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Molecular cloning of the human Substantia innominata : characterization of a brain large mRNA Boyes, Barry Edward 1990

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MOLECULAR CLONING OF THE HUMAN SUBSTANTIA INNOMINATA: CHARACTERIZATION OF A BRAIN LARGE mRNA By BARRY EDWARD BOYES B.Sc. (Hon.)/ The University of Alberta, 1982 M.Sc, The University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES NEUROSCIENCE PROGRAM We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1990 (c) Barry Edward Boyes, 1990 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) i i Abstract Brain tissue samples were collected from individuals with histologically and biochemically confirmed Alzheimer's Disease (AD), as well as from a group of individuals without any signs of neurological disease (NNC). Ribonucleic acid (RNA) was extracted from these tissues, characterized by several chemical methods, and the yields were compared between AD and NNC groups. High molecular weight RNA could be effectively extracted from frozen postmortem human brain. In comparison to the NNC group, tissue RNA levels were reduced in the AD hippocampus, but not in the temporal cortex or substantia innominata (SI). No difference in the physical integrity of the RNA was apparent between AD and NNC groups. A high complexity complementary deoxyribonucleic acid (cDNA) library was prepared using RNA extracted from the NNC SI. Differential hybridization screening using a variety of cDNA probes was employed to identify mRNAs expressed di f f e r e n t i a l l y between AD and NNC tissue, and between SI and other human tissues. Many selected mRNAs were examined for specificity of expression in brain tissue and brain regions. The cDNA clone pSI3a-24 identified an mRNA, which, on Northern blot hybridization, was expressed in brain tissue but not in the other human tissues examined. The identified mRNA was unusually large, with a chain length estimated at 15,500 bases. Quantification of the brain tissue levels of this mRNA was carried out using a ribonuclease protection assay. Tissue levels were higher in the SI (40 pg/jug RNA) than in the temporal cortex (28.6 pg/Mg), and were lowest in the cerebellum (11.2 pg/M9)• Levels of the mRNA in temporal cortex samples were increased 29% in the AD group, relative to NNC. No significant difference in the SI tissue levels was observed between AD and NNC groups. Hybridization analysis of human genomic DNA indicated that the mRNA was encoded by a single copy gene. Sequence analysis of the f u l l 3 kilobases of cloned cDNA was completed. Computer database searches failed to identify any known nucleic acid sequence with homology to the cDNA. Examination of the cDNA sequence for potential polypeptide coding regions suggested that the corresponding mRNA has a 31 untranslated region of at least 3 kilobases. Table of Contents Abstract i i List of Tables iv List of Figures v-vi Abbreviations v i i Acknowledgements v i i i I. Introduction . ...^ 1-46 A. Anatomical Correlates of AD 4-16 B. Neurochemical Correlates of AD 16-24 C. Genetics of AD 24-30 D. Gene Expression in AD 30-42 E. Approaches to New Disease Markers of AD .... 42-46 II. Statement of Problem to be Addressed 47 III. Experimental 48-169 A. Isolation of Human Post Mortem Brain RNA ... 53-82 B. Molecular Cloning of the Substantia Innominata 83-106 C. Screening the cDNA Library 107-131 D. Characterization of a Brain cDNA Clone for a Large mRNA 132-171 IV. Conclusion 172-175 V. Bibliography 176-203 iv L ist of Tables Table 1. RNA Content in Human Brain Tissue 31 Table 2. Selected mRNAs Investigated in AD Brain ... 34 Table 3. Group Data for Collected Human Brains 68 Table 4. Group Data for Selected AD and NNC Cases Used for RNA Yields 75 Table 5. Summary of the Northern Blot Analyses of cDNA Clones 124 V List of Figures Figure 1. Dissection of Human Brain SI and Hippocampus 67 Figure 2. Mean Neocortical ChAT Levels of AD and NNC Cases 69 Figure 3. Chromatographic Analysis of DNA Contamination in RNA Preparations .. 71 Figure 4. Electrophoretic Analysis of Brain RNA ... 73 Figure 5. Northern Blot Analysis of Brain RNA 74 Figure 6. RNA Yields in AD and NNC Brain Regions .. 76 Figure 7. DNA Reagents Used for SI cDNA Cloning ... 