UBC Theses and Dissertations

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UBC Theses and Dissertations

Specific aspects of neurodegenerative disease Biro, Andrew J. 1989

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S P E C I F I C A S P E C T S O F N E U R O D E G E N E R A T I V E D I S E A S E by ANDREW J . BIRO B.Sc, Simon F r a s e r U n i v e r s i t y , 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES PHARMACOLOGY AND THERAPEUTICS We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1989 (c) Andrew J . B i r o , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T This t h e s i s i s broken i n t o four chapters. The f i r s t two chapters summarize two separate l i n e s of i n v e s t i g a t i o n i n t o the r o l e of a putative neurotoxin i n the pathogenesis of Huntington's Disease (HD). The t h i r d chapter o u t l i n e s an i n v e s t i g a t i o n of the putative r o l e of beta-N-methylamino-L-alanine (BMAA) i n the pathogenesis of amyotrophic l a t e r a l s c l e r o s i s (ALS), while the f i n a l chapter d e t a i l s a post-mortem i n v e s t i g a t i o n of the contents of biogenic amines and amino acids i n the brain of a man who died of a f a m i l i a l form of parkinsonism. Chapter I i s a d e s c r i p t i o n of a chromatographic technique developed to i s o l a t e q u i n o l i n i c a c i d (QA), an endogenous compound implicated i n the pathogenesis of HD, from deproteinized human sera. A cati o n exchange column was used to s e l e c t i v e l y i s o l a t e QA, which was eluted with 10 mM HCl. The eluted f r a c t i o n s were analyzed by uv spectrometry to i s o l a t e and quantify QA. Once the f r a c t i o n s corresponding the e l u t i o n of authentic QA were i s o l a t e d , concentrated and the excess HCl removed, the f r a c t i o n s were added to growing f e t a l r a t s t r i a t a l explant c u l t u r e s as an assay of ne u r o t o x i c i t y . Since HD involves the s e l e c t i v e degeneration of GABAergic neurons i n the striatum, the a c t i v i t y of glutamic a c i d decarboxylase, the f i n a l enzyme i n the synthesis of GABA, was used to determine the v i a b i l i t y of the c u l t u r e s . Unfortunately, the method was confounded by the contamination of a l l e f f l u e n t s by compounds o r i g i n a t i n g from the c a t i o n exchange i i Abstract (Continued) r e s i n , which were discovered to be neurotoxic to the s t r i a t a l c u l t u r e s , and as a r e s u l t the i n v e s t i g a t i o n had to be abandoned. Chapter II describes an i n v e s t i g a t i o n designed to fu r t h e r c h a r a c t e r i z e the nature of neurotox i c i t y observed i n the sera obtained from p a t i e n t s with HD (Perry et a l . 1987). Compounds with the capacity to s e l e c t i v e l y stimulate neurons at the N-methyl-D-aspartate (NMDA) receptor have been implicated i n a v a r i e t y of neurodegenerative disorders, i n c l u d i n g HD. Sel e c t i v e antagonists at the NMDA receptor have been shown to protect neurons from the degenerative e f f e c t s of such " e x c i t o t o x i n s " . The i n v e s t i g a t i o n described used MK-801, a potent non-competitive NMDA antagonist, i n an attempt to protect f e t a l r a t s t r i a t a l c u l t u r e s from the neurodegenerative e f f e c t s of the sera obtained from HD pa t i e n t s . The r e s u l t s obtained were equivocal. No evidence was obtained to support a r o l e of the NMDA receptor i n the mediation of the neur o t o x i c i t y , and i n addition the neurodegenerative e f f e c t s of HD sera were not reproduced i n the present i n v e s t i g a t i o n . A v a r i e t y of possible explanations f o r the apparent discrepancy are suggested. Chapter I I I describes an experiment intended to produce an animal model of ALS based on the observations by Spencer e t a l . 1987 that chronic o r a l administration of BMAA i n monkeys produced the h i s t o l o g i c a l and behavioural c h a r a c t e r i s t i c s of t h i s disease. In the present i n v e s t i g a t i o n s y n t hetic D,L-BMAA was given by gavage to mice over an eleven week period. Since BMAA i s known to act at the NMDA receptor, a subset of the mice were also given MK-801 i n an e f f o r t t o protect them i i i Abstract (Continued) from any del e t e r i o u s e f f e c t s based on the a c t i o n of BMAA at t h i s receptor. The animals were s a c r i f i c e d at the end of the experiment, and biochemical analyses were performed on the s t r i a t a and c o r t i c e s of the animals. In add i t i o n , neuropathological studies were performed on the s p i n a l cords, basal ganglia and r e l a t e d s t r u c t u r e s . The r e s u l t s i n d i c a t e d no biochemical or neuropathological abnormality as a r e s u l t of BMAA administration. Chapter IV describes a post-mortem i n v e s t i g a t i o n of a man who was a member of a we l l described pedigree which c a r r i e s an autosomal dominant form of parkinsonism. The object of the i n v e s t i g a t i o n was to determine post-mortem l e v e l s of dopamine, noradrenaline, serotonin and t h e i r metabolites, i n addi t i o n to amino acids i n various regions of br a i n . Although c o n f l i c t i n g evidence was obtained during l i f e , neuropathological f i n d i n g s and the present neurochemical analyses confirm the degeneration of the n i g r o s t r i a t a l dopaminergic t r a c t , c h a r a c t e r i s t i c of parkinsonism, i n t h i s man. iv T A B L E O F C O N T E N T S A b s t r a c t i i L i s t o f T a b l e s v i i i L i s t o f F i g u r e s x Acknowledgements x i A b b r e v i a t i o n s x i i D e d i c a t i o n xv CHAPTER 1 1 ATTEMPTED ISOLATION OF A PUTATIVE CIRCULATING NEUROTOXIN IN HUNTINGTON'S DISEASE I n t r o d u c t i o n 1 O b j e c t i v e 9 H y p o t h e s i s 9 E x p e r i m e n t a l O u t l i n e 9 Methods 10 C o l l e c t i o n o f S e r a 10 P r e p a r a t i o n o f S e r a 11 Chromatography 12 c u l t u r e o f F e t a l Rat S t r i a t a l E x p l a n t s 14 D e t e r m i n a t i o n o f GAD Enzyme A c t i v i t y . . . 16 R e s u l t s 17 D i s c u s s i o n 21 v T a b l e o f C o n t e n t s (Continued) CHAPTER I I 23 A TRIAL OF THE NON-COMPETITIVE NMDA ANTAGONIST MK-801 IN THE PROTECTION OF FETAL RAT STRIATAL EXPLANTS FROM A PUTATIVE CIRCULATING NEUROTOXIN IN HUNTINGTON'S DISEASE I n t r o d u c t i o n 23 O b j e c t i v e 25 H y p o t h e s i s 25 E x p e r i m e n t a l O u t l i n e 26 Methods 26 R e s u l t s 27 D i s c u s s i o n 32 R e f e r e n c e s 35 CHAPTER I I I 42 INVESTIGATION OF THE PUTATIVE ROLE OF BETA-N-METHYLAMINO-L-ALANINE (BMAA) IN THE PATHOGENESIS OF AMYOTROPHIC LATERAL SCLEROSIS AND THE PARKINSON-DEMENTIA SYNDROME OF GUAM I n t r o d u c t i o n 42 O b j e c t i v e 45 H y p o t h e s i s 45 E x p e r i m e n t a l O u t l i n e 46 Methods 47 S y n t h e s i s o f D,L-BMAA 47 A d m i n i s t r a t i o n o f D,L-BMAA 48 A d m i n i s t r a t i o n o f MK-801 49 A n a l y s i s o f B i o g e n i c Amines 49 A n a l y s i s o f Amino A c i d s 51 N e u r o p a t h o l o g i c a l S t u d i e s 52 R e s u l t s 52 D i s c u s s i o n 57 R e f e r e n c e s 59 v i Table of Contents (Continued) CHAPTER IV 62 A CASE OF HEREDITARY MENTAL DEPRESSION AND PARKINSONISM POST-MORTEM STUDY OF BIOGENIC AMINES AND AMINO ACIDS IN BRAIN Introduction 62 Case H i s t o r y 66 Objective 72 Methods 73 Results 74 Discussion 86 References 94 Appendix 99 v i i LIST OF TABLES T a b l e I GAD a c t i v i t i e s o f f e t a l s t r i a t a l c u l t u r e s exposed t o HD and c o n t r o l s e r a , and Dowex 50W column e f f l u e n t s 20 T a b l e I I GAD a c t i v i t i e s o f f e t a l r a t s t r i a t a l c u l t u r e s exposed t o HD o r c o n t r o l s e r a , w i t h o r w i t h o u t c o n c u r r e n t e x p o s u r e t o MX-801....28 T a b l e I I I S t r i a t a l c o n t e n t s o f dopamine and i t s m e t a b o l i t e s i n mice t r e a t e d f o r 11 weeks w i t h D,L-BMAA, w i t h o r w i t h o u t c o n c u r r e n t a d m i n i s t r a t i o n o f MK-801 54 T a b l e IV C o n t e n t s o f t a u r i n e (TAU), a s p a r t a t e (ASP), g l u t a m a t e (GLU), g l u t a m i n e (GLN), g l y c i n e (GLY) and gamma-aminobutyric a c i d (GABA) i n c e r e b r a l c o r t i c e s o f mice t r e a t e d f o r 11 weeks w i t h D,L—BMAA, w i t h o r w i t h o u t c o n c u r r e n t a d m i n i s t r a t i o n o f MK-801 55 T a b l e V Summary o f CSF monoamine m e t a b o l i t e c o n c e n t r a t i o n s (ng/ml) ( P a t i e n t M892) 76 T a b l e VI Summary o f t a u r i n e and GABA c o n c e n t r a t i o n s i n CSF (umol/1) ( P a t i e n t M892) 77 T a b l e V I I E s t i m a t i o n o f dopamine c o n t e n t by HPLC 78 T a b l e V I I I E s t i m a t i o n o f h o m o v a n i l l i c a c i d c o n t e n t by HPLC 79 T a b l e IX E s t i m a t i o n o f n o r a d r e n a l i n e c o n t e n t by HPLC 80 T a b l e X E s t i m a t i o n o f s e r o t o n i n c o n t e n t by HPLC 81 T a b l e XI E s t i m a t i o n o f 5 - h y d r o x y i n d o l e a c e t i c a c i d c o n t e n t by HPLC 82 v i i i L i s t o f T a b l e s ( C o n t i n u e d ) T a b l e X I I E s t i m a t i o n o f t a u r i n e , g l u t a m i c a c i d , and GABA i n a u t o p s i e d b r a i n ( P a t i e n t M892) 83 T a b l e X I I I Summary o f abnormal b r a i n b i o g e n i c amine c o n c e n t r a t i o n s o b t a i n e d by HPLC ( P a t i e n t M892) 84 T a b l e XIV Summary abnormal b r a i n amino a c i d c o n c e n t r a t i o n s as d e t e r m i n e d by amino a c i d a n a l y s i s ( P a t i e n t H892) 85 T a b l e A - I Summary o f f a s t i n g plasma amino a c i d c o n c e n t r a t i o n s (umol/1) ( P a t i e n t M892) '. 99 T a b l e A - I I Summary o f CSF amino a c i d c o n c e n t r a t i o n s (umol/1) ( P a t i e n t M892)..100 T a b l e A - I I I E s t i m a t i o n o f amino compounds i n a u t o p s i e d b r a i n (umol/g wet weight) ( P a t i e n t M892) 102 T a b l e A-IV E s t i m a t i o n o f amino compounds i n a u t o p s i e d b r a i n ( C o n t r o l P a t i e n t s ) . 103 T a b l e A-v L i t e r a t u r e v a l u e s o f dopamine c o n c e n t r a t i o n s i n a u t o p s i e d human b r a i n 105 T a b l e A-VI L i t e r a t u r e v a l u e s o f h o m o v a n i l l i c a c i d c o n c e n t r a t i o n s i n a u t o p s i e d human b r a i n 107 T a b l e A - V I I L i t e r a t u r e v a l u e s o f n o r a d r e n a l i n e c o n c e n t r a t i o n s i n a u t o p s i e d human b r a i n 109 T a b l e A - V I I I L i t e r a t u r e v a l u e s o f s e r o t o n i n c o n c e n t r a t i o n s i n a u t o p s i e d human b r a i n 110 T a b l e A-IX L i t e r a t u r e v a l u e s o f 5 - h y d r o x y i n d o l e - a c e t i c a c i d c o n c e n t r a t i o n s i n a u t o p s i e d human b r a i n I l l LIST OF FIGURES F i g u r e 1 The m e t a b o l i c r o u t e from t r y p t o p h a n t o NAD The K y n u r e n i n e Pathway 7 F i g u r e 2 The s t r u c t u r e s o f v a r i o u s " e x c i t o t o x i c " compounds 8 F i g u r e 3 T y p i c a l QA p r o f i l e o f o p t i c a l d e n s i t y (268 nm) v s . f r a c t i o n s e l u t e d f rom a Dowex SOW c a t i o n exchange column 19 F i g u r e 4 A n t e r i o r h o r n c e l l s i n t h e s p i n a l c o r d o f a mouse t r e a t e d w i t h D,L-BMAA a l o n e and i n a c o n t r o l mouse 56 F i g u r e 5 P a r t i a l p e d i g r e e o f p a t i e n t M892 69 x A C K N O W L E D G E M E N T S I wish to thank Dr. T.L. Perry f o r giving me the opportunity to j o i n h i s research group and h i s continued f i n a n c i a l sponsorship. I would also l i k e to express my appreciation to Mrs. S h i r l e y Moller-Hansen, Ms. Karen Jones, and Mr. Martin Frouws f o r t h e i r expert t e c h n i c a l assistance and support. F i n a l l y , I o f f e r my sincere gratitude to Dr. D.V. Godin f o r his sound advice, steadfast encouragement, and most of a l l , h i s f r i e n d s h i p . x i ABBREVIATIONS 5-HIAA 5 - h y d r o x y i n d o l e - a c e t i c a c i d 5-HT 5-h y d r o x y t r y p t a m i n e ; s e r o t o n i n 5-HTP L-5-hydroxy t r y p t o p h a n AD A l z h e i m e r ' s d i s e a s e ALS amyotrophic l a t e r a l s c l e r o s i s ASP a s p a r t i c a c i d BMAA b e t a - N - m e t h y l a m i n o - L - a l a n i n e BOAA b e t a - o x a l y l a m i n o - L - a l a n i n e CNS c e n t r a l n e r vous system CSF c e r e b r o s p i n a l f l u i d CT c o m p u t e r i z e d tomography CYSTA c y s t a t h i o n i n e DA dopamine DDC dopa d e c a r b o x y l a s e DOPAC 3 , 4 - d i h y d r o x y - p h e n y l a c e t i c a c i d EAA e x c i t a t o r y amino a c i d GABA gamma-aminobutyric a c i d GABA—LYS ga m m a - a m i n o b u t y r y l - l y s i n e GAD 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 GC/MS gas chromatography/mass s p e c t r o m e t r y GLN g l u t a m i n e x i i Abbreviations (Continued) GLU glutamic acid GLY glycine GLYC-PEA glycerophosphoethanolamine HBSS Hank's balanced s a l t s o l u t i o n HCARN homocarnosine HD Huntington's disease HPLC : high performance l i q u i d chromatography HVA... homovanillic acid MAO monoamine oxidase MPTP l-methyl-4-phenyl-l, 2,3,6-tetrahydropyridine MSA mult i p l e system atrophy NA noradrenaline NAD nicotinamide adenine dinucleotide NMDA N-methyl-D-aspartate OPCA ol i v o p o n t o c e r e b e l l a r atrophy PD Parkinson's disease PEA phosphoethanolamine PET p o s i t r o n emission tomography QA q u i n o l i n i c a c i d QPRT q u i n o l i n i c a c i d phosphoribosyltransferase RFLP r e s t r i c t i o n fragment length polymorphism SD standard deviation SEM ....standard e r r o r of measurement x i i i Abbreviations (Continued) U taurine t y r o s i n e hydroxylase x i v DEDICATION To my wife Marlene, f o r your love, understanding and i n s p i r a t i o n . X V 1 C H A P T E R I ATTEMPTED ISOLATION OF A PUTATIVE CIRCULATING NEUROTOXIN IN HUNTINGTON'S DISEASE I n t r o d u c t i o n H u n t i n g t o n ' s d i s e a s e (HD) i s an autosomal dominant d i s o r d e r t h a t u s u a l l y b e g i n s i n m i d - l i f e and i s c h a r a c t e r i z e d by a p r o g r e s s i o n o f i n v o l u n t a r y c h o r e i f o r m movements, p s y c h o l o g i c a l changes, and dementia ( r e v i e w e d by M a r t i n 1984). T h i s g e n e t i c a l l y d e t e r m i n e d and p r o g r e s s i v e n e u r o d e g e n e r a t i v e c o n d i t i o n r e s u l t s i n t h e l o s s o f s p e c i f i c p o p u l a t i o n s o f neurons i n t h e s t r i a t u m and o t h e r r e g i o n s o f t h e b r a i n ( K o w a l l e t a l . 1987; R e i n e r e t a l . 1988). The d e a t h o f t h e s e neurons l e a d s t o a s u b s t a n t i a l d e c r e a s e i n t h e i n h i b i t o r y n e u r o t r a n s m i t t e r gamma-a m i n o b u t y r i c a c i d (GABA) i n t h e c a u d a t e n u c l e u s , putamen, g l o b u s p a l l i d u s , s u b s t a n t i a n i g r a and o c c i p i t a l c o r t e x ( P e r r y e t a l . 1973; 1982a; B i r d and I v e r s o n 1974). O t h e r b i o c h e m i c a l changes o b s e r v a b l e i n post-mortem t i s s u e i n c l u d e , b u t a r e n o t l i m i t e d t o , a d e c r e a s e i n t h e a c t i v i t y o f 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 (GAD), t h e f i n a l enzyme i n v o l v e d i n t h e b i o s y n t h e s i s o f GABA ( B i r d e t a l . 1973) and a d e c r e a s e i n t h e c o n c e n t r a t i o n o f s u b s t a n c e P i n t h e s t r i a t o p a l l i d a l and s t r i a t o n i g r a l pathways ( G a l e e t a l . 1978; Emson e t a l . 1980). F u r t h e r m o r e , t h e 1 r e l a t i v e c o n c e n t r a t i o n s o f b o t h s o m a t o s t a t i n and n e u r o p e p t i d e Y i n t h e s t r i a t u m a r e p a r a d o x i c a l l y i n c r e a s e d i n HD ( A r o n i n e t a l . 1983; Nemeroff e t a l . 1983; B e a l e t a l . 1 9 8 4 ) . M o r p h o l o g i c a l i n v e s t i g a t i o n o f p o s t -mortem HD b r a i n r e v e a l s s i g n i f i c a n t a t r o p h y o f t h e s t r i a t u m , moderate a t r o p h y o f t h e g l o b u s p a l l i d u s and thalamus, and v a r i a b l e a t r o p h y o f c o r t i c a l , l i m b i c and b r a i n s t e m s t r u c t u r e s ( V o n s a t t e l e t a l . 1 9 8 5 ) . D i s p r o p o r t i o n a t e - l o s s o f s m a l l and medium-sized s t r i a t a l neurons w i t h a r e l a t i v e s p a r i n g o f a s p i n y s t r i a t a l and l a r g e (macro) neurons a r e a m i c r o s c o p i c h a l l m a r k o f t h e post-mortem HD b r a i n ( F e r r a n t e e t a l . 1985; G r a v e l a n d e t a l . 1 9 8 5 ) . P o s s i b l e e x p l a n a t i o n s f o r t h e premature n e u r o n a l c e l l d e a t h i n HD have i n c l u d e d g e n e t i c a l l y d e t e r m i n e d abnormal DNA r e p a i r ( M o s h e l l e t a l . 1980) o r d e f e c t i v e c e l l membrane p r o t e i n s (Goetz e t a l . 1975; P e t t e g r e w e t a l . 1979; Gray e t a l . 1980) but more r e c e n t e v i d e n c e has n o t borne o u t t h e s e mechanisms ( r e v i e w e d by B e v e r s t o c k 1985; P e r r y e t a l . 1 9 8 5 ) . A n o t h e r h y p o t h e s i s f o r t h e mechanism o f s e l e c t i v e and prema t u r e n e u r o n a l d e a t h i n HD i n v o l v e s a n e u r o t o x i n , o f e i t h e r exogenous o r endogenous o r i g i n , w hich would b i n d o n l y t o neurons s p e c i f i c a l l y i n v o l v e d i n t h e p a t h o g e n e s i s o f t h i s d i s o r d e r ( C o y l e e t a l . 1978; o l n e y 1979; P e r r y e t a l . 1985; 1 9 8 7 ) . S e v e r a l compounds, a l l a n a l o g u e s o f g l u t a m i c o r a s p a r t i c a c i d , have been shown t o cause b i o c h e m i c a l and n e u r o p a t h o l o g i c a l changes s i m i l a r t o t h o s e f o u n d i n HD when i n j e c t e d i n t r a s t r i a t a l l y . These i n c l u d e k a i n i c a c i d , f o u nd i n t h e Japanese 2 seaweed Digenea simplex (Coyle and Schwarcz 1976; McGeer and McGeer 1976), i b o t e n i c a c i d , found i n the mushroom Amanita s t r o b i l i f o r m i s (Schwarcz et a l . 1979) and, more recent l y , q u i n o l i n i c a c i d (QA), a metabolite of tryptophan (Schwarcz et a l . 1983). Each of these compounds i s known to s p e c i f i c a l l y i n t e r a c t with e x c i t a t o r y amino a c i d (EAA) receptors (reviewed by Schwarcz et a l . 1985) which are thought to mediate t h e i r " e x c i t o t o x i c " actions (Olney et a l . 