laboratory  membranes  At  et  HC.  controls  by other  1982).  in  t o show d e c r e a s e d c o n c a n a v a l i n A - i n d u c e d  evidence  defect  of  n o t be c o n f i r m e d  Sumbilla  lymphocytes  present  functional  patients.  of  et  l a b e l l i n g with  h a s y e t t o be t e s t e d b y an i n d e p e n d e n t  mutant  been  reportedly  tion  membrane  reviewed  have a l s o been d e s c r i b e d .  although  claims  patients  spectra  a l . 1979; et  led to  compared  have  have  (Comings  when  and/or  thoroughly  characteristics  laboratories  techniques  grew  In  been  o f HC p a t i e n t s  f r o m HC p a t i e n t s  resonance  3 independent  fluorescence  patients  have  Fung e t a l . 1 9 8 2 ) .  Other  their  ghosts  (Butterfield  Cultured  These  (1985).  Membrane  ferent  medium.  94 -  of  authors, an  explain  or  abnormal why  the  certain  populations  is  the  neurons. The  gene  second  codes  for  degenerative been shown  hypothesis an  abnormal  conditions  t o have  regarding DNA  the e t i o l o g y  repair  mechanism.  ataxia-telangiectasia  associated defects  in their  of  HC The  inherited  and x e r o d e r m a DNA r e p a i r  that  mutant  neuronal  pigmentosum  mechanisms.  have Cells  -  from  these  patients  doses of r a d i a t i o n 1978;  Paterson  reported to  that  have  degrees  et  al.  retracted,  1979).  4 peripheral  (Arlett  of  than  In  cell  1979),  the  lines  cells  sensitivity when  increased  o r c h e m i c a l mutagens  gamma-irradiation  publication  an  95 -  a similar  rate  (Taylor  a l . 1975;  case  from  derived  9 out of to  mortality  11  et of  from  normal  HC c e l l  and no e v i d e n c e o f a n y e x c e s s  i r r a d i a t i o n damage  cultured  1980).  fibroblasts  o b t a i n e d by o t h e r Thus,  the bulk  (Arlett  investigators  suggests  DNA r e p a i r m e c h a n i s m s  i n HC c e l l s  DNA-damaging  Again,  repair,  i t would  vation of  that  t h e mutant  occurs  t h e most  likely  with  there  i n HC a f f e c t s  In  another  showed  varying  were  a  result  was  them more specific  only  also  Evan e t a l . 1 9 8 2 ) .  information  markedly  samples  i n t h e HC  i s no s p e c i f i c  render  later  blind-coded  negative  there  to reconcile this  an  enzyme  have  removing small  HC  sufficient neuronal  of  the  neuronal loss  such t o x i n s  might  metabolizing  defect  in  sensitive  to  defect with  certain  in  the  DNA  obser-  populations  to  be e x p l a i n e d  on c e r t a i n t y p e s  of  many  neurons.  HC  is  that  of  of  one  the  susceptible might HC.  code  putative  mutant  incapable of  it  form-  t h e HC gene m i g h t  and  years  an e x c l u s i v e  in  detoxifying  normal  t h e symptoms  by  to  thus  exposure  over  produce  allele  one  loss  e i t h e r endogenously  r e n d e r i n g them  Repeated  neurotoxin(s)  loss  neuronal  and  with  enzyme a c t i v i t y ,  neurotoxin(s).  amounts  The n o r m a l  heterozygotes,  subnormal  the  of  for  to a neurotoxin(s),  sources..  capable  neurotoxin(s). might  hypothesis  as a r e s u l t o f e x p o s u r e  o r from exogenous  for  for  gene  if  sensitive  neurons. Perhaps  ed,  be d i f f i c u l t  that  more  (1979)  f i n d i n g s . were  et a l . 1982;  which might  even  were  low  et a l .  al.  c o u l d be d e t e c t e d  latter  (Beverstock  of the evidence  agents.  The  et  tested  These  to  Andrews  controls.  lines  was r e p e a t e d  response  Arlett  HC p a t i e n t s  gamma-radiation.  experiment  HC,  in  allele,  completely neurons  eventually  cause  The s e l e c t i v i t y  localization  of  to  of  receptors  - 96  -  CH -CH -COOH 9  I  2  CH-COOH  Glutamic  acid  /  NH  2  CH H.C = C HC-CH - CH -COOH 3  Kainic  2  acid  HC CH-COOH 2 s / N H 0  ^"^V^COOH  Quinolinic  acid  \ ^COOH N  Figure acid,  1.  Chemical  and q u i n o l i n i c  structures acid.  of  glutamic  acid,  kainic  -  The 3  neurotoxin  compounds  are  changes t h a t These  capable  Digenea neurotoxic  (Coyle  et  1979;  Coyle  efferent  the s t r i a t u m , Although ism of  is  would an  has et  its  to  excitant  a potency al.  toxic  is  most  mechanism o f  toxicity  the  for  a  ticular  (Johnston  longer  Olney to 1971;  (Coyle  high-affinity  glutamate  period  propose  an  O l n e y 1974; toxic  that  action  of et  than  devoid  when- t e s t e d  suggest  by  body  on  rat  neurons,  of  et  in  KA  is  uptake  system  that  al.  1979).  time  Olney et would  be  Thus,  than  'excitotoxic' al.  it  necessary,  al.  are  desto  be  to  than  neuro-  1978).  These  be  to  death  this  in  of  gluta-  actions  receptor  concept,  of  sites  have  (Olney  a persistent  the  transported  observations  neuronal  According  at  it  lack  synaptic  persist  These  (McGeer  involved  to  KA  acid  a subset  appear  HC. but  effects  might  in  stud-  acid,  amino  al.  mechan-  such  death  et  the  the  that  glutamic  bind  may  other  et  afferents  1981),  latter  terminates  mechanism f o r  McGeer  hope  (Schwarcz  glutamate.  1979).  al.  is  does n o t  KA  pro-  striatum  neurons  neuronal  the  to  KA  HC  while  neuroexcitatory  believed and  1976;  the  excessive neuroexcitation KA.  Japanese  rats,  in  1).  spared.  as  of  striatum  1979),  of  are  (Beutler  that  al.  animals.  the  of  found  degenerate,  attention  al.  et  et  least  (Figure  of  cholinergic  tract,  CNS  striatum of  product  those  at  neuropathological  striatum  p o s s i b l e cause of  greater  derivatives  observations  mate r e c e p t o r s  the  the  and  neurons  the  mammalian  times  KA  properties  in  the  a  McGeer  GABAergic  nigrostriatal  elucidate  300  1978a;  striatal  found  is  resembling  h a s r e c e i v e d much  of  1979).  into  of  not  toxicity help  injected  al.  and  that  and q u i n o l i n i c a c i d  When  et  observation  injected into  acid,  Intrinsic  s u c h as t h e KA  HC when  closely  the  neurochemical  glutamic  Coyle  axons  by  of  effects  1983).  supported  producing  analog  a l . 1976;  -  glutamic a c i d ,  simplex.  duces  troyed,  of  (KA),  a structural  seaweed  is  resemble those of  are k a i n i c a c i d  KA,  ies  hypothesis  97  led  et-al. no  par-  activation  -  of  excitatory  the  capacity of  potential. with  The  tude  (McGeer  glutamic  condition, rat  when  al.  acid  and K A ,  striatum  which  toxicity  sensitive  by  at  is  2 orders  striatum,  neurotoxic  (Coyle  to. the  least  et  al.  alone  the  presence  of  of  this magni-  under  1979).  degenerative  of  an  co-administration  In  effects  in  in  manifest,  Lesions  observed  only  membrane  incompatible  become  required.  decorticate  of  to  are  tissue  KA  al.  culture  glutamate-containing  a  addition, of  et  such  only 1978).  of  cortical  strineurons  1979).  in  the  1984;  have  led  to  speculation  of  HC  (Olney  pathogenesis  1981;  Shoulson  s t r i a t u m not  cal  the  KA  is  KA  disturbances  the r a t  chronic  1983).  infusion unlike  of  that  In  et  al.  support  L-glutamate seen  that  i n HC  glutamate  1978; of  Olney  this  produces  i t s e l f may 1979;  contention  neurotoxic  (Mangano e t  al.  be  Sandis  the  effects  in  McBean  et  1983;  1985).  test  the  organized  1979),  In  of  of  morphologic  that  release  a state  (Campochiaro  finding  large  to  exceeding  resting  matured  et_al_.  have  pathway  toxicity  restores  These o b s e r v a t i o n s  To  the  has  (Whetsell  al_.  lead  shifts  innervation  cells  berg  and m a i n t a i n  neurotoxicity  neither  becomes  ionic  glutamatergic  KA-induced  involved  the  from  neuron. the  1978).  partially  the  atal  et  restore  corticostriatal  animals reduce  result  imbalance would  the for  -  would  to  ionic  of  that  glutamatergic  pathway i n  the  pumps  resulting  i s now. known  intact  Damage  membrane  continued survival It  of  receptors.  98  group of and  of  glutamate  a double-blind HC  patients.  excitatory its  attenuation  hypothesis,  amino  long-term of  the  and  Shoulson  and  placebo-controlled  Baclofen acids,  glutamatergic  trial  preferentially  including  administration  his  might  corticostriatal  of  (1983)  baclofen  blocks  glutamate provide  colleagues  the  function.  a  neuronal  (Potashner  chronic  in  1978;  pharmacologiShoulson's  -  group  intends  years  in  other  form  years  (when  to  approximately of the  code  of  effects appear that  the  of  of  glutamate  to  be  no  Another  creased  in  a  of  retinal  neurons  contain  anism,  an  present As  striatum of  of  be  glutamate  of  neurons  of  earlier,  animals.  This  metabolite  of  tryptophan  1985).  has  been  QA  including  man  there is  injected  into  (Schwarcz  et  the  al.  striatum  1983a;  in  from  or  a  the  the  et  al.  of  rats,  1984).  Rat  et  al.  1978b).  the  However,  the  the  agent  1983; it  brain  not  found  in  are  HC.  it  not  is  of in-  conceiv-  levels  of  uptake  the mech-  heightened might  can d e s t r o y  produces  1),  pathway  several  Moroni  shows  possible  contents  these,  (Figure  kynurenine of  of  there  be  disease.  which  (QA)  brains  the  endings,  combination with  is  involved  a defective  synaptic  certain  Yet  receptors  absolute  why  neurotoxic  it  However,  instance,  quinolinic acid  identified  (Wolfensberger  to  only  to  1973).  any  baclofen  explain  CNS  patients  another  in  in  HC  al.  3  several  not  the  of  take  be  to  cortex  patients  formed  fails  glutamate  for  HC.  that  For  is  of  is  glutamate  of  or  are s p e c i f i c a l l y  unrelated  receptors,  i n s u s c e p t i b l e neurons mentioned  et  to  will  hypothesis  cerebral (Perry  it  HC.  of  It  markedly  (Coyle in  baclofen  whether  that  of  or  permitted  symptoms  most  areas  these  be  known  is  subset  involved.  release  is  damage  a  abnormality  might  increased  and  period.  seen  glutamate  controls  glutamate  sensitivity  the  this  retina  e l s e w h e r e and t h a t with  not  it  is  sensitive  reports  the  neurotransmitter  HC  neural  from t h a t  that  in  placebo  will  hypothesis  the  problem  a  p r o g r e s s i o n of  is  susceptible  glutamate  most  who  during  the  process  either  before  glutamate  the  in high q u a n t i t i e s  able  prevent  neurodegenerative One  drugs  broken)  -  with  60 HC p a t i e n t s  is  slow or  A weakness  neurons.  therapy  anti-choreic  has h e l p e d t o  the  continue  99  et a  an  striatal endogenous  (Stone  et  mammalian  al.  1984),  species, and  kainate-like  differences  in  al.  when  lesion  susceptibil-  -  ity  to the n e u r o t o x i c  susceptible is  areas  effects  (Schwarcz  neuroexcitatory.  its  In  areas  (Perkins  neurotoxic  effect  of  is  conceivable  result  in  the  QA,  i s normal of  (Foster  1985).  b r a i n s of liminary  al.  quantifying  the  amounts  other  compounds.  of  theories  less  abnormal  DNA  sibility  that  might  responsible HC.  before  equivocal  of than  of  CNS  shows  variation  in  regional  probably  is  one  of  the  the  of  QA  patients.  QA  case f o r  in  the  However,  brain  when  etiology  of  HC,  generalized  examples  of  KA  and QA  may  exist  for  selective  an  as  to  the  a  there  the  be  yet  neuronal  degradative  to  to  unidentified and  in  in  pre-  by  QA w i t h  at the  several  hypothesis  defect, the  neurotoxin(s) the  the  system,  highlight  the  controls  Our  neurotoxin  serve  death  appears  complicated  membrane  the  could  measured  c o - e l u t i o n of  of  The  this  compared  been  those  repair.  brain  chromatographic  has  as by t h e  KA,  s l i g h t l y elevated  needs  liquid  sensi-  action.  c o n c l u s i o n s c a n be d r a w n .  human  as w e l l  even  itself  further  in  is  more  i t s neuroexcitatory  patients  performance  the  QA  as  HC  most of  being  and  the  action  phosphoribosyltransferase,  of  QA  one o f  physiological  again  HC  putamen  any  being  The  accumulation in  brains  using a high  appears  toms o f  QA  the  striatum  be r e l a t e d t o  seen  Content  o f QA p r e s e n t ,  Summarizing  be  in  amounts  small  of  autopsied  HC p a t i e n t s attempt,  to  losses  rat  Thus,  excessive  activity  caudate nucleus et  that  the  striatum  1983).  the  1983b).  regard, the  -  with  al.  appears  neuronal  because  enzyme f o r  al.  QA  It  unlikely,  with  et  QA,  et  this  s e n s i t i v i t y t o QA,  tive  of  100  consequent  or  of posthat  symp-  -  II)  101  -  OUTLINE OF  EXPERIMENTS  The p r e c e d i n g s e c t i o n s u g g e s t s t h a t a neurotoxin(s),  either  endogenously  evidence f o r the involvement cation,  would  agement  of  putative certain  HC  If  these  patients  might  be  neurotoxin foods,  then  proved  formed  of t h i s  be u s e f u l .  HC m i g h t  r e s u l t from  or of  exogenous  neurotoxin(s), could  greatly  improved.  t o be f r o m an e x o g e n o u s  simple exclusion of  origin.  as w e l l  be a c h i e v e d ,  the exposure  as i t s  the  genetically at r i s k ,  onset  of  symptoms  this  the p a r t i c u l a r  years,  man-  if  the  s o u r c e a n d was p r e s e n t dietary  d i e t - e x c l u s i o n therapy might  f o r several  identifi-  instance,  or might  even  in  substance(s)  m i g h t h a l t o r s l o w t h e p r o g r e s s i o n o f t h e d i s e a s e i n HC p a t i e n t s . viduals  Direct  the therapeutic For  to  For  indi-  to  delay  help  prevent  the disease  entirely. If  the putative  ous  pharmacological  its  removal,  neurotoxin strategies  might  become  restriction  fragment  of  o f t h e HC gene  carriers  tant  t o make r a p i d  w o u l d be h e l p f u l for  possible.  polymorphic  DNA  (Gusella  progress  t o have  preventing The marker  its  formation,  imminent for  a practical  preclinical  a preventive  treatment  hastening  availability  e t a l . 1 9 8 3 ) makes  i n developing  or  it  the  identification  e s p e c i a l l y impor-  therapy  to o f f e r  of  f o r HC.  It  i n d i v i d u a l s at  risk  r e c e i v e v e r y bad news.  The  of  analyses Neurol.  first  of  requirement, in  the  fasting  however,  pathogenesis plasma,  S c i . 67:351-358,  Perry  must of et  1985) observed  be  HC. al.  might  be an i m i n o a c i d .  proof During  eluted  involvement  quantitative  (Appendix  i n normal  c o l o r when r e a c t e d w i t h It  the  H,  Perry  i n 7 o f 2 8 HC p a t i e n t s  an unknown compound n o t p r e v i o u s l y f o u n d  compound y i e l d e d a y e l l o w it  for  vari-  t h e d i s e a s e , a b o u t 50% o f whom when t e s t e d w i l l  neurotoxin(s)  of  t u r n e d o u t t o be e n d o g e n o u s l y p r o d u c e d ,  from  amino et  al.  a  acid J.  trace  amounts  This  unknown  subjects.  ninhydrin,  of  suggesting  the chromatographic  system  that very  -  close of  to p r o l i n e  them.  It  circulating III,  and k a i n i c  seemed  tained  possible  in the blood  i n which r a t s from  acid,  were  of  -  b u t was c l e a r l y n o t  that  it  might  HC p a t i e n t s .  injected  HC p a t i e n t s ,  102  represent The  repeatedly  represents  our  identical  a neurotoxin  in vivo  with  rat  control  initial  with  study  to  that in  serum o r  attempt  either was  Section  serum o b -  investigate  this  possibility. The  next  culture  in  period of  the  of  techniques.  cultured  The  series  a medium  incubation,  rationale  striatum  of  of rats,  experiments  In  brief,  that  (Section  striatal  contained  these explants both  the  autopsied  might  indicate  patients. tent  area  the presence  Neurochemical  and GAD a c t i v i t y .  documented Urquhart  in  of  i n the s t r i a t u m of  e t a l . 1975;  Perry  or  of  reductions patients  1982a;  neonatal  experiments  was t h a t  resembled  a  then in  GABAergic  who d i e w i t h  HC  ex-  seen  the  in  results  the blood of  if  when  those  p r i n c i p a l l y that  Bird e t - a l .  After  explants,  patients,  of both  were  neurochemical^.  that  HC  tissue  rats  c o n t r o l . serum.  a c i r c u l a t i n g neurotoxin involved  t h e use o f  striatal  changes  brain  measurements Marked  from  and t n v i t r o  as t h e c u l t u r e d  neurochemical  brain  HC  were a n a l y s e d  p o s e d t o HC s e r u m showed same  involved  explants  either  the i n vivo  as w e l l  IV)  of  GABA  HC  con-  indices are well  (Perry  et  1973 and 1 9 7 4 ) .  al.  1973;  -  III)  The al.  J.  protocols Neurol.  Non-fasting  to avoid  had t a k e n  as  whole  was  limit  The v o l u m e  the  of the o r i g i n a l were  unlyophilized animal  HC  rats  were  patients  for  24  The  total In  were  or  injected control  volume  that  the  subjects.  days,  2,  sc with  subjects  given  191  in  final  protein-free  ml o f  1.8% N a C l ,  with  1  day  each  serum).  the i n j e c t i o n  with  of  7  filtrate  mem-  into old  for  80% of serum  serum  9% o f  used  for  filtrate.  6 day o l d  to  to  corresponding  litter-mate from  administered  serum f i l t r a t e  animal  untreated  a filtration  whole  were  f o r 37 c o n s e c u t i v e  that  original  (sc) with  serum i n j e c t e d  The  be r e f e r r e d  of the o r i g i n a l  randomly-assigned  a concentrated  once d a i l y  that  volumes  sub-  t h e HC o r c o n t r o l  reconstituted  injection  either  was a p p r o x i m a t e l y  Some o f  Injections  HC  a serum u l t r a f i l t r a t e  then  randomly-assigned  c o n c e n t r a t e d serum f i l t r a t e of  1,  from  or control  will  through  obtained  over  or  here.  collection.  