96 Figure 8. Summary of the cDNA Cloning 97 Figure 9. Electrophoretic Analysis of cDNA Products 100 Figure 10. Analysis of cDNA Insert Size 103 Figure 11. Histogram of cDNA Library Insert Size .. 104 Figure 12. Screening Strategies for the SI cDNA Library 116 Figure 13. Cerebellum cDNA Screening of the Library 118 Figure 14. Differential cDNA Screening of Selected cDNA Clones 120 Figure 15. Northern Blot Hybridization with Selected cDNA Clones 122 Figure 16. Northern Blot Analysis with Clone pSIac-24 127 Figure 17. Restriction Map of Clone pSIac-24 144 Figure 18. Southern Blot Analysis of Human DNA with Clone pSIac-24 144 v i Figure 19. Northern Blot Analysis of Brain Regional Expression of pSIac-24 mRNA 146 Figure 20. Northern Blot Analysis of Brain Regional Expression of pSIac-24 mRNA 147 Figure 21. Northern Blot Analysis of Rat Brain Expression of pSIac-24 mRNA 149 Figure 22. Nuclease Protection Assay of pSIac-24 mRNA 152 Figure 23. Electrophoretic Analysis of Nuclease-Resistant RNAs 153 Figure 24. Determination of the Tissue Levels of the mRNA in AD and NNC Brain Regions ... 156 Figure 25. DNA Sequence Determination Strategy of Clone pSIac-24 158 Figure 26. Sequence of pSIac-24 cDNA Clone 159-160 v i i Abbreviations AD Alzheimer's Disease AChE Acetylcholine esterase BSA Bovine serum albumin ChAT Choline acetyltransferase CNS Central nervous system cDNA Complementary deoxyribonucleic acid cRNA Complementary ribonucleic acid DS Down's syndrome dNTP Deoxynucleotidyl triphosphate EAA Excitatory amino acid EDTA Ethylenediamine tetracetic acid GABA Gama-aminobutyric acid GFAP G l i a l f i b r i l l a r y acidic protein HPLC High performance liquid chromatography kb Kilobases (1000 x nucleotides) kbp Kilobase pairs (1000 x nucleotide base pairs) kdal Kilodaltons (1000 x molecular weight) LC Locus ceruleus mRNA Messenger ribonucleic acid nBM Nucleus basalis of Meynert NF Neurofilament NFT Neurofibrillary tangles NGF Nerve growth factor NMDA N-methyl-D-aspartate NNC Neurologically normal control OAc Acetate PD Parkinson's disease PHF Paired helical filaments polyA RNA Polyadenylated ribonucleic acid RFLP Restriction fragment length polymorphism rRNA Ribosomal ribonucleic acid SDS Sodium dodecylsulfate SI Substantia innominata SSC Sodium citrate/sodium chloride (1 x is 0.15 M sodium chloride and 0.015 M sodium citrate) tRNA Transfer ribonucleic acid j8-APP /3-Amyloid precursor polypeptide v i i i Acknowledgements Many people were helpful to me during the course of the experiments described in this thesis. It would have been impossible to carry out this work on human brain without the donation of tissues by families of the deceased subjects. Financial support was generously supplied by the ADRDA, Inc. (Chicago), the Woodward Foundation, and the Medical Research Council of Canada. Many members of the Kinsmen Lab aided me in collecting specimens and acquiring relevant medical histories, most notably Margaret Druhan. I owe Margaret for many other things, including nagging me as necessary. I was greatly helped in learning to handle brain tissues by Drs. W. Staines, K. Sato, T. Beach, and J.R. O'Kusky. My lab sessions with Professor Pat McGeer remain unforgetable, as do the late night debates in his cigar-smoke f i l l e d office. Completion of this thesis was greatly aided by Dr. Douglas Walker, who reminded me periodically that I couldn't stay a student forever. Herculean patience has been exercised over my course of studies by my thesis supervisor, Professor Edith McGeer, my wife Wanda, and daughter Erin. I've had a lot of patient teachers, for which I am grateful. In particular I would like to thank Mr. D.J. Boyes, Dr. P. Kebarle, Dr. F. Cantwell, Dr. CM. Kay, Dr. S.C. Sung, and Dr. E.G. McGeer. 1 I. INTRODUCTION The investigation of human neurodegenerative diseases has a natural progression through the stages of an i n i t i a l description of a functional disorder, followed by anatomical investigation, then later to a chemical description. Before the turn of the century, there was l i t t l e evidence that the functional disorder characteristic of dementia had a clear anatomical basis. Alzheimer's description of the histopathology of a young victim of dementia (Alzheimer, 1907), established the existence of a distinct disease entity. The definitive diagnosis of dementia of the Alzheimer type (Alzheimer's Disease, AD) i s s t i l l dependent on the presence of the hallmark histological