1971). Q u i n o l i n i c a c i d i s a member of the kynurenine pathway which forms a major synthetic route to nicotinamide adenine d i n u c l e o t i d e (NAD) (Figure 1) (Stone and Connick 1985), i s present i n normal b r a i n at micromolar concentrations, comparable with those of other amines such as noradrenaline (Wolfensberger et a l . 1983; Moroni et a l . 1984a). Q u i n o l i n i c a c i d has a r e g i o n a l d i s t r i b u t i o n i n b r a i n (Perkins and stone 1983) and i t s concentration increases with age (Moroni e t a l . 1984b). Quinolinate has been shown to have both neuroexcitatory (Stone and Perkins 1981; Perkins and stone 1982; 1983) and neurotoxic c a p a b i l i t i e s (Schwarcz et a l . 1983). Both the neuroexcitatory and neurotoxic c h a r a c t e r i s t i c s of QA appear to be mediated by the N-methyl-D-aspartate (NMDA) receptor since the act i o n i s s e n s i t i v e to NMDA receptor 2+ antagonists (McLennan 1984; Perkins and Stone 1983) and Mg (Lehmann et a l . 1983), an inorganic ion known to be involved i n the unique voltage-dependent gating mechanism of t h i s ionophore (Ault et a l . 1980; Scatton and Lehmann 1983; Nowak et a l . 1984). Most importantly, the 3 i n t r a s t r i a t a l i n j e c t i o n o f QA has p r o d u c e d an a n i m a l model which i s a c l o s e r r e p l i c a t i o n o f HD t h a n t h a t p r o d u c e d by a s i m i l a r i n j e c t i o n o f k a i n i c a c i d o r i b o t e n i c a c i d (Schwarcz e t a l . 1983; 1984). Q u i n o l i n i c a c i d more p r e c i s e l y r e p r o d u c e s t h e n e u r o c h e m i c a l changes found i n HD i n t h a t QA l e a v e s s o m a t o s t a t i n and n e u r o p e p t i d e Y neurons r e l a t i v e l y i n t a c t ( B e a l e t a l . 1986; B e a l e t a l . 1989). A l t h o u g h i t appears t h a t QA i n an a t t r a c t i v e c a n d i d a t e f o r a c a u s a t i v e n e u r o t o x i n i n HD, t h e e x p e r i m e n t a l e v i d e n c e i s i n c o n c l u s i v e . Heyes e t a l . (1985) r e p o r t e d no i n c r e a s e d u r i n a r y e x c r e t i o n o f QA by HD p a t i e n t s . The a c t i v i t y o f t h e m e t a b o l i c enzyme r e s p o n s i b l e f o r t h e d e s t r u c t i o n o f QA, q u i n o l i n i c a c i d p h o s p h o r i b o s y l t r a n s f e r a s e (QPRT), i s no t d e f i c i e n t i n HD ( F o s t e r and Schwarcz 1985), a l t h o u g h t h e a c t i v i t y o f t h e m e t a b o l i c enzyme o f QA s y n t h e s i s , 3 - h y d r o x y a n t h r a n i l a t e oxygenase (3-HAO), i s e l e v a t e d i n t h e b r a i n s o f HD p a t i e n t s (Schwarcz e t a l . 1988a). Recent e v i d e n c e i n d i c a t e s no e x c e s s o f QA i n t h e putamen, f r o n t a l c o r t e x (Reynolds e t a l . 1988) o r c e r e b r o s p i n a l f l u i d (Schwarcz e t a l . 1988b) o f HD p a t i e n t s , a l t h o u g h i t would seem p l a u s i b l e t h a t l o n g - t e r m e x p o s u r e t o o n l y s l i g h t l y " h y p e r - p h y s i o l o g i c a l " l e v e l s o f QA mi g h t be r e s p o n s i b l e f o r n e u r o n a l t o x i c i t y i n a s l o w l y d e g e n e r a t i v e d i s o r d e r such as HD ( W h e t s e l l and Schwarcz 1989). I n a d d i t i o n t o QA, t h e r e may e x i s t o t h e r as y e t u n i d e n t i f i e d e x c i t o t o x i n s . O t h e r compounds which have been i d e n t i f i e d i n b r a i n and have e x c i t o t o x i c c h a r a c t e r i s t i c s i n c l u d e L - c y s t e i n e s u l p h i n i c a c i d (Stone 4 et a l . 1987), pyroglutamic a c i d (McGeer and Singh 1984; Rieke et a l . 1984), L-homocysteic a c i d (Olney et a l . 1987a) and the dipeptide N-acetylaspartylglutamate (Stone et a l . 1987) (see Figure 2). Perry et a l . (1985) found that repeated subcutaneous i n j e c t i o n s of large amounts of serum from HD patients i n t o i n f a n t r a t s r e s u l t e d i n a small but s i g n i f i c a n t reduction (16%) i n t h e i r mean s t r i a t a l GABA content when they were l a t e r k i l l e d as adult animals. Pretreatment of the serum f r a c t i o n s by a p h y s i c a l u l t r a f i l t r a t i o n system which removed a l l molecules l a r g e r than 10,000 Daltons r e s u l t e d i n no decrease i n the s t r i a t a l GABA content of the experimental animals, even though the rats were t r e a t e d from an e a r l i e r age, f o r a longer period of time, and with the equivalent of a much la r g e r amount of HD serum. These studies suggested that a c i r c u l a t i n g neurotoxin e x i s t s i n HD patients and that i t i s t i g h t l y bound to plasma proteins, or l e s s l i k e l y , a large molecule. Continued experimentation by t h i s group using r a t s t r i a t a l explants (Perry e t a l . 1987) provided f u r t h e r evidence of the presence i n the sera of some HD patients of a compound t o x i c to GABAergic neurons. This research i n d i c a t e d that i n cul t u r e s grown i n 30% whole or deproteinized HD serum, the a c t i v i t y of the enzyme d i r e c t l y responsible f o r GABA synthesis, glutamic a c i d decarboxylase (GAD), was s i g n i f i c a n t l y reduced. Morphological changes i n s t r i a t a l neurons as a r e s u l t of HD serum exposure have also been reported (Renkawek and Kida 1986). 5 In p r e v i o u s work by P e r r y e t a l . ( u n p u b l i s h e d r e s u l t s ) , t r a c e amounts o f an unknown compound were d e t e c t e d i n t h e q u a n t i t a t i v e a n a l y s i s o f f a s t i n g plasma o f some HD p a t i e n t s . T h i s unknown compound y i e l d e d a y e l l o w c o l o u r when r e a c t e d w i t h n i n h y d r i n and was e l u t e d from a c a t i o n exchange system ( P e r r y e t a l . 1968) v e r y c l o s e t o p r o l i n e and k a i n i c a c i d , a l t h o u g h i t was c l e a r l y n o t i d e n t i c a l w i t h e i t h e r . H u n t i n g t o n ' s d i s e a s e a f f e c t s a p p r o x i m a t e l y 5 t o 10 i n 100,000 p e r s o n s . A l t h o u g h t h e gene r e s p o n s i b l e f o r HD has been l o c a l i z e d t o t h e d i s t a l end o f t h e s h o r t arm o f chromosome 4, and a l t h o u g h g e n e - c a r r i e r s c a n u s u a l l y be i d e n t i f i e d w i t h g r e a t a c c u r a c y w i t h a r e s t r i c t i o n f ragment l e n g t h polymorphism (RFLP) t e s t ( G u s e l l a e t a l . 1983;), t h e e x a c t n a t u r e o f t h e gene d e f e c t remains unknown. There i s c u r r e n t l y no e f f e c t i v e t r e a t m e n t f o r t h i s p r o g r e s s i v e l y d e g e n e r a t i v e d i s e a s e . I s o l a t i o n o r c h a r a c t e r i z a t i o n o f a n e u r o t o x i n i n v o l v e d i n t h e p a t h o g e n e s i s o f HD c o u l d o f f e r t r e a t m e n t s t r a t e g i e s w h i c h m i g h t d e l a y o r p r e v e n t t h e e f f e c t s o f t h i s d i s e a s e . 6 F i g u r e 1 ; T h e m e t a b o l i c r o u t e f r o m t r y p t o p h a n t o N A D i T h e K y n u r e n i n e P a t h w a y ( S t o n e and C o n n i c k 1985) tryptophan S-hydroiytryptamina tryptophan I pyrrolaaaV 0 C^C-CrtCMCOjM ! l M H J NH—~*'~ 05 C H J C H J -CHO 7 tryptamln. f ormylk ynuranlna formylaaa ^ cc ^  a COjH ^ ^ C - C H J C ^ M C O J H > 6 •nthranllle kynuranlna kynurtnlc acid acid 3-hydroiylaia J o - C M 2 t : C - C N J C H C O J H 3-hydreiykynut•nlna xanthurenic acid 1 . . OH 3-hydroi y anthr anlllc acid 3-hy dre i y ant hr anlla t • I oildaaa • C ^ - c o o i ? I r ^ V- 0 0 0 * 1 - - ^ . amlnomuconlc » fcatoadlplc plcellnlc acid y •lutarylCoA OCT 2 * 0rc*"_^ M. Hitlnellitlc acid nlcetlnlcacld ^ NAD Fig. 1. A summary of the metabolic pathways from triptophan, showing details of the kynurenine pathway. B6. vitamin B6 (pyridoxal CoA, coenzyme 5-phosphatc); A; N A D , nicotinamide-adenine dinuclcotide; QPRT. quinolinate phosphoribosyltransfenue. 7 Figure 2; T h e s t r u c t u r e s o f v a r i o u s " e x c i t o t o x i c " c o m p o u n d s H 3 C / C H 2 ^ O O H I CHo H 2 COOH K A I N I C A C I D N' H COOH P Y R O G L U T A M I C A C I D CH2-CH2-SO3H CH-COOH H O M O C X S T E I C A C I D H o - C H - C O O H * I N H j N H 2 CH: BETA-N-METHTLAMINO-L-ALANINE H 2 N ^ \ C 0 0 H I B O T E N I C A C I D N ^ C 0 0 H Q U I N O L I N I C A C I D C H ^ S O j H :H-C OOH NH' C Y S T E I N E S U L P H I N I C A C I D C H ^ — C H — C O O H I I NH NHo I 2 c=o COOH BSTA-N—OXALYXJVMINO—lj—ALANINE Objective The o b j e c t i v e of the i n v e s t i g a t i o n was to confirm or disprove the r o l e of q u i n o l i n i c a c i d i n the pathogenesis of Huntington's disease by the establishment of a chromatographic system which would r e l i a b l y i s o l a t e and quantify a l l authentic QA from deproteinized sera. Neurotoxicity of serum f r a c t i o n s separated by t h i s system would be assayed with a t i s s u e c u l t u r e model s i m i l a r to that used by Perry et a l . 1987. Hypothesis A neurotoxin i s present i n the serum of Huntington's disease p a t i e n t s which i s u n l i k e l y to be q u i n o l i n i c a c i d yet can be i s o l a t e d using appropriate chromatographic techniques. Experimental Outline I n i t i a l experimentation, using ion exchange chromatography on the deproteinized sera of drug-free c o n t r o l subjects and p a t i e n t s with HD, attempted to confirm or disprove the q u i n o l i n i c a c i d hypothesis of Huntington's disease. The f r a c t i o n s separated by ion exchange corresponding to the known e l u t i o n volume of authentic QA were c o l l e c t e d , concentrated and added to c u l t u r e d f e t a l r a t s t r i a t a l neurons. A f t e r appropriate periods of incubation, these neuronal 9 c u l t u r e s were teste d f o r the v i a b i l i t y of GABAergic neurons by the determination of the a c t i v i t y of the synthetic enzyme of GABA, glutamic a c i d decarboxylase. I f the f r a c t i o n s obtained from HD serum had no harmful e f f e c t s on c u l t u r e d s t r i a t a l neurons, t h i s study would have demonstrated that QA was not the causative neurotoxin. Thereafter, various other portions of column e f f l u e n t s , c o l l e c t e d on a f r a c t i o n c o l l e c t o r , were to be t e s t e d f o r p o s s i b l e t o x i c e f f e c t s on c u l t u r e d f e t a l r a t s t r i a t a l neurons. I f a p a r t i c u l a r serum f r a c t i o n proved to have neurotoxic e f f e c t s , attempts would be made to c h a r a c t e r i z e and i d e n t i f y the neurotoxic m a t e r i a l . Methods C o l l e c t i o n of Sera Venous blood was c o l l e c t e d from HD patients and c o n t r o l subjects on one or more occasions, when neither patients nor c o n t r o l subjects were r e c e i v i n g drugs of any s o r t . Each of the HD patients used i n the p r o j e c t had a w e l l documented h i s t o r y of the disease, with c l a s s i c a l signs and symptoms, and at l e a s t one parent with a h i s t o r y of HD. A l l c o n t r o l s and HD patients were age- and sex-matched. Between 200 and 300 ml of blood were c o l l e c t e d , generally from the a n t e c u b i t a l vein, using a cannula, p l a s t i c tube, and 20 ml syringes i n order to minimize any discomfort to patients and subjects. Blood donors were not f a s t i n g . 10 The purpose of the study was c a r e f u l l y explained to a l l p a r t i c i p a n t s i n the p r o j e c t and informed consent was obtained. Blood specimens were c o l l e c t e d without the use of anticoagulants and were allowed to stand f o r ca. 1 hr at 4^ 0 before being centrifuged to separate the serum. Sera were stored at -70 C u n t i l they were used e i t h e r unaltered, or i n modified form f o r i n t r o d u c t i o n i n t o t i s s u e c u l t u r e media. Preparation of Sera Sera used i n the q u i n o l i n i c a c i d i s o l a t i o n experiments were f i r s t d e proteinized with HC10 4 ( d e p r o t e i n i z a t i o n was shown not to eliminate n e u r o t o x i c i t y i n previous experiments (Perry et a l . 1987)). To each 10 ml of serum, 0.29 ml of col d 10 M HC10 4 was added dropwise, while the serum was continuously s t i r r i n g i n an i c e bath. The r e s u l t i n g t h i c k suspension of denatured proteins was then c e n t r i f u g e d at 10,000 x gr i n a 4°C r e f r i g e r a t e d centrifuge f o r 15 min. The c l e a r supernatant was c a r e f u l l y removed and KHCO-j c r y s t a l s were added, while the supernatant was continuously s t i r r i n g i n an i c e bath, u n t i l the pH reached 7.0. The r e s u l t i n g n e u t r a l i z e d and c h i l l e d s o l u t i o n was cen t r i f u g e d to remove the p r e c i p i t a t e d , i n s o l u b l e potassium perchlorate and the e s s e n t i a l l y p r o t e i n - f r e e supernatant was stored frozen at -70*C f o r subsequent chromatography and incorporation i n t o t i s s u e c u l t u r e media. 11 P r i o r t o use i n chromatography e x p e r i m e n t s , a l l d e p r o t e i n i z e d s e r a were c e n t r i f u g e d f o r 15 min a t 1 0 , 0 0 0 x gr t o remove any r e m a i n i n g m a t e r i a l p r e c i p i t a t e d by f r e e z i n g a t - 7 0 * c . The pH o f a l l s e r a was a d j u s t e d t o 2 . 0 w i t h t h e dropwise a d d i t i o n o f 1 M H C l b e f o r e b e i n g l o a d e d on t h e i o n exchange column. Chromatography I s o l a t i o n o f q u i n o l i n i c a c i d was a c c o m p l i s h e d by i o n exchange chromatography u s i n g t h e c a t i o n exchange r e s i n Dowex 50W ( 2 0 0 - 4 0 0 mesh, 8% c r o s s - l i n k a g e ) , columns o f 1 . 5 x 65 cm were po u r e d under g r a v i t y , t h e r e s i n h a v i n g been p r e v i o u s l y p r e p a r e d by s e q u e n t i a l washing w i t h 2 x 2 5 0 ml a c e t o n e , 5 x 2 5 0 ml d i s t i l l e d H 2 0 , 2 x 2 5 0 ml 2 M NaOH, 10 x 2 5 0 ml d i s t i l l e d H 2 0 , 2 x 2 5 0 ml 2 M HCl and 10 x 2 5 0 ml d i s t i l l e d H 2 0 i n a s i n t e r e d g l a s s f u n n e l . Columns were e q u i l i b r a t e d w i t h 10 mM H C l ( c a . 1 0 0 0 ml) a t a f l o w r a t e o f 1 ml/min. The p r e v i o u s l y a c i d i f i e d and d e p r o t e i n i z e d samples, s t a n d a r d s o r OA - s p i k e d samples (see above) were l o a d e d i n a known volume o f l e s s t h a n 10 ml and a l l o w e d t o e n t e r t h e r e s i n under g r a v i t y . Once l o a d e d , t h e column s u r f a c e was c o v e r e d w i t h 10 mM H C l and t h e pump s t a r t e d . Ten ml f r a c t i o n s were c o l l e c t e d t h r o u g h o u t t h e e x p e r i m e n t s . 12 Quinolinate was i s o l a t e d and q u a n t i f i e d by uv spectroscopy on the basis of strong UV absorption by QA at 268 nm, with an e x t i n c t i o n c o e f f i c i e n t at t h i s wavelength of 3.9 x 10 1/mol/cm (Wolfensberger 1983). Samples i s o l a t e d by t h i s technique were compared to authentic QA by ascending paper chromatography using a mixture of butanol/acetic acid/H 2 0 i n a r a t i o of 12:3:5 r e s p e c t i v e l y . Deproteinized sera from c o n t r o l subjects and HD pati e n t s were subjected to ion exchange and the f r a c t i o n s corresponding to the e l u t i o n of authentic QA were i s o l a t e d . Excess HCl was removed by mul t i p l e co-evaporations with d i s t i l l e d H 20, under vacuum with the ambient temperature c o n s i s t e n t l y kept below 50°C. The minute residue remaining was r e c o n s t i t u t e d i n the i d e n t i c a l volume of horse serum as human serum c o l l e c t e d f o r each experiment, and s t e r i l i z e d by passing through a Nalgene f i l t e r u n i t (0.20 urn). Further c h a r a c t e r i z a t i o n of the e l u t i o n p r o f i l e of the c a t i o n exchange chromatography was accomplished by the separation of known amino a c i d standards i n c l u d i n g taurine, aspartate, p r o l i n e and alanine. The e l u t i o n gradient used i n t h i s system consisted of a s e r i e s of li t h i u m c i t r a t e b u f f e r s mixed i n a multi-chamber gradient former modified from a w e l l e s t a b l i s h e d system used i n amino a c i d a n a l y s i s (Perry et a l . 1968). The gradient consisted of l i t h i u m c i t r a t e buffers (0.2 M) i n the fo l l o w i n g proportions and a c i d i t i e s : 100 ml pH 2.80, 4 x 100 ml pH 3.80 and 100 ml pH 6.10. Samples from 10 ml f r a c t i o n s 13 c o l l e c t e d t h r o u g h o u t e a c h e x p e r i m e n t were s p o t t e d on p a p e r and d e v e l o p e d w i t h a m i x t u r e o f n i n h y d r i n (0.2%) and l u t i d i n e (1.0%) i n e t h a n o l i n o r d e r t o a l l o w v i s u a l i z a t i o n and i d e n t i f i c a t i o n . C u l t u r e o f F e t a l Rat S t r i a t a l E x p l a n t s C u l t u r e s o f f e t a l r a t s t r i a t a were e s t a b l i s h e d f o r t h e n e u r o t o x i c i t y s t u d i e s e s s e n t i a l l y as p r e v i o u s l y d e s c r i b e d ( P e r r y e t a l . 1987) w i t h some minor m o d i f i c a t i o n s . The b r a i n s were removed from day 19 l i t t e r - m a t e W i s t a r f e t a l r a t pups, p l a c e d i n t o a P e t r i d i s h c o n t a i n i n g Hank's b a l a n c e d s a l t s o l u t i o n (HBSS) and k e p t on i c e . U s i n g a d i s s e c t i n g m i c r o s c o p e , t h e l e f t and r i g h t s t r i a t a were e x c i s e d and p l a c e d i n t o a n o t h e r P e t r i d i s h c o n t a i n i n g c u l t u r e medium (see b e l o w ) , a l s o k e p t on i c e . W i t h f i n e s c a l p e l s each o f t h e i n d i v i d u a l s t r i a t a was 3 c u t i n t o 7-10 p i e c e s o f t i s s u e , m e a s u r i n g c a . 