was l e f t  to obtain  subcutaneously  o f HC o r c o n t r o l  Experiment  or c o n t r o l  was  and were  blood  any a l t e r a t i o n  the f i l t r a t e  in Experiment  consecutive  injected  valent  daltons,  briefly  et  h i s t o r y of the d i s o r d e r .  to  c r was p a s s e d  H (Perry  subjects  of the patients  serum s p e c i m e n .  so  family  The l a t t e r  i n j e c t i o n was c o n c e n t r a t e d 2 - f o l d  For. i n j e c t i o n s Wistar  of  lyophilized, filtrates  1,  10,000  be p r e s e n t e d  t h e 96 h p r i o r  serum.  i n Appendix  control  none  has not undergone  2.  filtrates  from  and a p r o v e n  during  to obtain  Experiment volume  obtained  f o r Experiment  brane o f e x c l u s i o n  and w i l l  any drug e f f e c t s ,  serum t h a t  serum)  are detailed  1985)  symptoms  any d r u g s  b l o o d was c e n t r i f u g e d (henceforth,  experiment  blood  classical  an e f f o r t  jects  this  IN VIVO STUDIES  S c i . 6_7:351-358,  venous  patients with In  for  103 -  either  once  body  daily weight.  e a c h a n i m a l was 52 m l . litter-mate from  days.  Wistar  either  HC  The t o t a l  rats  patients volume  of  r e c e i v e d was 7 6 . 5 ml ( t h e e q u i -  An a d d i t i o n a l schedule  control  and volume  group being  of  rats  identical  -  to  that  of  the concentrated  as h y p e r o s m o l a r After turbed revealed serum brains freezing  in  they  rapidly  procedure,  GABA  chromatographic 1,  only  months  the entire one-half  frozen  40 s of  death,  ively.  As shown  cantly  decreased  Experiment 2 ,  differ  undis-  this  period  injected  dislocation,  liquid  nitrogen.  et a l . 1981b).  After  warmed  served  left  with  HC  and  This  was n e c e s s a r y t o p r e v e n t  gradually  was m e a s u r e d  rat  on  described  the  rapid  any  20 s  post-  immersion  to - 1 0 ° C to  an  amino  (Perry  acid  allow  analyser  et a l . 1981b).  f o r GABA  of the striatum  was u s e d  f o r GABA d e t e r m i n a t i o n ,  was u s e d f o r m e a s u r e m e n t s  results  not  were  in  during rats  cervical  were  was u s e d  b y t h e method  control  (Perry  by  the  animals  rats.  experiments  in  of  striatum  ivity  and  group  Observations  was  and  the brains  both  disorders  Sacrifice  removed  from old.  movement  system p r e v i o u s l y  half  In  or  content  other  The  This  for  o f both h a l v e s o f t h e s t r i a t u m .  Striatal  2,  5  within  nitrogen,  dissection  filtrates.  rats  i n c r e a s e s i n GABA c o n t e n t  liquid  iment  were  ultrafiltrate.  were  mortem  injection,  no b e h a v i o r a l  or  serum  -  c o n t r o l s f o r t h e serum f i l t r a t e - i n j e c t e d  the f i n a l  until  104  o f Fonnum  (16%)  of choline acetyl transferase  1  and 2 a r e shown  i n Tables  t h e mean GABA c o n t e n t  in rats  however,  serum  1,  In  In  a  Exper-  Experiment while (CAT)  the act-  (1975).  of Experiments in Table  analysis.  using  mean  injected striatal  filtrate-injected  with  and 2  respect-  was s l i g h t l y b u t  signifi-  whole  GABA c o n t e n t  groups  were  serum  1  from  and CAT  similar.  s i g n i f i c a n t l y from those of the hyperosmolar  NaCl  HC  patients.  activity  These group.  of  values  HC did  -  Table  1.  Striatal  GABA c o n t e n t  S o u r c e o f whole serum  105  -  in rats  i n j e c t e d w i t h whole  Age o f r a t s during d a i l y i n j e c t i o n s (days)  serum  GABA c o n t e n t (ymol/g wet weight)  8 HC p a t i e n t s  5 t o 29  2.44 ± 0.09+ (18)  8 control  5 t o 29  2.90 ± 0.16 (12)  subjects  GABA v a l u e s a r e i n mean ± S E M , w i t h t h e number o f a n i m a l s examined n e u r o c h e m i c a l l y i n d i c a t e d i n b r a c k e t s . P < 0.02.  Table  2.  GABA c o n t e n t injected with  S o u r c e o f serum  14 HC  concentrated  in striatum of  serum  subjects  1 . 8 % NaCl  CAT a c t i v i t y (nmol/10 min/ mg p r o t e i n )  1 t o 37  2.51 ± 0.13 (16)  38.0 ± 2.1 (15)  1 t o 37  2.27 ± 0 . 0 9 (15)  40.0 ± 2.9 (13)  1 t o 37  2.55 ± 0.15 (16)  44.4 ± 2.9 (14)  .. .  -  .  • .  .  N e u r o c h e m i c a l v a l u e s a r e shown as mean ± SEM. The a n i m a l s examined i s i n d i c a t e d i n b r a c k e t s . None o f d i f f e r s i g n i f i c a n t l y (P = 0 . 0 5 ) .  There and  2.  are 2  possible  Firstly,  there  Secondly, cule or  of  if  interpretations may  a neurotoxin  over  that  have  10,000 daltons  it  d i d not  been  was p r e s e n t ,  i t may h a v e b e e n a s m a l l  teins  rats  filtrates  Age o f r a t s GABA c o n t e n t during d a i l y (ymol/g wet w e i g h t ) injections (days)..  patients  19 c o n t r o l  and CAT a c t i v i t y  of  no  reach  the  results  neurotoxin  i t may h a v e  (and t h e r e f o r e molecule  the  that  removed  shown  present  been  either  by t h e  was s o t i g h t l y  ultrafiltrate.  The  number o f t h e means  in in  Tables HC  1  serum.  a large  mole-  ultrafiltration),  bound  second  t o serum  pro-  interpretation  -  might tent  explain  the f i n d i n g  when w h o l e  ultrafiUrate (Experiment In  containing  daily  longer  period of  impractical trol)  rats  extensive.  effects The  ate  significant  (Experiment molecules  time,  been to  a more  obtained treat  serum,  1),  less  rats  or  longer  occurred. to  but  decrease  i n GABA  no d i f f e r e n c e  than  10,000  adverse  effects  being  experiments  reduction  Growth  HC  with  rate  con-  when  daltons  discontinued, i n Experiment  with  in  had been h a d been  serum  was  treated  used  serum. whole  animals  GABA  a  for  a  content  However, serum  and l o s s  with  continued  in striatal  was s l o w e r  controls,  it  (HC  or  i n whole of  was con-  serum-  body  f u r was  recovered  rapidly  2 d i d not  show  any  side  injections.  whole  immunological  tissue cultures  the  periods  ultrafiUrate  encountered  presumably  with  Animals  the rats  injections  given  uninjected  Once t h e i n j e c t i o n s w e r e normally.  if  animals  for  when c o m p a r e d  if  impressive  in  from the p r o t e i n - f r e e  these  but  i s possible that  dose o f w h o l e  gained weight  ment,  it  b e c a u s e many d e a t h s  exposed  and  only  -  2).  larger  have  a small  s e r u m was u s e d  Experiment 1,  might  of  106  serum  in nature,  vitro.  i n our  in vivo  prompted  us t o  experiiniti-  - 107 IV)  There ments  are several  as o p p o s e d  to hinder  into the b r a i n , of  the  possible The  in  vivo  STUDIES  doing of  striatal serum.  A higher  encountering  (Section  tissue  experiments  activity  to  i n these  large  The  in vitro  amounts  described  below  of  or  GABA c o n t e n t  respective  25% f r o m  of  immunological  blood-brain  the  experiments. putative  were  designed  HC p a t i e n t s  explants.  HC-exposed  reduced  control  controls  of  were  living  of r a t s t r i a t a l  when t h e s e v a l u e s w e r e  barrier,  Direct  neurotoxin  (or  increased)  Values  deemed  unchanged.  test  would  exposalso  Each  experiment with  compared  one f r o m  All  o f t h e HC p a t i e n t s  and  a l l h a d symptoms  is  (or  tested  for several  d i d not  differ some  are described  t h e HC  subjects  in Table  in Table  were  8.  obtained  adult-onset  formed b l i n d e d w i t h t h e samples c o d e d .  from  a  family history  Relevant  3, while Except cases.  to  be  content i n GAD present those  of  more  than  experiments  were  non-medicated control  subject.  patients  who s u p p l i e d CSF  two j u v e n i l e  Experiments  HC  of the disorder,  descriptions of  the patients for  by  not  neurotoxin.  a g e and s e x - m a t c h e d  years.  GABA  increase)  was deemed  Further,  had a p o s i t i v e  or  by a t l e a s t 2 5 % f r o m  that  specimen  a non-medicated  who s u p p l i e d s e r a a r e shown specimens  a  whether  reduce  A decrease  explants  values.  to  designed to e l u c i d a t e the c h a r a c t e r i s t i c s of the p u t a t i v e  all  concentration  t h e presumed  III).  experi-  t h e p a s s a g e o f some p e r i p h e r a l l y - i n j e c t e d s u b s t a n c e s  t o s e r u m o r CSF f r o m  and GAD a c t i v i t y  patient  culture  in vitro.  exposure  the  without  i s not present  striatum  to  injections  in vitro  encountered  w h i c h i s known  ure  advantages  to i n vivo  s e r u m c a n be u s e d toxicities  TISSUE CULTURE  were  HC  patients,  always  per-  -  Table 3 .  C h a r a c t e r i s t i c s of  108  HC p a t i e n t s whose s e r a w e r e u s e d  tissue culture ;ient  Juvenile or adult-onset  in  experiments  Age (years)  Duration of symptoms ( y e a r s )  Experiment number  7 9.5 8.5 5 3 2.5  1, 9, 2, 5 3 , 16 6 8 11 12 15  48 32 38 45 58 35 13 28  Adult Adult Adult Adult Adult Adult Juvenile Juvenile  GW LB JT JC DE JW BX JS  -  v  9  14  A l l t h e p a t i e n t s l i s t e d above a r e m a l e s . C h a r a c t e r i s t i c s of HC p a t i e n t s who s u p p l i e d CSF s p e c i m e n s a r e shown i n T a b l e 8 .  IV.i)  Methods  B r a i n s w e r e r e m o v e d f r o m newborn W i s t a r placed in a Petri dissecting  dish  c o n t a i n i n g Hanks'  microscope,  half-striatum,  about  the 7  right  to  10  and  less  than  balanced s a l t  left  pieces  rats  of  striata tissue  h o l d and  solution.  were were  24  Under  excised. cut,  were the  From  each  each  measuring  3 approximately explants.  1  mm .  Henceforth,  Three e x p l a n t s  ed w i t h r a t  tail  ed i n t o a K i m a x below)  was  rubber  stopper.  were  these  p l a c e d on a g l a s s  collagen (Bornstein glass  test-tube  introduced All  into  degrees from the h o r i z o n t a l p l a c e d on a r o t a t i n g  (15  each  culture  tissues  1958).  x 160 mm).  test-tube,  tubes were plane  drum w h e r e  The  for  will  previously  cover  slip  was t h e n  ml o f  the  first  24  they remained at  to  slip  unit  placed stationary  the  referred  cover  0.7  and  be  h,  the  growth was at  medium  (see  with  angle  and s u b s e q u e n t l y same a n g l e .  coat-  insert-  capped an  as  of  a 5  were  Incubation  t e m p e r a t u r e was m a i n t a i n e d a t 37° C . Fifteen in Table  4.  to  20 n e o n a t a l  This  rats  were t y p i c a l l y used i n  p r o v i d e d enough e x p l a n t s  for  60 t o  each experiment 80 c u l t u r e t u b e s .  shown The  -  Table 4.  C h a r a c t e r i s t i c s of  P r o p o r t i o n ( v / v ) and t y p e o f human s e r u m o r CSF i n medium  109  -  tissue culture  experiments  Expt number  Name o f patient  1 3 15 16  GW JT JS JT  1, 1, 1 1,  3  2 3 5 6 8 9 11 12 15 16  LB JT LB JC DE GW JW BX JS JT  1, 1, 1, 1 1, 1 1, 1, 1, 1,  2, 3 3 ,3 3 ,3 3 3 3 3  15% w h o l e s e r u m  30% whole serum  Duration of exposure of explants to medium ( w e e k s ) 2, 3  15% d e p r o t e i n i z e d  serum  8 9 11 15 16 • ,  DE GW JW JS JT  1, 1, 1, 1, 1,  3 3 3 3 3  15% d e p r o t e i n i z e d f i l t e r e d serum  and  12  BX  1,  3  7 10 10 13  M X Y Z  1 2 2 2  15% CSF  5  3  E x p e r i m e n t 14 i s n o t shown on t h i s T a b l e w h i l e E x p e r i m e n t s 5 and 16 a r e d e s c r i b e d f u r t h e r i n t h e t e x t . P a t i e n t GW was t e s t e d 3 t i m e s ( E x p e r i m e n t s 1 , 9 and 1 4 ) , e a c h t i m e a g a i n s t a different control. P a t i e n t s LB ( E x p e r i m e n t s 2 and 5) and J T (Experiments 3 and 16) w e r e t e s t e d t w i c e a g a i n s t different controls. T h e r e i s no E x p e r i m e n t 4 .  experiments order  listed  in  Table  i n w h i c h t h e y were  For  the  periments  first  7  contained  days  4  are  numbered  according  to  the  chronological  performed. of  70% by  incubation, volume  of  the  nutrient  a mixture  of  fluid  Eagle's  used f o r  all  ex-  minimum e s s e n t i a l  -  medium  (with  Earle's  gentamicin, medium  salt,  and was  (v/v)  was  but  2 mM i n  composed  After using  incubation  a phase-contrast  extended for  for  growth  7  the next stage df  and  tubes  containing  a number o f  sets,  depending  was  then  incubated  f r o m HC p a t i e n t s sisted (with cin,  of  Earle's  but  and was 2 mM i n  um was u s e d , utilised  remaining  chemicals  the  and  explants  magnification.  and  dark  yg/ml  3 0 % of horse  was  Only  necrotic  design of  a  In  mixture  the serum  determined  explants  areas  without  with  were  used  divided  into  human  excluded serum or  (Table 4 ) .  containing  cases,  Eagle's  glutamine),  were  experiment  medium  all  of  explants  the  appropriate  subjects.  of  growing  added g l u t a m i c a c i d .  of  of  insignificant  an  h o r s e s e r u m was  15% ( v / v )  The  state lOx  healthy  with  volume  salt,  the  on t h e  or c o n t r o l  70% by  The  20  experiment.  Culture  set  serum.  microscope at  the  acid.  0.5% glucose,  Laboratories.  days,  processes  glutamine),  glutamic  horse  used were p u r c h a s e d f r o m G i b c o  -  without  added  of  110  the  serum  nutrient  minimum  0.5% g l u c o s e ,  CSF,  the the  medium.  20  In  remaining  or  fluid  essential yg/ml  Where 3 0 % by v o l u m e  from  Each  con-  medium gentami-  human  ser-  experiments  that  15%  of  CSF  was  made  up  by  harvested  or  horse serum. After were until each  a further  incubated with they  were  explant  placing  it  with  in  These  Explants  for  containing  for  fine  Hanks' to  days  of  a fresh  ready  then t r a n s f e r r e d utes.  7  incubation,  solution  harvesting. forceps  balanced a fresh  'washes'  were  salt  to  rid  either media  consisted of  collagen-coated for  the  to  be  5  explants into used  a  scraping slip,  Explants  for of  once  cover  minutes.  solution  transferred  Those  were  respective  balanced s a l t  then  acid.  its  solution  designed  M perchloric  their  Harvesting  from  Hanks'  GABA a n a l y s i s w e r e  0.4  of  explants  another  adhering  GAD  off and were  5 minmedium.  a homogenization for  week  analysis  tube were  - Ill -  f r o z e n and s t o r e d a t - 7 0 C . urement of  o f GABA c o n t e n t ,  GAD  the  tissue  were performed Some  procedures,  experiments  (Table  deproteinization  deproteinize  were  pooled  pooled  f o r each  for  meas-  determination  with  the  exception  thick  4)  were  would  done  remove  molecules  with  most  deproteinized  large  tightly  supernatant insoluble explants  Potassium  until  bicarbonate  perchlorate)  in culture.  deproteinized  solution  t h e pH was 7 . 0 .  potassium  After  serum  was  acid  c r y s t a l s were This  in  another  mixed  with  removed  order  to  was a d d e d  or  CSF's  used  in  the  then  other  avoid  to  15  the  perchlorate  the  samTo  filter  of  min-  ions  (as  effects  the  unit  experiment  The  stirred  supernatant  ingredients  of  the  dropwise  lOOOOg f o r  detrimental  a Nalgene  types  serum  in a  added  excess  other  in  i n an i c e - b a t h .  at  centrifugation, the  serum  t o serum p r o t e i n s .  was c e n t r i f u g e d  medium and s t e r i l i z e d b y p a s s i n g i t t h r o u g h sera  harvesting,  molecules from  bound  0 . 2 9 ml o f 10 N p e r c h l o r i c  homogeneous  of  conditions.  dislodge small  serum,  at 4 ° C .  The  were  t o 10 ml o f s e r u m w h i c h was s t i r r e d c o n t i n u o u s l y  resulting utes  culture sterile  or would  manner  explants  and 10 t o 15 e x p l a n t s  under  that  ples,  t o 11  activity.  All  hope  Seven  on  of  the  culture  ( 0 . 2 0 um).  were  similarly  sterilized. Other  types  of  experiments  performed  will  be  detailed  in  the  next  section. Analysis means  of  reaction small  of  GABA  a modified products  amounts  of  content  was as d e s c r i b e d  amino  acid  amino  acids with  o f GABA.  analyser  GAD a c t i v i t y  that  in Perry uses  the  o-phthalaldehyde was d e t e r m i n e d  et  (1979b),  except  was  increased to 0.1  that  the  specific  uCi/vimole.  GAD  activity activity  of  (1982c),  fluorescence for detection  by  of of  the very  as d e s c r i b e d b y P e r r y r  aj_.  al.  of  L-[l-  explants  14  et  -i  CJ-glutamic was e x p r e s s e d  acid as  -  vimol/h/g p r o t e i n ,  w h i l e GABA c o n t e n t  not  p o s s i b l e t o e x p r e s s GABA c o n t e n t  ent  in explants  proteinized. of  explants  offset  because  It (7  samples  was h o p e d  t o 11)  the difference  5 shows  posed t o  15 % d e p r o t e i n i z e d  tive  of  material  were  three  weeks, that  the  of  same  in  had b e e n  o r 30% w h o l e exposure  of  GAD  t h e GABA c o n t e n t s  of  same c o n t r o l  serum s i m i l a r l y v a r i e d  GABA c o n t e n t  pres-  to  be d e -  from  a  pool would  after  of  the  after  one week.  randomly.  This even  On t h e o t h e r 30% c o n t r o l  three  different  the control  explants,  medium. to  and GABA c o n t e n t  a c t i v i t i e s of  weeks  15% d e p r o t e i n i z e d  experiments,  had f i r s t  activity  serum f r o m  exposed  declined to  GAD  8.  