changes in the postmortem brain, as originally described by Alzheimer. Increasingly, research effort on AD i s concentrating on the chemical description. The functional disorder, most notably the progressive loss of intellectual a b i l i t y and memory, has been well documented. Similarly, the basic facts of the anatomy of the disease have been well characterized, although there i s s t i l l much to be learned about the quantitative pathology. Based on the functional and histopathological characteristics of AD, i t i s known that this is a relatively common disease, affecting as many as 10% of 2 the population over the age of 65 (Evans et a l . , 1989; Heston et a l . , 1981; Khachaturian, 1985), but occasionally appearing in much younger persons. In dementia patients over age 65 years, a f u l l half of those coming to autopsy can be seen to exhibit Alzheimer-type pathology (Tomlinson et a l . , 1970). It is well established that the incidence of the disease increases with age, and at age 85 as many as 50% of the population may be c l i n i c a l l y affected. This disease thus represents a severe threat to the quality of l i f e of aged individuals. The c r i t e r i a for the c l i n i c a l diagnosis of AD have been standardized, as described by McKhann et a l . (1984). The c l i n i c a l diagnosis i s exclusive in nature, arriving at probable AD after elimination of other probable causes of dementia, the most common of which i s multi-infarct dementia. The definitive diagnosis of AD can only be made by the c l i n i c a l observation of probable AD combined with the postmortem histological observation of a profusion of extracellular neuritic (senile) plaques and intracellular neurofibrillary tangles (NFTs). The descriptive chemistry of AD has progressed a great deal in the last 20 years. A number of neurochemical abnormalities are observed in the postmortem AD brain. Those involving the cholinergic system have been confirmed in many centers, and have a r e l i a b i l i t y for postmortem diagnosis approaching histological observations. As yet, however, the 3 chemical basis of this disease, like most other neurodegenerative diseases, remains unknown. With the aid of descriptive neurochemical and biochemical-neuroanatomical analyses, something has been learned of the c e l l types that are vulnerable to loss, but no conclusions can be drawn about the mechanism leading to c e l l dysfunction and death. In the past decade much of the basic research on AD has focused on several key areas, namely, 1) the occurrence, composition, and biogenesis of the pathognomic protein accumulations called neurofibrillary tangles and amyloid plaques, 2) delineation of the vulnerable neuronal types, 3) attempts to correlate abnormalities in identified CNS pathways with the relevant behavioral functions, 4) the creation of an animal model of the disease, and 5) a wide ranging search for additional functional, anatomical, chemical, and genetic markers of the disease. These are very broad categories, but these generalizations underscore the requirement for further clues in the search for the disease mechanism. It has become axiomatic that recombinant DNA technologies have revolutionized the a b i l i t y to investigate human diseases. In AD, gene expression w i l l be altered, relative to the normal state, as a cause or a consequence of the formation of the plaques and tangles and neuronal degeneration, as well as the various cellular processes that accompany these events. This suggests that the application of recombinant DNA methods to study gene expression in AD would serve a useful purpose. In 4 recent years work has begun on the study of the molecular genetics of the disease, and of gene expression in the AD affected brain. Work in this area has established the genetic susceptibility of AD within certain families, and i s playing a c r i t i c a l role in understanding the biochemistry of the aberrant protein accumulations which form the plaques and NFTs. Recombinant DNA methods w i l l contribute to the search for further biological markers of the disease. This thesis describes a series of experiments directed to the goal of defining genes expressed differentially in AD affected and neurologically normal brains. This search was focused on the substantia innominata (SI), a brain region containing the neocortically