1 mm , r e f e r r e d t o hence as e x p l a n t s . Three t o f o u r s t r i a t a l e x p l a n t s were p l a c e d on one o f t h r e e c o l l a g e n - c o a t e d ( B o r n s t e i n 1958) 10 mm g l a s s c o v e r s l i p s i n 35 mm d i s p o s a b l e P e t r i p l a t e s . Thus, each 35 mm P e t r i p l a t e c o n t a i n e d 12 t o 16 s t r i a t a l e x p l a n t s . The c o v e r s l i p s had been p r e v i o u s l y c o a t e d w i t h r a t t a i l c o l l a g e n and c o n d i t i o n e d o v e r n i g h t w i t h c u l t u r e medium. To a l l o w t h e e x p l a n t s t i m e t o a t t a c h t o t h e c o l l a g e n - c o a t e d c o v e r s l i p s , c u l t u r e medium was added o n l y d r o p w i s e f o r t h e f i r s t two days a f t e r e x p l a n t a t i o n b e f o r e b e i n g f l o o d e d w i t h 1.5 ml o f t h e n u t r i e n t c u l t u r e 14 medium on the t h i r d day. incubation was maintained at 37 c i n a moist environment of 95% and 5% o 2 . During the f i r s t 7 days a f t e r explantation, the c u l t u r e n u t r i e n t medium used to s t a r t the growth of s t r i a t a l explants contained 70% (v/v) of a mixture of Eagle's minimum e s s e n t i a l medium (with E a r l e ' s s a l t s and glutamine, but without sodium bicarbonate), 0.5% glucose, 26.2 mM i n added sodium bicarbonate. The remaining 30% of the c u l t u r e medium was composed of he a t - i n a c t i v a t e d horse serum. A l l chemicals and horse serum were t i s s u e c u l t u r e grade obtained from Gibco Laboratories (Grand Island, NY). A f t e r the i n i t i a l 7 days of incubation, the v i a b i l i t y of the explants was determined v i s u a l l y using a phase-contrast microscope at lOx m a g n i f i c a t i o n . Explants with large, dark n e c r o t i c areas and without extended growth processes were removed from the pool of c u l t u r e s used fo r the remainder of the studies of sera from HD p a t i e n t s and c o n t r o l s . T y p i c a l l y , s t r i a t a from 12 to 18 f e t a l r a t s , y i e l d i n g ca. 100 to 180 explants were used f o r each comparison of sera. P e t r i p l a t e s containing healthy, growing explants were di v i d e d i n t o a number of groups, depending upon the design of each experiment. Each group was incubated f o r a furt h e r 7 days with c u l t u r e medium as described above except that the horse serum was replaced with 30% serum obtained from HD patients or c o n t r o l subjects. A l l c u l t u r e media were 15 s t e r i l i z e d by passing them through a Nalgene f i l t e r u n i t (0.20 um) and a l l t i s s u e c u l t u r e procedures, with the exception of harvesting, were performed under s t e r i l e conditions. Explants were harvested two weeks a f t e r d i s s e c t i o n , and one week a f t e r exposure to the experimental condi t i o n s . Harvesting consisted of three r i n s e s of the P e t r i d i s h with i c e - c o l d HBSS to remove contaminating medium, before the collagen-bound explants were scraped o f f with a rubber policeman i n t o 2 ml Eppendorf tubes. Forty to 60 explants were pooled f o r the determination of GAD enzyme a c t i v i t y and were stored frozen at -70 c u n t i l analyzed. Determination of GAD Enzyme A c t i v i t y GAD enzyme a c t i v i t y was determined by measuring the rate of 14 14 formation of l a b e l l e d C<>2 from L - [ l - C]glutamic a c i d as previously described (Perry et a l . 1987). A l l determinations were the average of 14 du p l i c a t e measurements and were expressed i n nmol C0 2 formed/hr/explant. The large number of explants i n each group assayed should have minimized v a r i a b i l i t i e s due to d i f f e r e n c e s i n explant s i z e . In comparison studies, p r o t e i n concentration was determined by the Lowry method (Lowry et a l . 1951), using bovine serum albumin as a standard. 16 Results Repeated e x p e r i m e n t a t i o n w i t h d i f f e r e n t c o n d i t i o n s u l t i m a t e l y p r o d u c e d a r e l i a b l e and r e p r o d u c i b l e c h r o m a t o g r a p h i c system which c o u l d be u s e d f o r t h e i s o l a t i o n , q u a n t i f i c a t i o n and f u l l r e c o v e r y o f QA from samples o f d e p r o t e i n i z e d human s e r a . The e l u t i o n p r o f i l e o f QA from t h e Dowex 50W column u s i n g 10 mM HCl as t h e e l u t i o n s o l v e n t i s shown i n F i g u r e 3 . The u v a b s o r b i n g peak r e p r o d u c i b l y e l u t e d a t a volume o f 3 0 0 t o 4 5 0 ml ( c o r r e s p o n d i n g t o 5 t o 7 . 5 h r ) . When t h i s m a t e r i a l was p o o l e d and t h e c o n c e n t r a t i o n o f QA d e t e r m i n e d from t h e e x t i n c t i o n c o e f f i c i e n t , 100% r e c o v e r y o f QA from b o t h QA s t a n d a r d s and QA-spiked, d e p r o t e i n i z e d human s e r a samples was o b t a i n e d . A f t e r t h e e l u t i o n p r o f i l e o f QA was e s t a b l i s h e d w i t h c o n f i d e n c e , m u l t i p l e c o - e v a p o r a t i o n s w i t h d i s t i l l e d w a ter were used t o remove t h e HCl u s e d i n t h e c h r o m a t o g r a p h i c p r o c e d u r e . The e f f e c t i v e n e s s o f t h i s p r o c e d u r e was e n s u r e d w i t h t h e use o f c o n t r o l f l a s k s c o n t a i n i n g o n l y 10 mM H C l . The r e s u l t s o f t h e f i r s t p r e l i m i n a r y s t u d i e s a r e shown i n T a b l e I . I t s h o u l d be n o t e d t h a t t h e GAD a c t i v i t y v a l u e s a r e e x t r e m e l y low i n b o t h t h e c o n t r o l and HD groups r e l a t i v e t o GAD a c t i v i t i e s o b t a i n e d r o u t i n e l y i n p r e v i o u s e x p e r i m e n t s w i t h o u t c h r o m a t o g r a p h i c s e p a r a t i o n o f s e r a , i n o r d e r t o d e t e r m i n e i f some unknown agent was e l u t e d from t h e Dowex 50W r e s i n under t h e i s o l a t i o n c o n d i t i o n s used, f u r t h e r e x p e r i m e n t s 17 included the a n a l y s i s of GAD a c t i v i t i e s of neurons exposed to horse serum containing any trace material remaining a f t e r extensive co-evaporation with d i s t i l l e d water to remove HCl. These r e s u l t s are also shown i n Table I. In a fur t h e r s e r i e s of experiments, a b u f f e r system was developed which would separate amino compounds from deproteinized plasma i n much the same manner as that developed by Perry et a l . (1968) and s t i l l used r o u t i n e l y f o r t h i s purpose. The e l u t i o n p r o f i l e which r e s u l t e d from the gradient described above corresponded to the p r o f i l e obtained from the Technicon amino analyzer system. 18 Figure 3: T y p i c a l Q A p r o f i l e of o p t i c a l d e n s i t y (268 nm) v s . f r a c t i o n s e l u t e d from a Dowex 50W c a t i o n exchange column 19 Table I: GAD a c t i v i t i e s of f e t a l s t r i a t a l c u l t u r e s exposed to HD and c o n t r o l sera, and Dowex 50W column e f f l u e n t s E x p t : E x p e r i m e n t a l Groups: GAD A c t i v i t y : ( n m o l / h r / e x p l a n t ) 1 C o n t r o l : (S2987b) 0.022 HD: (S2984b) 0.025 2 C o n t r o l : (S2986b) 0.024 HD: (S2985b) 0.031 9 C o n t r o l : (S2950b) 0.110 HD: (2943b) 0.070 H C l Wash from QA peak a r e a 0.110 10 C o n t r o l : 30% HS + H 20 0.210 T e s t : 30% HS + HCl wash 0.060 20 Discussion Although a system of separation and q u a n t i f i c a t i o n of QA was developed, the o b j e c t i v e of the p r o j e c t was not reached due to the t o x i c i t y of a l l e f f l u e n t s eluted not only from Dowex 50W r e s i n but also from a number of other commercially a v a i l a b l e c a t i o n exchange r e s i n s ( r e s u l t s not shown). I t would seem that some u n i d e n t i f i e d material was removed by washing of these r e s i n s with 10 mM HCl. I t was observed that a majority of these exchange r e s i n s release coloured compounds, e s p e c i a l l y i n the i n i t i a l stages of the matrix preparation. While these coloured materials were not obvious during the separation procedures, the extreme s e n s i t i v i t y of c u l t u r e d explants to a large v a r i e t y of materials precluded the use of f e t a l s t r i a t a l explants as an assay of HD serum n e u r o t o x i c i t y i n t h i s manner. Although a chromatographic system was a l s o developed which would r e l i a b l y separate a number of known amino compounds, the projected d i f f i c u l t y i n removing a l l contaminating l i t h i u m s a l t s without simultaneously removing any putative neurotoxin present, together with the above considerations, necessitated the abandonment of the p r o j e c t . The approach o u t l i n e d i n t h i s p r o j e c t would remain f e a s i b l e i f a c a t i o n exchange r e s i n without s i m i l a r neurotoxic p r o p e r t i e s , or a novel method of separation could be found. In l i g h t of the development of s e n s i t i v e systems f o r the q u a n t i f i c a t i o n of QA which u t i l i z e gas 21 chromatography c o u p l e d w i t h mass s p e c t r o m e t r y (GC\MS) ( W o l f e n s b e r g e r e t a l . 1983; Mor o n i e t a l . 1986; Heyes and Markey 1988; R e y n o l d s e t a l . 1988), t h e a n a l y s i s o f serum f r a c t i o n s w i t h GC\MS s h o u l d r e l i a b l y d e t e c t any d i f f e r e n c e s between normal and HD p a t i e n t s . I n any e v e n t , t h e p r o b a b i l i t y o f Q A i n c i r c u l a t i n g plasma b e i n g r e s p o n s i b l e f o r t h e n e u r o d e g e n e r a t i o n o b s e r v e d i n HD i s low, due t o i t s p o o r p e n e t r a t i o n o f t h e b l o o d - b r a i n b a r r i e r ( F o s t e r e t a l . 1984; K i t t and S p e c t o r 1987), normal e x c r e t i o n i n HD (Heyes e t a l . 1985) and t h e p r e s e n c e o f normal c o n c e n t r a t i o n s and a c t i v i t i e s o f Q P R T ( t h e c a t a b o l i c enzyme o f Q A ) i n t h e b l o o d o f HD p a t i e n t s ( F o s t e r and Schwarcz 1985). 22 CHAPTER I I A TRIAL OF THE NON-COMPETITIVE NMDA ANTAGONIST MK-801 IN THE PROTECTION OF FETAL RAT STRIATAL EXPLANTS FROM A PUTATIVE CIRCULATING NEUROTOXIN IN HUNTINGTON'S DISEASE I n t r o d u c t i o n A l t h o u g h v a r i o u s p h a r m a c o l o g i c a l i n t e r v e n t i o n s have been attempted i n HD, none have been v e r y s u c c e s s f u l t o dat e ( r e v i e w e d by P e r r y 1987). Most agents which have undergone c l i n i c a l t r i a l s i n HD have been GABA an a l o g u e s o r s u b s t a n c e s t a r g e t e d a t i n c r e a s i n g l e v e l s o f endogenous GABA. Compounds which have been i n v e s t i g a t e d t o d a t e i n c l u d e , muscimol ( S h o u l s o n e t a l . 1978), a m i n o - o x y a c e t i c a c i d ( P e r r y e t a l . 1980), i s o n i a z i d ( P e r r y e t a l . 1979; 1982b), gamma-acetylenic GABA ( T e l l e t a l . 1981), gamma-vinyl GABA ( S c i g l i a n o e t a l . 1984), THIP ( F o s t e r e t a l . 1983), and p r o g a b i d e (Marsden and Sheeny 1981; M o r s e l l i e t a l . 1980). R e c e n t l y , s h o u l s o n e t a l . (1989) att e m p t e d t o slow t h e f u n c t i o n a l d e c l i n e i n HD w i t h a c l i n i c a l t r i a l o f b a c l o f e n , a p u t a t i v e GABAg a g o n i s t ( H i l l and Bowery 1981) which i n h i b i t s t h e c o r t i c o s t r i a t a l r e l e a s e o f g l u t a m a t e and a s p a r t a t e ( P o t a s h n e r 1979; M i t c h e l l 1980). U n f o r t u n a t e l y , a l l o f t h e s e a g e n t s have p r o v e n t o be d i s a p p o i n t i n g i n th e t r e a t m e n t o f HD. 23 S i n c e t h e i n t r o d u c t i o n o f t h e e x c i t o t o x i c h y p o t h e s i s o f n e u r o d e g e n e r a t i v e d i s e a s e ( O l n e y 1974) and t h e subsequent h y p o t h e s i s o f a s i m i l a r mechanism i n HD ( C o y l e and Schwarcz 1976; r e v i e w e d by Schwarcz and S h o u l s o n 1987), a g e n t s have been sought which would a c t t o s p e c i f i c a l l y i n t e r f e r e w i t h t h i s p r o c e s s . C o n c u r r e n t i n v e s t i g a t i o n o f t h e m e d i a t o r s o f e x c i t a t o r y amino a c i d t r a n s m i s s i o n have y i e l d e d a w e a l t h o f i n f o r m a t i o n r e g a r d i n g a group o f e x c i t a t o r y amino a c i d (EAA) r e c e p t o r s , namely t h e NMDA, k a i n a t e and q u i s q u a l a t e r e c e p t o r t y p e s ( r e v i e w e d by Monaghan e t a l . 1989; MacDermott and Da l e 1987; Fagg e t a l . 1986) . I n p a r t i c u l a r , t h e NMDA r e c e p t o r has c e r t a i n p r o p e r t i e s which a r e h i g h l y s u g g e s t i v e o f a r o l e i n t h e p a t h o g e n e s i s o f c e r t a i n n e u r o d e g e n e r a t i v e c o n d i t i o n s , i n c l u d i n g HD (Rothman and O l n e y 1987). These r e c e p t o r c h a r a c t e r i s t i c s i n c l u d e a unique v o l t a g e - d e p e n d e n t b l o c k 2+ by Mg and r e l a t i v e l y n o n - s p e c i f i c a c t i o n as a c a t i o n l o n o p h o r e 2+ ( i n c l u d i n g Ca ) . There i s a now a g r e a t d e a l o f i n f o r m a t i o n i m p l i c a t i n g NMDA-mediated a c t i o n s i n n e u r o t o x i c i t y (Rothman and O l n e y 1987; C h o i 1988) and EAA a g o n i s t s and a n t a g o n i s t s i n g e n e r a l ( r e v i e w e d by Wroblewski and Danysz 1989; Watkins and Olverman 1987; Kemp e t a l . 1987) . The o b s e r v a t i o n i n post-mortem HD b r a i n o f major r e d u c t i o n s (93%) o f NMDA r e c e p t o r b i n d i n g i n t h e putamen (Young e t a l . 1988) and glu t a m a t e r e c e p t o r b i n d i n g i n t h e ca u d a t e and putamen (Greenamyre e t a l . 1985), s u p p o r t a r o l e f o r NMDA r e c e p t o r - m e d i a t e d n e u r o t o x i c i t y i n HD. 24 The p o t e n t i a l therapeutic b e n e f i t of EAA antagonists has been suggested (Meldrum 1985; Schwarcz and Meldrum 1985) and warrants f u r t h e r i n v e s t i g a t i o n . MK-801 ((+)-5-methyl-10,ll-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) i s a potent non-competitive NMDA receptor antagonist (Wong et a l . 1986; Huettner and Bean 1988; Kemp et a l . 1987). The a b i l i t y of t h i s agent to cross the blood-brain b a r r i e r (Foster et a l . 1987) has obvious therapeutic advantages. I t s e f f i c a c y i n the p r o t e c t i o n of neurons i n v i t r o (Olney et a l . 1987b; Rondouin et a l . 1988; Hahn et a l . 1988) and i n v i v o (Foster e t a l . 1987; 1988; Beal et a l . 1988) against a v a r i e t y of NMDA agonists, i n c l u d i n g QA, i s we l l documented. Objective The o b j e c t i v e of the i n v e s t i g a t i o n was to determine the e f f i c a c y of the non-competitive NMDA receptor antagonist MK-801 i n the protection of f e t a l s t r i a t a l explants from the degenerative e f f e c t s of HD sera. Hypothesis The putative neurotoxin suggested by experiments performed by Perry e t a l . (1985; 1987) produces neurodegeneration v i a an e x c i t o t o x i c mechanism mediated by receptors of the NMDA-type. 25 Experimental Outline A s e r i e s of matched experiments were performed to assess the neuroprotective e f f i c a c y of MK-801. s t r i a t a l explants were grown i n cu l t u r e under standard conditions f o r 1 week a f t e r which the pool was div i d e d i n t o two groups, one exposed to HO sera and the other to con t r o l sera. Each of these 2 groups was furth e r separated i n h a l f , one exposed to 30% serum alone and the other to 30% serum containing MK-801. The concentration of MK-801 used i n the experiments was assessed as the maximum concentration which d i d not cause s i g n i f i c a n t d e l e t e r i o u s e f f e c t s on the v i a b i l i t y of explants i n c u l t u r e . Methods The explantation, growth, harvest and general a n a l y s i s of the f e t a l s t r i a t a l t i s s u e i n v i t r o was performed as pr e v i o u s l y described i n Chapter I. MK-801 was dis s o l v e d i n d i s t i l l e d water and s t e r i l i z e d by f i l t r a t i o n through a Nalgene f i l t e r u n i t (0.2 um). The minor d i l u t i o n of growth medium which r e s u l t e d from the a d d i t i o n of MK-801 was dup l i c a t e d i n c o n t r o l cultures by the ad d i t i o n of an i d e n t i c a l volume of s t e r i l e , d i s t i l l e d water. 26 Results A summary of the r e s u l t s obtained i s presented i n Table I I . I t can be seen that no obvious trends i n the a c t i v i t i e s of GAD with or without the ad d i t i o n of MK-801 could be detected. In some cases, the ad d i t i o n of MK-801 r e s u l t e d i n a decrease i n GAD a c t i v i t i e s while i n others an increase was observed. Moreover, the decrease i n GAD a c t i v i t y i n the presence of 30% HD sera was not confirmed, as GAD a c t i v i t i e s were found to be e i t h e r increased or decreased r e l a t i v e to c o n t r o l values. Although GAD a c t i v i t i e s were comparable i n magnitude to pr e v i o u s l y reported values (Perry et a l . 1987) which ranged from 8 to 78 umol/hr/g p r o t e i n (see Experiment 31B), no consistent r e s u l t s were obtained i n t h i s more recent set of experiments. 27 T a b l e H i GAD a c t i v i t i e s o f f e t a l r a t s t r i a t a l c u l t u r e s exposed t o HD o r c o n t r o l s e r a , w i t h o r w i t h o u t c o n c u r r e n t e x p o s u r e t o MK-801. E x p t : E x p e r i m e n t a l Groups; GAD A c t i v i t y : ( n m o l / h r / e x p l a n t ) C o n t r o l : (S2950b) 0.200 HD: (S2943b) 0.140 C o n t r o l + MK-801 (0.1 uM) 0.290 HD + MK-801 (0.1 uM) 0.190 C o n t r o l : (S2950b) 0.410 HD: (S2943b) 0.320 C o n t r o l + MK-801 (0.1 uM) 0.440 HD + MK-801 (0.1 UM) 0.230 13 C o n t r o l : (S2950b) 0.400 HD: (S2951b) 0.450 C o n t r o l + MK-801 (0.1 uM) 0.340 HD + MK-801 (0.1 UM) 0.400 28 GAD a c t i v i t i e s o f f e t a l r a t s t r i a t a l c u l t u r e s exposed t o HD o r c o n t r o l s e r a , w i t h o r w i t h o u t c o n c u r r e n t exposure t o MK-801. E x p t ; E x p e r i m e n t a l Groupst GAD A c t i v i t y ; ( r a n o l / h r / e x p l a n t ) 23 C o n t r o l : (S3030b) 0.140 HD: (S3029b) 0.120 C o n t r o l + MK-801 (0.2 uM) 0.160 HD + MK-801 (0.2 uM) 0.180 26 C o n t r o l : (S3016b) 0.080 HD: (S3014b) 0.090 C o n t r o l + MK-801 (0.2 uM) 0.130 HD + MK-801 (0.2UM) 0.140 27 C o n t r o l : (S2986b) 0.190 HD: (S2985b) 0.440 C o n t r o l + MK-801 (0.2 uM) 0.160 HD + MK-801 (0.2 uM) 0.240 29 GAD a c t i v i t i e s o f f e t a l r a t s t r i a t a l c u l t u r e s e x p osed t o HD o r c o n t r o l s e r a , w i t h o r w i t h o u t c o n c u r r e n t exposure t o MK - 801 . E x p t : E x p e r i m e n t a l Groups: GAD A c t i v i t y : ( n m o l / h r / e x p l a n t ) 28 C o n t r o l : (S3016b) 0.190 HD: (S3013b) 0.360 C o n t r o l + MK-801 (0.2 UM) 0.150 HD + MK-801 (0.2 UM) 0.360 30 C o n t r o l : (S3015b) 0.190 HD: (S2984b) 0.160 C o n t r o l + MK-801 (0.2 uM) 0.130 HD + MK-801 (0.2 UM) 0.150 31 C o n t r o l : (S2987b) 0.270 HD: (S2985b) 0.220 C o n t r o l + MK-801 (0.2 uM) 0.440 HD + MK-801 (0.2 UM) 0.160 30 GAD a c t i v i t i e s o f f e t a l r a t s t r i a t a l c u l t u r e s exposed t o HD o r c o n t r o l s e r a , w i t h o r w i t h o u t c o n c u r r e n t e x p o s u r e t o MK-801. E x p t : E x p e r i m e n t a l Groups; GAD A c t i v i t y ; ( n m o l / h r / e x p l a n t ) 31B C o n t r o l : (S2987b) 17.1 * HD: (S2985b) 12.9 C o n t r o l + MK-801 (0.2 uM) 40.8 HD + MK-801 (0.2 UM) 14.5 *The GAD a c t i v i t i e s a r e e x p r e s s e d as umol/hr/g p r o t e i n . 