than  growth  explants  of  protein  and D i s c u s s i o n  i n Experiment  exposed  analysis  of  t o a p a r t i c u l a r c u l t u r e medium  values  to the growth  and a c t u a l l y  t h e amount  I t was  the e x p l a n t s .  Results  and c o n t i n u e d  had been added  tents  plants  t h r e e weeks  survival  exposed  the i n d i v i d u a l obtained  of  f o r GABA  i n s i z e s between  explants  higher after  i n terms  t h e measurement  similarly  striatal  -  was c a l c u l a t e d as n m o l / e x p l a n t .  used  that  IV.ii) Table  112  sets  of  of  control  explants  ex-  subject was  serum  human  GABA for  con-  one  treatment serum.  were  indica-  though  hand,  explants  of  in In  exposed  or ex-  other to  the  -  Table  5. of  Individual rat striatal  113  -  v a l u e s o f GAD a c t i v i t y explants  and GABA  content  exposed t o 30% whole serum  o r 15 % d e p r o t e i n i z e d s e r u m o f E x p e r i m e n t 8 Serum u s e d  GAD a c t i v i t y (ymol/h/g p r o t e i n ) Exposure:  GABA c o n t e n t (nmol/explant)  1 week  Control HC  (30% whole) (30% whole)  47.3 26.6  0.102 0.175  Control HC  (15% d e p r o t e i n i z e d ) (15% d e p r o t e i n i z e d )  50.5 35.1  0.197 0.127  Exposure:  3 weeks  Control HC  (30% whole) (30% whole)  66.1 37.0  0.102 0.040  Control HC  (15% d e p r o t e i n i z e d ) (15% d e p r o t e i n i z e d )  58.3 39.8  0.050 0.016  -  It as  should  nmol/explant.  plants  The hope  was t h a t  measurement  if  explants  from  a particular  another,  this  would  of  values.  indicative absolute  the  Another  of  differences  level  of.GABA  in  have  possible  the v i a b i l i t y  -  t h e GABA c o n t e n t  offset  from that GABA  that  would  However,  of  be e m p h a s i z e d  114  pool  of  to  a variety  t h e c a t a b o l i c enzyme GABA t r a n s a m i n a s e ,  from p u t r e s c i n e o f GABA i n Very error  durations  earlier  of  medium. ments  were  time  was an i n c r e a s e  explants  that  to  explants  those  tion  had u n d e r g o n e  f o r postmortem  death-to-freezing (Perry  et  ability duration  size of  contents  explants  is  as  that  The  could affect  the  activity of  the  GABA  content  the  a l . 1981b).  i n GABA c o n t e n t s  Thus,  for  rinsed  one o f  of explants  of the washing process of  only  the  washes  15,  two  exposed  the  a minute.  between  1.5  washing  before  to  the  rises  have  was f o u n d  that  3 fold  2  factors  that when  ( n = 2)  when  h  situa-  increasing  after to  in  compared  the  r a p i d l y with  been  such to  resembles  maximum  same  Experi-  coverslip It  of  different  rinse,  process,  contributing  could well  to  This  stable  explants.  harvesting  a quick  off  quickly.  a  For the  ( a s was t h e c a s e f o r  GABA c o n t e n t  reaching  source  five-minutes  explants  than  ten minutes  where  intervals,  of  possible  Experiment  explants  was l e s s  content  had b e e n  brains,  In  duration  the  another  explants.  two  consisted of  scraping  i n GABA  used  total  perchlorate  that  involved  were  the other  between  of  perchlorate.  ten minutes  while  elapsed  in  had  procedure  they were p l a c e d i n t o there  GABA  as t h e f o r m a t i o n  that  we d i s c o v e r e d  o f GABA c o n t e n t s  placed  washing  to 14),  15),.  experiments  One was o f  1  in  quantitations  influences.  as w e l l  a r e two f a c t o r s  (Experiment  in the determination  explants  the  1980),  using  ex-  explants.  significantly  in  of  of  explants.  recently  procedure, the  (Seiler  in  a pool  individual  in erroneous  neurons  was e x p r e s s e d  GABA f r o m  differed  error  GABAergic  i s subject  of  explants  between  resulted  source  of  size  of  the  differences  death varii n the  - 115  The  random  explants plants  variability  precluded  exposed  any  to  HC  or  to  follow will  Figure  2  indicates  sera  activities  of  from  30% HC  serum  weeks  of  exposure  in  the  is  that  Experiment the  other  to  Figure  a 2  due  to  It  from  patient  from  HC  and 3 ,  shows  patients GAD  the  activity  but  subsequently  The  results  of  these  were r e m i n i s c e n t o f  the  a  rats  consistent  15)  JT  and  GW  3  these impairs  second  pos-  obtained  onset,  of  and  is  since  to  in  those  apparent  HC was  GAD  or  of  with  appeared of  of  1  that  (a  to  results  after  results  juvenile  diagnosis  LB, (see  Table  all  initially  two  having  of  immediately  the  activities  returned  not  that  of  to  for  the  explants  treating  tested  serum  contain  the  inaccurate  for  exposed (one  to  or  was  experiments  'excitotoxic'  each  with  controls.  time  experiments).  their  sera  or t h r e e weeks)  explants  appropriate  twice,  respective  increased in explants  normal,  early  4  were  time p e r i o d s  compared w i t h  was  is  The  interpretation  GAD  not  exposed  sera.  activity  neurons  were  and  as %  a substance(s)  The  results  explants.  expressed  GAD  One  (Experiment  consequence of  when  in  ex-  12.  r e d u c e d compared to c o n t r o l s at Table 6  reason  patient's  may be  GAD  patient)  the  control  between  explants  control  contains  below).  control  that  appropriate  patients  reason,  and  of  contents  striatal  reduction  pools  a c t i v i t y of  compared  discussed  patients  different  of  GABAergic  the  this  experiments.  The  GABA  t h e GAD  striatal  juvenile-onset  shows  a  juvenile-onset  i n Experiment  30%sera against  is  neurotoxin.  the p a t i e n t  the  11  HC  experiments. be  another  putative  (a  10  unlikely from  12  of  cultured  interpretation  to  different of  For  when  produced 10  of  activities  patients,  serum f r o m  v i a b i l i t y of  sible  GAD  exposed  that  serum.  c o n s i s t only of  HC  explants  content  comparisons  control  the  show  results  GABA  meaningful  discussion  30% w h o l e  in  -  tested.  15% w h o l e In  were  serum  Experiments  1  exposed  to  HC  decreased r e l a t i v e  to  controls.  were  because  hypothesis  of  interesting, Olney  (see S e c t i o n  serum,  they I.ii).  -  Figure  2.  GAD  activities  from  HC p a t i e n t s  tive  controls.  above  each  is  3 weeks  in  no  this  since  the  the  used.  Thus,  4 . 3 %(n  =  17).  refers  exposure has  sampling  and  for  the  each  previous  coefficient the  represents  of  coefficient  to  as %  of  the None  of  unrelated of  their  of  the  variation  of to  respec-  The  the  GAD this  GAD  number There  comparisons  significance, activity thesis,  analytical for  serum  experiment.  statistical  determination  variation  30%whole  comparison.  14. for  work  of  one  number  analysed  for  exposed  expressed  Experiment  been  error from  to  -  explants  3 weeks,  triangle  triangle  However,  for  1  Each  section  unknown. exist  for  of  116  is data  technique  analyses  is  -  117  200  150  12  % of 12  controls  6 A  11  14  50  1  8 11 15 16 A  1  2 A 15  A  6  A  A  1 week Duration o f  9 A  3 weeks exposure  Normal  controls  It  was  as  though  neurotoxin more  in  HC  prolonged  Further  GAD  possible  tive  with  activity for  that  medium, are  Table of  this  in for  CSF It  of  present  in  neurotoxin  such  low  for  from  3  one  of  the  volume  the  of  of  patients  the  GAD  is  absent  of  use  of  CSF,  3 of  serum from  those  either  had  no  effect  30 % b y  volume  the medium,  serum)  in  on of  tissue  alternatively, patients to  that  affect  GAD  CSF  the  (or,  culture which  culture.  8 would  are  the  serum p r o t e i n s  or  up t o 2 weeks was i n a d e q u a t e  pro-  explants.  Table  4  is  puta-  in  the  It  the  of  in  decreased  of  volume  explants  in  concentration  material  e x c l u s i o n of  results  from in  to  a  15%  resulted  incubation.  while  activity  with  or  concentrations  deproteinized  growth  HC,  of  while  activity.  as  sometimes  detrimental  complete  well  unchanged  explants,  putative  explants,  represented  as 15% b y 4  the  neuronal  as  week  high  possible  concentrations  a period of  with  6),  were  results  patients,  e n t a i l e d the  required  of  GABAergic  that  of  striatal  similarly  after  activity  was n o t  30% by  7),  values  random  of  (Table  i n c r e a s e i n GAD  f r o m HC  The  because t h i s  putative  explants  CSF  explants.  is  (Table  concentrations  concentrations  activity  serum  patients  an i n i t i a l  8.  apparently  present  of  matter,  neurotoxin  from  low  depressed  encountered  seemingly  lower  The e f f e c t s  in  15% w h o l e  s u p p r e s s i n g GAD  with  in  also  -  GAD  However,  15% s e r u m  d u c e d no c h a n g e o r  shown  to  deproteinized  these  neurotoxin  patients  with  were  that  exposure  resulted  activity.  controls  118  stimulated  exposure  had been  increased  effect,  serum  experiments  serum t h a t  from  initial  -  If  suggest that  it  exposure GAD  a  that was of  activity.  -  Table  119  -  6. E f f e c t s o f 15% w h o l e s e r u m f r o m HC p a t i e n t s on GAD a c t i v i t y o f r a t s t r i a t a l e x p l a n t s  Experiment number  HC p a t i e n t  1  GW  Duration of exposure of e x p l a n t s (weeks)  GAD  1 2 5  3  JT  1 3  15  JS  1 3  16  JT  1 3  t '  + + + n.d. + +  A d e c r e a s e i n GAD a c t i v i t y o f e x p l a n t s e x p o s e d t o HC s e r u m o f a t l e a s t 25 % f r o m t h a t o f c o n t r o l v a l u e s i s shown as+ , w h i l e an i n c r e a s e o f a t l e a s t 2 5 % i s i n d i c a t e d a s + . Values that d i d n o t d i f f e r by more t h a n 2 5 % a r e shown a s =. Non-determined samples are r e p r e s e n t e d by n . d .  Table 7.  Experiment number  8  E f f e c t s o f 15 % d e p r o t e i n i z e d s e r u m f r o m HC on GAD a c t i v i t y o f r a t s t r i a t a l e x p l a n t s HC p a t i e n t  patients  Duration of exposure of e x p l a n t s (weeks)  GAD  DE  1  +  3  +  9  GW  1 3  + +  11  JW  1 3  =  15  JS  1 3  -t-  1 3  4.  16  JT  Refer  to Table  6 for  legend.  +  -  Table 8.  Effects.of  15% CSF  of Experiment number  rat  120  -  f r o m HC p a t i e n t s  striatal  activity  explants  Duration o f symptoms . (years)  HC P a t i e n t , Age ( y e a r s )  on GAD  Duration of exposure of e x p l a n t s (weeks)  GAD  1  +  7  M,  49  6  10  X,  48  Stage  I  2  ' 10  Y,  29  Stage  I  2  13  Z,  34  Stage  II  2  =  R e f e r to Table 6 f o r of I h a v e no i n f o r m a t i o n the legend. t h e d u r a t i o n o f symptoms o f p a t i e n t s X , Y and Z , e x c e p t for t h e i r stage of the d i s e a s e . P a t i e n t s M, Y and Z a r e f e m a l e s , while patient X i s a male. Experiments  5  and  16  represent  characteristics  of  the  putative  our  attempts  neurotoxin  in  to  serum  examine from  HC  further  some  patients.  For  o  experiment against  5,  some  a dialysate  albumin.  Four  Another  portion  described The  in  protein  volume  of  changes of  tration protein  in  the  fraction  the  concentration  H. left  the  h  of  were  applied  Experiment  results. which  16,  patient  Whole failed  activities  dialysate  at  explants  at  the  were  s e r u m was n o t  also  pass  the  of  explants,  h  medium w e r e  made.  the  filter  prior to  a 15%  to  use.  original  and  (v/v)  and  unit  reconconcen-  reconstituted  tested  at  15%  (v/v)  tested).  dialysed  to  48  serum  filtrate  filtrate  for  bovine  -70°C  the  serum,  C  of  through  as  JT  4  was r e c o n s t i t u t e d  as w e l l to  at  5% ( v / v )  stored  filter  serum,  (dialysed  dialysed  passed  was  on t h e  serum of  the  was  filtrate  dialysed  the  GAD  serum  was  containing  intervals  behind  In  fraction  reduced  0.9% NaCl,  The  i n t h e medium  protein all  The  from  collected  original  medium.  F i g u r e 3 shows  patients,  12  fractions,  fractions  stituted  at  Appendix  protein  serum  medium o f  in 0.9%NaCl.  stituted  the  serum,  filter, while  and t h e  recon-  c o l l e c t e d from HC  serum  HC  filtrate  -  had no e f f e c t .  Thus,  it  would  appear  i n w h o l e s e r u m f r o m HC p a t i e n t s sult It  is  in  accord with  suggests  that  if  large molecule of tightly  bound  to  the  putative  from  HC  also  free  posed that  of  shown  is  This  in  is  that  the in  vivo  or  the  a protein  Table  7,  the  small serum  rat  it  present should  HC  that  was  use  of  unchanged  from  controls.  effect  the  two  the  remove If  it  dislodge  alternatives.would determine  whether  large  activity  were  that  these  neurotoxin,  were results  then  it  serum either were would  n e u r o t o x i n was a l a r g e m o l e c u l e , o r a p r o t e i n t h a t  III).  of  neurotoxin  so  it.  A  be or  appear  to  explants  ex-  compared a  volume  to  large  be r e d u c e d .  a  not  and  of  increased, decreased, due  to  supernatant  being  1 5 % by  a  is  molecules,  decreased  as  re-  either  molecule that  easily  most  putative  deproteinized  is  deproteinized  GAD  patients the  serum,  a small  This  (Section  p e r c h l o r a t e - i n s o l u b l e , would  activities If  HC  cannot  in  present  serum f i l t r a t e .  and t o  molecules. from  in  these  neurotoxin  experiments  is  dialysis  p o s s i b i l i t y of  i n GAD  putative  still  putative  the  present  between  procedure  medium r e s u l t e d  of  the  differentiating  neurotoxin  controls,  in  protein  that  absent  10,000 d a l t o n s ,  protein-bound  or  of  neurotoxin  over  deproteinized  molecule,  was  -  s e r u m by c h e m i c a l d e n a t u r a t i o n ,  patients.  to  a  serum  p o s s i b l e means o f deproteinize  that  121  As the or  concentration  unlikely  that  the  was p e r c h l o r a t e - i n s o l u b l e .  - 122  120  "  T  <u  5100  •-• <u V. B  +-> C o s- o <+- co u o. •o X II LU £ I/) o c cu i - •—  80  O-T-  tn s_o -•-> cy c IT) TJ o  Q.JZ fO X CO M—T3 O o C (/) O irt (/I E cu cn S_ s_ Co O a. X 0)  60 A •  40  --  •r- (/>  >  •r4J  3 LO  O -f  o s- •o irj rO c > <0 a  < o  20  LO  6 3 Scu l/l  a>  o JC  E 3  i_ 4) V)  E 3 i_ CU  CU  w> ^>>  •(->  OJ •M O i. O.  •a QJ  X  o <r> t/1  C  o u  Figure 3. GAD a c t i v i t i e s o f e x p l a n t s e x p o s e d t o various f o r m s o f HC s e r u m f o r 1 week, e x p r e s s e d as % o f their respective controls. Closed triangles are values from Experiment 5 , w h i l e open t r i a n g l e s are v a l u e s from Experiment 1 6 . D i a l y s e d s e r u m was n o t t e s t e d i n E x p e r i m e n t 1 6 .  -  Finally,  two  experiments  dose-response  relationships  of  For  explants.  was  filtered  on t h e then  as  filter  the  pH  bicarbonate. duced.  such the to  manner  these  or  as  the  the  HC  to  for  exp.lants  explants  20,  to  exposed to  its  was  which  treated low  GAD  the  these  extensive  properties  sustaining  GAD  activity  control  serum  in of  treated  in  centripotassium  volume  were  in  serum  in  were  either  all  exposed  of  a manner  How-  the  around  protein  in  7  ±  1  original it  Comparisons way w e r e  con-  control  the that  the  done  harvested.  were  this  pro-  of  was  original  Explants  explants.  and  perchlorate  treatment  such  water,  with  the  50 y m o l / h / g  behind  After  s e r a from  (they  about  with  7.0  of  120%.  activity  The  (v/v)  subject  left  Reconstitution  explants  with  a mean o f  1/12  activity  control  potassium to  8 0 and  its  and  the  or  acid. to  possible  and GAD  volume  up  test  fraction  perchloric  brought  altered  HC  protein  0 . 9 3»NaCl.  50,  medium,  original  removed  to  patient  concentrations  experiments).  of  The  with  with  after  very  the  lyophilized  30,  explants  compared  other  capable  10,  had  s e r u m had p r e s u m a b l y  was  no  between therefore  useful. In  whole  Experiment s e r u m was  centrations.  It  16,  added  instead to  was f o u n d  serum were e q u a l l y r e d u c e d all  then  final  week,  of  patient  of  allow  1  sets  pmol/h/g p r o t e i n ,  not  above  designed  an HC  H.  supernatant  was  were  serum i n  half  centrifugation  medium o f  media  all  longer  to  mentioned  supernatant  nutrient  trol  of  HC  Appendix  s e r u m and r e c o n s t i t u t e d  a  ever,  as  Another  The  original  in  16)  serum from  was r e c o n s t i t u t e d  deproteinized  fugation,  14,  -  and  between  experiment described  (14  123  three  media.  concentrations,  the  of  using  medium  that (over when  the  sera  that  as e i t h e r GAD  60%)  at  compared  had  7.5%,  a c t i v i t i e s of one to  or the  undergone 15%,  or  explants  t h r e e weeks  of  corresponding  alteration,  30% (v/v) exposed  conto  HC  incubation  at  control  serum  -  In summary, Firstly, is  the r e s u l t s of  30 %whole  detrimental  putative or,  GABAergic  neurotoxin  -  the various experiments  serum from  to  124  HC p a t i e n t s neurons  may be e i t h e r  in  appears  cultured  a small  to rat  suggest the contain  a material  that  striatal  explants.  The  molecule t i g h t l y  f a r - l e s s - l i k e l y , a molecule l a r g e r than  following.  bound  to  10,000 daltons which  is  protein, perchlor-  ate-soluble. As b r i e f l y our  results,  neurotoxin GABAergic of  especially  tive,  neurons  neurons  appears  deproteinized)  Further clude  of  it  possible that  is  absent rat  procedures  were  30% HC  serum.  