projecting cholinergic neurons of the nucleus basalis of Meynert (nBM). As w i l l be reviewed in the following sections, this group of neurons is of particular interest because of their well documented vulnerability in AD, and the good correlation between abnormalities of cortical cholinergic function and the c l i n i c a l status of the affected individual. A . ANATOMICAL CORRELATES OF AD At autopsy, on gross examination, the AD brain may present signs of cortical atrophy including decreased brain weight, sulcal widening, and ventricular dilatation. However, this is frequently not remarkable, in comparison with normal age related changes. The hallmark pathological findings in the AD cortex are the profusion of argyrophilic neuritic plaques and NFTs seen with various silver stains. Although both 5 structures can be found in the normal aged brain, the density of these structures in certain regions of the AD brain i s several orders of magnitude greater that seen in tissue from unaffected persons (Wilcock and E s i r i , 1982), and the density of occurrence correlates with the degree of cognitive impairment (as f i r s t detailed by Roth et a l . , 1967). Additional histological features of AD include marked intraneuronal granulovacuolar changes (Woodard, 1962). Structural Features of AD Plagues and Tangles At the microscopic level, the morphology of the proteinaceous accumulations, which are characteristic of AD, has been extensively studied. The plaque has been observed to be present in several discernable forms, ranging from a diffuse accumulation of dystrophic neurites and g l i a l elements (the immature or primitive plaque), through a structure with a well established core of amyloid, surrounded with a rim of neurites, g l i a l elements, and occasional g l i a l c e l l bodies (the classical or mature plaque), to the "burned-out" plaque, a structure up to 200 jitm in diameter composed of a solid core of amyloid with few peripheral neurites. It has long been thought that these various forms of the plaque represent a continuum, reflecting the age of the structure. Considerable information has been acquired in the last 5 years on the biochemistry of the major protein component of the core amyloid, as w i l l be described below. One of the consequences of information on the amyloid core polypeptide has been the 6 provision of specific immunochemical reagents for i t s detection. The use of such reagents yields a different view of the plaque from that obtained by silver stains, and has suggested that the form of the amyloid plaque varies with the regional and laminar distribution of these structures (Majocha et a l . , 1988). The microscopic features of the NFT have also been well described. At the ultrastructural level, the term paired helical filaments (PHF) was coined to describe the fibrous structures having a periodicity of 80 nm with a 22 nm maxima and a 10 nm minima (Kidd, 1963; Terry, 1963). It appears that the PHF are composed of pairs of 10 nm filaments wound in a double helical structure (Wisnewski et a l . , 1976). The apparent insolubility of the core components of this structure confounded attempts at direct protein chemical analysis (Selkoe et a l . , 1982). The NFTs are observed as both massive accumulations within the perikaryon, as well as less dense deposits (wisps) within the dystrophic neurites that contribute to the senile plaques. A great deal of effort has been invested in the immunochemical characterization of components associated with PHFs, both in situ as well as in a semi-purified form. A serious d i f f i c u l t y in the immunochemical characterization of the PHFs resides in resolving PHF associated epitopes from epitopes which are authentically part of the core PHF structure. The search for PHF components has concentrated on 7 proteins comprising the normal neuronal cytoskeletal apparatus. Immunochemical evidence suggests the association of the PHF with the high molecular weight neurofilament proteins (Dahl et a l . , 1982; Miller et a l . , 1986), the intermediate filament protein vimentin (Yen et a l . , 1983), the microtubule associated protein-2 (MAP-2: Kosik et a l . , 1984; Yen et a l . , 1987), and the tau polypeptides. The most intensively characterized cytoskeletal components associated with the PHFs are the tau polypeptides. The tau polypeptides are a group of