32 C o n t r o l : (S2987b) 0.150 (same s e r a HD: (S2985b) 0.160 as E x p t 31) HD + MK-801 (0.2 uM) 0.160 33 C o n t r o l : (S2987b) 0.330 HD: (S2985b) 0.330 C o n t r o l + MK-801 (0.2 uM) 0.270 HD + MK-801 (0.2 UM) 0.260 31 Discussion The r e s u l t s obtained i n t h i s s e r i e s of experiments were s u b s t a n t i a l l y d i f f e r e n t from e a r l i e r r e s u l t s reported by Perry et a l . (1987). More s p e c i f i c a l l y , the present r e s u l t s f a i l e d to duplicate the p r e v i o u s l y found decrease i n GAD a c t i v i t y of r a t f e t a l s t r i a t a l cultures when exposed to 30% HD serum r e l a t i v e to 30% normal human serum. In a thorough comparison of techniques used i n both experimental protocols, a few major d i f f e r e n c e s must be mentioned. Previous r e s u l t s were obtained a f t e r the explants were grown i n a r o l l i n g tube apparatus (Kim and T u n n i c l i f f 1974). This system r e s u l t e d i n the constant mixing of the growth medium which ensured a homogeneous environment f o r a l l explants, throughout the experiment. There could have been l o c a l inhomogeneities using the present method which might have confounded r e s u l t s . Other major d i f f e r e n c e s included the a d d i t i o n of gentamicin (20 ug/ml) to the growth medium, the use of newborn r a t pups, and the v i s u a l i d e n t i f i c a t i o n and removal of compromised explants from the experimental pool during the l a s t week of experimentation i n the previous study (Perry et a l . 1987). Gentamicin was not included i n the present seri e s of experiments i n order to minimize t o x i c i nfluences on the growing c u l t u r e s , provided a s t e r i l e environment could be maintained. Because a wide spectrum anti-mycotic and a n t i - b i o t i c (10,000 u n i t s P e n i c i l l i n G, 10,000 meg Streptomycin s u l f a t e , and 25 meg Fungizone per 100 ml medium) was used during the explantation process, preliminary f i n d i n g s suggested 32 that t h i s m o d i f i c a t i o n would not confound r e s u l t s , since the o v e r a l l GAD a c t i v i t i e s obtained with the modified sera, remained the same as those obtained with gentamicin. However, the increased opportunity f o r i n f e c t i o n s i n the c u l t u r e s might have r e s u l t e d i n uncontrolled v a r i a b i l i t i e s i n the v i a b i l i t y of the cultures due to b a c t e r i a l and/or fungal contamination, although c u l t u r e s were examined f o r gross contamination and removed from the experimental pool when t h i s was i n d i c a t e d . Although the use of day 19 f e t a l r a t pups should have improved r e s u l t s due to increased v i a b i l i t y of the younger t i s s u e , the process of an a d d i t i o n a l s e l e c t i o n of explants used i n e a r l i e r work (Perry et a l . 1987) may have had s i g n i f i c a n t e f f e c t s . In the present and previous experiments, explants were examined a f t e r one week i n growth medium containing 30% horse serum. Explants which appeared t o have large n e c r o t i c areas or had f a i l e d to grow extended processes were p h y s i c a l l y removed from the experimental pools. In the e a r l i e r published work (Perry et a l . 1987), t h i s process was repeated a f t e r the e n t i r e experiment was completed, at the time of c e l l harvest. This process eliminated a l l explants which had died during the week or more of exposure to the experimental sera. Since the assay was designed to t e s t t o x i c i t y , which i n the extreme r e s u l t s i n death of t i s s u e s i n c u l t u r e , t h i s process was eliminated. This may explain to some degree the decreases of o v e r a l l GAD a c t i v i t y observed i n some cul t u r e s compared t o the e a r l i e r r e s u l t s . 33 Future experiments along s i m i l a r l i n e s should include consideration of d i f f e r e n c e s i n HD s e v e r i t y and attempts should be made to assess the f u n c t i o n a l c a p a c i t i e s of HD patients with a r a t i n g scale (Shoulson 1981). other independent d i s p a r i t i e s such as r e n a l and hepatic problems, which might r e s u l t i n the release of p o t e n t i a l l y t o x i c substances i n t o the bloodstream of e i t h e r c o n t r o l or HD subjects should a l s o be considered. 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Watkins JC and Olverman HJ (1987) Trends N e u r o s c i . , 10:265-272. Wexler NS, Young AB, T a n z i RE, T r a v e r s H, S t a r o s t a - R u b i n s t e i n S, Penney JB, S n o d g r a s s SR, S h o u l s o n I , Gomez F, Ramos A r r o y o MA, Penchaszadeh GK, Moreno H, Gibbons K, F a r y n i a r z A, Hobbs W, Anderson MA, B o n n i l l a E, C o n n e a l l y PM, and G u s e l l a J F (1987) Na t u r e , 326:194-197. W h e t s e l l WO J r and Schwarcz R (1989) N e u r o s c i . L e t t . , 97:271-275. 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 AC, W h e t s e l l WO J r , and Schwarcz R (1983) N e u r o s c i . L e t t . , 41:247-252. 40 Wong EHF, Kemp JA, P r i e s t l y T, K n i g h t AR, Woodruff GN, and I v e r s o n L L (1986) P r o c . N a t l . Acad. S c i . (USA), 83:7104-7108. Wr o b l e w s k i J T and Danysz W (1989) Ann. Rev. P h a r m a c o l . T o x i c o l . , 29:441-474. Young AB, Greenamyre J T , H o l l i n g s w o r t h Z, A l b i n R, D'Amato C, S h o u l s o n I , and Penney JB (1988) s c i e n c e , 241:981-983. 41 CHAPTER I I I INVESTIGATION OF TBE PUTATIVE ROLE OF BETA-N-METBYLAMINO—L —ALANINE (BMAA) IN THE PATBOGENESIS OF AMYOTROPBIC LATERAL SCLEROSIS AND TBE PARKINSON-DEMENTIA SYNDROME OF GUAM I n t r o d u c t i o n A m y o t r o p h i c l a t e r a l s c l e r o s i s (ALS) i s a r e l e n t l e s s l y p r o g r e s s i v e n e u r o l o g i c a l d i s o r d e r which r e s u l t s i n d r a m a t i c motor neuron d e g e n e r a t i o n . Upper motor weakness appears e a r l y i n t h e c o u r s e o f the d i s e a s e , b u t muscle weakness p r o g r e s s e s s t e a d i l y and becomes w i d e s p r e a d and s y m m e t r i c a l . Most v i c t i m s d i e from r e s p i r a t o r y f a i l u r e o r r e l a t e d c a u s e s . The p a t h o l o g i c a l h a l l m a r k s o f ALS i n c l u d e d e g e n e r a t i o n o f a n t e r i o r h o r n c e l l s and t h e i r p e r i p h e r a l motor axons which l e a d s t o n e u r o g e n i c muscle a t r o p h y ( B o n d u e l l 1975). B i o c h e m i c a l changes i n ALS i n c l u d e o b s e r v a t i o n s o f c e r e b r a l hypometabolism ( D a l a k a s e t a l . 1987) and a g e n e r a l i z e d d e f e c t i n gl u t a m a t e m e t a b o l i s m ( P l a i t a k i s and C a r o s c i o 1987) w h i c h may be a s s o c i a t e d w i t h a l t e r e d l e v e l s o f g l u t a m a t e i n b r a i n ( P e r r y e t a l . 1987; P l a i t a k i s e t a l . 1988) and s p i n a l c o r d ( P l a i t a k i s e t a l . 1988). E p i d e m i o l o g i c a l and g e n e t i c s t u d i e s s u g g e s t t h e e x i s t e n c e o f a t l e a s t t h r e e forms o f ALS ( K u r l a n d and Brody 1975). W h i l e t h e g r e a t e s t p r e v a l e n c e (90-95%) o f c a s e s appears t o be s p o r a d i c , a s m a l l b u t 42 s i g n i f i c a n t number a r e b e l i e v e d t o be g e n e t i c a l l y l i n k e d i n an autosomal dominant f a s h i o n . I n a d d i t i o n , t h e r e appears t o be a s p e c i a l form o f ALS which i s combined w i t h P a r k i n s o n i s m and dementia (PD) and i s i s o l a t e d g e o g r a p h i c a l l y t o t h e M a r i a n a s I s l a n d s o f t h e s o u t h w e s t e r n P a c i f i c ( K u r l a n d and M o l g a a r d 1982), t h e K i i p e n i n s u l a o f Japan (Yase 1979) and West New Guinea, I n d o n e s i a (Gajdusek 1979). W h i l e s p o r a d i c ALS has a p r e v a l e n c e r a t e o f 5 t o 7 p e r 100,000, t h e i n c i d e n c e o f t h e Guamanian form among t h e i n d i g e n o u s p e o p l e o f Guam (Chamorros) was much h i g h e r . D u r i n g t h e p a s t 30 y e a r s , however, t h e o c c u r r e n c e o f t h e Guamanian form o f ALS has dropped s t e a d i l y and t h e u n u s u a l l y h i g h r e g i o n a l c o n c e n t r a t i o n has a l l b u t d i s a p p e a r e d ( G a r r u t o e t a l . 1985; P l a t o e t a l . 1986). A l t h o u g h many h y p o t h e s e s have been p r o p o s e d f o r t h e a e t i o l o g i c a l l i n k s f o r ALS, no c a u s a t i v e f a c t o r has y e t been d e m o n s t r a t e d . Two c u r r e n t h y p o t h e s e s r e g a r d i n g t h e Guamanian form o f ALS i n c l u d e a m i n e r a l d e f i c i e n c y r e s u l t i n g i n m e t a l i n t o x i c a t i o n (Yase 1979; G a r r u t o e t a l . 1985; 1986) and c y c a d i n g e s t i o n r e s u l t i n g i n motor system n e u r o t o x i c i t y ( W h i t i n g 1963; K u r l a n d and M o l g a a r d 1982; Spencer 1987; Spen c e r e t a l . 1987a). The sec o n d h y p o t h e s i s i s c o n c e r n e d w i t h t h e consumption o f a f l o u r made o f t h e t o x i c seeds o f t h e f a l s e sago palm, Cycas c i r c i n a l i s . T h i s p l a n t s p e c i e s i s w e l l d i s t r i b u t e d t h r o u g h o u t t h e a r e a s o f t h e s o u t h w e s t e r n P a c i f i c showing a h i g h p r e v a l e n c e o f ALS. A l t h o u g h a c o n s i d e r a b l e amount o f r e s e a r c h has been d e v o t e d t o i n v e s t i g a t i n g t h e 43 p o s s i b l e r o l e of the cycad i n ALS, most e f f o r t s were d i r e c t e d toward examining the putative r o l e of cycasin i n the disease. Another plant t o x i n , beta-N-methylamino-L-alanine (BMAA) present i n the seeds at a concentration of 0.015% (Nunn et a l . 1968), has received l i t t l e a t t e n t i o n . Twenty years a f t e r i t s i n i t i a l i s o l a t i o n (Vega and B e l l 1967), Spencer et a l . (1987b) reported that chronic o r a l administration of synthetic L-BMAA to male macaques induced signs of c o r t i c o s p i n a l dysfunction, as w e l l as chromatolytic and degenerative changes of motor neurons i n the c e r e b r a l cortex and s p i n a l cord s i m i l a r to those observed i n ALS. However biochemical data were not reported. In the foregoing studies, o r a l administration of BMAA ensured that the c o n t r i b u t i o n of any p o t e n t i a l l y neurotoxic metabolites, i n c l u d i n g those formed as a r e s u l t of the a c t i o n of i n t e s t i n a l b a c t e r i a , would not be overlooked. Beta-N-methylamino-L-alanine i s chemically s i m i l a r to beta-N-oxalylamino-L-alanine (BOAA) (Figure 2). Beta-N-oxalylamino-L-alanine i s a neurotoxic amino a c i d found i n the c h i c k l i n g pea Lathyrus sativus which seems to be responsible f o r the development of a form of i r r e v e r s i b l e s p a s t i c paraparesis known as lathyrism (Spencer et a l . 1984; 1986; Hugon et a l . 1988). The L-isomers of both BMAA and BOAA have both been shown to be neurotoxic, while the D-isomers were non-active i n a number of animal models (Nunn et a l . 1987; Polsky et a l . 1972; Rao and Sharma 1966). Experiments i n v i t r o have demonstrated th a t both plant toxins act on glutamate receptors of mammalian neurons, BMAA binding to 44 NMDA r e c e p t o r s (Nunn e t a l . 1987) w h i l e BOAA b i n d s t o t h e non-NMDA typ e g l u t a m a t e r e c e p t o r s (Ross e t a l . 1987). W h i l e BMAA d i f f e r s f rom t h e m a j o r i t y o f e x c i t a t o r y amino a c i d s i n i t s l a c k o f an omega a c i d i c m o i e t y (see F i g u r e 2 ) , r e c e n t e x p e r i m e n t s have s u g g e s t e d t h a t BMAA i n t e r a c t s e i t h e r n o n - c o v a l e n t l y (Weiss and C h o i 1988) o r c o v a l e n t l y (Morz 1989) w i t h b i c a r b o n a t e a n i o n s t o produce a m o l e c u l a r c o n f o r m a t i o n w h i c h a c t i v a t e s NMDA r e c e p t o r s . O b j e c t i v e The o b j e c t i v e o f t h i s i n v e s t i g a t i o n was t o c o n f i r m o r d i s p r o v e the r o l e o f b e t a - N - m e t h y l a m i n o - L - a l a n i n e (BMAA) i n t h e p a t h o g e n e s i s o f the Guamanian form o f ALS u s i n g mice i n s t e a d o f p r i m a t e s , and s e c o n d l y , t o f u r t h e r a s s e s s t h e n e u r o p r o t e c t i v e e f f i c a c y o f MK-801 i n t h i s p u t a t i v e a n i m a l model o f ALS. Hypothesis E x c e s s i v e o r a l consumption o f BMAA i s r e s p o n s i b l e f o r t h e c l i n i c a l , b e h a v i o u r a l and n e u r o p a t h o l o g i c a l changes seen i n t h e Guamanian form o f ALS and ALS/PD. Due t o t h e s p e c i f i c a c t i o n o f BMAA on NMDA-type g l u t a m a t e r e c e p t o r s , t h e n o n - c o m p e t i t i v e NMDA a n t a g o n i s t MK-801 s h o u l d have n e u r o p r o t e c t i v e e f f e c t s i n a n i m a l s t r e a t e d w i t h BMAA. 45 E x p e r i m e n t a l O u t l i n e M i c e were d i v i d e d i n t o 4 groups a t t h e b e g i n n i n g o f t h e e x p e r i m e n t . Two groups r e c e i v e d D,L-BMAAHCl w h i l e t h e two r e m a i n i n g groups r e c e i v e d d i s t i l l e d w a t e r . A l l f o u r groups r e c e i v e d e i t h e r D,L—BMAAHCl o r d i s t i l l e d water by gavage on t h e same s c h e d u l e . MK-801 was a d m i n i s t e r e d t o one o f t h e 2 groups o f BMAA-treated mice, and one o f the 2 groups o f c o n t r o l mice d a i l y by subcutaneous i n j e c t i o n . A t t h e end o f 11 weeks o f BMAA a d m i n i s t r a t i o n , mice were k i l l e d by c e r v i c a l d i s l o c a t i o n , and t h e i r s t r i a t a and motor c o r t i c e s were d i s s e c t e d o u t and f r o z e n f o r b i o c h e m i c a l s t u d i e s . R e s i d u a l b r a i n t i s s u e and v e r t e b r a l columns ( c o n t a i n i n g t h e s p i n a l c o r d ) were f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n s o l u t i o n f o r n e u r o p a t h o l o g i c a l e x a m i n a t i o n . C o n t e n t s o f dopamine, i t s m e t a b o l i t e s 3 , 4 - d i h y d r o x y - p h e n y l a c e t i c a c i d (DOPAC) and h o m o v a n i l l i c a c i d (HVA), and o f n o r a d r e n a l i n e (NA), s e r o t o n i n (5-HT) and 5 - h y d r o x y i n d o l e - a c e t i c a c i d (5-HIAA) were measured i n i n d i v i d u a l s t r i a t a by h i g h performance l i q u i d c hromatography (HPLC) w i t h e l e c t r o c h e m i c a l d e t e c t i o n ( P e r r y e t a l . 1985) (see b e l o w ) . C o n t e n t s o f amino a c i d s w h i c h a c t as n e u r o t r a n s m i t t e r s o r n e u r o m o d u l a t o r s were d e t e r m i n e d i n i n d i v i d u a l mouse c o r t i c e s by automated amino a c i d chromatography ( P e r r y e t a l . 1968; 1981) (see b e l o w ) . 46 s t a t i s t i c a l a n a l y s i s o f r e s u l t s was a c c o m p l i s h e d w i t h S t u d e n t ' s u n p a i r e d t t e s t . A l l b i o c h e m i c a l and h i s t o l o g i c a l s t u d i e s were c a r r i e d o u t by i n v e s t i g a t o r s w i t h o u t knowledge as t o w h i c h t r e a t m e n t groups the mice were i n . S y n t h e s i s o f D,L,-BMAA D,L-BMAAHCl was s y n t h e s i z e d by Dr. T.L. P e r r y from 2 - a c e t a m i d o a c r y l i c a c i d (Sigma) and 30% aqueous methylamine as p r e v i o u s l y d e s c r i b e d (Vega e t a l . 1968). B r i e f l y , 2 - a c e t a m i d o a c r y l i c a c i d was d i s s o l v e d i n 30% methylamine f o r 72 h r . a t 40*C. E x c e s s methylamine was removed under vacuum and t h e r e a c t i o n m i x t u r e c o n c e n t r a t e d t o a s y r u p . D , L - a c e t a m i n o - m e t h y l a m i n o p r o p i o n i c a c i d was s e p a r a t e d as p r e c i p i t a t e d c r y s t a l s f o l l o w i n g t h e a d d i t i o n o f e t h a n o l . T h i s compound was d e a c e t y l a t e d by r e f l u x i n g f o r 2 h r . a t 100° c i n 2.5 M HCl and c o n c e n t r a t e d under vacuum. E x c e s s HCl was removed as an a z e o t r o p e by s e v e r a l c o - e v a p o r a t i o n s w i t h added, d i s t i l l e d H 20. The f i l t e r e d and c o n c e n t r a t e d s o l u t i o n y i e l d e d D,L-BMAAHCl when r e c r y s t a l l i z e d from aqueous e t h a n o l . The t w i c e r e - c r y s t a l l i z e d compound had a m e l t i n g p o i n t ( w i t h 0 d e c o m p o s i t i o n ) o f 178 C, gave a s i n g l e n i n h y d r i n - r e a c t i v e peak when chromatographed on an a u t o m a t i c amino a c i d a n a l y z e r , and a s i n g l e n i n h y d r i n - p o s i t i v e s p o t when chromatographed on p a p e r . N u c l e a r magnetic r e s o n a n c e s p e c t r o s c o p y gave s i g n a l s i d e n t i c a l t o t h o s e o f p u r e BMAA and 47 d i s p l a y e d no e v i d e n c e o f i m p u r i t i e s . o p t i c a l r o t a t i o n measurements c o n f i r m e d an e q u a l d i s t r i b u t i o n o f o p t i c a l i s o m e r s . A d m i n i s t r a t i o n of P,L-BMAA Gavage f e e d i n g s o f D,L-BMAAHCl (100 mg/ml d i s s o l v e d i n B^O) were g i v e n t h r o u g h a p o l y e t h y l e n e tube w i t h o u t a n e s t h e s i a and w i t h o u t m e c h a n i c a l i n j u r y t o t h e a n i m a l s . BMAA-treated mice r e c e i v e d 500 mg/kg d a i l y f o r 18 days, t h e n 500 mg/kg e v e r y o t h e r day o v e r 28 days, and f i n a l l y 1000 mg/kg e v e r y o t h e r day o v e r a f u r t h e r 30 d a y s . The t o t a l dose o f D,L-BMAAHC1 r e c e i v e d by each a n i m a l o v e r t h e 11 week p e r i o d was 31.0 g/kg, h a l f o f which was t h e L - i s o m e r ( i n d i c a t e d by o p t i c a l r o t a t i o n measurements). As a r e s u l t o f BMAA a d m i n i s t r a t i o n , mice r e c e i v e d a t o t a l o f 15.5 g/kg o f L-BMAAHCl d u r i n g 11 weeks, c o r r e s p o n d i n g t o c a . 435 mg p e r mouse. Based upon a maximal L-BMAA c o n t e n t o f a p p r o x i m a t e l y 100 mg p e r 100 g o f u n p r o c e s s e d Cycas c i r c i n a l i s f l o u r (Duncan e t a l . 1988), e a c h mouse would have had t o consume o v e r 400 g o f u n p r o c e s s e d c y c a d seeds t o a c h i e v e t h i s dosage. A d m i n i s t r a t i o n of MK-801 I n an e f f o r t t o p r o t e c t t h e BMAA-treated mice a g a i n s t any p o s s i b l e n e u r o t o x i c e f f e c t s o f BMAA, one o f t h e 2 groups o f BMAA-treated mice and one o f t h e 2 groups o f c o n t r o l mice, were i n j e c t e d s u b c u t a n e o u s l y once d a i l y w i t h MK-801 (Merck, s h a r p & Dohme R e s e a r c h L a b o r a t o r i e s ) . MK-801 48 was d i s s o l v e d i n 0.9% NaCl and was given at a dosage of 2 mg/kg f o r the f i r s t 12 days, and t h e r e a f t e r at a dosage of 1 mg/kg. The higher dosage produced severe a t a x i a and excitement l a s t i n g f o r about 3 hours, s i m i l a r to symptoms reported f o r r a t s given MK-801 (Koek et a l . 