explants  of  lack  increases  a  growth  serum.  an e s s e n t i a l As  to  in  growth  impaired,  with  factor  factor  have  been  serum  activity would  of  for  re-  attrac-  (whole  HC-serum  certainly  a  viability  Although  15 % HC  of  of  the  controls.  GAD  Instead  a result,  might  since experiments  The  al.  on  not  explants  studies  1985)  to  using  to  to  with  exclude  i n CSF  of  actual  HC  due t o  serum might  microscopy neuronal  Experiments  or  or ex-  not  have  the  Golgi  also  be  disorders GAD  staining  released into  However,  the  absence  of  the  (for  blood  the  These  performed  reduction  a factor  probably argues  logically in-  deterioration.  should  neurodegenerative  p o s s i b i l i t y that  degeneration.  HC p a t i e n t s  to  electron  us.  other  the  e x p o s e d t o HC s e r u m was undergone  exposed  identify  available  patients  Alzheimer's)  substance  HC  striatal  produced  serum from  had  from  with  a c t i v i t y when c o m p a r e d  experiments  et  neurons  obtained  possible interpretation  such i n c r e a s e s .  (Graveland  plants  i s another  those  unlikely,  morphological  using  be  there  sometimes  explants.  accounted f o r  ple,  may  i n r e d u c e d GAD  this  posed  above,  being present,  GABAergic  sulting  a l l u d e d to  examin  exafter  inhibitor  against this p o s s i b i l i t y .  -  V)  The vide in  in  CONCLUSIONS  the i n v i v o  and t i s s u e  suggestive but not c o n c l u s i v e evidence  the  appears larly  r e s u l t s obtained  125 -  if  serum  of  HC  patients.  t o be a u s e f u l  technique  the recommendations  corporated.  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J.  central  membranes 38:1699-1708,  Stevens S, Bridges abnormal radiation  U r q u h a r t N, P e r r y T L , H a n s e n S , K e n n e d y J , GABA c o n t e n t and g l u t a m i c a c i d d e c a r b o x y l a s e a c t i v i t y i n b r a i n o f H u n t i n g t o n ' s c h o r e a p a t i e n t s and c o n t r o l s u b j e c t s , J . Neurochem. 2 4 : 1 0 7 1 - 1 0 7 5 , 1975. Went L N , ease,  Der V I i s MV, B r u y n GW, P a r e n t a l Lancet i : 1 1 0 0 - 1 1 0 2 , 1984.  transmission in Huntington's  dis-  W o l f e n s b e r g e r M, A m s l e r U , Cuenod M, F o s t e r A C , W h e t s e l l WO J r . , S c h w a r c z R, I d e n t i f i c a t i o n o f q u i n o l i n i c a c i d i n r a t and human b r a i n t i s s u e , Neuros c i . L e t t . 41:247-252, 1983. Young A B , S h o u l s o n I , P e n n e y J B , S t a r o s t a - R u b i n s t e i n S , Gomez F , T r a v e r s H , R a m o s - A r r o y o MA, S n o d g r a s s R, B o n i l l a E, M o r e n o H , W e x l e r NS, Huntington's disease in Venezuela: N e u r o l o g i c f e a t u r e s and f u n c t i o n a l d e c l i n e , Neurol. 36:244-249, 1986.  -  CHAPTER 3 :  133 -  DIALYSIS  ENCEPHALOPATHY  ABSTRACT  The  absence o f  cephalopathy  (DE)  sociated  with  brain  patients  of  abnormalities of  DE.  tal  suggests  the  loss  who d i e w i t h  of  of  dentate  in brains  abnormally  were  of  and s e r o t o n i n high  might  10 p a t i e n t s  nucleus,  for  the  neurochemical symptoms  and o c c i p i -  caudate n u c l e u s ,  and m e d i a l -  Pyridoxal  i n the f r o n t a l  unasin  who d i e d w i t h  acetyl transferase  present  present  en-  in frontal  t h e DE p a t i e n t s , were  changes,  be  We l o o k e d  s i g n i f i c a n t l y reduced  regions.  levels  activity  phosphate  was d e -  contents  but metabolites  i n normal  were  of  nor-  amounts.  Aluminum  matter  patients  cortical  gray  role  aluminum  of  DE. In  tempted  an e f f o r t  to  p o s s i b i l i t y that rats  though  clarify  t o p r o d u c e a model this  as t h o s e o b s e r v e d  elevation  of  aluminum  we e m p l o y e d  administration Thus, in  brains  in dialysis  neurochemical  disease.  by 25-35% i n 3 c o r t i c a l  dopamine  in  were  the  Choline  reduced  abnormalities  populations,  DE p a t i e n t s .  adrenaline,  the  neuronal  cerebellar cortex,  substantially  with  of  in the autopsied  thalamus  creased  neuropathological the p o s s i b i l i t y that  Mean GABA c o n t e n t s  cortex,  dorsal  specific  in  the causative  of the disease i n r a t s , metal  might  i n human the  the question  DE was n o t c l a r i f i e d  produce  patients.  brains  heminephrectomy,  in attempts  of  of  by our  using AlfOH)^ to  we  at-  explore changes  However,  produce  rats  treated  5/6 n e p h r e c t o m y ,  we f a i l e d with  and/or  excretion.of  o r n o t aluminum  experiments.  DE,  t h e same n e u r o c h e m i c a l  to decrease the renal  as t o whether  in  to  AUOH)^, chronic  even  lithium  aluminum.  i s the causative  factor  -  I)  INTRODUCTION  I.i) In  1972,  gressive,  Alfrey  fatal  syndrome,  dialysis  s i n c e been o b s e r v e d stitutes  et  renal  a l . 1976a; The  conjunction occur  with  et  prominent  Initially,  during  disturbances bination sist. paranoid display  be  thinking, seizures, ensues,  incidence  failure  related  of  et  continous  dementia  psychoses,  to  of  DE  population  and s h a r p  lobes  dialysis As  (Alfrey  and  usually  to  undergoing of  dialysis  arrest.  (EEG)  activity, et a l . apparFlendrig the  manifesting  speech  as a c o m -  abnormalities  In  changes  as  patients  and m y o c l o n i c the onset  (Chui  per-  such  addition,  jerks.  of  symp-  et a l . 1980).  to  0.75% o f  (Lang  et  a l . 1979),  et  In  changes  Hughes  0.6  (Alfrey  Flendrig  abnormality  progresses,  after  f o r 60 c a s e s  dialysis  with  and s p i k e  apraxia,  be between  patients  speech  personality  a few months  was r e p o r t e d  con-  speech  T h e EEG  has  it  et a l . 1972;  and d e l i r i u m a r e s e e n . motor  This  a n d t y p i c a l l y more  the disease  pro-  et a l . 1982).  1978;  (Alfrey  and d y s p h a s i a .  develops,  appears  the duration  waves  twitching,  occurring  times,  and i n c r e a s i n g l y s e v e r e ,  dyspraxia  asterixis,  Prior  a mild  are intermittent  a l . 1976).  where  e t a l . 1973;  1982;  and a t  a  dementia,  the world  Mahurkar  i s usually  frontal  following  dialysis  throughout  of  patients.  electroencephalograph^  slow  symptoms  or  Sideman  DE  defect,  the  a mean o f 6 . 3 m o n t h s  The renal  become  progressive  soon  toms;  these  syndrome  for chronically dialysed  hesistancy,  speech  of d y s a r t h r i a , A  symptom  over  Burks  centres  e t a l . 1976;  or immediately  a l . 1976a;  Death  the  clinical  (DE)  et a l . 1973;  i n c l u d e . paroxysmal  especially  ent  (PIatts  by. s t a m m e r i n g ,  which  1980).  failure  noticeable  characterized  encephalopathy  cause o f death  Chokroverty  first  a distinct  i n 5 c h r o n i c a l l y hemodialysed  i n many r e n a l  a significant  end-stage  Perspectives  et a l . described  encephalopathy  termed  134 -  a l . 1976;  the  chronic  and m i g h t  Schreeder  et  -  al.  1977;  duration to  Berseth  of hemodialysis  7.5 years,  with  Although also  been  1980).  as  with  amounts  In  al  of  in  DE  brains  DE  renal  the  failure  have  been  Diffuse  also  and n u c l e a r  neuronal  observed  were  also  have  the  months  hemodialysis,  course  of  dialysis  never  DE h a s  chronic  (Sideman  hemoet  has a l s o , been  been  et  been al.  al.  report-  hemodialysed,  g e l s have  (Etheridge  Routine  In t h e i r of  but  to  administered  to  1978;  features,  Nathan  e_t  original  included  electron  et al.(1980), and/or  patients  and a s t r o c y t e  from  those  Alfrey  abnormalities  microscopic 32 h a d b e e n was  observasubjected  of  DE.  changes.  counts, of  other  fluid  e t c . ) have  To  lesions, In  living  analyses  cytoplasmic  by B u r k s  date,  and have  (e.g.  not y i e l d e d  not c l a r i f i e d computed  et a l . 1981).  glucose,  results  c h r o n i c a l l y hemodialysed  et a l .  histological  DE p a t i e n t s ,  (Parkinson  since  hemodialysed,  (e.g.  reported  in  demonstrated  been  changes  proliferation  any d i s t i n c t i v e features  who h a d n e v e r  the neuronal  non-specific  and c e r e b r o s p i n a l cell  publication,  gliosis  s c a n s h a v e b e e n d e s c r i b e d as n o r m a l  contents,  neuropathological  neuropathological  degeneration  Similarly,  discerned  blood  significantly  clinical  by Chui  i n uremic  considered  not  tomography  urea  from 6  was c o n s i d e r e d n o n - s p e c i f i c f o r t h e d i s e a s e ,  DE.  shrinkage)  the  The s t u d y  the o r i g i n of the c l i n i c a l  and  of  on t h e a b s e n c e  of 4 p a t i e n t s .  and who d i d n o t h a v e  studies  who h a v e  unrewarding.  in only 4 c a s e s , but t h i s  (1976)  the  t h e syndrome  aluminum h y d r o x i d e  Of t h e 6 0 c a s e s s u r v e y e d  t h e y were  in  peritoneal  absorption  that  et a l . 1980).  early  on  ranged  on l o n g - t e r m  literature,  severity  commented  autopsy.  axial  very  60 c a s e s r e v e a l e d  o f symptoms  (Chui  patients  patients  phosphate  of  (1972)  tions.  to  of  Griswold et a l . 1983).  spite  studies  to the onset  patients  of oral  intestinal  a_h 1 9 8 0 ;  to  in  well  in patients  reduce  the  confined  I n t h e more r e c e n t  whom l a r g e  et  usually  as  prior  -  An a n a l y s i s  a mean o f 2 . 5 y e a r s  reported  dialysis,  ed  et a l . 1978).  135  that  patients  protein differ who d i d  - 136 not  have  DE ( A l f r e y e t a l . 1 9 7 2 ;  Brain Section  neurochemical  I.ii),  activities synthetic tively,  have  been  of glutamic enzymes  these  The  treatment  the  disease,  by  intravenous  et  al.  DE  As  recently,  remission of  although  not  et  all  arrest  the progression  al.  1982). of  (Parkinson  (McGeer  from  et a l . 1981).  i n the course  (Nadel  e t a l . 1976;  even  of  B.  the  to  dimercaprol,  the  of  has  intravenous  this  been  intervention  than  treat.  et - a l .  chelating  reported  substantial  However,  have 1976).  Pogglitsch  use  afford  Sweeney  Med. J .  by  of  improved  and v i t a m i n s ,  a l . 1980;  transplantation or  which can  temporarily,  achieved  benefit  symptoms,  deaths,  et a l . 1976).  (Editorial,  et  Renal  patients.  albeit  has been  respec-  4 DE  Early  anticonvulsants,  ( A c k r i 11  patients  anoxic  the  or  to com-  must o c c u r  at  e a r l y stage of the d i s o r d e r .  DE  appears  centres to  (Flendrig  led  symptoms  desferoxamine  (Milne  recovery  of  of  administration  the disease  found  acetyl transferase,  unsatisfactory.  levodopa,  (see  e t a l . 1977)  nucleus  a n d had s l o w  progresses,  contents  and a c e t y l c h o l i n e  and caudate  c a n be r a p i d l y ,  the course of  agent  et  (GABA)  1978).  aluminum (Perry  in the b r a i n  is generally  dexamethasone,  administration  made  enzymes  Alfrey  and c h o l i n e  acid  comatose  the. disease  f a i l e d to a l t e r  a very  A single report  cortex  defect  than  administration of benzodiazepines  penicillamine,  plete  were  the speech  1985).  1981),  other  DE,  decarboxylase  o f both  of  in  y-aminobutyric  patients  decrease the a c t i v i t y  More  acid  for  et a l . 1975;  lacking.  t o be l o w i n c e r e b r a l  However,  all  studies  Barratt  have  to  a l . 1978).  easier  reported  decrease et  be  a  the  a l . 1976a; This  t o aluminum b e i n g  to  lowering  prevent of  concentration Platts  observation,  et  to  the  incidence  of  of  aluminum  in  a l . 1977;  as w e l l  Berkseth  as o t h e r s  A DE  when  the et  number  efforts  dialysate a l . 1978;  (see s e c t i o n  i m p l i c a t e d as t h e c a u s a t i v e a g e n t  of  of  DE.  I.ii),  renal were fluid Rozas has  -137  I.ii) Earlier Thurston were  Etiology  observations  e t a l . 1972)  Alfrey  et  al.  patients  dying with  patients  was 4  and  10  times  correlation  that  m u s c l e and bone  tion  were  1978;  to  be t h e c a u s e o f  previous  report  (McLaughlin  t h e most  comprises Despite  be  i t s prevalence,  rather al.  because  inert.  excreted known  most  Also,  it of  In  by  that  tion  of Al  ly,  that  addition,  t h e normal  metal  hemodialysis,  patients  earth's  with  A l can e n t e r  crust  occuring  tract  (Kaehny  already  patients,  Cartier Al  et- a l .  supported  a rapidly  common et  compromised  the c i r c u l a t i o n from  renal  to  to  a n d more function  the d i a l y s a t e  It  1984).  thought  present Poole  t o be  However,  DE.  hazard,  were  et a l . 1957;  et a l . 1977),  pro-  for  al.  (Campbell  impervious  for  element.  compounds  1977a).  by a  factory  reported  et- - a l .  mat-  intoxication  powder  appears  a  accumula-  Al  an e n v i r o n m e n t a l  i n the blood  DE  of  (Boegman  Al  of  gray  has been  i s b y no means Recker  most  of  brain  developing  considered  i t s absorption  (Kaehny e t a l . 1977a; in  the  barrier  later  brains  demonstrated  1976b; that  (Al)  orally,  not u n l i k e those since reported  any e x c e s s A l  the i n t e s t i n a l  of  i n an A l  and t h e t h i r d  intestinal  kidney  the  also  findings  al.  Al gels  dialysed  content  et  1972;  of gray matter  authors  Similar  al_.  o f aluminum  p o s s i b i l i t y was i m m e d i a t e l y  has n o t been  to  content  and t h e A l  the n a t u r a l l y  the  formidable barrier  1965).  These  et a l . 1962),  8% o f  et  in  non-demented  (Flendrig  symptoms  abundant  approximately  primarily  of  contents  leading to the suggestion  This  gressive encephalopathy with is  that  o f an i n d i v i d u a l who h a d w o r k e d  more t h a n 13 y e a r s  Al  Al  the Al  patients.  by o t h e r s  DE.  Clarkson  concentrations  that  duration  i n t h e DE  reported  the  controls.  Dewberry e t a l . 1 9 8 0 ) ,  might  to  than  normal  1971;  bone  examine  greater  dialysis  ter,  al_.  aluminum  receiving phosphate-binding  They f o u n d  of  between  soon  patients  DE.  et  serum and/or  (1976)  times  : The p o s s i b l e r o l e o f  (Parsons  that  increased in renal  led  o f DE  -  a et  promptly  it  i s now  the  absorp-  significantwho  a r e on  (Kaehny e t a l .  - 138  1977b;  Kovalchik  et  (1977b)  have measured  and h a v e  estimated  dialysis  session.  That for  the  Al  can  ate with  water  et  increased  DE of  dialysate The  been  DE  in  of  almost  is  not  dialysis  1977).  have  from the et  in  membrane  with  of  is  exposed  al.  of  to  200  1978).  in  reveal  was  less  higher  appears a high  renal  in  centres  50 u g / L ,  to  a  al.  where  of  mark-  correl-  the  high  Kingdom  the the  of  1976a;  United  while  concentration  a  incidence  et  the  per  support  containing  (Flendrig  survey  than  water  that  al.  body  studies  with  ug/L  the  strong  dialysate  A  those  into  et  membrane,  furnished  DE  centres  Kaehny  dialysis  taken  has  cases of  renal  excess  fluid  Al  the  Epidemiological  be  et  Indeed,  across  6 mg o f  article  by  Flendrig  an  of  ordinarily  been  In  Al  of  et  traced  a cathodic  (1976a)  low  concentration was  al.  water  was  Al  con-  incidence  metal  to  the  (Park-  removed  by of  syndrome to  conventional centres has  et  al.  1977;  where  been  r e s t r i c t oral  f l u i d b y means o f  Platts  patients  content.  to  be  gradual  protection  6  When  greatly  elevated.  dissolution  system a g a i n s t  the  of  2  Al  corrosion  in  tank.  a number  taken  reported  Al  found  used as a f l o c c u l a t i n g agent  the  dialysis  a l . 1976a;  area  part  reliably  incidence of  tions  of  those to  1982).  transfer  DE.  unknown  contamination  i s commonly  the  found  analysed,  Al  al.  In  Elliot  the d i a l y s a t e heating  et  in  al.  1981).  was  source  Al  distribution  1978;  the  et  dialysis  Al  usually  anodes w h i c h were  and  of  Al,  fascinating  developing  the  content.  was  of  an a v e r a g e  progressively  inson et a l . A  of  al.  that  centration  Al  King  rate  traverse  have  concentration  showed  that  geographical  patients  Berseth  1978;  the  causative role  e d l y uneven  DE,  al.  -  water  intake  d e i o n i s e r s or Berkseth  et  purification  softening  outbreaks  reduced Al  i n water  or  of  DE  have  eliminated  and  to  remove  'reverse al.  methods  1978),  (Platts  occurred,  when  precau-  trace  osmosis' although  plants  metals  (Flendrig this  has  -  not  been  evident  in  every  139 -  instance  (Burks  et  a l ,  1976;  Dewberry  et  al.  1980). Oral  Al  intake  intoxication use o f A l  than  gels  is  al.  reduced  Griswold  At  other  centration However, when t h i s  uncertainty  et  oral  that  cancer  as  than  to  of  view  DE,  et  al.  with  brain of  is also with  renal  the Al  last  content  the  of Al  than A l  have  of  DE  was r e p o r t e d  DE  never  (Etheridge  (McKinney  (Buge  et  et  to  be  et  the d i a l y s i s  resumed  DE,  Al  al. con-  al.  1978).  and a g a i n  abated  there  the  hepatic  blood-brain  three  conditions, might in  it  some  studies  have  same  finding,  disease coma,  h a s been  but  (Crapper  or  barrier  be t h e this  is still  many  in Alzheimer's  the  evidence  metastamight  be  suggested  result  of  condition,  c o r r e l a t i n g high  i n the d i a l y s a t e ,  suggested  heavy  gels  i n DE,  in  barrier  who h a v e  Al  Although  DE  universal  Nevertheless,  function  when  of  in-  rather  et a l . 