homologous phosphoproteins with a molecular weight of 55 to 65 kdal. The presence of PHF associated tau proteins has been well characterized by immunochemical means (Grundke-Iqbal et a l . , 1986, 1988; Delacourte and Defossez, 1986; Kosik et a l . , 1986; Goedert, et a l . , 1988; Papasozomenos, 1989a,b and references therein). One of the known functions of the tau proteins i s in the regulation of microtubule assembly (Drubin and Kirschner 1986). Immunochemical and immunohistochemical evidences suggest that the phosphorylation state of tau in the perikaryon may be different in AD than in normal brain (Grundke-Iqbal et a l . , 1986b, 1988; Wood et a l . , 1986), although not a l l workers in the f i e l d find these observations convincing (Goedert et a l . , 1988). The resistance of tau polypeptide to release during proteolytic treatment suggests that i t i s tightly associated with the PHF core and may be an integral component (Wischik et a l . , 1988). Many of the 8 antibodies for other cytoskeletal components have been found to cross-react with isolated tau polypeptides. To date, tau is the only component for which there i s good chemical evidence for a "tight" PHF association. On the basis of microscopic comparison with the normal neuronal cytoskeletal apparatus, the PHFs are structurally distinct from intermediate filaments, neurofilaments, or microtubule apparatus (Wisnewski, et a l . , 1984). On a morphological basis i t is therefore unclear what the relationship i s between the normal neuronal cytoskeleton, the NFTs, and the presence of normal cytoskeletal proteins within, or at least tightly associated with, the PHFs. Nevertheless, the fibrous nature of the NFTs has encouraged speculation that a significant feature of the pathophysiology of AD i s the accumulation of cytoskeletal components within affected neurons (Gajdusek, 1985). The accumulation of protein mass within c e l l bodies could lead to, or be caused by, a failure of axoplasmic transport, which could then lead to the demise of the c e l l s . Other lines of evidence which indicate the importance of cytoskeletal elements in AD pathology have been reviewed recently by Matsuyama and Jarvik (1989). Brain Regional Pathology A considerable literature exists on the regional distribution of plaques and NFTs within the brain. Generally, these intracellular accumulations can be observed throughout the cortex, but are most frequently observed in the allocortex 9 (hippocampus, associated limbic structures), as well as certain neocortical regions. The cortical topographic density of NFTs appears highest in the medial temporal lobe structures (eg. amygdala, hippocampus, uncus, and parahippocampal gyrus) and temporal neocortex, followed by association cortical areas including parietal and frontal cortical divisions, and i s least in the primary sensory areas (Pearson et a l . , 1985; E s i r i et a l . , 1990). In the most affected areas of neocortex there appears to be a preferential involvement of pyramidal neurons in layers III and V, with a generally greater density in layer V (Pearson et a l . , 1985). Patches of increased NFT density are observed in longitudinal strips of affected neocortex, the occurrence of which i s in register in layers III and V, perhaps suggesting a vertical mobility of the pathology. This selective laminar pathology would be consistent with a loss of pyramidal projection neurons subserving short intracortical connections (reviewed by Morrison et a l . , 1988), and has been thought to contribute to a global cortical disconnection syndrome. This could, in part, explain the c l i n i c a l presentation of agnosias and apraxias in advanced cases (Terry and Katzman, 1983). In contrast to the neocortical laminar appearance of NFTs, a clear pattern of lamination of plaques i s not common, although some preference for layers II, III and V has been reported (Pearson et a l . , 1985; Rogers and Morrison, 1985). This dissociation of plaque and NFT densities has also been demonstrated in other AD affected brain regions (Kalus et a l . , 1989; Braak et a l . , 1989). In the Guamanian ALS-Parkinsonian-Dementia complex, as well as in the Steele-Richardson-Olszewski syndrome (progressive supranuclear palsy), a similar dissociation of plaque and tangle pathology has been noted, with the primary microscopic lesion being the NFTs (Hirano