1988). Mice l o s t weight and a few died u n t i l the MK-801 d a i l y dosage was reduced to 1 mg/kg. At the lower dosage, ataxia and excitement were reduced, and no f u r t h e r f a t a l i t i e s r e s u l t e d from MK-801 administration. A n a l y s i s of Biogenic Amines Contents of DA, DOPAC, HVA, and of NA, 5-HT and 5-HIAA were measured i n i n d i v i d u a l s t r i a t a by HPLC with electrochemical detection, as p r e v i o u s l y described (Perry et a l . 1985), with a few minor mod i f i c a t i o n s . B r i e f l y , mouse s t r i a t a were homogenized i n ca. 1 ml of 0.1 M HC104 containing 0.4 M NaHS03. Homogenates were ce n t r i f u g e d at 10,000 x g f o r 15 min at 4 aC and the r e s u l t i n g supernatants were f i l t e r e d through a M i l l i p o r e SJHV f i l t e r (0.45 um). Between 5 and 25 u l of each sample were i n j e c t e d i n t o the HPLC system with e x t e r n a l standards i n j e c t e d every second to f o r t h sample of striatum to compensate f o r s l i g h t changes i n system s e n s i t i v i t y . The HPLC system consisted of a reverse-phase chromatographic column (60 x 4.6 mm ODS H y p e r s i l , dp 3um) and an LC-4A amperometric detector ( B i o a n a l y t i c a l Systems) with a glassy carbon el e c t r o d e . The p o t e n t i a l was set at +0.7 V with respect to an Ag/AgCl reference 49 e l e c t r o d e . A two c h a n n e l r e c o r d e r s e t a t 2 and 20 nA f u l l s c a l e d e f l e c t i o n was u s e d t o a l l o w q u a n t i f i c a t i o n o f a l l compounds, t h e c o n c e n t r a t i o n o f w h i c h o f t e n d i f f e r e d by more t h a n an o r d e r o f magnitude. The m o b i l e phase c o n s i s t e d o f an aqueous s o l u t i o n o f NaH 2PO^ (0.1 M, c o n t a i n i n g 60 mg Na 2EDTA and 200 mg sodium o c t y l s u l f a t e p e r l i t e r ) . The pH was a d j u s t e d t o 3.85 4 0.01 w i t h 3 M H 3 P 0 4 , and t h e f l o w r a t e was 1.0 ml/min. I n d o l e s (5-HT and 5-HIAA) were a n a l y z e d w i t h t h e a d d i t i o n o f 10% MeOH and a c i d i f i c a t i o n o f t h e m o b i l e phase t o pH 3.60 i n o r d e r t o d e c r e a s e t h e r e t e n t i o n time o f t h e s e compounds. B r a i n c o n t e n t s of compounds were c a l c u l a t e d from t h e a m p l i t u d e o f t h e peak d e f l e c t i o n s w h ich t h e y e x h i b i t e d upon chromatograms. 50 A n a l y s i s of Amino Acids C o n t e n t s o f amino a c i d s were d e t e r m i n e d i n i n d i v i d u a l mouse c o r t i c e s by a u t o m a t i c amino a c i d chromatography ( P e r r y e t a l . 1 9 6 8 ; 1 9 8 1 ) . T h i s system employed l i t h i u m c i t r a t e b u f f e r s i n a c o n t i n u o u s b u f f e r g r a d i e n t , f o r use on s i n g l e c a t i o n exchange r e s i n columns o f a T e c h n i c o n amino a c i d a n a l y z e r . The t e c h n i q u e a l l o w e d adequate r e s o l u t i o n o f a l a r g e number o f amino a c i d s , up t o and i n c l u d i n g a r g i n i n e , i n a s i n g l e c h r o m a t o g r a p h i c r u n o f c a . 2 1 h o u r s . The c a . 80 x 0 . 6 cm g l a s s columns were f i l l e d a t room temperature w i t h a c a t i o n exchange r e s i n ( P C - 6 A , P i e r c e C h e m i c a l s o r Durrum DC - 6 A , Benson P o l y m e r i c I n c . ) a f t e r t h e r e s i n was washed s u c c e s s i v e l y w i t h a c e t o n e , d i s t i l l e d H 2o, 6 M n i t r i c a c i d , d i s t i l l e d H 2o, 2 M LiOH and d i s t i l l e d H-jO. B e f o r e each c h r o m a t o g r a p h i c r u n , t h e r e s i n was r e g e n e r a t e d by pumping 0 . 2 M LiOH t h r o u g h t h e column f o r 30 min, f o l l o w e d by 0 . 2 M l i t h i u m c i t r a t e b u f f e r (pH 2 . 8 0 ) f o r a f u r t h e r 90 min. The columns was m a i n t a i n e d a t 70*C t h r o u g h o u t t h e r e g e n e r a t i o n p r o c e s s . Once t h e samples were l o a d e d , e l u t i o n b u f f e r was pumped t h r o u g h t h e columns a t a c o n s t a n t f l o w r a t e o f 0 . 5 ml/min. A nine-chambered A u t o g r a d was u s e d t o s u p p l y t h e g r a d i e n t e l u t i o n b u f f e r . Temperature o f t h e columns were m a i n t a i n e d a t 35 C f o r t h e f i r s t 6 . 5 h r , and t h e r e a f t e r a t 70*C f o r t h e r e m a i n d e r o f t h e r u n . The column e f f l u e n t s c o n t a i n i n g t h e s e p a r a t e d amino compounds were r e a c t e d w i t h n i n h y d r i n and t h e r e s u l t i n g c o l o u r e d n i n h y d r i n - p o s i t i v e compounds were p a s s e d t h r o u g h 51 c o l o r i m e t e r s . Q u a n t i f i c a t i o n o f t h e amino a c i d s was a c c o m p l i s h e d by t h e co m p a r i s o n o f t h e t o t a l a r e a s under t r a c i n g s o f o p t i c a l d e n s i t y (570 and 440 mu) w i t h r e s p e c t t o known, a u t h e n t i c s t a n d a r d s . I n a l l c a s e s , n o r l e u c i n e was added as an i n t e r n a l s t a n d a r d t o m i n i m i z e s m a l l f l u c t u a t i o n s i n c o l o r i m e t r i c d e t e r m i n a t i o n s . N e u r o p a t h o l o g i c a l S t u d i e s N e u r o p a t h o l o g y was per f o r m e d by Dr. C a t h e r i n e B e r g e r o n , Department o f P a t h o l o g y , U n i v e r s i t y o f T o r o n t o . These s t u d i e s were a c c o m p l i s h e d by m i c r o s c o p i c e x a m i n a t i o n o f t h e f o r m a l i n - f i x e d t i s s u e s o f t h e b r a i n and s p i n a l c o r d s o f t h e e x p e r i m e n t a l a n i m a l s . F i v e m i c r o n t h i c k s e c t i o n s were o b t a i n e d a f t e r p a r a f f i n embedding and were s t a i n e d w i t h h e m a t o x y l i n - e o s i n / l u x o l f a s t b l u e . S e l e c t e d s e c t i o n s were l a t e r s t a i n e d w i t h c r e s y l v i o l e t and B i e l s c h o w s k y ' s s i l v e r method. R e s u l t s A l l mice g i v e n D,L-BMAAHCl m a i n t a i n e d t h e i r w e i g h t and e x h i b i t e d no b e h a v i o u r a l a b n o r m a l i t i e s t h r o u g h o u t t h e e x p e r i m e n t . BMAA was d e t e c t a b l e i n t h e b r a i n and l i v e r f o r up t o 48 h o u r s a f t e r d o s i n g . BMAA c o n c e n t r a t i o n s i n l i v e r v a r i e d from 0.1 t o 0.3 mM, and i n b r a i n from 0.1 t o 0.15 mM, 24 h o u r s a f t e r BMAAHCl doses o f 500 mg/kg. F o r t y - e i g h t h ours a f t e r a dosage o f 1000 mg/kg, l i v e r and b r a i n BMAA c o n c e n t r a t i o n s were 0.2 and 0.5 mM, r e s p e c t i v e l y . Presumably t h e compound measured was 52 the L-isomer, since the D-isomer would not be expected to be reabsorbed w e l l from the glomerular f i l t r a t e ( S i l b e r n a g l 1988). Mean contents of the catecholamines DA, HVA and DOPAC, and the indoles 5-HT and 5-HIAA were not a l t e r e d i n the s t r i a t a of the BMAA-tre a t e d mice as compared to c o n t r o l mice (Table I I I ) . The mean contents of aspartate and glutamate were not s i g n i f i c a n t l y decreased i n the c o r t i c e s of mice given BMAA alone as compared to con t r o l s (Table IV). Mean glycine l e v e l s were s i g n i f i c a n t l y lower i n the c o r t i c e s of mice given BMAA (with or without MK-801) than i n con t r o l s (Table IV). Microscopic examination of br a i n and s p i n a l cord showed no pa t h o l o g i c a l changes i n any of the animals. S p e c i f i c a l l y , c e r e b r a l cortex, hippocampus, striatum and substantia nigra were normal, with no evidence of neuronal l o s s , g l i o s i s or n e u r o f i b r i l l a r y degeneration. The s p i n a l cords, examined i n two d i f f e r e n t segments, revealed no abnormalities. A n t e r i o r horn c e l l s (Figure 4) were present i n normal numbers, with no evidence of chromatolysis and no intracytoplasmic i n c l u s i o n s or n e u r o f i b r i l l a r y tangles. Axonal swellings were not seen, and the c o r t i c o s p i n a l t r a c t s were normal throughout the neuraxis. 53 Table I I I : 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 a n d i t s m e t a b o l i t e s i n m i c e t r e a t e d f o r 1 1 w e e k s w i t h D,L-BMAA, w i t h o r w i t h o u t c o n c u r r e n t a d m i n i s t r a t i o n o f M K - 8 0 1 . Compound Controls BHAA BMAA • MK-801 MK-801 Dopaiine 12.97 i 0.84 (8) 14.29 ! 0.65 (11) 1S.09 ! 0.82 (12) 12.35 ! 0.72 (8) DOPAC 1.30 t 0.09 (9) 1.32 i 0.11 (12) 1.30 t 0.05 (12) 0.97 ! 0.05 § (7) HVA 1.44 i 0.05 (8) 1.75 ! 0.15 (12) 1.50 t 0.06 (12) 1.20 t 0.08 «• (8) Values dean ! SEM) are expressed in ug/g vet weight, with nuiber of aniials shown in parentheses. Significantly different froa values for control aice (unpaired t test) t P < 0.01, « P < 0.02 54 T a b l e I V : Contents of taurine ( T A U ) , aspartate ( A S P ) , glutamate ( G L U ) , glutamine ( G L N ) , g l y c i n e ( G L T ) and gamma-aminobutyric a c i d ( G A B A ) i n c e r e b r a l c o r t i c e s of mice t r e a t e d f o r 11 weeks with D , L - B M A A , with or without concurrent administration of MK-801. Aiino Compound Controls BMAA BMAA • UK-801 MK-801 (8) (12) (12) (8) TAU 12.61 1 0.6S 12.19 ! 0.30 12.66 i 0.40 12.24 1 0.50 ASP 3.82 1 0.13 3.81 ! 0.08 3.47 1 0.09 3.62 1 0.13 6LU 1S.60 1 0.24 15.00 • 0.25 15.14 t 0.22 15.22 i 0.41 GLN 7.27 1 0.26 7.12 1 0.34 7.84 J 0.17 7.32 10.32 6LV 0.79 1 0.04 0.69 1 0.03** 0.65 1 0.03* - 0.75 1 0.02 GABA 3.13 1 0.27 3.03 1 0.07 2.91 1 0.14 3.06 1 0.16 Values dean ! SEH) are expressed in uaol/g wet weight, with naiber of aniaals shown in parentheses. Significantly different f r o i value for control l i c e (unpaired t test) t P < 0.01, « P < 0.05. 55 Figure 4: A n t e r i o r horn c e l l s i n the s p i n a l cords of a mouse treated with D,L-BMAA alone and i n a c o n t r o l mouse. A B BMAA-alone Control No h i s t o l o g i c a l changes other than scattered dark neurons r e s u l t i n g from manipulation of the t i s s u e p r i o r t o f i x a t i o n are present i n e i t h e r A or B. C r e s y l v i o l e t , o r i g i n a l m a g n i f i c a t i o n x 400. 56 D i s c u s s i o n R ecent o b s e r v a t i o n s o f a d e c r e a s e i n g l u t a m a t e ( P e r r y e t a l . 1987) and a s p a r t a t e ( P l a i t a k i s e t a l . 1988) i n a u t o p s i e d b r a i n and s p i n a l c o r d o f s p o r a d i c ALS v i c t i m s have n o t been d u p l i c a t e d by t h e s e e x p e r i m e n t a l r e s u l t s , a l t h o u g h t h e r e a r e no r e p o r t s o f measurements o f t h e s e compounds i n t h e Guamanian form o f ALS. B i o c h e m i c a l a n a l y s i s o f dopamine and i t s m e t a b o l i t e s r e v e a l e d no changes s u g g e s t i v e o f damage t o 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 n e u r o n s . Moreover, m i c r o s c o p i c e x a m i n a t i o n o f t h e b r a i n and s p i n a l c o r d s o f BMAA-treated mice f a i l e d t o i n d i c a t e any h i s t o l o g i c a l a l t e r a t i o n s i n d i c a t i v e o f e i t h e r ALS o r PD. Thus, r e p e a t e d l a r g e doses o f D,L-BMAA t o mice o v e r an 11 week p e r i o d f a i l e d t o pr o d u c e any d e t e c t a b l e b e h a v i o u r a l , b i o c h e m i c a l o r n e u r o p a t h o l o g i c a l c h a n ges. Repeated a d m i n i s t r a t i o n o f l a r g e amounts o f D,L-BMAA o r a l l y t o mice does n o t produce an a n i m a l model o f ALS o r P a r k i n s o n ' s d i s e a s e . I t i s p o s s i b l e t h a t r o d e n t s a r e not s u s c e p t i b l e t o t h e n e u r o t o x i c e f f e c t s o f BMAA, whereas p r i m a t e s a r e , o r t h a t BMAA must be a d m i n i s t e r e d f o r a much l o n g e r p e r i o d t o produce any e f f e c t . However, o u r e x p e r i m e n t does n o t l e n d s u p p o r t t o t h e h y p o t h e s i s t h a t BMAA d e r i v e d from c y c a d seeds cau s e d t h e ALS/PD f o r m e r l y so common on Guam. I t i s s t i l l p o s s i b l e , o f c o u r s e , t h a t some compound u n r e l a t e d t o BMAA was p r e s e n t i n c y c a d seeds and 57 caused the Guamanian disease, our r e s u l t s do not suggest that sporadic ALS or i d i o p a t h i c Parkinson's disease are caused by u n i d e n t i f i e d d i e t a r y sources of BMAA. A p o s i t i v e r e s u l t of the experiment i s the demonstration that mice can t o l e r a t e d a i l y i n j e c t i o n s of MK-801 of 1 mg/kg f o r at l e a s t 11 weeks without any permanent behavioural e f f e c t s or b r a i n biochemical changes i n the parameters measured. E f f e c t i v e antagonism of NMDA receptors i n the human b r a i n might be of c l i n i c a l value i n the treatment of ne u r o l o g i c a l disorders such as Huntington's disease, i n which neurons appear to be damaged by e x c i t o t o x i c a c t i o n mediated by these receptors (Young et a l . 1988). The s i g n i f i c a n t decreases i n gly c i n e concentrations i n the striatum of the mice treated with BMAA alone, as wel l as BMAA and MK-801 are unexplained, but i t should be emphasized that g l y c i n e i s known to regulate a subset of NMDA re c o g n i t i o n s i t e s (Monaghan et a l . 1988; Johnson and Ascher 1987; Bonhaus et a l . 1987; B e r t o l i n o et a l . 1987; Mayer et a l . 1989) and modulates MK-801 binding (Wong et a l . 1987). The action of glycine as a neuromodulator has not been p r e c i s e l y characterized; however, the i d e n t i f i c a t i o n of a new cl a s s of antagonists at the s t r y c h n i n e - i n s e n s i t i v e modulatory g l y c i n e receptor (Kemp et a l . 1988; Huettner 1989; Kessler et a l . 1989) should f a c i l i t a t e the c l a r i f i c a t i o n of the f u n c t i o n a l r o l e of the g l y c i n e b i n d i n g - s i t e . 58 References B e r t l o i n o M, V i c i n i S, M a z z e t t a J , and C o s t a E (1988) N e u r o s c i . L e t t . , 84:351-355. B o n d u e l l e M (1975) I n : Handbook o f C l i n i c a l N e u r o l o g y ; V i n k e n P and Bruyn GW (Eds.) N o r t h - H o l l a n d , Amsterdam. Bonhuas DW, Burge BC, and McNamara JO (1987) E u r . J . Pha r m a c o l . , 142:489-490. Dal a k a s MC, e t a l . (1987) Ann. N e u r o l . , 22:580-586. Duncan MW, K o p i n I J , G a r r u t o RM, L a v i n e L, and Markey SP (1988) L a n c e t , ii:631-632. 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Young AB, Greenamyre JT, H o l l i n g s w o r t h Z, A l b i n R, D'Amato C, Sh o u l s o n I, and Penney JB (1988) S c i e n c e , 241:981-983. 61 CHAPTER IV A CASE OF HEREDITARY MENTAL DEPRESSION AND PARKINSONISM: POST-MORTEM STUDY OF BIOGENIC AMINES AND AMINO ACIDS IN BRAIN I n t r o d u c t i o n P a r k i n s o n ' s d i s e a s e (PO) i s a c h r o n i c , d e g e n e r a t i v e and p r o g r e s s i v e n e u r o l o g i c a l c o n d i t i o n o f t h e c e n t r a l n e r vous system (CNS). F i r s t d e s c r i b e d by James P a r k i n s o n i n 1817, PD i s v e r y common w i t h a worl d w i d e p r e v a l e n c e o f 1 i n 1000 p e r s o n s . The g e n e r a l c l i n i c a l f e a t u r e s o f PD i n c l u d e : tremor ( s h a k i n g due t o a l t e r n a t i n g c o n t r a c t i o n s (3 t o 5 p e r second) o f a muscle group and i t s a n t a g o n i s t ) w h i c h d e c r e a s e s w i t h v o l u n t a r y movement; p l a s t i c (cogwheel) r i g i d i t y o r s t i f f n e s s o f t h e s k e l e t a l muscles due t o i n c r e a s e d muscle t o n e ; a k i n e s i a r e s u l t i n g i n a p o v e r t y o f spontaneous a c t i o n s , masked f a c i a l e x p r e s s i o n ; and d i s t u r b a n c e o f p o s t u r e , which u s u a l l y o c c u r s l a t e i n t h e c o u r s e o f t h e d i s e a s e and i s c h a r a c t e r i z e d by an i n a b i l i t y t o m a i n t a i n an u p r i g h t p o s i t i o n o f t h e upper body w h i l e s t a n d i n g o r w a l k i n g ( H o r n y k i e w i c z 1986). I n a d d i t i o n t o motor d i s t u r b a n c e s , a f f e c t i v e d i s o r d e r s and demen t i a a r e t h e most c o n s i s t e n t l y n o t e d b e h a v i o u r a l a b n o r m a l i t i e s o f PD, r e p o r t e d t o o c c u r i n a l a r g e p e r c e n t a g e o f c a s e s (Benson 1984). A l t h o u g h t h e i n c i d e n c e o f ment a l d e p r e s s i o n i s s i g n i f i c a n t l y h i g h e r i n p a t i e n t s w i t h PD t h a n i n t h e normal p o p u l a t i o n (Mayeux e t a l . 1984), t h e 62 d e g r e e o f t h e d e p r e s s i o n does n o t appear t o be r e l a t e d t o t h e d u r a t i o n o r s e v e r i t y o f t h e d i s e a s e p r o c e s s (Warburton 1967). P a r k i n s o n ' s d i s e a s e i s r e g a r d e d as a c l a s s i c example o f a d i s t u r b a n c e o f b a s a l g a n g l i o n f u n c t i o n . T h i s n e u r o d e g e n e r a t i v e d i s e a s e r e s u l t s i n t h e s p e c i f i c and c h a r a c t e r i s t i c l o s s o f m e l a n i n - c o n t a i n i n g neurons i n t h e compact zone o f th e s u b s t a n t i a n i g r a and, t o a l e s s e r e x t e n t , o t h e r m e l a n i n - c o n t a i n i n g b r a i n stem n u c l e i ( H a s s l e r 1938). T h i s d e g e n e r a t i o n i s a s s o c i a t e d w i t h t h e f o r m a t i o n o f c h a r a c t e r i s t i c e o s i n o p h i l i c c y t o p l a s m i c i n c l u s i o n s , r e f e r r e d t o as Lewy b o d i e s (Forno 1986; A l v o r d and F o r n o 1987), and r e s u l t s i n a s e v e r e r e d u c t i o n o f a l l p r e s y n a p t i c n e u r o c h e m i c a l i n d i c e s o f d o p a m i n e - c o n t a i n i n g neurons i n a l l t e l e n c e p h a l i c a r e a s n o r m a l l y r e c e i v i n g d o p a m i n e r g i c i n n e r v a t i o n ( r e v i e w e d by H o r n y k i e w i c z 1966; 1973; 1979). The s i g n i f i c a n t r e d u c t i o n o f DA ca n be d i r e c t l y a t t r i b u t e d t o th e l o s s o f d o p a m i n e r g i c c e l l b o d i e s i n t h e s u b s t a n t i a n i g r a and a d j o i n i n g a r e a s , and c o r r e l a t e s w i t h t h e s e v e r i t y o f p a r k i n s o n i a n d i s a b i l i t y , e s p e c i a l l y t h a t o f a k i n e s i a (Bernheimer e t a l . 1973). The r e d u c t i o n o f DA i s w e l l documented i n the c a u d a t e n u c l e u s , putamen, s u b s t a n t i a n i g r a and g l o b u s p a l l i d u s ( E h r i n g e r and H o r n y k i e w i c z 1960; Bernheimer e t a l . 