1979).  has  that  of Al  in  the  of  Since  blood-brain  DE.  source  Masselot et a l . 1978).  failure,  seen  i n c i d e n c e o f DE when t h e d i a l y s i s  other  symptoms  present  1979).  because  discontinued  be e l e v a t e d  the  metals  disappeared  usage  important  discontinued  importance.  to  of the epidemiological  to high c o n c e n t r a t i o n s  was  (Buge e t a l . 1 9 7 8 ;  Al  t h e c a u s e o f DE ( A r i e f f In  usage  etiological  of  permeability  incidence  indicating a role  brain  in patients  amounts  was r e i n t r o d u c e d ,  its  (Arieff  The  Al  i s mainly  impaired renal  symptoms  and i n p a t i e n t s  the elevated  creased  Al  t h e symptoms  abnormal  DE  be a l e s s  incidence of  large  Al  oral  and o r a l  much e v i d e n c e  a l . 1976),  tic  where  to  This  t h e uneven  a l . 1983).  centres,  the content  without  with  p r a c t i c e was s t o p p e d  Despite  shown  et  was r e d u c e d  when  fluid.  b u t who consume  i n centres  1982).  considered  in individuals with  hemodialysed, 1978;  dialysis  contrasts  has been r e p o r t e d been  is generally  metals  incidence of  DE  and t h e s t r i k i n g r e d u c t i o n  in  f l u i d has been d e i o n i s e d , A r i e f f  might  play  n o t been e x c l u d e d  a  role,  but  pointed  as t h e c a u s a t i v e  factor.  (1981)  out  that  Others  -  (Gacek e t  al.  sate might  be  Animal and  its  can  be  when  in  rats  Al)  were  In  aluminium  higher  dose  1979).  At  in  study, or  levels,  s e r u m and b r a i n A l , ability  flicker.  No  (15/16)  AlfOHjg,  less  1  rats  chow  jections, period rats  of In  the  of  when  to  pH o f  compared  that  the  dialy-  5/6  not  were  fed  brain  with  to these  rats  more  mg/kg  function  had  by  showed  Arieff  et  for  matter  than of  4 Al  Al  were  chloride  fed  Al  the  no  as  elemental  gastric  et  al. oral  intubation  mortality  at  the  (Bowdler  et  al.  (1979)  elevations  in  activity',  a  when  dry  weight.  retained and  containing  increased  from  Mayor  al.  in the  given alone.  behavioral  to  subtotally  diet  content  compound  observed  as  well  administered  modest  months  pg/g  death,  increased s e n s i t i v i t y  al.  5  as  ' r a p i d general  and  rats  Similar-  (Berlyne  a high  reported  aluminum  authors  rats,  gastric distension  gray  amounts  (150  (A1(0H)3)  activity,  rats  higher  renal  to  anorexia.  subcutaneously  the r a t s  by  to  salts  Al  intoxication  Prior  nephrectomised  Al  of  nephrectomised  and  authors  noted  (5/6)  toxicity  fatal  lethargy,  mg/kg/day,  rotorod  that  water.  hydroxide  same  CNS  drinking  intact  were  weight  that  the  but  1650  demonstrate  reported  bleeding,  with  the  although  injections,  et  brains  parathyroid Over  a 25  changes  in  of inday the  group.  general, of  to  associated with  supplemented  either  role  s u c h as t h e  partially  when  maintain  dry  demonstrated  fed  in  function,  200  to  (1972)  in  and s t a t e d  to  300 mg/kg  (1980)  factors  observed  doses,  i l l effects  ug/g  days  rats  often  nephrectomised  than  8  aluminium of  lower  decreased  al.  intraperitoneal ly,  doses  the  et  added  was  renal  chloride  daily  that  performed  periorbital  normal  another  twice  to  were  intoxication  injected  1972).  3  salts  developed  with  been  Berlyne  within  2% A l  fatal  have  i n DE.  produced to  cautioned  -  important.  role  1  have  experiments  these r a t s ly,  1979)  140  Al  the in  animal DE.  experiments  Where  described  behavioral  tests  above were  have  not  performed  clarified on  animals  - 141  intoxicated not It  with  Al  compounds,  conspicuous,  or  were  is  possible  tion  with  role  of  brain  this  that Al  metal  not  studies  alterations  clearly relying  accumulation, in  readily reconciled with  DE,  since  -  in  related on  would  to  those  behavioral not  behavioral  the symptomatology  behavior  be  DE  seen  either in  alterations,  helpful  changes of  were  in  in  patients.  in  conjunc-  elucidating  animals  patients.  DE  absent,  might  not  the be  -  II)  The  Burks  unassociated  present  in  than  the  brains  measurement  decided  to  of  examine  t h e hope  that  findings  of  effective  treatments  After to  a  that  rats.  It  seemed  furnish question  treated  abnormalities  gels.  of  might  critical  Since been  lacking  be f o u n d . lead  the  to  in  of  might  studies  this  be  (other  condition, DE  we  patients  in  was a p o s s i b i l i t y  the  development  changes  same  rather  f o r or against  hemodialysis  It  neurochemical  populations  brains  using AHOH)^,  produce  many  that  et  of  that more  DE.  neurochemical  since  i n DE ( A l f r e y  neurochemical  autopsied  might with  neuronal  the neurochemical  might  evidence  major  the  the disease  l i k e l y that  better is  of  compound  by r e p e a t e d  binding Al  for patients some  of  have  change(s)  abnormalities  e t a l . 1980) s u g g e s t s  patients.  content)  specific  this  DE  loss  neurochemical^  a r a t model  bility  This  some  Chui  the  of  Al  determining  produce  might  with  EXPERIMENTS  neuropathological  e t a l . 1976;  changes  -  OUTLINE OF  absence of s p e c i f i c  aj_. 1 9 7 2 ;  142  renal  of  to  we  explore  neurochemical than  failure large  role  patients amounts  possi-  changes  of are of  attempted  the  behavioral  a causative  and a r e g i v e n  DE,  in  end-points Al  in  DE.  currently phosphate-  -  III)  143  HUMAN STUDIES  Ill.i) The of  methodologies  DE p a t i e n t s  1043-1048, DE  with  the  Ten  seizures,  characteristic  teristic  after  phosphate-binding the c o n t r o l The ites  of  phosphate enzyme choline  disorder.  highly  Further  determined.  the cofactor glutamic  acid  Brain Al content  when  compared  to  control  was a l s o  and o c c i p i t a l  is  interesting,  s i n c e t h e language  in  DE  benzodiazepines, receptors  be  reversed  which  (Baestrup  have  with  and a l l  and  died the  with  charac-  chronic  renal  had i n g e s t e d as w e l l  Al  a s on  of  and c o n t e n t s  enzymes,  GABA, of  including  (GAD).  enzyme  pyridoxal-5the  Enzyme  for  metabol-  synthetic  activity  acetylcholine,  of was  and D i s c u s s i o n  1),  and i n  the  recognition  in 6 regions reduction  the thalamus.  d i s o r d e r as w e l l  temporarily  et a l . 1982).  of  examined.  (Table  cortex  lacked  contents  s i g n i f i c a n t l y reduced  subjects  symptoms  patients  died  45:  I.  included  biosynthetic  brains  dyspraxia,  on t h e p a t i e n t s ,  i n Appendix  several  2  they  had  decarboxylase  40% i n f r o n t a l  can often  but  hemodialysis,  111. i i ) R e s u l t s were  DE,  and s e r o t o n i n , for  the  of  the  Neurochem.  dementia,  remaining  subjects  performed  noradrenaline  Mean GABA c o n t e n t s  The  of  8 had c l e a r - c u t  changes,  information  are a v a i l a b l e  acetyl transferase,  EEG  10  examination  (Perry et a l . J .  studied:  repeated  measurements  GABA,  I  suggestive All  by  gels.  (PLP),  for  language  disorder.  dopamine,  were  typical  subjects,  various  i n Appendix  patients  treatment  Method  the neurochemical  myoclonus,  symptoms  language  failure  for  are d e t a i l e d  1985).  neurological  used  -  by sites  linked  DE  being This  as t h e EEG  intravenous  of  brains  at  least  observation  changes  administration  functionally  to  of GABA  -  T a b l e 1.  Regional  144  GABA c o n t e n t s control  -  of autopsied b r a i n  i n DE and  patients  Patients  Frontal cortex  Occipital cortex  Cerebellar cortex  Caudate nucleus  Putamen  Controls  1.67*0.09 (26)  1.83*0.11 (25)  1.65*0.08 (26)  2.90*0.14 (32)  2.91*0.20 (19)  DE  0.96*0.04* (10)  0.99*0.06* (10)  1.18±0.10f (10)  1.91*0.11* (10)  2.35*0.20 (10)  Globus Pallidus  Substantia nigra  Nucleus Accumbens  Thalamus  Dentate Nucleus  Controls  7.32*0.40 (16)  6.05*0.26 (28)  4.15*0.29 (23)  2.05*0.12 (35)  4.77*0.23 (18)  DE  6.40*0.35 (10)  5.53*0.37 (10)  3.35*0.30 (9)  1.20*0.10* (10)  3.86±0.28§ (10)  V a l u e s (mean * SEM) a r e e x p r e s s e d i n y m o l / g w e t w e i g h t , w i t h number o f b r a i n s e x a m i n e d shown i n p a r e n t h e s e s . M e d i a l - d o r s a l region of t h e t h a l a m u s was a n a l y s e d . Values t h a t d i f f e r s i g n i f i c a n t l y from those of c o n t r o l s : *P < 0 . 0 0 1 ; + P < 0 . 0 1 ; 5 P < 0 . 0 5 .  Why a r e GABA c o n t e n t s sibility  i s that  this  GABAergic neurons, However, not that  any  had e l e v a t e d  reliably  measured  DE  patients  GABA c o n t e n t s tion, trol  of  the autopsied  brain  who d i e d  with  as m a r k e d  who d i e d  is  severely  brains  acid  of  at  analyser.  normal of  brain  (brain  of  chorea  of  DE  i n DE ?  possible  have  death.  Urea  results levels,  cortex urea  of  generally  explanation  since 8 of  urea  pos-  (Perry et a l . 1973).  uremia,  Our  One  selective loss  patients  the remaining  the f r o n t a l  uremic  regions  Another  due t o  contents  as t h a t  we c o m p a r e d GABA c o n t e n t s patients  brain  is a result  changes.  urea  on o u r amino  were  finding  i n GABA c o n t e n t  patients  two  neurochemical  neuropathological  the reduction  in several  as i s t h e c a s e i n H u n t i n g t o n ' s  examinations  shown  reduced  t h e 10  contents show the  i n the  reduction  i n a group  In of  greater  DE  c a n be  that  8 patients.  contents  is  10  of  addicon-  than  10  -  times  normal)  with  urea contents. were  almost  pared that to  to  a group  1.67  with  ( 1 . 6 4 ± 0 . 0 9 y.mol/g  i n GABA c o n t e n t  neither  wet w e i g h t  brains,  mean  in the brains  low t o normal  o f w h i c h had  in uremic  ± SEM). of  DE  brain  It  brains, thus  patients  DE, com-  appeared  was n o t due  uremia. Yet  the  another  p o s s i b i l i t y f o r the reduction  a c t i v i t y o f GAD,  bility  cannot  likelihood that  PLP,  after  by  about  50% i n uremic  and i f of  GABA t h a t  than  but t h i s  PLP  l e v e l s were  was m a r k e d l y  DE  brains  (Table  s i n c e PLP  3)  GABA  PLP  this  If  PLP  only  also serves  that  were  reported  contents  dopamine brain  as t h e c o f a c t o r  were  f o r the optimal enzyme for  (Table  and s e r o t o n i n contains  for  t h e enzyme e s s e n t i a l f o r t h e p r o d u c t i o n  since GABA  contents  in 3 regions  reasonable  aromatic  while  t h e normal  for  the r e -  be r e d u c e d  and putamen, Also,  brain  reduced  f o r t h e GABA d e f i c i e n c y , cortex  is  hemodialysed  account to  further  t o be r e d u c e d  catabolic  could  A  re-  in the  have been  required  possi-  DE p a t i e n t s  amounts  (the  in the c o r t e x .  t h e DE  of  that  markedly  b r a i n PLP c o n t e n t s  this  the f r o n t a l  noradrenaline,  imply  been  or not they  t o be an e x p l a n a t i o n  reduced  of  have  as a c o f a c t o r ) ,  i n both  often  in the brains  cofactor  brain  is  is  This  (McGeer e t a l . 1 9 7 6 ) .  aminotransferase  We d i d f i n d  reduced  the metabolites  of  i n DE  i s reduced.  in deficient  et a l . 1983).  for  is unlikely  of  whether  content  activity  dying  concentrations  amounts  contents.  2),  boxylase,  GAD  i s present  patients,  Lacour  greater GAD  PLP  also requires  GABA  content  since  anoxic  o f GAD,  Plasma  (Stone e t a l . 1975;  duced  prolonged  the cofactor  DE p a t i e n t s .  activity  tested,  i n GABA  enzyme f o r G A B A ,  f o r t h e d e c r e a s e i n GABA c o n t e n t  of  i n DE,  the synthetic  be r e a d i l y  duced i n b r a i n  PLP,  who d i e d  in the 2 groups,  ± 0.09 i n non-uremic  the reduction  -  o f 26 p a t i e n t s  Mean GABA c o n t e n t s  identical  145  L-amino  amounts acid  of these 3 biogenic  of of  decaramines.  -146  Table 2.  Pyridoxal  phosphate  Subjects  content  Frontal  -  o f a u t o p s i e d b r a i n o f DE  patients  Putamen  cortex  Controls  2.96 ± 0.24 (H)  2.10 ± 0.24 (10)  DE  1.53 ± 0 . 1 0 * (10)  1.41  ± 0.28 § (10)  V a l u e s (mean ± SEM) a r e e x p r e s s e d i n n m o l / g w e t w e i g h t , with t h e number o f s a m p l e s a n a l y s e d i n d i c a t e d i n b r a c k e t s . Values that d i f f e r from c o n t r o l v a l u e s : *P < 0.001; § P < 0 . 0 5 . Table 3 .  Contents  o f monoamine of  DE  metabolites  in autopsied  brain  patients  Patients  Frontal cortex  Controls  0.39 ± 0.07 (13)  0.47  DE  0.37 ± 0.17 (10)  0.66 ± 0.14 (10)  Caudate nucleus  Putamen  MHPG ± 0.10 (8)  0.52 ± 0.12 (13) 0.71  ± 0.14 (9)  HVA Controls DE  0.79 ± 0.31 (13)  22.3  0.95 ± 0.09 (10)  23.4  ± 2.48  31.5  ± 3.63 (15)  30.8  ± 2.02 (10)  (8) ± 2.14 (10)  5-HIAA Controls  0.61  3.35 ± 0.69  (13)  (8)  0.89 ± 0.13 (10)  3.56 ± 0.53 (10)  ' DE  ± 0.10  4.88 ± 0.59 (15) 5.07  ± 0.67 (10)  V a l u e s (mean ± SEM) a r e i n n m o l / g w e t w e i g h t , w i t h t h e number of b r a i n s examined i n d i c a t e d i n b r a c k e t s . None o f t h e DE v a l ues differ significantly from those of controls. MHPG (3-methoxy-4-hydroxyphenylethylene g l y c o l ) i s the metabolite of n o r a d r e n a l i n e , HVA ( h o m o v a n i l l i c a c i d ) i s t h e m e t a b o l i t e o f o f dopamine, w h i l e 5-HIAA (5-hydroxy-indoleacetic acid) i s the metabolite of s e r o t o n i n .  - 147 -  Table areas  4  of  shows  a  cerebral  activity  was  of  cortex  equal  and i n t h e 8 w i t h ity  in  the  DE  small  the  decrease in  syndrome.  was  et  al.  1980;  disease  (Perry  et  al.  1983).  Choline  less  the  or  DE  1.07  ±  the  CAT  activity  reduction some  in  of  d e c r e a s e d CAT  patients  3  enzyme  symptoms  observed  in  DE,  activ-  in  Alzheimer's  with  Parkinson's  in cerebral  cortex  patients Temporal cortex  0.05  1.01  (16) DE  that  activity  Frontal cortex  Controls  with  However, than  mean The  i n demented  acetyl transferase of  in  2 patients  marked  1983),  35%)  patients.  clear-cut  patients  Patients  DE  in  (Perry  4.  10  to  magnitude  disease  Table  the  (25  0.64 ± 0.08 (12)  ± 0.08 (15)  0.76  0.73 ± 0 . 0 9 + (10)  Occipital cortex  0.42 ± (10)  ± 0.15 (9)  0.07  V a l u e s (mean ± SEM) a r e e x p r e s s e d a s n m o l / 1 0 min/mg protein. Figures in brackets i n d i c a t e the number of brains examined. Values that d i f f e r from c o n t r o l s : 5 P < 0 . 0 5 ; + P < 0 . 0 1 .  The  Al  subjects  content  was  For  the  10  4.2  yg/g  dry  ing  from  11  magnitude Cartier  et  5.6  DE  ±  of 0.6  the  yg/g  patients, weight  to as al.  t o be a f e a t u r e  56.2 that  (P<  of  the  found  dry  matter  compared  This by  Thus,  the with  frontal  cortex  a range  of  a r e a had a mean A l to  increase  other Al  of  weight,  same b r a i n  0.001  yg/g.  1978).  gray  controls, in the  DE  investigators  accumulation  the d i s e a s e , although  not  in  2.0  patients (Flendrig of  15  to  DE  control  9.9  content  Student's  brains  of  of  yg/g. 21.8  t-test), was et  of  the  al.  patients  n e c e s s a r i l y an e t i o l o g i c a l  ±  rangsame 1976; seems one.  -  In regions also  summary, of  DE  observed  noradrenaline, over  of  these  we  found  patients. in  3  biogenic  -  mean GABA c o n t e n t s A small  cortical  dopamine  148  decrease  areas.  in  Normal  and  serotonin,  amines  in the  DE  to the  reduced  enzymic  contents  suggest brain  be  that  are not  of the  in  several  activity the  of  CAT  was  metabolites  synthesis  different  brain  from  and  of  turn-  normal.  -  IV)  149 -  ANIMAL  IV. i ) 5  groups  taneously tated of  o f young  once  daily  suspension  Al-treated  adult  of  for varying A1(0H)3  rats,  in  shown on T a b l e 5 , w i l l  Table 5 .  which  to  left  forth, of  either  of The  various  sex were  time  with  rationale  a  in the Results  having  5  agigroups  c h a r a c t e r i s t i c s are  and D i s c u s s i o n  given to aluminum-injected Extent of nephrectomy  subcu-  continuously  for  experimental  injected  section.  animals Duration of L i C l pretreatment  150 mg e l e m e n t a l A l / k g d a i l y f o r 49 days  None  None  200 mg e l e m e n t a l A l / k g d a i l y f o r 14 d a y s  None  None  200 mg e l e m e n t a l A l / k g d a i l y f o r 14 d a y s , s t a r t i n g 14 days a f t e r nephrectomy  5/6  None  200 mg e l e m e n t a l A l / k g d a i l y f o r 3 or 5 days, s t a r t i n g 24 h a f t e r nephrectomy  5/6  16 w e e k s  2 0 0 mg e l e m e n t a l A l / k g d a i l y f o r 30 d a y s , s t a r t i n g 24 h a f t e r nephrectomy  1/2  16 w e e k s  injections,  kidney  this  the  be c l a r i f i e d  which o n l y t h e r i g h t k i d n e y the  of  periods  A1(0H)3 dosage  E  Method  rats  in water.  Treatments  Experimental group  Prior  Wistar  EXPERIMENTS  (Piatt  extent  of  suspension i n j e c t e d  et  some  rats  (Group E ) ,  underwent  or the r i g h t  a l . 1952) were  nephrectomy (20 ml/kg)  will  surgical  removed  kidney (Groups  be r e f e r r e d  was s u f f i c i e n t  nephrectomies,  to  to  as w e l l  provide  as 2/3 o f  C and D;  as 5 / 6 ) .  in  The  a daily  hencevolume dose  of  -  elemental  A l o f 150 mg/kg  D,  or E rats.  or  non-operated,  NaCl  i n Group A r a t s ,  Appropriate were  groups  injected  f o r t h e same p e r i o d s o f After  the  last  were  within  Brains  a l l o w e d t o thaw  i n Groups  similarly  with  brains  nitrogen  then  subcutaneously  injection,  and t h e i r  increase  rats,  saline  dislocation,  postmortem  of control  or  cervical  any  and o f 2 0 0 mg/kg  equal  B,  C,  nephrectomised  volumes  of 0.9%  time.  Al(0H).j  40s.  150 -  were  i n GABA  frozen  removed  (Perry  were  sacrificed  and immersed  immediately  contents  to -10 C to allow  rats  to prevent  or  e t a l . 1981).  f o r accurate  in  by  liquid  minimize  Brains  dissection into  were  discrete  areas. The moved  entire  cerebellum  f o r assay  sagitally,  content, of  half,  portions  occipital At  of A l content.  and one h a l f  From t h e o t h e r  striatum  rats  (w/v)  were  analyses  described using GAD  enzyme  which acid  t h e method  sacrificed,  h a d been  analysed (Perry  the  was t h e n  subsequent  removed  first  re-  bisected midGABA  analysis.  of  i t s dopamine  f o r GAD a s s a y ,  and p o r t i o n s  brains  of rate  et a l . 1979).  blood  was a l s o  c o l l e c t e d f o r measure-  and c o n t a i n e r s previously  for r a t brain  of  soaked  a n d CAT enzyme  i n Appendix  rat  f o r storage ( f o r 24  o f samples f o r hours)  in 0.6%  any c o n t a m i n a t i n g A l .  content  employed  activity  were  were  concentrations.  o f GABA  f o r human  brain  at -70 C f o r  cortex  d i s o d i u m EDTA t o r e m o v e  Measurements  specimen  was d i s s e c t e d f o r m e a s u r e m e n t  the dissecting instruments Al  each  f o r CAT a s s a y .  ment o f s e r u m u r e a and A l  subsequent  from  The r e m a i n i n g  was s t o r e d  of f r o n t a l  cortex  t h e time  All  and b r a i n s t e m  frontal  I.  Dopamine described  cortices  formation  activity  of  were  content  by P e r r y  was m e a s u r e d  ^ C 0 2 from  performed  as  was d e t e r m i n e d et  a l .  using  L-[1-^C]  (1984).  a  method  glutamic  -  Two  different  The f i r s t brains  which  to  tion  increased with  brain  weight vials)  A,  used  of  this  which  To. a v o i d  measure  then  dissolved  of  rat  brain. human  yielded  blank  a relatively  reconstitution  contains  (Fisher  contents  was as d e s c r i b e d a b o v e f o r  method  this  Al  problem,  linear  large  Samples were  polyethylene  probably  amounts  a s e c o n d method  i n 2 ml o f  high  of the b r a i n ,  relatively  ( G r o u p D and E r a t s ) .  in plastic vials and w e r e  to  time a f t e r  glass  specimens  -  B and C r a t s )  However,  et a l . . 1980).  later  stant  I).  dissolution  (Sansoni for  were  (used f o r Groups  (Appendix  value, due  methods  151  Al  was e m p l o y e d dried  liquid  5% (w/v)  of  to  con-  scintilla-  tetramethylammonium  o  hydroxide 16 h ,  by h e a t i n g  Thus,  a t i s s u e d i s s o l u t i o n method w h i c h e m p l o y e d  relatively by  a t 90 C f o r 1 or 2 h .  adding  human  shorter  t i m e , was u s e d .  4 volumes  specimens.  Material  No.  technique  of  ethanol  Repeated  1577a  yielded  from  an A l  and w e r e  U.S.  value  Standards.  Al  measurements  an a l k a l i  analysed  for  Bovine  Liver  of  National  Bureau  of  dry-ashing  and w h i c h  for this material rat  frontal  Al  as done  took  A  by e i t h e r  diluted  with  the  this  non-certi-  by t h e National  cortices  a  Reference  Standards ± SD).  for  with  Standard  of  o f 2 . 8 ± 0 . 9 p g / g (mean  f i e d A l v a l u e o f 2 p.g/g i s g i v e n  of  The r e s u l t i n g s o l u t i o n s w e r e t h e n  analyses  the  instead  Bureau  method  of  were  done i n d u p l i c a t e . Serum u r e a n i t r o g e n analyser  (Astra  ity rate  method.  8,  c o n c e n t r a t i o n s were  Beckman  Instruments)  IV.ii) Lack  of  neurochemical  In tions  preliminary of  elected  Al Cl^ to  treat  in  in  (Group A  experiments, rats  rats  rats  an e n z y m a t i c  clinical  urease-conductiv-  daily  with  normal  renal  function  injected  rats) the subcutaneous  was f o l l o w e d  with  with  on a r o u t i n e  Results-and Discussion  changes  c h r o n i c a l l y with A1(0H)3  determined  by  severe  subcutaneous  injection tissue  injections  of  acidic  necrosis. of  We  a neutral  soluthus sus-  -  pension.of of  the  a depot Young  once ous  insoluble  AHOH)^.  of Al to provide  continous  exposure  Al/kg  daily  After  at  the  controls 30  adopted  days  a  ficed,  Al-treated Table A1(0H)3  injections, stance  the l a s t  motor  shows  d i d not e l e v a t e  vealed  no d i f f e r e n c e s  A),  or  between  no  mean  rats  backs,  changes  weight  became  for  i n an open  field No  solution,  developed they  1 5 0 mg  subcutane-  grew  normally,  49 d a y s .  The s a l i n e - i n -  gain  70  an  from  hour  behavior.  o f t h e 49 i n j e c t i o n s ,  with  saline  aggressive,  about  in  with  advantage  of the metal.  subcutaneously  their  rats  lasted  in  Group  brain  Al  Quantitation  o f GAD and C A T , differ  on  the  apparatus, differences  and  after  g.  frequently  being  were  sacri-  observed  and t e s t e d were  175  injections.  Before  a l l rats  to  on a  observed  for  rotatbetween  rats.  that  unaltered.  not  sites  incoordination.  mained  ties  injected Group  offered  t o l a r g e amounts  the A l - i n j e c t e d  Al  that  showed  and c o n t r o l 6  5,  corresponding  abnormalities  for  (Table  Although  a  rats  24 h a f t e r  drum  were  injection  had  boxing  g r o s s motor  rats  This  i n c r e a s i n g f r o m 68 t o 186 g o v e r  of  Saline-injected  ing  as A1(OH)3  mean w e i g h t  jected  female  f o r 49 d a y s .  scarring  their  -  relatively  litter-mate  elemental  152  between as w e l l  the Al  of  A  rats,  content, other  Al-treated as o f  the  and t h a t amino  acids  and c o n t r o l  striatal  and c o n t r o l  chronic  groups.  dopamine  administration  brain in rats.  GABA  content  the brain Cortical  content,  also  of rere-  activi-  similarly did  Table 6.  Neurochemical  Animals  parameters  Al content (ug/g dry weight)  Controls  6.1  Aluminuminjected  ± (12)  1.3  5.0 ± (H)  0.9  153  -  i n Group A r a t s  ±  0.08  239 ± 13 (12)  1.13  252 ± 11 (16)  1.11  (12) 2.52  ±  chronic Al  exposure  GAD a c t i v i t y CAT (nmol/lOmin/ (pmol/h/g protein) mg w e t w e i g h t  GABA c o n t e n t ( u m o l / g wet weight) 2.62  after  0.09  (16)  ±  Dopamine (ug/g wet weight) 12.7  0.07  ±  (10)  0.8  (12) 12.6  ± 0.06 (10)  ±  0.7  (12)  Rats number o f a n i m a l s i n b r a c k e t s . V a l u e s a r e mean ± SEM, w i t h were k i l l e d 24 h a f t e r the l a s t o f 49 d a i l y s c i n j e c t i o n s of A1(0H)3 i n a dosage providing 150 mg/kg/day of elemental Al. None o f t h e means f o r t h e 2 g r o u p s d i f f e r s i g n i f i c a n t l y ( t w o - t a i l e d Student's t - t e s t , P = 0.05). A l c o n t e n t was m e a s u r e d i n t h e c e r e b e l l u m , GABA c o n t e n t was d e t e r m i n e d i n w h o l e b r a i n m i n u s t h e c e r e b e l l u m , dopamine c o n c e n t r a t i o n was a n a l y s e d i n t h e s t r i a t u m , and GAD and CAT a c t i v i t i e s w e r e a s s a y e d i n t h e f r o n t a l and o c c i p i t a l cortices respectively. B r a i n a l u m i n u m was d e t e r m i n e d by t h e drya s h i n g EDTA m e t h o d .  Absorption In  o f • A1(OH)3 s u s p e n s i o n  view  treated  of  with  the  was i m p o r t a n t The  l a c k of  the  to  failure  to  relatively prove  of  Al  duce n e u r o c h e m i c a l changes rats  were  A1(OH)3 e q u i v a l e n t or  of  saline,  dose of vating  addition,  rats  was  body l o a d o f Group C r a t s  7  shows  injected  with  that  AHOH)^  have  in these given  daily of  for  had  the  A  rats,  (Group  accounted  for  subcutaneous  elemental days.  this  also  in  brains  of  Table  our  rats  5),  it  depots.  inability  to  pro-  rats.  i n c r e a s e d from Al  accumulation  indeed absorbed from subcutaneous  might  consecutive  i n j e c t i o n s were  Table  was  200 mg/kg  14  elemental Al the  Al(OH)3  for  to  Al  insoluble  that Al  absorption  Eighteen  obtain  and  No 150  Al  (Table  mortality to  5/6  5, was  200 mg/kg  subsequent  undergone  injections  of  Groups  either  B and  encountered.  i n the  planned  nephrectomies  The  hope o f  ele-  experiments. 14  days  C),  In  before  started. serum  A1(0HK,  Al  concentrations  regardless  of  were  whether  markedly or  not  elevated  they  had  in been  -  nephrectomised.  At autopsy,  of  injected  the  A1(0H)^  injection left  sites.  kidney  Presumably, this  7)  had h y p e r t r o p h i e d relatively  normal  were  at t h e time  tomy,  Table  7.  after  to  i s about  Evidence  Controls Group B Group C  the  2 fold  small  the remaining  large  as  had been  an  that than  that  for circulatory absorption  of  of  serum  at the  kidney.  by t h i s  after  in control  space  undamaged  regained  4 - 6 days  amounts  1/3  i n the 5/6 nephrectomised  ( T a b l e 8 shows  time,  urea  rats 5/6  con(Table  nephrec-  rats).  subcutaneously-  A1(0H)3  Elemental aluminum d o s e , 14 injections  None None 5/6  only  subcutaneous  by t h e o b s e r v a t i o n  4-6 times higher  Surgical pretreatment  (6) (6) (6)  as  function  injected Experimental group  in  surgery,  a size  renal  only  of s a c r i f i c e  serum u r e a  i n s p e c t i o n showed t h a t  strenghtened  elevated  -  unabsorbed  o n e month  p o s s i b i l i t y being  centrations  visual  remained  Also,  154  None 2 0 0 mg/kg 2 0 0 mg/kg nephrectomy  Serum aluminum (ug/L)  Serum urea (mmol/L)  5.8*4.2 69.3*4.6 60.8*10.7*  9.2*0.6 7.0*0.2 17.3*1.4*  Brain aluminum (yg/g dry weight 10.1*1.1 9.5*1.1 11.5*1.2  V a l u e s shown a r e mean ± S E M . The number o f r a t s a n a l y s e d a r e i n d i cated i n parentheses. B r a i n a l u m i n u m c o n t e n t was m e a s u r e d i n c e r e b e l l u m u s i n g t h e d r y - a s h i n g EDTA m e t h o d . Values that d i f f e r from controls:*P < 0.001.  Absence o f l a s t i n g n e p h r o t o x i c i t y Before and t h e i r (LiCl) of  commencing  Al  corresponding  chronically.  injections, controls)  Christensen  irreversible chronic renal  chow)  from  means  of  birth  until  impairing  in 1ithium-treated  were et  function,  rats  (Groups  fed large  al.  failure  16 w e e k s .  renal  some  (1983)  in rats  Since  this  rats  amounts  of  had r e p o r t e d  fed LiCl procedure  we r e p e a t e d  D and E  these  i n Table  lithium the  ( 4 0 mmol/kg appeared  5,  chloride  production of to  investigators*  ground offer  a  proce-  -  dure,  giving  LiCl  investigators, the to  instituted and  ure  (rats  were  r a t pups  a t 16 w e e k s . of  of treatment,  serum u r e a  10 w e e k s ,  animals  blood  content  were  the  end o f  rats  10  was c o l l e c t e d  weeks  impairment  had n o t been  group o f l i t h i u m - t r e a t e d  Rats  high  doses  four  Normal  LiCl  during At  of  the degree  of  were  treatment  10.1  (Table  diet  5 , group  orally for  D rats).  w h i c h we had o b s e r v e d tage  was l i k e l y  elapse Thus,  between beginning  daily  mental  with  both  groups.  of both  to  24  days,  The r a p i d i t y  h  either  after  an A1(0H) 3  4 months  of  suspension  further,  mmol/1 a t  while the  control hoped-for  to nephrectomise  life,  underwent  initiation  animals  to continue  meas-  impairment.  and 7 . 7  which  a substantial  and t h e  Injections  rate  a l l of  l e d us t o c o n c l u d e  saline.  The m o r t a l i t y  sexes,  o f the compensatory  surgery,  to  a  nephrectomy  by a l l o w i n g  nephrectomy  a n d 16  injections.  and then  i n Group C r a t s  partial 24  after  f o r the f i r s t  t o be a c h i e v e d  A l , or with  not possible  7  f o r A1(0H) 3  decided  was r e -  10  renal  Thus,  a g e 10  treatment  of rats  respectively,  We t h e n  from  t h e end o f vein  rats  dosage f o r  the l i t h i u m  the t a i l  achieved.  rats  foregoing  r a t chow  from  i n these  promptly  litter-mate  of L i C l  lithium-free  in  rats  i n j e c t e d w i t h A1(0H)3 Sixty  was  of  by t h e  LiCl/kg  had a mean s e r u m u r e a c o n c e n t r a t i o n o f 7 . 9 mmol/1.  renal  once  no d e a t h s  as an i n d i c a t i o n  a n d 16  6 0 mmol  was u n i m p a i r e d .  The mean s e r u m u r e a c o n c e n t r a t i o n s  used  and i n c o n s i d e r a b l y h i g h e r  f o r the next 4 weeks).  There  these  i n t h e dosage  f e d chow c o n t a i n i n g  and 8 0 mmol/kg  t h e growth  weeks  newborn  f o r the f i r s t  l a s t 6 weeks 12 w e e k s ,  to  155 -  were  continued  for only  due t o t h e number in this  period  were 5/6  that  nephrectomies hypertrophy  little  period of  given  p l a c e d on a  renal  injected  providing  had been  Al  of  advantime  injections.  subcutaneously  2 0 0 mg/kg  of  3 or 5 days. of  w a s much  to  deaths higher  eleIt  occuring f o r the  -  Al-treated Death Al  in  rats  the  (58%,  latter  22  of  group  38)  was  156  than  -  in  saline  presumably  due  to  uremia,  g r o u p was p r e s u m a b l y due t o c o m b i n e d u r e m i a and A l Seven  jection the  surviving  (i.e.  fifth  4  rats  days  injection  differences  were  were  sacrificed  post-nephrectomy), (i.e.  found  6 days  between  24h  while  brief  vate  Al  exposure content  to  Al  in the  these  tent,  even  though  either  with  Al  or  immediately brain,  the  brain  saline  was  after 9 were  post-nephrectomy). 2  nor  did  urea more  after it  the have  content than  The  14  any  times  while  7  26),  in  the  AHOH)^  in-  toxicity. the  third  sacrificed  24  nephrectomy effect  on  after  did  data  for  relativenot  ele-  b r a i n GABA  con-  nephrectomised of  h  significant  neurochemical  that  of  death  r e s u l t s show t h a t  5/6  in  (27%,  B e c a u s e no  sub-groups,  t h e 2 groups have been combined i n T a b l e 8 . ly  controls  rats  treated  non-nephrectomised  rats.  Table 8.  Effects after  of  A1(0H)2 or  5/6 n e p h r e c t o m y  saline  i n j e c t i o n s s t a r t e d 24 h  in LiCl-pretreated  Animals  Serum aluminum (ug/L)  Brain aluminum (ug/g d r y weight) _  Saline injected controls  19.8*5.2  1.85*0.19 (12)  57.4*13.4 (12)  76.1*17.5 (12)  2.53*0.07 (12)  Aluminum Group D rats  75.2*6.9  1.63*0.14  36.8*7.1 (16)  56.0*11.4  2.61*0.07  (16)  (16)  (5)  (9)  (16)  Serum urea (mmol/L)  rats  Brain urea ( p m o l / g wet weight)  Brain GABA (pmol/g wet weight).  V a l u e s shown a r e mean * SEM, w i t h number o f a n i m a l s i n b r a c k e t s . R a t s w e r e k i l l e d 24 h a f t e r t h e l a s t o f 3 s c i n j e c t i o n s o f s a l i n e , o r o f A 1 ( 0 H ) 3 i n a d o s a g e p r o v i d i n g 200 m g / k g / d a y o f elemental A l . B r a i n a l u m i n u m c o n t e n t was m e a s u r e d b y t h e t e t r a methylammonium h y d r o x i d e m e t h o d . Brain urea content in nonn e p h r e c t o m i s e d r a t s i s < 4 pmol/g wet w e i g h t , w h i l e serum u r e a l e v e l f o r n o n - n e p h r e c t o m i s e d r a t s i s 9 . 2 * 0 . 6 mmol/L ( n = 6 ) . Values that d i f f e r from c o n t r o l s : * P < 0 . 0 0 1 .  -  It with  seemed less  whether  severe  brain  (n = 30)  subcutaneously mg/kg/day • o f (n = 1 4 ) . days,  therefore  either  elemental  Injections  cerebellar Al  to high the  (2.24 ± 0.25)  5,  (mean  the b r a i n s of the A l - t r e a t e d  Group  it  lethal  possible  remaining  determine  E,  was no  n = 16),  to  injected  provide  or  with  200  saline  c o n t i n u e d d a i l y f o r 30  the l a s t  injection.  post-operative  increased  No GABA  lead  to  (P<  These  mortality.  0.05,  (3.20 ± 0 . 3 1 ug/g dry weight)  group.  to  animals  and w e r e  sufficient  24 h a f t e r  ± SEM).  in  1 ithium-pretreated  nephrectomy  nephrectomy,  slightly  less  c o n c e n t r a t i o n s might  The  and t h e r e  was o n l y group  make  serum A l  24 h a f t e r  thrived,  be  suspension  sacrificed  content  thus  unilateral  (Table  started  might  metal.  A1(0H)3  Al  in the A l - t r e a t e d  controls  and  underwent  and t h e a n i m a l s w e r e  t-test) to  of  -  treatment  impairment  uptake  with  Al  exposure  heminephrectomised r a t s Mean  that  renal  or not longer  substantial rats  possible  157  when  Student's compared  d e f i c i e n c y was e v i d e n t  in  -  V)  The not Al  question  answered in  the  as t o w h e t h e r  by o u r animal  brains  heminephrectomy, attempts not  to  of  nitude  of  treated  Al(0H)-j,.  the report tion,  blood Al  A1(0H)3  given  brain  Al  given  in this  dry  (from  It  i s possible that  sources  (Arieff  or  have  rats  of  Analytical  vials  reagents,  in  A l compound,  more  successful Al  was  in  clearly  deposited • subof  whether  mag-  or not  1 et  a  a l . 1979;  cleaning ambient spectral  with air  than  et  a l .  1980).  disodium  by  appropriate of  Al  EDTA),  (Minczewski  and c h e m i c a l  dif-  levels.  difficult  leaching  not  reported  Al  of  of  4 ug/g  technical  tissue  and l a c k  were  were  a r e made  include  func-  elevated  values  encountered  materials  contamination  are both  markedly  Mayor  of  renal  less  Al  example,  injections  ug/g d r y w e i g h t ) ,  measurements  in  For  normal  control  interferences,  dust  with  generally  studies  careful  i n producing  compounds.  