1963; 1973; H o r n y k i e w i c z 1963; Fahn e t a l . 1971; Rinne e t a l . 1974; L l o y d e t a l . 1975). The r e d u c t i o n o f DA i s i n d e p e n d e n t o f t h e a e t i o l o g y o f PD, t h e o n l y d e t e r m i n a n t b e i n g t h e d e g r e e o f n e u r o n a l c e l l l o s s i n t h e zona compacta o f t h e s u b s t a n t i a n i g r a (Bernheimer e t a l . 1973); however, i t has been e s t i m a t e d t h a t a 63 l o s s o f a t l e a s t 80% must o c c u r b e f o r e t h e o n s e t o f c l i n i c a l symptoms ( H o r n y k i e w i c z 1982). The l o s s o f DA neurons and t h e i m p a i r e d a b i l i t y o f t h e s t r i a t u m t o s y n t h e s i z e and s t o r e DA i n P a r k i n s o n ' s d i s e a s e has r e c e n t l y been d e m o n s t r a t e d w i t h p o s i t r o n e m i s s i o n tomography (PET) u s i n g [ 1 8F]-L-DOPA (Lee n d e r s e t a l . 1986; M a r t i n e t a l . 1986). By t h e p r e s e n t l y a c c e p t e d c l i n i c a l and m o r p h o l o g i c a l c r i t e r i a , PD can be s u b d i v i d e d i n t o f o u r main a e t i o l o g i c a l c a t e g o r i e s : a) i d i o p a t h i c P a r k i n s o n i s m ; b) p o s t e n c e p h a l i t i c form, a s e q u e l a o f von Economo's e n c e p h a l i t i s l e t h a r g i c a ; c) a r t e r i o s c l e r o t i c form, as p a r t o f a more g e n e r a l i z e d v a s c u l a r e n c e p h a l o p a t h y ( H o r n y k i e w i c z 1986); and d) d r u g -i n d u c e d , r e s u l t i n g from t h e i n t r a v e n o u s abuse o f " d e s i g n e r d r u g s " c o n t a m i n a t e d w i t h t h e m e p e r i d i n e d e r i v a t i v e l - m e t h y l - 4 - p h e n y l -1 , 2 , 3 , 6 - t e t r a h y d r o p y r i d i n e (MPTP) ( D a v i s e t a l . 1979, L a n g s t o n e t a l . 1983). B i o c h e m i c a l changes i n PD i n c l u d e , b u t a r e not l i m i t e d t o , s i g n i f i c a n t r e d u c t i o n s i n t h e a c t i v i t y o f t y r o s i n e h y d r o x y l a s e (TH) and dopa d e c a r b o x y l a s e (DDC) (Rinne 1978; 1979) as w e l l as changes i n DA r e c e p t o r b i n d i n g ( e g . C o r t e s e t a l . 1989). C e r t a i n c h a r a c t e r i s t i c s o f n i g r o s t r i a t a l DA neurons may r e n d e r them p a r t i c u l a r l y v u l n e r a b l e t o damage from o x i d i z i n g r a d i c a l s ( F a r i e l l o and C a l a b r e s e 1988) and a r e d u c t i o n o f g l u t a t h i o n e (GSH) i n t h e s u b s t a n t i a n i g r a ( P e r r y e t a l . 1982) o f p a t i e n t s d y i n g w i t h PD has been r e p o r t e d . The o b s e r v a t i o n o f MPTP-induced p a r k i n s o n i s m t o g e t h e r w i t h e p i d e m i o l o g i c a l e v i d e n c e (Barbeau and Roy 1985; A q u i l o n i u s and H a r t v i g 1986; Schoenberg 1987) 64 s u g g e s t a p o s s i b l e r o l e o f e n v i r o n m e n t a l t o x i n s i n t h e e t i o l o g y o f PD (see Tanner 1989) . P e r r y e t a l . (1975) d e s c r i b e d a n e u r o p s y c h i a t r i c d i s o r d e r o c c u r r i n g i n t h r e e s u c c e s s i v e g e n e r a t i o n s , c h a r a c t e r i z e d by p r o g r e s s i v e m e n t a l d e p r e s s i o n , p a r k i n s o n i s m and d e a t h w i t h i n f o u r t o s i x y e a r s o f the o n s e t o f symptoms, u s u a l l y from r e s p i r a t o r y f a i l u r e . T h i s r e p o r t ( P e r r y e t a l . 1975) was t h e f i r s t a c c o u n t o f an auto s o m a l dominant form o f p a r k i n s o n i s m a s s o c i a t e d w i t h c h r o n i c d e p r e s s i v e i l l n e s s . I n a d d i t i o n t o c l a s s i c h i s t o l o g i c a l f i n d i n g s o f PD, i n c l u d i n g d e p i g m e n t a t i o n o f the s u b s t a n t i a n i g r a and e x t e n s i v e l o s s e s o f neurons i n t h e zona compacta and zona r e t i c u l a r i s , a marked d e f i c i e n c y o f t h e amino a c i d t a u r i n e was found i n t h e b l o o d , c e r e b r o s p i n a l f l u i d (CSF) and b r a i n o f a member o f t h i s p e d i g r e e ( P e r r y 1976). A l t h o u g h a c o r r e l a t i o n between a d e f i c i e n c y i n t a u r i n e and t h e c l i n i c a l syndrome was h y p o t h e s i z e d ( P e r r y e t a l . 1975; P e r r y 1976) t h i s was not s u b s t a n t i a t e d by o t h e r s i n two o t h e r u n r e l a t e d p e d i g r e e s (Purdy e t a l . 1979; Roy e t a l . 1988) and c l i n i c a l t r i a l s o f o r a l t a u r i n e and/or p y r i d o x i n e u n f o r t u n a t e l y showed no s i g n i f i c a n t b e n e f i t ( P e r r y 1976; 1981). O t h e r f a m i l y p e d i g r e e s w i t h a s i m i l a r syndrome have been i d e n t i f i e d , a l l c h a r a c t e r i z e d by autosomal dominant i n h e r i t a n c e , symptoms o f p a r k i n s o n i s m , d e p r e s s i o n , and r e s p i r a t o r y i n s u f f i c i e n c y (Purdy e t a l . 1979; Tune e t a l . 1982; Roy e t a l . 1983; Barbeau and Roy 1984; Roy e t a l . 1988). 65 Case His t o r y The subject of the present study (M892) was a man 58 years of age at the time of h i s death i n 1989. He had complained over the l a s t f i v e to f i v e and one h a l f years of l i f e of a general f e e l i n g of malaise, loss of i n t e r e s t , concentration and v i g o r . A loss of appetite had contributed to a s u b s t a n t i a l los s of weight. Although t h i s p a t i e n t had no obvious f e e l i n g s of sadness, repeated examinations by a v a r i e t y of p r a c t i t i o n e r s i d e n t i f i e d some vegetative signs suggestive of mild depression. No i n d i c a t i o n of psychosis was evident and no d e t e r i o r a t i o n i n memory was observed. Some tremor was noted i n h i s hands which occurred p r i m a r i l y at r e s t but d i d continue somewhat during voluntary a c t i v i t y . He presented with l i t t l e f a c i a l expression and impaired eye movements (bli n k r a t e , down gaze, saccadic v e r t i c a l movement) although n e u r o l o g i c a l t e s t s i n d i c a t e d that h i s c r a n i a l nerves were normal. Computerized tomography (CT) with contrast was normal. P o s i t r o n 18 18 emission tomography (PET) scans with [ FJ-DOPA i n d i c a t e d decreased [ F] a c t i v i t y i n the striatum but l e v e l s were greater than those i n an 18 i n d i v i d u a l with mild to moderate PD. PET observations of [ F] a c t i v i t y i n the putamen of t h i s p a t i e n t (M892) was s i g n i f i c a n t l y higher than i n p a t i e n t s with PD. The p a t i e n t ' s family h i s t o r y i n d i c a t e d that h i s f a t h e r had died at the age of 40 from drowning as a possible r e s u l t of s u i c i d e . Two of his paternal r e l a t i v e s (an aunt and uncle) died of a progressive 66 n e u r o l o g i c a l disorder characterized by a l o s s of energy, c l i n i c a l depression and parkinsonian features. Four of 5 c h i l d r e n of the p a t e r n a l aunt s u f f e r e d from a s i m i l a r problem (a p a r t i a l pedigree i s shown i n Figure 5). Assessment i n the Department of Medical Genetics at the U n i v e r s i t y of B r i t i s h Columbia (U.B.C.) concluded that, i n a l l l i k e l i h o o d , h i s father was a heterozygote who passed the gene responsible f o r the progressive n e u r o l o g i c a l c o n d i t i o n to t h i s p a t i e n t . The gene appears to be transmitted i n an autosomal dominant fashion and therefore each of the patient's s i b l i n g s would have a one i n two chance of developing a s i m i l a r c o n d i t i o n . Since c e n t r a l r e s p i r a t o r y f a i l u r e may be a terminal event i n t h i s disease, the p a t i e n t was assessed i n the Respiratory Sleep Disorder C l i n i c , U.B.C. where mild hypercapnia during sleep and a r t e r i a l blood gas c h a r a c t e r i s t i c s c o n s i s t e n t with c e n t r a l a l v e o l a r h y poventilation was diagnosed. Routine q u a n t i t a t i v e amino a c i d a n a l y s i s completed at the time of the p a t i e n t ' s f i r s t assessment i n 1986 i n d i c a t e d no s i g n i f i c a n t abnormalities i n plasma (see Appendix, Table A- I ) , and s p e c i f i c a l l y d i d not show reduced l e v e l s of taurine (Appendix, Table A-Z) as d i d h i s f i r s t cousin (Perry et a l . 1975). Analyses of h i s c e r e b r o s p i n a l f l u i d (CSF) (Tables V and VI, and Appendix, Table A-II) d i d r e v e a l s e v e r a l consistent (excluding changes as a r e s u l t of pharmacological intervention) abnormalities i n c l u d i n g : a) Moderately elevated p r o t e i n concentration; 67 b) The l e v e l of homovanillic a c i d was s i g n i f i c a n t l y reduced (range 3.1 to 7.3 ng/ml; normal = 37 £ 19); c) The l e v e l of 5-hydroxyindoleacetic a c i d was low (range 3.7 to 8.8 ng/ml; normal = 20 £ 12); d) The concentration of gamma-aminobutyric a c i d was very low (range 3 to 19 nmol/ml; normal = 84 £ 36) . However, none of the CSF analyses i n d i c a t e d abnormally low taurine concentrations (Table V I ) . 68 F i g u r e 5 : P a r t i a l p e d i g r e e o f p a t i e n t M 8 9 2 . Pharmacological interventions considered at t h i s time included the use of t r i c y c l i c antidepressants or monoamine oxidase (MAO) i n h i b i t o r s to t r e a t h i s depression, the use of L-DOPA with Carbidopa to t r e a t p o s s i b l e parkinsonian symptoms, and i s o n i a z i d to increase b r a i n GABA content. The p a t i e n t was i n i t i a l l y placed on a m i t r i p t y l i n e (to a maximum dosage of 150 mg/day) which r e s u l t e d i n signs of improvement, i n c l u d i n g an enhanced appetite and associated weight gain, decreased daytime sleepiness and improved communicativeness. A m i t r i p t y l i n e was gradually discontinued due to some side e f f e c t s and to o f f e r an opportunity to attempt a c l i n i c a l t r i a l of i s o n i a z i d . The t r i a l of i s o n i a z i d was attempted f o r three weeks with dosages of up to 750 mg twice d a i l y . The p a t i e n t began to d e t e r i o r a t e soon a f t e r d i s c o n t i n u a t i o n of a m i t r i p t y l i n e , but before administration of i s o n i a z i d . As a r e s u l t , a m i t r i p t y l i n e was resumed i n c r e a s i n g the dose to 250 mg/day at bedtime. Analysis of CSF and f a s t i n g plasma was done a f t e r each change i n pharmacological i n t e r v e n t i o n . These r e s u l t s are summarized i n Tables V, VI and Appendix, Tables A-I and A - I I . In order to explore the p o s s i b i l i t y that a serotonin d e f i c i e n c y was causing impaired dopaminergic a c t i v i t y i n t h i s i n d i v i d u a l , a c l i n i c a l t r i a l of L - 5-hydroxytryptophan (5-HTP) together with a p e r i p h e r a l decarboxylase i n h i b i t o r added to the a m i t r i p t y l i n e regimen was i n i t i a t e d . This t r i a l was fraught with problems since the p a t i e n t responded to 100 mg of 5-HTP 30 min a f t e r 100 mg carbidopa with nausea, 70 vomiting and weakness which p e r s i s t e d throughout the day. The patient was h o s p i t a l i z e d i n order to r a p i d l y increase the 5-HTP dosage to 500 mg b . i . d . and assess the e f f i c a c y of t h i s treatment. Although plasma l e v e l s of 5-HTP were s i g n i f i c a n t l y increased and some short-term improvement was observed, administration was terminated due to a lack of any l a s t i n g c l i n i c a l b e n e f i t , i n ad d i t i o n to the problems mentioned above. During the l a s t year of the patient's l i f e , he was maintained on a m i t r i p t y l i n e u n t i l p o s t u r a l hypotension became a s i g n i f i c a n t problem and a short t r i a l with f l u o x e t i n e was attempted. A d e t e r i o r a t i o n of his neu r o l o g i c a l c o n d i t i o n r e s u l t e d i n the replacement of f l u o x e t i n e with n o r t r i p t y l i n e which improved h i s postu r a l hypotension and returned h i s neu r o l o g i c a l status to a l e v e l s i m i l a r to that found with a m i t r i p t y l i n e . The p a t i e n t remained apathetic and presented i n much the same way as a year before. A short time l a t e r , he developed a pneumonia which d i d not respond to a n t i b i o t i c treatment, and u l t i m a t e l y l e d to h i s death i n March of 1989. 71 Within hours of death the patient's body was transported to U.B.C. where h i s b r a i n was removed and bisected s a g i t a l l y . One h a l f of the b r a i n was frozen on dry i c e (death-to-freezing i n t e r v a l was 6 hrs and 20 min) while the other h a l f was examined f o r neuropathology. The neuropathologist's report noted the following c h a r a c t e r i s t i c s : a) Marked p a l l o r of the substantia nigra; b) Suggestion of atrophy of the superior c e r e b e l l a r vermis; In a d d i t i o n , microscopic examination revealed f u r t h e r observations, i n c l u d i n g : c) A r t e r i a l and neuronal l o s s , and g l i o s i s i n the thalamus; d) Patchy and quite severe neuronal los s from the substantia n i g r a , i n c l u d i n g extensive g l i o s i s and some f o c a l c o l l e c t i o n s of melanin, presumably i n macrophages; e) No Lewy bodies or n e u r o f i b r i l l a r y tangles were present i n substantia nigra; f) Neurons of the caudate, putamen, globus p a l l i d u s , and median raphe nucleus appeared normal; g) Neurons of the locus caeruleus, medulla, superior c e r e b e l l a r vermis, dentate nucleus, and t h o r a c i c s p i n a l cord were unremarkable. F i n a l P a t h o l o g i c a l Diagnosis: Brain showing degeneration of the substantia nigra and thalamus. 72 Objective T h e o b j e c t o f t h i s i n v e s t i g a t i o n was t o d e t e r m i n e t h e post-mortem c o n c e n t r a t i o n s o f dopamine (DA), i t s m e t a b o l i t e s h o m o v a n i l l i c a c i d (HVA) and 3 , 4 - d i h y d r o x y - p h e n y l a c e t i c a c i d (DOPAC), t h e i n d o l e s s e r o t o n i n (5-HT) and 5 - h y d r o x y i n d o l e - a c e t i c a c i d (5-HIAA), and o f n o r a d r e n a l i n e (NA) and i t s m e t a b o l i t e 3 - m e t h o x y - 4 - h y d r o x y p h e n e t h y l e n e g l y c o l ( M H P G ) w i t h i n t h e b a s a l g a n g l i a and o t h e r r e l e v a n t b r a i n a r e a s o f a r e c e n t l y d e c e a s e d p a t i e n t (M892) who was a member o f a p e d i g r e e d i s p l a y i n g c l i n i c a l s i g n s o f p a r k i n s o n i s m w i t h c h r o n i c d e p r e s s i o n . Amino a c i d s n o t a f f e c t e d by th e r e l a t i v e l y l o n g d e a t h - t o - f r e e z i n g i n t e r v a l e n c o u n t e r e d w i t h t h i s p a t i e n t w i l l be e s t i m a t e d by q u a n t i t a t i v e amino a c i d a n a l y s i s ( P e r r y e t a l . 1968). Methods The b r a i n s o f b o t h t h e p a t i e n t M892 (PD w i t h c h r o n i c d e p r e s s i o n ) and h i s b r o t h e r M793, who showed no e v i d e n c e o f any n e u r o l o g i c a l a b n o r m a l i t y and d i e d a t t h e age o f 51 as a r e s u l t o f t e r m i n a l c a n c e r o f th e a d r e n a l g l a n d , were o b t a i n e d a t au t o p s y . The d e a t h - t o - f r e e z i n g i n t e r v a l s were 6 h o u r s , 20 min and 20 hours f o r M892 and M793, r e s p e c t i v e l y . N e u r o p a t h o l o g i c a l e x a m i n a t i o n was p e r f o r m e d a t t h e time o f a u t o p s y . One h a l f o f t h e b r a i n was f r o z e n i m m e d i a t e l y and s t o r e d a t -70*c f o r b i o c h e m i c a l a n a l y s i s , w h i l e t h e o t h e r h a l f was f i x e d i n f o r m a l i n . B r a i n s were p a r t i a l l y thawed ( t o c a . -10°C) f o r r e g i o n a l d i s s e c t i o n . Samples were prepared and analyzed by HPLC and amino acid chromatography as described i n chapter I I I . Results The r e s u l t s obtained by the HPLC determination of biogenic amine concentrations are shown i n Tables VII to XI. The values obtained by automated amino a c i d analyses of autopsied b r a i n are shown i n Table XII and Appendix, Table A - I I I . The r e s u l t s of HPLC analysis i n d i c a t e that DA and HVA were s u b s t a n t i a l l y reduced i n the caudate nucleus, putamen, substantia nigra and globus p a l l i d u s of the patient (M892) r e l a t i v e to both h i s brother (M793) and l i t e r a t u r e values f o r other c o n t r o l subjects (Appendix, Tables A-V and A-VI). i n addition, HVA concentrations were low i n the f r o n t a l and o c c i p i t a l cortex as w e l l as the thalamus. Although l i t e r a t u r e values f o r NA i n autopsied b r a i n are scarce (see below and Appendix, Table A-VII), NA contents of the caudate, putamen, globus p a l l i d u s and thalamus were low i n comparison to a c o n t r o l subject (M793). In addition, the serotonin concentrations of the f r o n t a l and o c c i p i t a l cortex, caudate, putamen, and hippocampus were also found to be low. 74 C o n c e n t r a t i o n s o f e i g h t amino compounds ( g l y c e r o p h o s p h o e t h a n o l a m i n e (GLYC-PEA), t a u r i n e (TAU), phosphoethanolamine (PEA), g l u t a m i c a c i d (GLU), g l u t a m i n e (GLN), c y s t a t h i o n i n e (CYSTA), homocarnosine (HCARN), and gamma-aminobutyryl-l y s i n e (GABA-LYS)) i n t i s s u e s o b t a i n e d i m m e d i a t e l y a f t e r d e a t h o r a f t e r 48 h o u r s i n s i m u l a t e d m o r t u a r y c o n d i t i o n s were f o u n d n o t t o d i f f e r ( P e r r y e t a l . 1981). A l t h o u g h GABA c o n c e n t r a t i o n s r i s e r a p i d l y i n t h e f i r s t two hours a f t e r d e a t h , t h e y r e m a i n s t a b l e f o r t h e f o l l o w i n g 48 ho u r s ( P e r r y e t a l . 1981). S i n c e c o n t r o l v a l u e s f o r GABA c o n t e n t s were o b t a i n e d from c o n t r o l s u b j e c t s w i t h d e a t h - t o - f r e e z i n g i n t e r v a l s o f g r e a t e r t h a n 1 hour and no t more t h a n 30 hours ( P e r r y e t a l . 1985a; 1987b) (Appendix, T a b l e A - I V ) , t h e y can be f a i r l y compared t o t h e v a l u e s o b t a i n e d f o r t h e p a t i e n t . The most ma r k e d l y abnormal r e s u l t s (see T a b l e X I I and Appendix, T a b l e A - I I I ) were a s i g n i f i c a n t r e d u c t i o n (P < 0.05) i n l e v e l s o f g l u t a m i c a c i d i n v i r t u a l l y a l l b r a i n a r e a s i n v e s t i g a t e d ( w i t h t h e e x c e p t i o n o f t h e g l o b u s p a l l i d u s and d e n t a t e n u c l e u s ) and a s i g n i f i c a n t r e d u c t i o n (P < 0.05) i n t h e c o n c e n t r a t i o n s o f t h e amino compounds GLYC-PEA, TAU, PEA, GLU, and GABA i n t h e s u b s t a n t i a i n n o m i n a t a . s i n c e g l u t a m a t e c o n t e n t s were o b t a i n e d from a number o f d i s c r e t e a n a t o m i c a l a r e a s t h e v a l u e s a r e u n d o u b t e d l y o f b i o l o g i c a l s i g n i f i c a n c e . The f a c t t h a t v i r t u a l l y a l l amino compounds q u a n t i f i e d i n t h e s u b s t a n t i a i n n o m i n a t a were low s u g g e s t s t h a t t h e s e r e s u l t s be i n t e r p r e t e d w i t h some s k e p t i c i s m . These v a l u e s may be i n d i c a t i v e o f an e r r o r i n d i s s e c t i o n r a t h e r t h a n a g r o s s n e u r o c h e m i c a l d e f e c t i n t h i s a n a t o m i c a l r e g i o n , o t h e r s t a t i s t i c a l l y s i g n i f i c a n t a b n o r m a l i t i e s (P < 0.05) b u t which have q u e s t i o n a b l e b i o l o g i c a l r e l e v a n c e i n c l u d e d , a d e c r e a s e o f GLYC-PEA i n t h e hippocampus, a d e c r e a s e o f PEA and GABA i n t h e s u b s t a n t i a n i g r a , and an i n c r e a s e o f GABA-LYS c o n c e n t r a t i o n s i n the putamen. 76 T a b l e V: Summary o f CSP monoamine m e t a b o l i t e c o n c e n t r a t i o n s ( n g / m l ) . THERAPY Compound Controls* Mean i S.D. None 7-9-86 8-25-86 A. 3-18-87 8. 5-19-87 C. D. 11-29-87 7-13-88 E. 8-9-81 HHPG 7.9 ! 2.9 9.9 7.6 5.1 (HO 6.2 8.6 8.1 OOPAC ' 1.0 1 0.5 1.8 (NO 2.0 (NO (NO trace 1.5 HVA 36.8 i 18.9 5.0 7.3 6.0 4.7 4.2 3.1 4.2 5-HIAA 20.7 t 12.5 7.6 8.8 4.7 7.5 21.1 3.7 4.6 Therapies: A. Aiit r i p t y l i n e (200 ag/day) 8. Off Aaitriptyline for 3 weeks, Isoniazid (20 ag/kg/day) C. Aaitriptyline (200 ag/day), L-5-HTP (500 ag b.i.d.), Carbidopa (100 ag b.i.d.) D. Aaitriptyline (250 ag/day) E. Fluoxetine (40 ag/day) * 107 control subjects, aost of thea ill with various neurological diseases, and receiving drugs. These are NOT noraal adults. However, diseases known to involve abnoraalities in brain aonoaaines (e.g. Parkinson's disease), and patients receiving drugs known to alter cerebral aonaaines (e.g. L-D0PA, carbidopa, L-tryptophan, aonoaaine oxidase inhibitors, and t r i c y c l i c antidepressants) have been excluded froa this control group. Abbreviations: HHP6 3-aethoxy-4-hydroxyphenethyleneglycol D0PAC 3,4-dihydroxy-phenylacetic acid HVA hoaovanillic acid 5-HIAA 5-hydroxyindole-acetic acid NC not calculated 76a Table VI; summary of CSF amino a c i d concentrations (umol / 1 ) . Pharmacological Intervention: Compound Control Adults None A. B. Mean J S.D. 7-9-86 8-25-86 3-18-87 5-19-87 Taurine 6.9 2.3 5.5 4.6 5.4 4.5 GABA (mol/1) 84 36 19 9 5 73 t Compound Control Adults C. 0. E. Mean ! S.D. 11-29-87 7-13-88 8-9-88 Taurine 6.9 2.3 5.8 5.5 4.8 6ABA (naol/1) 84 36 26 < 3 5 5-HTP = 0.2 Protein = 0.77 g/1 Treatments: A. Aaitriptyline (200 ag/day) B. Off Aaitriptyline for 3 weeks, Isoniazid (20 ag/kg/day) C. Aaitriptyline (200 ag/day), L-5-HTP (500 ag b.i.d.), Carbidopa (100 ag b.i.d.) D. Aaitriptyline (250 ag/day) E. Fluoxetine (40 ag/day) Abbreviations: 5-HTP 5-hydroxytryptophan 6ABA gaaaa-aainobutyric acid 77 Table VII; Estimation of dopamine content by HPLC. Brain Region: Frontal Cortex O c c i p i t a l Cortex Cerebellar Cortex Caudate Putamen Substantia Nigra Substantia Innominata Nucleus Accumbens Globus P a l l i d u s I Globus P a l l i d u s II Thalamus Dentate Tissue Control M793 ng/g 38.7 30.5 9.9 333.5 517.4 112.6 229.3 44.7 145.2 294.2 9.1 DA Patient M892 ng/g 31.6 16.1 7.3 36.9 44.7 87.4 215.5 285.1 70.2 37.0 29.6 12.9 7 8 Table v i i i ; Estimation of homovanillic a c i d content by HPLC. Tissue HVA Control M793 Patient M892 Brain Region: ng/g ng/g Frontal Cortex 712 .7 33 .5 O c c i p i t a l Cortex 245 .8 50 .2 Cerebellar Cortex 218 .3 45 .0 Caudate 3589 .6 137. .0 Putamen 5089 .0 445 .9 Substantia Nigra 1567 .3 369 .1 Substantia Innominata 2934 .0 1982 .8 Nucleus Accumbens 1191 .0 1857 .6 Globus P a l l i d u s I 3142 .3 682 .6 Globus P a l l i d u s II 5192 .4 929 .1 Thalamus 636 .1 125 .9 Dentate 219 .6 56 .0 79 T a b l e IX: E s t i m a t i o n o f n o r a d r e n a l i n e c o n t e n t by HPLC. Brain Region: Fro n t a l Cortex O c c i p i t a l Cortex Cerebellar Cortex Caudate Putamen Substantia Nigra Substantia Innominata Nucleus Accumbens Thalamus Dentate Tissue Control M793 ng/g 117.4 85.2 36.3 59.9 146.4 93.3 59.3 145.6 97.0 76.1 NA Patient M892 ng/g 86.0 109.4 52.2 96.5 129.5 157.8 132.3 1264.7 50.0 33.9 8 0 Table X; Estimation of serotonin content by HPLC. Brain Region: Frontal Cortex O c c i p i t a l Cortex Cerebellar Cortex Caudate Putamen Substantia Nigra Substantia Innominata Nucleus Accumbens Globus P a l l i d u s I Globus P a l l i d u s II Thalamus Dentate Tissue Control M793 ng/g 20.5 30.8 26.0 363.6 192.8 362.6 275.4 198.7 108.6 154.1 110.6 24.3 5-HT Patient M892 ng/g 8.2 3.5 19.1 130.8 81.0 331.8 412.4 382.4 44.1 82.8 108.1 11.3 81 T a b l e X I ; E s t i m a t i o n o f 5 - h y d r o x y i n d o l e a c e t i c a c i d c o n t e n t b y H P L C . Brain Region: Frontal Cortex O c c i p i t a l Cortex Cerebellar Cortex Caudate Putamen Substantia Nigra Substantia Innominata Nucleus Accumbens Globus P a l l i d u s I Globus P a l l i d u s II Thalamus Dentate Tissue Control M793 ng/g 389.0 432.5 114.1 1317.3 1582.6 2362.5 1142.0 1009.0 2757.6 2376.0 794.4 499.1 5-HIAA Patient M892 ng/g 37.4 162.1 58.5 468.5 515.8 2486.3 1435.4 675.3 694.1 909.3 551.6 351.9 82 Table XII; Estimation of t a u r i n e , glutamate and G A B A i n autopsied b r a i n (Patient M 8 9 2 ) . Taurine Glutamic acid GABA Brain Region: H892 Controls H8S2 Controls H892 Controls Frontal Cortex .73 1.03 i 0.38 4.43* 8.28 ! 1.S6 1.35 1.64 I 0.45 Occipital Cortex .67 0.93 J 0.37 5.49* 8.31 i 1.22 1.46 1.80 i 0.53 Cerebellar Cortex 2.26 2.65 + 1.02 5.39* 9.05 i 1.63 0.98 1.65 1 0.41 Caudate .94 1.25 i 0.46 5.68* 10.27 i 1.78 2.31 2.87 i 0.86 Putaien 1.07 1.30 ! 0.39 7.35* 11.29 i 2.38 2.30 2.94 ! 0.83 Substantia Nigra .75 1.05 i 0.34 2.54* 5.51 i 1.36 1.86* 6.00 i 1.26 Substantia Innominata .38* 1.05 t 0.37 1.57* 6.45 i 1.37 1.60* 5.84 i 1.11 Nucleus Accumbens .65 0.83 i 0.32 2.22* 6.81 i 1.40 1.55 4.15 i 1.39 61obus Pallidus M I .96 1.26 ! 0.28 3.12 5.91 i 1.88 5.24 7.32 i 1.60 Thalamus (HD) .58 0.87 • 0.27 4.76* 9.11 ! 1.83 0.62 1.94 * 0.73 Dentate .87 1.14 ! 0.42 2.42 4.77 i 1.48 2.91 4.77 t 0.98 Hippocampus 1.33 1.77 i 0.55 5.56* 9.83 • 1.81 1.65 1.93 i 0.47 * Significantly different from controls (P < 0.05). 83 T a b l e X I I I ; S u m m a r y o f a b n o r m a l b r a i n b i o g e n i c a m i n e v a l u e s o b t a i n e d b y HPLC ( P a t i e n t M892). Brain Region: DA HVA NA 5-HT 5-HI/ Frontal Cortex - < z < < Occipital Cortex < < z < < Cerebellar Cortex = < : r Caudate «< « < z < < Putaaen «< «< = < Substantia Nigra < < z -Substantia Innoainata z < - z Nucleus Accuabens > »> > s Globus Pallidus I < «< < , «< Globus Pallidus II <« «< < «< Thalamus s < z : Dentate s < = - = Hippocaipus : < < = saie as control < s l i g h t l y decreased or the saee as control < decreased f r o i control « < significantly decreased f r o i control > s l i g h t l y increased or the saie as control > increased froa control > » significantly increased froa control 84 T a b l e X I V ; S u m m a r y o f a b n o r m a l b r a i n m o n o a m i n e v a l u e s o b t a i n e d b y a m i n o a c i d a n a l y s i s ( P a t i e n t M892). Brain Region: GLYC-PEA TAU PEA GLU 6LN CYSTA GABA GABA-LYS Frontal Cortex Occipital Cortex -Cerebellar Cortex Caudate Putaien Substantia Nigra Substantia Innoainata Nucleus Accuabens Globus Pallidus I • II Thalamus Dentate Hippocaapus < < < < < < < «< «< < < < < «< «< «< «< «< «< «< «< < < < <« «< < < < < < > > > = saae as control < slightly decreased or the saae as control < decreased froa control (greater than 1 SD) « < significantly decreased froa control (greater than 2 SD) > slightly increased or the saae as control > increased froa control (greater than 1 SD) » > significantly increased froa control (greater than 2 SD) 85 Discussion F o r t h e p u r p o s e s o f comparison, t h e summaries o f s e v e r a l i n v e s t i g a t i o n s o f t h e c a t e c h o l a m i n e and i n d o l e c o n t e n t s o f t h e b a s a l g a n g l i a and s u r r o u n d i n g n u c l e i a r e g i v e n i n t h e Appendix, T a b l e s A-v t o A-IX. The r e s u l t s o f t h e a n a l y s i s o f DOPAC and MHPG c o n c e n t r a t i o n s were n o t i n c l u d e d i n t h e p r e s e n t i n v e s t i g a t i o n due t o u n r e l i a b l e r e s u l t s o b t a i n e d from t h e HPLC d e t e r m i n a t i o n o f t h e s e compounds. The peak c o r r e s p o n d i n g t o MHPG was c o n s i s t e n t l y o b s c u r e d by t h e e l u t i o n o f t h e v e r y l a r g e s o l v e n t f r o n t i n human b r a i n samples. The d e t e r m i n a t i o n o f DOPAC c o n c e n t r a t i o n was u n r e l i a b l e due t o a f a i l u r e i n r e p r o d u c i b l y d e t e c t i n g t h i s compound i n t h e a u t o p s i e d b r a i n samples. A l t h o u g h DOPAC s t a n d a r d s and DOPAC-spiked samples had c h a r a c t e r i s t i c , r e l i a b l e r e t e n t i o n t i m e s and peak s i z e s , t h e DOPAC peaks i n many samples were o b s e r v e d t o be h i g h l y v a r i a b l e o r s i m p l y were n o t e v i d e n t . The re a s o n s f o r t h i s u n e x p e c t e d d i f f i c u l t y a r e u n e x p l a i n e d and s u r p r i s i n g , e s p e c i a l l y s i n c e DOPAC c o n c e n t r a t i o n s have been r e l i a b l y q u a n t i f i e d w i t h t h e same equipment and t e c h n i q u e s i n p r e v i o u s i n v e s t i g a t i o n s (see C h a p t e r I I I ) . The HPLC s t u d i e s i n d i c a t e a r e d u c t i o n o f DA and HVA i n ar e a s c h a r a c t e r i s t i c o f PD and c o n f i r m c l i n i c a l and n e u r o p a t h o l o g i c a l o b s e r v a t i o n s s u g g e s t i v e o f p a r k i n s o n i s m i n t h i s p a t i e n t (see T a b l e X I I I ) . The v a l u e s o b t a i n e d a r e g e n e r a l l y comparable t o r e p o r t e d c o n c e n t r a t i o n s ( E b i n g e r e t a l . 1987; J a v o y - A g i d e t a l . 1989; Her r e g o d t s 87 e t a l . 1989; R inne and Sonninen 1973; Fahn e t a l . 1971; L l o y d e t a l . 1975; A d o l f s s o n e t a l . 1978; G i l b e r t e t a l . 1988; P e r r y e t a l . 1985b) a l t h o u g h t h e c o n t r o l ( M 7 9 3 ) DA v a l u e s a r e lower t h a n most c o n c e n t r a t i o n r a n g e s o b t a i n e d f o r t h i s compound (see Appendix, T a b l e s A-V t o A - I X ) . W h i l e a r e l a t i v e two t o t e n - f o l d d e c r e a s e i n c o n t r o l DA c o n c e n t r a t i o n s was o b t a i n e d i n t h i s p r e s e n t HPLC s t u d y w i t h r e s p e c t t o l i t e r a t u r e v a l u e s , a f u r t h e r t e n - f o l d d e c r e a s e i n DA c o n t e n t remained between the c u r r e n t c o n t r o l ( M 7 9 3 ) and t h e p a t i e n t (M892) i n t h e c a u d a t e and putamen, and a f o u r - f o l d d e c r e a s e i n t h e g l o b u s p a l l i d u s . The p o s s i b i l i t y o f a s y s t e m a t i c e r r o r i n t r o d u c e d t h r o u g h i n a c c u r a t e DA s t a n d a r d p r e p a r a t i o n was i n v e s t i g a t e d and e v e n t u a l l y e l i m i n a t e d when new DA s t a n d a r d s p r o d u c e d i d e n t i c a l peaks as t h o s e p r e p a r e d f o r t h e p r e s e n t i n v e s t i g a t i o n . In a d d i t i o n , t h e DA s t a n d a r d peak s i z e s were comparable t o t h o s e o b t a i n e d o v e r t h e l a s t few y e a r s o f HPLC d e t e r m i n a t i o n s o f b i o g e n i c amines i n t h i s l a b o r a t o r y . Repeated a n a l y s e s o f t h e c a u d a t e and putamen o b t a i n e d from t h e p a t i e n t ' s b r o t h e r M793 i n d i c a t e d a s u b s t a n t i a l d e c r e a s e (90%) o f DA i n t h e s e a r e a s when compared t o l i t e r a t u r e v a l u e s . S i n c e t h e p r e s e n t c o n t r o l ( M 7 9 3 ) c o n c e n t r a t i o n s o f t h e o t h e r c a t e c h o l a m i n e s and i n d o l e s a r e v e r y s i m i l a r t o t h o s e i n t h e l i t e r a t u r e , a p l a u s i b l e c o n c l u s i o n i s t h a t t h e p a t i e n t ' s b r o t h e r ( M 7 9 3 ) was i n f a c t a p r esymptomatic c a r r i e r o f t h e gene r e s p o n s i b l e f o r t h i s c o n d i t i o n . The d e c r e a s e s i n DA c o n c e n t r a t i o n s o b s e r v e d i n t h e p a t i e n t ' s b r o t h e r ( M 7 9 3 ) were r e s t r i c t e d t o t h o s e a n a t o m i c a l a r e a s e x p e c t e d t o be compromised i n PD. s i n c e t h e symptoms o f t h i s d i s e a s e o n l y became 88 evident i n the p a t i e n t at the age of 54, the low DA contents observed i n hi s brother may i n d i c a t e the f i n a l stages of presymptomatic degeneration of dopaminergic n i g r o s t r i a t a l neurons i n t h i s man. This conclusion i s supported by the high HVA:DA r a t i o i n the caudate, putamen and substantia n i g r a (14, 16, and 15 r e s p e c t i v e l y ) of the c o n t r o l which would i n d i c a t e a very high, pre-synaptic biochemical compensatory mechanism i n the - c o n t r o l subject (Hornykiewicz and Kish 1986). A lower HVA:DA r a t i o i n the pa t i e n t M892 i s not s u r p r i s i n g since the degeneration of the dopaminergic n i g r o s t r i a t a l system had r e s u l t e d i n a lo s s of almost 99% of DA concentrations as compared to l i t e r a t u r e values, and compensatory mechanisms would have c e r t a i n l y f a i l e d by t h i s p o i n t . The i n a b i l i t y to recognize p o s s i b l e parkinsonian feature i n the patie n t ' s brother i s conceivable, e s p e c i a l l y i n l i g h t of the d i f f i c u l t y i n reaching a diagnosis of PD i n the present patient from s t r i c t l y c l i n i c a l features. The contents of 5-HT and 5-HIAA were found to be low i n the caudate, putamen, and both anatomical regions of the globus p a l l i d u s (Tables VIII and IX). These r e s u l t s are i n agreement with the CSF analyses performed during the patient's l i f e . In summary, the HPLC r e s u l t s i n d i c a t e d a s i g n i f i c a n t reduction of DA, HVA, 5-HT, and 5-HIAA i n the caudate nucleus, putamen, and globus p a l l i d u s . The observations of low DA and HVA contents i n the caudate nucleus, putamen, and globus p a l l i d u s , together with the 89 neuropathological observation of pronounced degeneration i n the substantia n i g r a of t h i s p a t i e n t (M892), are both c o n s i s t e n t with the diagnosis of PD, although they are i n contrast to the PET scan observations obtained during l i f e . Since the PET scan was obtained over two years before the death of t h i s patient, a r a p i d degenerative los s of dopaminergic neurons i n the terminal stages of t h i s disease may not have been evident at the time of the scan. The most s i g n i f i c a n t r e s u l t obtained from the analyses of amino acids i n the post-mortem samples i n d i c a t e d (see Table XIV and Appendix, Table A-III) th a t the contents of glutamic a c i d were s i g n i f i c a n t l y reduced i n the majority of the brain regions analyzed. The values obtained f o r the substantia innominata are questionable, e s p e c i a l l y due to the nature of the broad neurochemical d e f i c i e n c i e s observed, i n a d d i t i o n to the extreme d i f f i c u l t y i n i d e n t i f y i n g and d i s s e c t i n g these h i g h l y s p e c i f i c areas i n b r a i n . These a n a l y t i c a l studies have in d i c a t e d a s i g n i f i c a n t neurochemical malfunction of the caudate, putamen, and globus p a l l i d u s . The neurochemical d e f i c i e n c i e s i n the basal ganglia would account f o r the observations co n s i s t e n t with parkinsonism, although i t should be noted th a t Lewy bodies, a c h a r a c t e r i s t i c neuropathological hallmark of PD (Forno 1986) were not found i n t h i s p a t i e n t (M892). The present r e s u l t s , i n a d d i t i o n to the neurochemical analyses performed during the pat i e n t ' s l i f e and the neuropathological f i n d i n g s , are i n d i c a t i v e of a profound degeneration of the basal ganglia ( i n c l u d i n g the caudate nucleus, putamen, and substantia nigra) and thalamus of the p a t i e n t M892. Degeneration of the thalamus would r e s u l t i n disturbances of depth perception and verbal s k i l l s , since l e s i o n s of the r i g h t thalamus cause d i f f i c u l t i e s with s p a t i a l r e l a t i o n s h i p s and l e f t thalamic lesions r e s u l t i n a form of aphasia (DeMyer 1988). While present i n some of the other members o f ' t h i s pedigree, neither aphasia nor a l t e r e d s p a t i a l d i s c r i m i n a t i o n were evident i n the present p a t i e n t . The h i g h l y s i g n i f i c a n t d e f i c i e n c y of glutamic a c i d i s unexplained, but the decrease i n t h i s e x c i t a t o r y neurotransmitter i s very l i k e l y of f u n c t i o n a l relevance, considering the number of s p e c i f i c neuronal pathways u t i l i z i n g e x c i t a t o r y amino acids (Cotman et a l . 1987). Disorders of glutamatergic transmission have been implicated i n a v a r i e t y of neurodegenerative diseases, i n c l u d i n g Alzheimer's disease (Marangos et a l . 1987; Procter et a l . 1988), amyotrophic l a t e r a l s c l e r o s i s (Perry et a l . 1987b; P l a i t a k i s et a l . 1988), ol i v o p o n t o c e r e b e l l a r atrophy ( P l a i t a k i s et a l . 1982), and Huntington's disease (Perry, submitted). Due to the nature of the broad neurochemical changes observed i n t h i s p a t i e n t , i n a d d i t i o n to low CSF l e v e l s of HVA and 5-HIAA during l i f e and s i g n i f i c a n t behavioural and p h y s i o l o g i c a l changes, the diagnosis of m u l t i p l e system atrophy (MSA) with autonomic f a i l u r e seems conceivable (Polinsky et a l . 1988), although chronic autonomic i n s u f f i c i e n c y can a l s o occur with parkinsonism and overlaps with MSA (Aminoff and Wilcox 91 1971; Bannister and Oppenheimer 1972). Parkinson's disease p a t i e n t s have been reported to have p a t h o l o g i c a l abnormalities of neural areas involved i n autonomic r e g u l a t i o n , p a r t i c u l a r l y the hypothalamus (Langston and Forno 1978; Javoy-Agid et a l . 