developed  although  biological  and A l  employed  one o r d e r  this  elevations,  Al  interferences  was e l e v a t e d  of  Al),  Bowdler  (despite  suspension  subcutaneous  unreliable  of  failure  number  about  analytical  This  we  of  administration  rats  Al  elevation  though  claims that  in  sources  Al.  with  a t l e a s t one o f t h e s e  Important  of  been  weight),  of  produce  i n DE was  7).  treated  modest  a l . 1979;  resulted  plastic  with  factor  lithium  the AHOH)^  elemental  values  of contamination,  contamination 1982).  et  measurements  standards. glass  as  More  control  others  Accurate  to  (340 ug/g d r y  study.  which  (Table  unspecified  mg/kg/day  by  ficulties  of  of  to even  chronic  concentration  e t a l . (1972)  an  content  weight  claim  in the brains  (150  of  injected subcutaneously  by B e r l y n e  and  and/or  absorption  investigators  accumulation  with  excretion  t h e s e r a t s were n e p h r e c t o m i s e d Other  i s the causative We f a i l e d  the renal  In f a c t ,  in rats  or not A l  5/6 n e p h r e c t o m y ,  due t o t h e l a c k  cutaneously.  CONCLUSIONS  experiments.  rats  decrease  158 -  from Al  et a l .  (Goode  et  - 159 al.  1977;  , Slavin  tent of control ashing  (Table  hydroxide  from h e a t - a c t i v a t e d  our  finding  content  i n Bovine  6),  Liver  Standard  the tetramethylammonium  because renal injected,  blood  deposited  Al  permanent  et  a l . 1983),  accumulation  as w e l l  determine  whether  phosphate-binding  tious,  the  answer  especially  who consume  although  causes  it  Also,  these s u b j e c t s can o f f s e t  is  of  i s supported  by  3 to 5  in r a t brain  either  the large barrier  was c l e a r l y  Chronic  will  elevation to  of  DE.  Kosik  as t o w h e t h e r  of with if  answer  hemodialysed regard  to  A l proved gels  assumed  or prevent  This  or  other  then  needed  to  patients  normal  perhaps  renal  Al  a n d CAT  administration  should  t h e normal  labora-  not brain  failure  t o be t o x i c ,  as a n t a c i d s that  renal  to  et a l . 1983;  i s urgently  the oral  alter  be n e c e s s a r y  u s e some  et a l . 1980;  of Al  a n d was p r e -  insufficient  It to  amounts  t h e d e f i c i e n c i e s o f GABA c o n t e n t  Al-containing  generally  leaching  but of only  s u c h as b o n e .  the question  treatment  Al gels.  to excrete  i n the r a t , or (Yates  tetra-  N o . 1 5 7 7 a , o f an A l  EDTA m e t h o d ,  we m e a s u r e d .  the rabbit  a s t h e symptoms  be m o d i f i e d ,  dividuals  failure  con-  method.  tissue, rats  the  due t o  Material,  Al  by t h e d r y -  with  explanation  to cross the blood-brain  i n these  i n DE p a t i e n t s  activity,  should  to  obtained  Al accumulation  adequate  parameters  renal  a n i m a l , f o r example  Yokel  Reference  i n some o t h e r  concentrations  A l was d e t e r m i n e d  This  hydroxide  was s t i l l  the neurochemical  establish tory  function  mean b r a i n  8 ) , was p r o b a b l y  t o produce  or because A l f a i l e d  ferentially  of  (Table  higher  to that  i n the v i a l s .  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Trace Element Res. 5 : 4 6 7 - 4 7 4 , 1983.  alum-  -  CHAPTER 4:  165 -  HALLERV0RDEN-5PATZ  SYNDROME  ABSTRACT  We  examined  for  patients  who d i e d  tents  cystine  of  pallidus  were  dioxygenase, reduced  a result may  sive  and o f  elevated  that  pallidus  of  l o c a l l y i n the globus  an  iron  i n the globus  cysteine  and o f  membranes  to cause  den-Spatz  syndrome.  iron  both  to  brain  in the  Activity  cysteine  of  for  disease. free  morphological  the  The  that  changes  that  observed  in  as  cysteine  increase  damage  was  cysteine  syndrome  combination  radicals  globus  cysteine  Accumulated  local  2  con-  sulfinate,  We p r o p o s e  dioxygenase.  of  patient,  in Hallervorden-Spatz  accounting  may g e n e r a t e  the t y p i c a l  patients.  one  disulfide  controls.  cysteine  pallidus  in this  autopsied In  mixed  of  of cysteine  chelator, pallidus  those  converts  the  syndrome.  glutathione-cysteine  2 SD a b o v e  t h e enzyme  as  abnormalities  Hallervorden-Spatz  of decreased a c t i v i t y  serve  content  with  i n the globus  accumulates  neurochemical  of  in  iron  excesneuronal  Hallervor-  -  I)  First  described  syndrome  (HSS)  symptoms cular  is  sias  (Pooling  also  present  The  e t a l . 1974; with  (Hallervorden  with  onset  tures  after  (GP)  t h e main  (Pooling  diffuse  axonal  swellings  localised  (both  substantia  innominata,  An  the pars  perhaps  of  GP,  neurons  1985).  one  of  patient  The d i s e a s e an a v e r a g e  i n the  of  globus  are reduced  non-nucleated  others  throughout.  and r e t i c u l a t a ) ,  gives  Loss rise  brain  degeneration  (Dooling  et  al.  involvement locus of  in  struc-  While the autopsied  had n e u r o n a l  probably  a l .  are scattered  and c e r e b e l l u m .  nigra  and  1974; of the  coeruleus,  neurons  i n the  to the parkinson-  patients.  neuropathological  the f e r r i c  the  compacta  hippocampus,  s e e n i n some  interesting  to  et  although  i n death  or oval  axons  (1985)  p a t i e n t s can  o r second decade  e t a l . 1985) have r e p o r t e d  compacta o f t h e s u b s t a n t i a symptoms  a l .  hyperkine-  feature.  a r e seen  area,  can a l s o o c c u r .  et  only  and round  of  e t a l . 1979).  changes  this  mus-  et a l . 1985).  (Elejalde  as s w o l l e n  areas  Jankovic  nigra  In  postures,  (Jankovic  and c u l m i n a t e s  i s observed,  by Perry  substantia  ian  course  The  In a d d i t i o n ,  et a l . 1974),  (Jankovic  disorder.  types  in the f i r s t  neuropathological  brain  studied  V a k i l i e t a l . 1977;  pars  reported  identifiable  of other  a patient  Pooling  et a l . 1974).  gliosis  ('spheroids')  Degeneration of  downhill  and o t h e r  are a prominent  t h e a p p e a r a n c e o f symptoms  autopsy,  number,  et a l . 1922;  by d y s t o n i c  disease  i s usually  Hallervorden-Spatz  neurological  et a l . 1977).  Parkinson's  o f symptoms  a relentless  pallidus  choreoathetosis  of  a t a g e 55 h a s b e e n  a l . 1922),  characterized  Vakiii  symptoms  et  recessive  i n d i c a t i v e of dementia  life  11 y e a r s  autosomal  extrapyramidal,  age o f o n s e t  follows  rare  INTRODUCTION  (Hallervorden  or s p a s t i c i t y ,  changes  At  a  are mainly  rigidity  Mental  i n 1922  166 -  form  (Dooling  finding et  is  a l . 1974;  the accumulation Vakili  et  of  iron,  a l . 1977),  in  -  the  affected  report 1420  brain  (Vakili  a mean  iron content  compare  normal  adult  appears  with  to  be e l e v a t e d  area a dark  ation  with  t h e naked  reportedly  of  the unique  extracellular  of  combination  550 t o  4-fold  color  et  neuroaxonal  Jankovic  iron retention  in  in the basal  copper  and  zinc  Non-protein et a l . 1983).  (Park  findings intra  and  et  al.  1975;  death,  the  diagno-  presentation, ganglia  of the  examin-  1979).  Before  of  on g r o s s  (Swaiman  GP  These  content  and e x c e s s i v e  the  et a l . 1985).  the c l i n i c a l  1979)  f o r GP  neuropathological  swellings  pigments  al.  iron  of  al.  one  between  content  increased  contents  on p o s t - m o r t e m  iron  the iron  The  in the cerebrospinal f l u i d  of  et  ug/g dry weight  Thus,  (Goldberg  contents  f o r 3 HSS p a t i e n t s .  1070  iron,  the GP,  (Goldberg  i s r e a d i l y observed to  GP  For  iron  report  i n HSS.  that  the  o f HSS c a n be s u s p e c t e d f r o m  evidence of high  of  HSS.  as h a v i n g  et a l . 1972).  to  in  die with  a second  In a d d i t i o n  of  e t a l . 1977;  who  ug/g dry weight  HSS i s b a s e d  deposition  Seitelberger sis  eye.  increased  diagnosis  2  brown  i r o n may be e l e v a t e d  The  while  (Volkl  -  2 patients  contents  human b r a i n  this  bound  lists  o f 1911  iron  gives  are  patients  ug/g dry w e i g h t ,  values  GP  of  e t a l . 1977)  and 2280  gives  areas  167  as w e l l  as d e t e r m i n e d  as  by  from  intra-  59 venous et  injection  a l . 1979;  computed aK  of  Fe  Dooling  tomography  et  a  narrow  a l . 1980).  may p r o v i d e  field-of-view  Demonstration  further  support  of  of  collimator lucency  (Elejalde  i n t h e GP  the diagnosis  (Dooling  by e_t  1980). There  oxamine,  i s as y e t a heavy  no e f f e c t i v e  metal  well-advanced  symptoms,  though  iron  al.  and  serum  1974).  L-DOPA  chelator,  for  was t e s t e d  but i t d i d not a l t e r  levels  decreased  The p a r k i n s o n i a n  and c a r b i d o p a  treatment  therapy,  3-fold  symptoms but t h i s  of  patients  for  HSS.  Desfer-  i n one p a t i e n t  with  the course of the disease,  even  during some  a year  with  the treatment  patients  response  respond  (Dooling  e_t  initially  to  i s not maintained  (Jankovic  -  et  al.  single  little  (Jankovic vanillic similar  et  The  al.  acid, to  known  of  subject  revealed  projection,  marked  with  one o f  This  the  those seen  in  is  that  one  cysteine  was  presenting  dopamine  (over  of  HSS  present  in  symptoms 95%)  dopamine  in  in  the  the  metabolites  dopamine.  Such  unknown.  in  the  as  areas  of  homo-  changes  are  disease.  The  GP,  mainly  limbic  reduction  is  A  nigrostri-  parallel  of  HSS.  were  accompanied by  idiopathic Parkinson's  prominent  diagnosis might  'accumulation of of  of  changes  cause of  well  as  the  the  marked  neuron-  accumulation  of  uncertain.  ly-confirmed  GP  whose  preservation  2 major  We had t h e o p p o r t u n i t y  changes  neurochemical  loss  relative  1985).  especially  the  studied,  b i o l o g i c a l b a s i s of  loss,  iron,  is  post-mortem  parkinsonian,  al  -  1985).  Very  atal  168  be  of  to  study  HSS.  present  2 patients  We  attempted  in  these  glutathione-cysteine  patient,  dioxygenase  and  reduction  i n both  c h a n g e s may c a u s e t h e n e u r o n a l  mixed  of  patients. loss  the  2  who to  died with examine  subjects.  disulfide, activity  We p r o p o s e  the We  the  the  neurochemical  found  and o f  of  s e e n i n t h e GP o f  a pathological-  localised  cystine, catabolic  hypothesis  patients  with  in  the  enzyme  that HSS.  these  -  II)  The HSS  2 patients  Case  Neurol. to  1  h a s been  Jankovic  the courtesy  et a l . (1985).  abnormalities  only  to  as w e l l  without  acid  abnormalities  36  of  system w i t h  putamen,  a l . Ann.  2,  whose  of  Massachusetts,  E.  Bird  axons,  loss,  et  was made  and 2 .  available  i s described  o f HSS C a s e 1  iron  et  (Perry  brain  examination  neuronal  deposition)  a l . 1985),  and i n c l u d e d t h e s u b s t a n t i a  as  controls  neurological  atrophy  i n the brain  acids  in  that  cystine,  of  were  in  using  sulfur  reveal-  t h e same nigra  in  confined abnor-  and c a u d a t e  activity  t h e method  of  from  acids,  patients  J.  (Table  These mixed were  h a d no  study.  included that  regions  died  Y-aminobutyric  were used f o r  i n the striatum  of the  con-  using  the  1),  analyses  included  disulfide.  Contents  quantified  by  a  HPLC  (Perry et a l . 1984).  was a n a l y s e d  Misra  were  who  T h e OPCA p a t i e n t s  performed  Appendix  subjects  some  amino  that  brain  from  but  and g l u t a t h i o n e - c y s t e i n e  and i t s m e t a b o l i t e s  dioxygenase  mostly  (OPCA).  areas  several  detailed  were  diseases,  electrochemical detection  Cysteine  J  HSS C a s e  i n the metabolism  system  f o r GABA,  dopamine  1  Appendix  Dr.  measurements  amino  chromatographic those  as HSS C a s e s  in  olivopontocerebellar  Neurochemical  of  used  any e v i d e n c e  of  here  detail  i n HSS C a s e 2 ( J a n k o v i c  (GABA), o r o f i r o n ,  tents  to  as t h e G P . brains  died with  known  in  w e r e more w i d e s p r e a d ,  Autopsied  who  while  be r e f e r r e d  while of  EXPERIMENTS  Neuropathological  (swollen  the GP,  malities nucleus  HSS w i l l  1985),  -  OUTLINE OF  described  18:482-489,  us t h r o u g h  ed  with  169  (1979),  i n the GP,  i n which  frontal  amounts  of  cortex  and  radioactive  35 cysteine  sulfinate  quantified.  Assays  of  t h e GP w e r e  2,  only  2  formed were  studied  areas  of  from  done  L-[  blind  for cysteine  t h e GP w e r e  S]-cysteine  by  in t r i p l i c a t e . dioxygenase  analysed,  brain For  activity,  due t o  homogenates  HSS C a s e while  insufficient  1,  3  are areas  f o r HSS quantities  Case of  -  material To  being assess  dioxygenase calculated weight assumed in  Although  possible  (Volkl  t h e GP of  ammonium  form,  GP  from  sulfate  mixture  as a c o f a c t o r  (1975). A,  than  the cortex  choline  could  concentrations  Lowry e t a l . ( 1 9 5 1 ) ,  as  iron  activity  in  form  the c a l c u l a t e d  was c h o s e n  here  present  mM (we  enzyme  iron  are thought  was  content/dry  t o be 1 . 4 3  measured  adding  cysteine  the normal  value.  t o be m a i n l y as t h e  i n t h e normal  of  of  exogenous reaction  dioxygenase.  activity  i s measured  times  was a l r e a d y  and o c c i p i t a l  method,  We t h e n  on  GP h o m o g e n a t e s  e t a l . 1977)  is in  was d e t e r m i n e d cortices,  using  incubated  with  n o t be a n a l y s e d  in brain  using bovine  in  because  homogenates  of  X-100 Fonnum  [^C]-acetyl-coenzyme formed.  Brain  insufficient  areas  quantities  subjects.  were  serum albumin  0.5% Triton  t h e method  the acetylcholine  t i s s u e w e r e a v a i l a b l e f r o m b o t h HSS a n d c o n t r o l Protein  of  this  sulfate  content  i n normal  HSS p a t i e n t s  for cysteine  frontal  and r a d i o a c t i v i t y  other of  In  compound  iron  expressed  subjects,  a n d 10  ammonium  acetyl transferase of  Vakili  5 control 5,  iron  were  80%water).  2.5,  ferrous  of  (which  i n t h e GP o f  iron since this  homogenates  values  contains  form of  Choline  increased  e t a l . 1972;  iron deposits  ferric  of  effect  the concentration  published  brain)  homogenates  ferrous  the  the  activity,  that  -  available.  from  of  170  measured  by t h e  as s t a n d a r d .  \  method  -  .171  III)  Quantitative showed mixed  an e l e v a t i o n disulfide  1).  The  more  than  2  nucleus,  brain  freezing  of  the GP, of  HSS C a s e  butes  to  jected  for  mean  1  control  rapid  elevated interval  levels  neither  analysis  glutathione antemortem  rapidly-frozen (Perry  nucleus,  of  to  that  interval  A  after  high  brain  of  contrideath-to-  cystine  mixed  of  death, one  also  long  of  death-to-  products,  b i o p s i e s o f human  not  measurements  proteins  artifactually  cau-  were  the  because,  death.  were  regions  compounds  breakdown  after  meas-  disulfide are sub-  et a l . 1971). and f r e e z i n g  placed at 4°C u n t i l  patient's  elevation  its  1  (Table  brain  dentate  these  frozen  Case  areas  death-to-freezing  Breakdown  in  other  is critical  measured  result  these  was c o m p a r a b l e  HSS C a s e 2 ' s d e a t h  of  in this  to  in  In  of  HSS  t h e putamen  c y s t i n e nor g l u t a t h i o n e - c y s t e i n e  d e t e c t a b l e when  between  The  It  in  cortices,  from  glutathione-cysteine  compounds  are r a p i d l y  a l . 1981).  can t h e r e f o r e  In f a c t ,  interval  that  and o f  values.  which  autolysis  et  cystine  h,  samples  extent,  contents  brains.  in brains  and t h e b o d y h a d n o t b e e n  contents  control  was 3 . 5  h,  ible  both  and c e r e b e l l a r  from  (Perry  t o amino a c i d  The  the  of  controls.  undergoes  normally  amounts  cystine  a lesser  different  is cysteine  are  the  the brain  of  nigra),  which  urements.  of  and, to  occipital  c y s t i n e be made o n l y  freezing  analyses  and s u b s t a n t i a  intervals  glutathione  RESULTS  the contents  above  (frontal,  significantly the  in  SD  acid  of  elevations  analysed date  amino  -  brain  had been  of  4 h after  his brain death.  so e x t e n s i v e  of c y s t i n e or g l u t a t h i o n e - c y s t e i n e  c o u l d n o t be a s c e r t a i n e d a c c u r a t e l y .  was 7  Autolysis  that  any p o s s -  mixed  disulfide  -172  Table  1.  -  B r a i n c y s t i n e and g l u t a t h i o n e - c y s t e i n e Hallervorden-Spatz Caudate  Putamen  Controls  0.05 ± 0.04 (41)  Cystine 0.05 ± 0.04 (25)  Case 1  HSS C a s e 1  mixed  0.41 ± 0.15  0.43 ± 0.17 (39)  (19) 1.02  0.65  0.50  0.16  disulfide  0.34 ± 0.08 (23)  0.32 ± 0.14 (37)  0.12 ± 0 . 0 8 (44)  0.10 ± 0.07 (20) 0.45  Glutathione-cysteine Controls  Substantia nigra  Globus Pallidus  0.14  0.08  disulfide in  syndrome  Patients  HSS  mixed  0.68  V a l u e s a r e mean ± SD i n y m o l / g w e t w e i g h t . The number o f c o n t r o l s a m p l e s i s shown i n p a r e n t h e s e s . The d e a t h - t o - f r e e z i n g interval f o r e v e r y . c o n t r o l s p e c i m e n was l e s s t h a n 4 . 0 h , w h i l e t h a t f o r HSS C a s e 1 was 3 . 5 h . HSS v a l u e s i n c r e a s e d > 2 SD o f c o n t r o l s a r e underlined.  