1984), a l b e i t experimental studies i n animals suggest that a s i n g l e CNS l e s i o n i s not l i k e l y to r e s u l t i n autonomic f a i l u r e and o r t h o s t a t i c hypotension (Talman 1985). The major features of MSA ( i n i t i a l l y termed the Shy-Drager syndrome) include o r t h o s t a t i c hypotension, u r i n a r y incontinence, anhydrosis, p u p i l l a r y changes and impotency (Shy and Drager 1960; Banninster et a l . 1977; Spokes et a l . 1979) i n ad d i t i o n to neuronal c e l l loss and g l i o s i s i n the striatum, c e r e b e l l a r cortex, and pontine n u c l e i (Shy and Drager 1960). However, the observations of brainstem neuropathology i n MSA, in c l u d i n g c e l l losses i n the i n t e r m e d i o l a t e r a l c e l l column of the sp i n a l cord, are i n contrast to the neuropathological changes observed i n t h i s p a t i e n t . In ad d i t i o n , the pat i e n t (M892) i n i t i a l l y underwent pharmacotherapy f o r hypertension, and the o r t h o s t a t i c hypotension encountered l a t e r i n the treatment of t h i s p a t i e n t was l i k e l y an adverse e f f e c t of t r i c y c l i c antidepressant therapy. Moreover, MSA does not include symptoms of c e n t r a l r e s p i r a t o r y i n s u f f i c i e n c y . Other mu l t i p l e system atrophies which have parkinsonian features include s t r i a t o n i g r a l degeneration, progressive supranuclear palsy (PSP) and the oliv o p o n t o c e r e b e l l a r atrophies (OPCA) ( R o l l e r 1987), but none are associated with autonomic nervous system f a i l u r e . Thus, none of the 92 m u l t i p l e system atrophies seem to account f o r a l l the behavioural and p h y s i o l o g i c a l symptoms seen i n t h i s case. The c h a r a c t e r i s t i c s of the co n d i t i o n which a f f l i c t e d t h i s patient are, i n general, consistent with those p r e v i o u s l y described (Perry et a l . 1975; Purdy et a l . 1979; Roy et a l . 1988), although there were biochemical d i f f e r e n c e s i n c l u d i n g normal l e v e l s of taurine i n blood, CSF, and bra i n as we l l as cont r a s t i n g behavioural c h a r a c t e r i s t i c s , i n c l u d i n g l e s s severe depression, and improved s p a t i a l d i s c r i m i n a t i o n than i n other patients previously observed i n the same family pedigree (Perry et a l . 1975). The pharmacological interventions attempted throughout the course of the patient's i l l n e s s were l a r g e l y unsuccessful, although some moderate improvements were noted during t r i c y c l i c antidepressant therapy, many of which were l i k e l y due to the appetite stimulating properties of a m i t r i p t y l i n e . None of the drug therapies a l t e r e d CSF l e v e l s of MHPG, DOPAC or HVA, and while i s o n i a z i d and, to a l e s s e r extent 5-HTP treatment, were shown to e f f e c t i v e l y increase CSF GABA concentrations, no s i g n i f i c a n t n e u r o l o g i c a l improvement was noted as a r e s u l t of t h i s increase. Moreover, the administration of 5-HTP, which l e d to increases of plasma and CSF 5-HTP, and a s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.05) increase i n CSF content of 5-HIAA (and presumably 5-HT), d i d not r e s u l t i n any l a s t i n g c l i n i c a l improvement. In view of the neuropathological and neurochemical evidence which confirmed losses of dopaminergic neurons i n the basal 93 g a n g l i a o f t h i s p a t i e n t , t r e a t m e n t w i t h L-DOPA might have r e s u l t e d i n some c l i n i c a l improvement and s h o u l d be att e m p t e d i n f u t u r e c a s e s which may o c c u r i n t h i s f a m i l y p e d i g r e e . In c o n c l u s i o n , t h e most l i k e l y d i a g n o s i s o f t h e n e u r o l o g i c a l c o n d i t i o n which a f f l i c t e d t h i s p a t i e n t (M892) i s o f a d i s t i n c t c o n d i t i o n , c h a r a c t e r i z e d by f e a t u r e s o f f a m i l i a l p a r k i n s o n i s m , a p a t h y o r d e p r e s s i o n , w e i g h t l o s s , and c e n t r a l h y p o v e n t i l a t i o n . 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Mean • S.D. 7-3-86 9-29-86 5-19-87 11-27 Taurine 56 14 37 47 31 47 Pbosphoethanolaaine traceM trace trace trace trace Urea 4431 1565 7060 8330 4450 4320 Aspartic acid trace>l trace trace trace 0 Hydroxyproline 1 1 1 13 2 2 Threonine 139 28 141 168 144 97 Serine 105 28 94 107 110 82 Asparagine 58 13 85 95 83 (NO Glutaaic acid 23 11 15 20 18 68 61utaaine 624 118 791 830 633 641 Proline 181 63 311 384 329 427 Glycine 222 55 267 290 378 267 Alanine 367 97 474 576 609 335 Citrulline 33 10 59 65 47 48 a-Aaino-n-butyric acid 23 8 32 8 (NO 15 Valine 233 41 291 315 269 227 Cystine 48 14 61 62 43 65 Methionine 22 5 28 28 21 16 Isoleucine 60 12 86 89 81 78 Leucine 116 22 163 149 153 127 Tyrosine 53 13 63 61 67 46 Cystinylglycine 12 4 24 15 (NO (NO Phenylalanine 50 9 62 59 56 42 Tryptophan 39 11 42 51 27 46 Ethanolaaine I 3 trace trace trace trace Ornithine 54 15 58 61 113 58 Lysine 193 34 239 235 207 154 €-M-Methyllysine 6 6 (NO (NO (NO 1 Histidine 90 14 99 86 71 54 1-Hethylhistidine 4 8 7 3 4 1 3-Hethylhistidine 2 2 4 4 1 1 Arginine 85 24 117 128 88 82 Treatments: 5-HTP = 22 A. Off Aaitriptyline for 3 weeks, Isoniazid (20 •g/kg/day) B. Aaitriptyline (200 ag/day), L-5-HTP (500 ag b.i.d.), Carbidopa (100 ag b.i.d.) L-5-HTP L-5-hydroxytryptophan 101 Appendix T a b l e A - I I : Summary of CSF amino a c i d concentrations (umol/1) (Patient M892) Pharmacological Intervention: Coipound Control Adults None A. B. Mean ! S.D. 7-9-86 8-25-86 3-18-87 5-19-87 Taurine 6.9 2.3 5.5 4.6 5.4 4.5 Phosphoethanolaaine 4.3 1.8 4.3 2.7 2.1 2.5 Urea 4481 1758 10345 7255 6125 6280 Aspartic acid 0>trace 0 0 0 0 Threonine , 31.5 8.7 41.0 48.7 44.2 40.7 Serine 27.0 5.6 23.5 23.9 24.8 24.2 Asparagine 6.9 2.9 10.3 13.3 10.6 4.3 Glutaiic acid 0.7 0.6 0.1 0.1 trace trace Glutaiine 587.3 85.8 734.9 748.2 906.4 688.9 Proline 0 0 0 0 0 Glycine 6.3 2.8 10.1 8.6 9.0 10.5 Alanine 32.6 11.4 74.8 65.1 81.2 77.3 Citrulline 1.8 1.1 5.0 4.9 3.7 1.1 a-Aaino-n-butyric acid 2.7 1.7 4.8 8.3 1.4 4.8 Valine 17.9 5.9 28.7 32.3 28.0 24.7 Cystine 0>trace 0 0 0 0 Methionine 2.9 1.1 4.5 4.9 3.7 • 2.0 Isoleucine 4.7 1.6 7.9 7.1 7.3 5.2 leucine 12.8 3.7 17.9 20.8 20.6 17.8 Tyrosine 8.4 2.8 8.8 8.6 9.5 8.0 Phenylalanine 9.0 3.1 12.2 13.9 11.0 10.4 Tryptophan traceM 0.1 0.5 trace trace Ethanolaaine 15.1 5.8 4.6 3.1 1.9 (NO Ornithine 4.6 1.8 2.6 2.0 3.4 13.7 * Lysine 26.7 6.3 31.6 30.8 38.2 30.4 E-N-Nethyllysine 0.9 1.5 (NO (NO (NO (NO Histidine 13.3 2.8 14.6 16.3 13.5 12.4 1-Methylhistidine 0 > 0.3 0 0.3 0 0 3-Hethylhistidine 0 > 0.3 0.5 0 trace trace Hoaocarnosine 1.3 1.5 0 0 0 trace GABA-lys 0.2 0.4 0 0 0 trace Arginine 20.3 4.7 26.2 23.8 23.8 16.7 6ABA (mol/1) 84 36 19 9 5 73 » Treatments: A. Aaitriptyline (200 ag/day) B. Off Aaitriptyline for 3 weeks, Isoniazid (20 ag/kg/day) (NO Not Calculated 102 T a b l e A - I I ; ( c o n t i n u e d ) . Appendix Pharmacological Intervention: Coapound Control Adults C. D. E. Hean • S.D. 11-29-87 7-13-88 8-9-88 Taurine 6.9 2.3 5.8 5.5 4.8 Phosphoethanolaaine 4.3 1.8 3.1 1.6 2.2 Urea 4481 1758 4754 6440 6820 Aspartic acid 0>trace 0 trace 0 Threonine 31.5 8.7 37.6 35.8 39.0 Serine 27.0 5.6 22.3 20.5 21.7 Asparagine 6.9 2.9 11.6 8.0 8.6 Slutaaic acid 0.7 0.6 0.1 0.1 0.2 Glutaaine S87.3 85.8 663.7 767.0 668.7 Proline 0 0 0 0 Glycine 6.3 2.8 10.0 7.0 8.8 Alanine 32.6 11.4 83.5 60.7 58.7 Citrul l i n e 1.8 1.1 2.8 1.0 2.2 a-Aaino-n-butyric acid 2.7 1.7 1.0 0.7 2.8 Valine 17.9 5.9 28.6 21.7 23.2 Cystine 0>trace 0 trace trace Hethionine 2.9 1.1 1.9 1.6 1.6 Isoleucine 4.7 1.6 7.9 6.1 7.6 Leucine 12.8 3.7 19.9 16.3 ' 20.8 Tyrosine 8.4 2.8 6.7 6.1 5.8 Phenylalanine 9.0 3.1 8.2 9.4 9.1 Tryptophan traceM 0.2 0.2 0.2 Ethanolaaine 1S.1 5.8 5.5 3.2 2.6 Ornithine 4.6 1.8 3.8 3.4 1.6 Lysine 26.7 6.3 34.0 32.8 28.6 C-N-Hethyllysine 0.9 1.5 trace 0.1 0.1 Histidine 13.3 2.8 11.8 13.9 8.5 1-Hethylbistidine 0 > 0.3 0.1 trace 0 3-Hethylhistidine 0 > 0.3 0.1 0.2 trace Hoaocarnosine 1.3 1.5 0 trace trace 6ABA-lys 0.2 0.4 0 0 0 Arginine 20.3 4.7 25.3 28.3 22.3 GABA (naol/1) 84 36 26 * 3 5 5-HTP = 0.2 Protein s 0.77 g/1 Treataents: C. Aaitriptyline (200 ag/day), L-5-HTP (500 ag b.i.d.), Carbidopa (100 ag b.i.d.) D. Aaitriptyline (250 ag/day) E. Fluoxetine (40 ag/day) v 5-HTP 5-hydroxytryptophan 103 Table A - I I I : Estimation of amino compounds i n autopsied b r a i n (umol/g wet wt) (Patient M892). Appendix Patient H892 Brain Region: GLYC-PEA TAU PEA Frontal Cortex .436 .732 .928 Occipital Cortex .423 .673 .661 Cerebellar Cortex .796 2.255 .962 Caudate .677 .940 .689 Putaien .525 1.072 .661 Substantia Nigra .983 .748 .191* Substantia Innoiinata .336* .381* .108* Nucleus Accuabens .801 .648 .316 Globus Pallidus 1*11 .997 .957 .371 Thalaius (HD) .983 .575 .254 Dentate 1.162 .867 .151 Hippocaipus .680* 1.328 1.006 Aaino Compound 6LU 6LN CYSTA 6ABA HCARN 6ABA-L) 4.432* 3.312 .466 1.350 .143 .010 5.490* 4.482 2.187 1.461 .278 .060 5.385* 4.767 .267 .981 .437 .034 5.683* 5.228 .531 2.312 .166 .012 7.352* 5.300 1.060 2.303 .623 .127* 2.535* 3.547 .637 1.856* .441 .155 1.570* 1.560* .194 1.595* .168 .020 2.221* 2.561 .344 1.550 .230 .014 3.117 6.540 1.369 5.245 .637 .069 4.755* 3.180 .979 .617 .360 .119 2.416 3.275 1.114 2.912 1.170 .436 5.563* 4.245 .443 1.650 .262 .032 * Significantly different froa controls (P < 0.05). Abbreviations: GLYC-PEA glycerophosphoethanolaaine TAU taurine PEA phosphoethanolaaine GLU glutaaic acid 6LN glutaaine CYSTA cystathionine GABA gaaaa-aainobutyric acid HCARN hoaocarnosine 6A8A-LYS gaaaa-aainobutyryl-lysine 104 Appendix T a b l e A-IV: E s t i m a t i o n o f a m i n o c o m p o u n d s i n a u t o p s i e d b r a i n ( u m o l / g w e t w t ) ( C o n t r o l s u b j e c t s ) . Brain Region: TAU 6LU GLN 6ABA HCARN ** Frontal Cortex 1.03 i 0.38 (30) 8.28 f 1.56 (31) 4.64 ! 1.51 (27) 1.64 f 0.45 (31) 0.27 i 0.18 (26) Occipital Cortex 0.93 ! 0.37 (28) 8.31 ! 1.22 (28) 4.98 i 2.74 (24) 1.80 i 0.53 (28) 0.41 ! 0.32 (25) Cerebellar Cortex 2.65 i 1.02 '(26) 9.05 • 1.63 (26) 5.81 i 1.59 (22) 1.65 i 0.41 (26) 0.59 • 0.32 (26) Caudate 1.25 i 0.46 (33) 10.27 t 1.78 (33) 4.46 i 2.21 (29) 2.87 i 0.86 (33) 0.21 i 0.18 (31) Putaien 1.30 i 0.39 (19) 11.29 i 2.38 (18) 4.04 i 1.87 (18) 2.94 i 0.83 (19) 0.45 i 0.20 (17) Substantia Nigra 1.05 i 0.34 (33) 5.51 i 1.36 (32) 4.11 i 1.82 (27) 6.00 i 1.26 (33) 0.79 t 0.33 (29) Substantia Innoainata * 1.15 i 0.37 (8) 6.45 i 1.37 (B) 5.39 : 1.58 (8) 5.84 i 1.11 (8) 0.52 • 0.18 (8) Nucleus Accuabens 0.83 i 0.32 (16) 6.81 i 1.40 (16) 4.31 1 1.92 (19) 4.15 i 1.39 (23) 0.26 ! 0.15 (15) 61obus Pallidus I+II 1.26 i 0.28 (16) 5.91 i 1.88 (16) 5.97 ! 2.17 (15) 7.32 i 1.60 (16) 0.72 i 0.37 (16) Thalaaus (HD) 0.87 i 0.27 (21) 9.11 ! 1.83 (19) 4.27 ! 1.27 (14) 1.94 ! 0.73 (21) 0.64 i 0.32 (20) Dentate 1.14 ! 0.42 (IB) 4.77 ! 1.48 (IB) 4.27 i 1.20 (14) 4.77 i 0.98 (IB) 1.38 t 0.46 (18) Hippocaapus * 1.77 i 0.55 (11) 9.83 i 1.81 (11) 4.96 i 1.25 (11) 1.93 i 0.47 (11) 0.29 i 0.18 (11) Values (aean 1 SD) are expresed in uaol/g vet weight with nuaber of subjects in parentheses. Abbreviations: TAU (taurine), 6LU (glutaaic acid), 6LN (glutaaine), 6ABA (gaaaa-aainobutyric acid), HCARN (hoaocarnosine). Perry et a l . 1987a, * Perry et a l . 1987b, «* Perry 1982 105 Appendix Table A-IV: Continued. Brain Region: 6LYC-PEA # PEA CYSTA 6ABA-LYS Frontal Cortex 0.89 i 0.32 (29) 1.83 i 0.51 (25) 0.55 i 0.53 (25) 0.01 1 0.02 » (18) Occipital Cortex 0.77 J 0.36 (27) 1.08 i 0.43 (24) 1.65 i 1.25 (24) 0.02 ! 0.03 ** (15) Cerebellar Cortex 1.00 i 0.41 (26) 1.69 i 0.45 (25) 0.51 ! 0.61 (25) 0.03 i 0.03 (25) Caudate 0.97 ! 0.40 (33) 1.45 t 0.51 (30) 0.73 • 0.44 (30) 0.02 i 0.02 (30) Putaien 0.80 i 0.31 (19) 0.96 i 0.44 (16) 1.19 ! 0.68 (16) 0.04 t 0.04 (16) Substantia Nigra 1.34 i 0.46 (33) 0.79 i 0.23 (27) 1.16 i 0.74 (27) 0.07 t 0.08 « (20) Substantia Innoiinata ** 1.49 i 0.35 (7) 1.01 • 0.25 (7) 0.63 t 0.32 (8) . 0.01 ! 0.02 (8) Nucleus Accuabens 0.87 * 0.52 (16) 0.89 t 0.34 (15) 0.64 i 0.39 (15) trace (15) Globus Pallidus I+1I 1.12 ! 0.52 (16) 0.79 t 0.40 (15) 1.54 ! 0.74 (15) 0.05 i 0.06 (15) Thalamus (HD) 0.95 i 0.41 (21) 0.58 i 0.21 (20) 1.23 t 0.70 (20) 0.05 ! 0.05 (20) Dentate 1.94 i 1.87 (19) 0.36 t 0.17 (17) 1.41 ! 0.99 (17) 0.11 t 0.12 (17) Hippocaapus 1.26 i 0.27 (9) 1.03 i 0.26 (10) 0.52 i 0.34 (11) 0.01 ! 0.02 (11) Values (aean ! SD) are expresed in uaol/g vet veigbt with nuaber of subjects in parentheses. Abbreviations: 6LYC-PEA (glycerophosphoethanolaaine), PEA (phosphoethanolaaine), CYSTA (cystathionine), 6ABA-LYS (gaaaa-aainobutyryl-lysine). Perry 1982 except * Perry et a l . 1988, »» Perry et a l . 1987a 106 Appendix Table A-V; L i t e r a t u r e v a l u e s o f d o p a m i n e c o n c e n t r a t i o n s i n a u t o p s i e d h u m a n b r a i n . Reference: Control Subjects Brain Region: A. B. C. D. (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) Frontal Cortex 6.7 i 1.3 10.4 ! 2.6 190 • SO * Occipital Cortex 4.2 ! 1.3 Cerebellar Cortex 230 i 60 Caudate 4027 i S73 I 1370 ! 230 Putaien 5B13 t 767 t l 2340 i 450 Substantia Nigra 900 i 350 540 i 90 Substantia Innominata Nucleus Accuibens Globus Pallidus I 950 t 110 •> Globus Pallidus II Thalamus 310 i 70 Dentate 10.9 ! 2.6 Hippocampus 10.9 ! 3.9 A. Gilbert et a l . 1988 I average caudate, I t average putamen B. Javoy-Agid et a l . 1989 C. Herregodts et a l . 1989 D. Rinne and Sonninen 1973 * unspecified cerebral cortex, ** combined globus pallidus Values are given as mean ! SD Globus pallidus I: internal, II: external 107 Appendix Table V: Continued. References: Control Subjects Brain Region: E. F. 6. H. (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) Frontal Cortex 70 i 130 Occipital Cortex 30 ! 50 Cerebellar Cortex 50 * 70 i 80 Caudate 3380 i 790 4060 • 470 1570 t 970 3340 i 680 Putaien 3720 i 870 5060 i 390 1450 i 1100 5220 i 2130 Substantia Nigra 950 ! 130 Substantia Innominata Nucleus Accumbens Globus Pallidus I 420 t 460 ** 61obus Pallidus II 420 i 80 Thalamus 50 i 50 Dentate Hippocampus 50 t 40 E. Fahn et a l . 1971 F. Lloyd et a l . 1975 • standard deviation not specified 6. Adolfsson et a l . 1978 ** combined globus pallidus H. Perry et a l . 1985 Values are given as mean ! SD. Globus pallidus I: internal, II: external 108 Table A-VI: L i t e r a t u r e v a l u e s o f h o m o v a n i l l i c a c i d c o n c e n t r a t i o n s i n a u t o p s i e d h u m a n b r a i n . Appendix References: A. (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) Control Subjects Brain Region: . B. C. 0. Frontal Cortex 2320 ! 290 1B20 i 680 90 i 40 » Occipital Cortex 1200 i 300 Cerebellar Cortex 30 i 20 Caudate 4S00 ! 690 t 2590 i 310 Putaien 7703 i 1053 I t 4820 i 450 Substantia Nigra 4200 ! 990 1660 ! 170 Substantia Innoainata Nucleus Accuabens 61obus Pallidus I 3100 i 420 ** Globus Pallidus II Thalaaus . 300 i 60 Dentate 2730 i 730 Hippocaapus 2550 ! 460 A. Gilbert et a l . 1988 t average caudate, I I average putaaen B. Javoy-Agid et a l . 1989 C. Herregodts et a l . 1989 D. Rinne and Sonninen 1973 * unspecified cerebral cortex, •* coabined globus pallidus Values are given as aean ! SD. Globus pallidus I: internal, II: external 109 Table VI: Continued. Appendix References: Control Subjects Brain Region: E. F. B. H. (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) Frontal Cortex 190 i 50 40 t 60 Occipital Cortex Cerebellar Cortex 80 J 20 50 ** 80 i 130 Caudate 3310 i 250 2920 i 370 3370 ! 1640 1870 i 180 Putaien 4950 i 550 4920 i 320 7070 • 2840 2920 t 260 Substantia Nigra 570 i 160 1670 •* 1790 i 180 Substantia Innominata Nucleus Accuibens Globus Pallidus I 2570 i 190 « 4110 • 1960 * 1610 i 360 61obus Pallidus II 3510 i 310 1950 ! 380 Thalamus 570 t 160 410 ! 290 130 i 40 Dentate Hippocampus 230 i 50 160 • 140 E. 6ottfries et a l . 1965 tcombined globus pallidus F. Lloyd et a l . 1975 ** standard deviation not specified G. Adolfsson et a l . 1978 * combined globus pallidus H. Hornykiewicz et a l . 1968 Values are given as mean t SD. Globus pallidus I: internal, II: external 110 Table A-VII; L i t e r a t u r e v a l u e s o f n o r a d r e n a l i n e c o n c e n t r a t i o n s i n a u t o p s i e d h u m a n b r a i n . Appendix References: Control Subjects Brain Region: A. B. C. D. (piol/ig Pr-) (ng/g vet vt) (ng/g vet vt) (ng/g vet vt) Frontal Cortex 11.8 i 1.2 41.1 i 4.3 19.2 i 1.4 Occipital Cortex 17.6 i 2.S Cerebellar Cortex Caudate 0.21 1 0.11 149.6 • 16.9 Putaeen 0.17 ! 0.13 245.0 i 24.4 Substantia Nigra 1.51 i 0.64 Substantia Innoainata Nucleus Accuabens 0.20 ! 0.08 Globus Pallidus I 0.41 i 0.23 Globus Pallidus II 0.33 i 0.06 Thalaaus 152.4 ! 23.4 Dentate 22.4 1 6.5 Hippocaapus 0.22 i 0.09 19.3 ! 4.3 27.6 i 1.9 A. Ebinger et a l . 1987 B. Javoy-Agid et a l . 1989 C. Herregodts et a l . 1989 D. Reinikainen et a l . 1988 Values are given as aean i SD. Globus pallidus I: internal, II: external 111 Appendix Table A-VIII: L i t e r a t u r e v a l u e s o f s e r o t o n i n c o n c e n t r a t i o n s i n a u t o p s i e d h u m a n b r a i n . Control Subjects Brain Region: References: A. (piol/ig Pr-) (ng/g wet wt) (ng/g wet wt) (piol/ag Pr-) Frontal Cortex 31.8 i 1.8 2.4 ! 0.3 Occipital Cortex 2.6 i 0.4 Cerebellar Cortex Caudate 7.99 ! 2.42 178.1 t 10.2 246 • 30 i 25.0 i 4.0 Putaien 4.36 • 2.72 222.5 ! 13.8 317 ! 50 It 14.0 i 2.0 Substantia Nigra 17.93 ! 3.27 820 t 270 26.0 i 4.0 Substantia Innoiinata Nucleus Accuabens 1.79 i 0.27 26.0 i 4.0 61obus Pallidus I 10.91 t 1.56 12.0 J 1.0 Globus Pallidus II 9.47 i 3.23 Thalaaus 135.5 i 9.7 Dentate 6.7 i 2.5 Hippocaapus 3.25 i 0.56 75.1 ! 5.3 117,000 J 83,000 6.6 t 1.1 A. Ebinger et a l . 1987 B. Reinikainen, K.J. et a l . 1988 C. Gilbert et a l . 1988, I average caudate, II average putaaen D. Korpi et a l . 1986 * unspecified globus pallidus Values are given as aean i SD. Globus pallidus I: internal, II: external 112 Appendix T a b l e A-IX: L i t e r a t u r e v a l u e s o f 5 - h y d r o x y i n d o l e - a c e t i c a c i d c o n c e n t r a t i o n s i n a u t o p s i e d h u m a n b r a i n . Control Subjects Brain Region: References: A. (ng/g vet vt.) B. (ng/g vet vt.) C. (ng/g vet vt) D. (naol/mg Pr-) Frontal Cortex Occipital Cortex Cerebellar Cortex Caudate Putamen Substantia Nigra Substantia Innominata Nucleus Accuibens Globus Pallidus I Globus Pallidus II Thalaaus Dentate Hippocaapus 134.8 i 18.6 550.3 i 55.4 909.2 • 61.1 1462.4 i 107.2 255.0 I 18.3 174 t 37 228 i 90 553 t 11 t 960 i 147 II 2600 t 450 8.8 • 0.9 9.9 t 1.0 40.0 • 6.0 39.0 • 5.0 139.0 i 17.0 76.0 i 10.0 49.0 i 10.0 * 21.0 ! 5.0 31.0 i 3.0 All values are mean i SEN. A. Reinikainen et a l . 1988 B. Javoy-Agid et a l . 1989 C. Gilbert et a l . 1988, I average caudate, II average putamen D. Korpi et a l . 1986 * unspecified globus pallidus Globus pallidus I: internal, II: external 113 

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