The  only  patients nigra  amino  was a m a r k e d  (Table  (Perry the  other  et  2).  in  Table  2  2  rise  rapidly  a stable  intervals  were  found  i n the brains  i n GABA c o n t e n t  contents  reaching  death-to-freezing  shown  abnormality  reduction  GABA  a l • 1981),  acid  hours  of or  2  t h e 2 HSS  i n t h e GP a n d t h e  substantia  in  maximum  the  of  human  after  patients  greater,  the  brains  about  and a l l values  after  death  2 hours. of  Since  the  controls  be  reliably  could  compared. The both  and  of  HSS p a t i e n t s .  subjects of  activity  was 1 . 7 7  0 . 8 2 (mean 0.92).  of  cysteine While  dioxygenase  t h e mean  enzyme a c t i v i t y  ± 0 . 4 0 ymol/h/mg 3 separate  HSS C a s e  2  areas  was f o u n d  protein, of  had c y s t e i n e  to  i n t h e GP  HSS C a s e  t h e GP w h i c h dioxygenase  be l o w i n t h e GP  1  of  25  h a d a mean  had v a l u e s  activity  of  of 0.97  of  control activity  0.44,  1.11  pmol/h/mg  -  Table  2.  173  B r a i n GABA c o n t e n t  Patients  Caudate  Controls  2.90 ± 0.81 (32)  -  in Hallervorden-Spatz  Putamen  2.91  syndrome  Globus Pallidus  ± 0.87 (19)  7.32  ± (16)  Substantia nigra  1.60  6.05 ± 1.36 (28)  HSS  Case 1  4.83  5.28  2.55  0.42  HSS  Case 2  1.33  2.79  2.50  1.95  V a l u e s (mean ± c o n t r o l s shown underlined.  SD) a r e i n y m o l / g w e t w e i g h t , w i t h t h e number of in parentheses. HSS v a l u e s d e c r e a s e d > 2 SD a r e  protein,  w h i c h was t h e mean o f  1.16).  Thus,  creased  by  other  more  hand,  men o f  both  ±  enzyme than  2  cysteine  brains  SD),  SD  from  were w i t h i n  had  and  activity  an  linear  In  in  which  the  GP  mean  2 SD o f  of  the  of  for of  analysed  HSS  the  GP  the  frontal  controls  analysis  GP  both  of  (Figure  death-to-freezing  regression  enzyme a c t i v i t y w i t h  areas  activities  average  changes of  experiments  in  the  dioxygenase  patients  Control (mean  the  2 separate  of  to  10  times  was  de-  controls.  On  the  cortex  of  GP,  and  puta-  1)..  interval these  the  and  patients  of  brains  increasing death-to-freezing  up  (0.77  6.5  ±  7.2  h  revealed  no  intervals.  normal  iron  content  (as  2+ Fe  )  teine  was  added  deoxygenase  Choline duced  in  homogenates  was o b s e r v e d  acetyl transferase  the  cerebral  3).  Contents  were  comparable  cortex  o f "dopamine  death-to-freezing Only t i s s u e s from obtained  to  to  those  and in  intervals HSS  (Figure  of  HSS  its  significant  was  Case  slightly 1,  as  metabolites  a  small  for  the  However,  inhibition  of  cys-  2).  activity  Case 1 were  f r o m HSS C a s e 2 .  no  group brain  analysed this  compared  in of  the  because  insignificantly to  controls  striatum  control  were  latter  but  of  case  1  the  (Table  4).  i n s u f f i c i e n t material  was  short  s u b j e c t had been  in  (Table  whom  equally  adults  HSS  re-  studied  - 174  -  Frontal Cortex  H  V Ol «** o C  Globus PalUdus  3-j  Q.  •^-E  2  «i — c o — e  • • • •  o  C o n t r o l s H S 3 C o n t r o l s (18) patients (13)  1.  Figure ates  from  patients. ± 2 SD  for  Enzyme a c t i v i t y 3  regions  Horizontal the  of  of  autopsied  bars  controls.  t h e o p e n s q u a r e HSS  Case  cysteine  indicate The  2.  solid  HSS patients  dioxygenase  brain means, square  of  in  control  and  Controls (25)  HSS patients  tissue  homogen-  adults  and  vertical  indicates  HSS  bars Case  HSS  indicate 1,  and  -  100  175  -  -r-  80  --  60  --  (/) >> 1— o •r— s_ >  •f—'  c  o  ym  u u ncu o N  C  CU r O c -a •r-~ cu VI 0) Ol cu c Sra Q . JC X c_> cu  40  20  ±  0  | 1  — f  1  2.5  1  5  10  M u l t i p l e s o f c o n t r o l amount i r o n i n homogenates Figure  2.  activity  as  Each p o i n t mean  ±  points  SEM.  % change a  function  represents Analysis  in  cysteine  of  increasing  5 analyses. of  variance  are s t a t i s t i c a l l y s i g n i f i c a n t  of  dioxygenase brain  Vertical shows  enzyme  iron bars  that  from each  content. shown  none other.  of  are the  - 176 (Jankovic low  et a l .  1985), and d o p a m i n e and HVA c o n t e n t s w e r e f o u n d  i n h i s caudate  nucleus  and putamen  (dopamine  contents  i n both  t o be  very  areas  were  r e d u c e d b y more t h a n 9E%).  Table  3.  Choline  acetyl transferase  Hallervorden-Spatz Patients  Frontal  Controls  HSS  1.07  Case 1  activity  in  disease  cortex  Occipital  ± 0.20  0.64  cortex  ±  0.28  (16)  (12)  0.79  0.51  Enzyme a c t i v i t y i s e x p r e s s e d a s nmol/10 min/mg p r o t e i n . Cont r o l v a l u e s a r e mean ± S D , w i t h number o f c o n t r o l s i n d i c a t e d in parentheses.  Table  4.  Dopamine  Patients  Caudate Dopamine  Controls  and m e t a b o l i t e s  (4)  HSS C a s e 1  3.34  ±0.68 2.84  .  i n t h e s t r i a t u m o f HSS C a s e 1  nucleus  .DOPAC 0.70  ±0.70 0.73  Putamen HVA 5.20  Dopamine 5.22  ±1.96  ±2.13  4.31  6.90  C o n c e n t r a t i o n s o f dopamine and i t s m e t a b o l i t e s yg/g wet w e i g h t . C o n t r o l v a l u e s a r e mean ± S D .  are  DOPAC 0.14  ±0.05  HVA 6.05  ±1.42  0.74  expressed  13.0  in  -  IV)  Little  is  HSS  except  that  the  disease.  iron  salts  reactive lipids  are  1982;  strated  that  persistent  increase  lipid can  injections  were  and  the  serve to  life,  resulting disulfide  analysed. 2,  The  ing  of same  of  this  is  being  the and  al.  rat  of GP  and  might  freezing  the  contents  glutathione have his  whereby  of  membrane  undergo  decom-  and c e l l  (1984)  death  have  demon-  cortex  which  putamen  can  of  enzymes  cause  be  et GP of  al. of  pre-  been  present  Case  1  seems during  and  the  tissue  was  GP  of  HSS  prevented  our  brain the  death  thiol-  It  cystine  in  1.  reductases  1983).  both  mixed  Case  s u c h as  HSS  autopsied  after  HSS  disulfide  when  brain  since  a-tocopherol.  in v i v o ,  in  acids  of  with  glutathione-cysteine  (Schneider  accumulated  died  process  cerebral  nucleotide-dependent  high  a  integrity,  into  and  have  unstable,  et  basis  interesting,  fatty  the a n t i o x i d a n t  form  who  peroxidation,  cystine  reduced  and  GP  membrane  lipid  is cysteine,  had  the  Triggs  in  biological  peroxidation,  chloride  abnormally  in  of  or  patients  of  latter,  1984).  abnormality  delay  possible increase.  of  the  increased  d i s e a s e might  (dimethylcysteine) used  The  and  of  polyunsaturated  animals with  cysteine  c y s t e i n e were  agent  the  significant  in the  GP  lipid  disruption  contents  the  If  lamine  of  al.  changes  content  ferrous  cysteine in  although  demonstrating  ogy  of  cystine  keep c y s t i n e  l i k e l y that  Case  et  transhydrogenases  highly  mixed  the  to  the  s i g n i f i c a n t l y elevated  The r e d u c e d f o r m o f disulfide  iron  oxidize  lead  by p r e t r e a t i n g  disulfide  in  in  inducers  Halliwell  that  neurochemical  peroxides.  seizures  We f o u n d  the  -  DISCUSSION  retained  potent  This  (Slater  is  radicals  form  position.  about  iron  The  free  to  vented  known  177  i n the  in  HSS  during  be e x p l a i n e d . in  chemical  treatment  of  life,  Firstly, structure.  heavy  metal  some o f  cysteine  the  neuropathol-  resembles  Penicillamine is poisoning.  Since  penicila  the  chelatstruc-  -  tural  elements  present The  p e n i c i l l a m i n e which  in cysteine,  high  r e s u l t i n g complexes,  teristic  excessive  penicillamine of  of  cysteine  other  can a l s o  those  of  to  i r o n , were e l e v a t e d  caudate  as  important  HSS p a t i e n t s  rats  (Karlsen  ized  brain  aspect  i s that  t o x i c when p r e s e n t  nigra iron  such  with  with  HSS.  as c o p p e r ,  HSS.  of  HSS  were  iron.  to the charac-  of  is significant  contents  Since  elevation metal  that  ions  Goldberg  in  addition  I n t h e same  article,  in non-pallidal patients  were  preferentially  cysteine,  accumulation  although  (Olney  atrophy.  In  radicals,  vitro,  areas  such  reduced  when  distributed  (Saez  Malondialdehyde  and  a single  causing  them  It to  acid,  can produce  autoxidation  and h y d r o x y  et a l . 1982).  amino  i n t h e GP c a n be  i n j e c t i o n of cysteine  a l . 1971)  undergoes  life  radicals  release  general-  to  generate  detectable  can a l s o d i s r u p t  to  the  glutathione,  by  integrATP  and  (Saez e t a l . 1 9 8 2 ) .  production  have r e p o r t e d  somes was s t i m u l a t e d and c y s t e i n e became  thiyl  during  an e n d o g e n o u s  et  cysteine  including  hepatocytes,  dehydrogenase  dialdehyde  cysteine  or mice  isolated  (1983)  of  e t a l . 1981)  spin resonance  al.  it  patients  Thus,  electron  lactate  patients  are also  chelate  c a l c i u m and m a n g a n e s e ,  amounts.  free  of  zinc,  iron  to  i n the accumulation  in high  reactive  ity  though  in  act  could lead  heavy metals  regard,  i n t h e GP o f  controls,  seen  result  copper,  and s u b s t a n t i a  the GP,  iron  this  might  ions  i n the GP.  Another of  of  that  allow c h e l a t i o n of metal  within  also  showed  -  the l a t t e r  other  In  that  (1979)  nucleus  to  localised  chelate  reported  et a l .  compared  trapped  iron.  a l . (1979)  as  if  of  accumulation  et  Goldberg  levels  i n t h e GP s h o u l d  than  178  that  i s an i n d e x  malondialdehyde  by f e r r o u s a very  production  of  iron  potent  or  inducer  lipid  production  cysteine. of  lipid  induced by t h e c o m b i n a t i o n  those r e s u l t i n g from the i n d i v i d u a l  peroxidation.  in rat liver  The c o m b i n a t i o n peroxidation,  g r o s s l y exceeded  actions of these  Searle  agents.  et  microof  iron  and m a l o n t h e sum o f  -179  The  high  cysteine  content  accumulation,  would  brain  p o s s i b l y by  region,  be n e u r o n a l  death  Cysteine olic et the  route ible the  1983).  2  HSS  cal  deficient  cysteine  of  them  is  (Misra  inhibit  gested  that  et-al.  1974;  addition  the  normal  GP,  appears  cysteine et  HSS,  Although GP  of  ion  was  no  that  form)  is  up  the  to  cysteine at  In  the  cystine  would  in  this  result  could  be  in  the  low  in  1983), Case  the  GP  of  the  major  is  poss-  it  1  metab-  (Schneider  represents  al. HSS  GP.  tissues  excess  another  important  dioxygenase  requires  is  that  resulted  from  iron  for  the  dioxygenase disorder  accumulates  and  ion  iron  staining  presume  that  in  abnormal  (if low  it  the of  GP  It  there  is  present  be a  disease cystinosis as  cystine  of  the the  therefore  activity  d e f i c i e n c y might  is iron  with  enzyme.  enzyme  sug-  there  content  mainly  a  (Dooling  iron  is  as  content  to  the  deposits  whether  form  in which  recessive  ferrous  homogenates  of  content  learn  ferric  expected  inhibition  neurochemi-  the  forms  in  the  to  histological of  ferric  both  excessive  reasonable  times  autosomal  are  be  responsible  look  et of  iron  measured  10  elevated  itself  brain  and  and  was to  enzyme  is mainly  it  significant  Rather,  metabolism. 1983),  at  to  conventional  HSS  ferrous  activity  enzyme  conceivable  the  ferrous  the  it  is  between  useful  al.  it  other  (Schneider  iron  i n the  enzymes  and  this  with  dioxygenase.  Cysteine  in the  enzyme  of  in  1977),  disease.  is  of  this  action  al.  unlikely  i n HSS  brain  of  The  s w e l l i n g s seen  in  et  there  ferrous  It  iron  radicals.  taurine  deficiency  and  free  coupled  peroxidation  3 sequential  metabolised  enzyme.  Vakili  When of  1979),  of  1,  lipid  of  the  GP  Case  first  cysteine  the  HSS  neuroaxonal  accumulation of  of  increase  activity  primary.  the  equilibrium  deposits.  der  the  dioxygenase in  the  to  is  GP  generation  the  Since  activity  abnormalities  might  GP  We f o u n d  patients.  cofactor  the  cysteine  that the cysteine  either  an  is  by w h i c h c y s t e i n e  If  the  dioxygenase  the  cetainly  and p e r h a p s  pathway.from  al.  almost  in  -  in  in the  primary. defect  in  (Schnei-  crystals  in  -180  the  kidney,  unaware in  conjunctiva,  of  any r e p o r t s  cystinosis.  intracellular in cystine  are  probably from  1983).  degradation.  cellular  presence i n t h e GP  dioxygenase tine,  putamen  1,  contents  from  this  dioxygenase  Case  were  was  showed also  immediately  pencillate  of  ent  tissue  i n HSS,  being  to  be  a  cystine  mixed  disorder  than  and t h e r e f o r e  fail-  separ-  (Schneider in  deficiency  is cysteine,  the  in cystinosis  disulfide  as t h e  of  an e n z y m i c  levels  structures  it  cystine  in  the  the  normal  et  al.  excessive  in  cysteine  rather  than  cys-  an  glutathione-cysteine  putamen  this  amino  of  HSS  brain  acid  of  mixed  Yet,  the  and  cysteine  The  probable  a n a l y s i s was p e r f o r m e d This  neuroaxonal  putamen  1.  region.  on a  c o u l d have been  swellings  histologically.  area  Case  alterations,  t o t h e GP.  where  observed  available,  in  adjacent  putamen,  were  and  no m o r p h o l o g i c a l  inconsistency i s that  staining  changes  lysosomes,  as w e l l  that  elevated  patient  bundles  or neuropathological  rather  membrane  1,  that  seen  p o r t i o n o f putamen  iron-positive  within  or  We a r e  in brain.  is  activity  reason f o r t h i s  HSS  suggests  it  cystine,  the elevated  storage  and l e u c o c y t e s .  believed  glutathione-cysteine  of  activity,  Table  disulfide  Also,  nodes  symptoms is  of  enzymes  of  which accumulates  In  cystinosis  associated with  The  lymph  neurological  However,  most  quantities  of  marrow,  compartmentalisation  ure  ated  bone  -  Because  more  and  of  distant  the  diffuse  insuffici-  from  the  GP  c o u l d n o t be a n a l y s e d . The  GABA  HSS  patients  This  region  reduction is  found  probably  contains  the  HSS  Case  result  GABAergic  n i g r a and i s a l s o t h e t e r m i n u s striatum.  i n both  t h e GP of  cell  1  (Table  2)  is  marked  bodies  f o r GABAergic  The i n c r e a s e d GABA c o n t e n t unexplained.  and s u b s t a n t i a  of  loss  of  neurons  projecting neurons  with  the caudate GABA  content  nigra  to  the  cell  nucleus was  of  in  our  the  GP.  substantia  bodies  in the  and putamen  normal  2  in  of  other  -  regions  of  this  and d e n t a t e HSS tion did  1,  with  striatal  dopamine  not  resemble  HSS  Case  2  symptoms  was  striatum, (Jankovic  (frontal,  early onset  in  and  brain  -  occipital  and  cerebellar  cortex,  nucleus).  Case  age)  the  patient's  181  et  al.  substantia  2,  suggestive  reported along  Case  or  to  with 1985).  nigra.  have  its who of  and  death  at  metabolites. had  an  deficiency  extensive  loss  of  HSS  1  showed  Case  age  In  this  unusually  parkinsonism  marked  the  of  19,  late  onset et  dopamine in  had  respect,  (Jankovic  neurons no  of  the  no HSS  (55  al • and  Case  years  1985). HVA  substantia  neuropathological  reduc1 of HSS  in  the nigra  changes  in  -  V)  Following ing  the etiology  A primary in  of  enzymic  accumulation  High  of  CONCLUSIONS  described in this  the autosomal  defect  recessive  in cysteine  cysteine,  -  which  combination  generating neuronal It  of  free  disorder  dioxygenase then  binds  a hypothesis HSS may be  activity iron  cysteine  radicals  and  that  iron,  result  however, in  lipid  of  lesion,  the  be r e c o g n i z e d disease  that  2 patients with  s i m i l a r neurochemical  suggest  appear,  antioxidant  drugs  further such  changes  t h e above  in  excessive  more  approaches  deterioration  might  o-tocopherol  in individuals genetically at r i s k  of  chelator  the disease,  Such  or  treatment  desferroxamine  amounts.  lethal and  although  one r e p o r t  t h e c o u r s e o f HSS i n a p a t i e n t  1974).  with  the  autopsy initial  mixture, subsequent  if  need  For  also  suggested  obtained  t o be  examined  our f i n d i n g s  i n s t a n c e , when  which  prevent  useful that  well-advanced  symptoms  by t r e a t m e n t  f o r the disease. be  many  pathological  confirmed,  B-carotene,  also  after  i s based on d a t a  be p r e v e n t e d  might  might  at  HSS b r a i n s  t o HSS.  symptoms  metal  found  Nevertheless,  free  alter  a  results  individually.  peroxidation  hypothesis  detoxify  stages  suggested.  i n t h e GP  becomes  necessarily reflect  defects.  as  radicals.  the  H S S , and t h a t  possible therapeutic  initially  that  may n o t  and f u r t h e r m o r e ,  from o n l y  heavy  regard-  death w i t h i n the GP. should  years  al.  work,  l e v e l s o f c y s t e i n e o r i r o n c a n be n e u r o t o x i c when p r e s e n t  The  for  from the f i n d i n g s  182  with  can serve  to  the  of  onset  The u s e o f t h e in its  disease  the  initial  use d i d n o t (Dooling  et  -  VI)  183 -  REFERENCES  D o o l i n g E C , R i c h a r d s o n E P , D a v i s K R , Computed t o m o g r a p h y Spatz d i s e a s e , N e u r o l . 30:1128-1130, 1980.  in Hallervorden-  D o o l i n g E C , S c h o e n e WC, R i c h a r d s o n E P , N e u r o l . 3 0 : 7 0 - 8 3 , 1974.  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