et a l . , 1961; Chen, 1986). This dissociation has the c r i t i c a l implication that the genesis of plaques and NFTs may not occur by a single mechanism. The high density of plaques and NFTs in the hippocampal formation has been suggested as a major substrate of the memory deficits in AD patients. A major projection pathway from the entorhinal cortex (via the parasubiculum) to the hippocampus is essentially eliminated, resulting in partial deafferentation. This has been referred to as hippocampal disconnection (Hyman et a l . , 1984; Ball et a l . , 1986). It has been suggested that the hippocampal pathology could, in i t s e l f , explain the memory de f i c i t of AD, in analogy with the classical memory deficits observed in persons with surgical hippocampectomy. In addition to the telencephalic occurrence of plaques and NFTs, a number of subcortical nuclei display marked accumulations, particularly the cells of the nBM, locus ceruleus (LC), and dorsal raphe complex. The neurochemical results of the pathological involvement of the aminergic neuronal groups w i l l be detailed separately in the next section. These three aminergic c e l l groups have the common characteristics of having widespread and relatively diffuse projections to cortical structures. Demonstration of a cholinergic abnormality in AD near the end of the 1970s (see below), intensified interest in the cells of origin for the cortical cholinergic projections. Lesion studies in animals (Hebb et a l . , 1963; Johnson et a l . , 1979) suggested a basal forebrain origin. The acetylcholinesterase (AChE) histochemical (reviewed by Fibiger, 1982) and choline acetyltransferase (ChAT) immunohistochemical mapping (Kimura et a l . , 1981; McGeer, 1984) of the cholinergic c e l l bodies in the basal forebrain was followed by the observation of Alzheimer-type histopathological changes and c e l l loss in this brain region (Whitehouse et a l . , 1982). These authors noted the presence of abundant NFTs and many shrunken magnocellular neurons displaying granulovacuolar degeneration. A subsequent study by McGeer et a l . (1984) defined the cholinergic neurons of this brain region on the basis of their large somal diameter; virtua l l y a l l of the neurons (> 95%) with diameters >20 pm displayed immunoreactivity for ChAT. Determination of the density of these magnocellular neurons indicated a marked loss (up to 75 %) of these cells in the AD brain. In the same study, i t was noted that there was a good correlation between the c e l l loss and the decrement in neocortical ChAT. These findings are in accordance with the results obtained by a number of other investigators, the results of which w i l l be described in more detail below. Abundant NFTs and associated pathological changes have been well documented in the noradrenergic neurons of the LC. An associated loss of the number of LC neurons i s also observed, when judged by either the number of pigmented cel l s , or by dopamine /3-hydroxylase stained neurons (reviewed by Mountjoy, 1986; see also, Burke et a l . , 1988; Zweig et a l . , 1988) . Attempts have been made to differentiate subgroups of AD with varying severity of the disorder, reflected by the bimodal distribution of LC c e l l loss (AD-1 and AD-2 groups, Bondareff et a l . , 1987). This distinction has been challenged by others, using similar methods (Mann et a l . , 1988). Studies of larger numbers of cases across the ages 50-90 may reveal the existence of such subgroups. The serotonergic neurons of the dorsal raphe display prominent AD pathological changes, including abundant NFTs (Hirano and Zimmerman, 1962; Perry and Perry, 1985; Zweig et a l . , 1988; Burke et a l . , 1989). The determination of c e l l loss of the somewhat dispersed serotonergic neurons is d i f f i c u l t , but a substantial loss (up to 50%) has been reported by many groups (Mann et a l . , 1984; Herrogodts et a l . , 1989; Burke et a l . , 1989; Ebinger et a l . , 1987; Yamamoto and Hirano, 1985; Zweig et a l . , 1988). Another site of prominent AD pathology i s in the olfactory system, described originally by Averback (1983) and E s i r i and Wilcock (1984). In these reports, NFTs and c e l l loss were observed in the anterior olfactory nucleus. These findings have been confirmed and extended by many others, as recently reviewed by Ferreya-Moyano and Barragan (1989). Pathological changes in the olfactory system correlate well with the c l i n i c a l observation of impaired olfaction in AD patients (Warner et a l . , 1986). Based on the overall pattern of AD pathological changes, certain generalizations have been made in order to direct research on the etiology of AD. It has been noted that there is a heavier involvement of brain regions with relatively direct connections to the olfactory system (Pearson et a l . , 1985; McGeer et a l . , 1986; Ferreya-Moyano and Barragan, 1989). On this basis, i t has been hypothesized that the pattern of pathology could be related to a retrograde degeneration, possibly resulting from an i n i t i a l route of entry of a toxic agent through the primary olfactory apparatus, a v i r a l etiology being the most frequently invoked (in analogy with Creutzfeldt-Jakob disease). Although an attractive hypothesis from the anatomic viewpoint, l i t t l e evidence has been forthcoming in support. In order to test this hypothesis, i t would be necessary to have information on two aspects of the disease about which nothing is presently known: 1) the nature of the toxic agent, and 2) the anatomical progression of the pathology. It has been noted that many of the nuclei which have 14 projections to the neocortex exhibit pathological changes in AD. As such, i t has been suggested that AD i s primarily a disease of the neocortex, which secondarily affects subcortical nuclei (eg., German et a l . , 1987). At the present time there appears to be too l i t t l e information available to draw conclusions about the relative times at which the cortex or subcortical nuclei exhibit pathological changes. Based on the positive correlation between the density of NFTs found in certain cortical regions and the degree of connection of these regions with the amygdala, E s i r i et a l . (1990) have advanced the proposition that the disease process may also spread along these reciprocal connections. Although a variety of brain regions exhibit the pathognomic histology of AD, many brain regions do not appear heavily involved. The cerebellar cortex is generally free of plaques and tangles, although diffuse amyloid deposits are found there (Wisniewski et a l . , 1989). Similarly, the basal ganglia i s not usually involved in AD cases without Parkinson Disease. The peripheral and autonomic nervous systems have not been observed to be affected in AD. Thus i t i s reasonable to conclude that the disease displays brain regional specificity. Similarly, there i s l i t t l e evidence that nonneural tissues are affected by the disease. Cl i n i c a l l y , this f i t s with the fact that AD patients can survive for relatively long periods after severe cognitive impairment has become manifest. 15 Non-neuronal Pathology In order to understand the pathological processes at work in AD, i t i s necessary to consider nonneuronal c e l l types which may have a role in neurodegeneration. These include the various g l i a l elements, vascular components, and ce l l s of the immune system. A number of studies have addressed the astrocytic response to degeneration, noting the hypertrophic and hyperplasic response. In AD brain, astrocytic gliosis has been studied by c e l l counts (30 % increase in affected areas, Hansen et a l . , 1988) and immunohistochemical staining with antisera for g l i a l f i b r i l l a r y acidic protein (GFAP). Immunohistochemical studies have noted an apparent increase in the strength of immunoreaction, as well as an increased number of stained astrocytes in both the neocortex (Schechter et a l . , 1980; Hansen et a l . , 1987) and hippocampus (Duffy et a l . , 1980; Probst et a l . , 1982). In a l l of these studies, the astrocytes were observed to be most numerous when in close proximity to plaques and NFTs. A marked increase in GFAP in tissue homogenates has been demonstrated by electrophoresis combined with densitometry (Panter et a l . , 1985), as well as by immunoassay (Delacourte, 1990) and by mRNA quantification (see Table 1, page 31). Several recent studies have detected a variety of immune system c e l l markers indicating an active immune response within the AD affected brain. The detection of human leucocyte antigen-D Region (HLA-DR) immunoreactive c e l l s has highlighted the involvement of the macrophages or activated microglia in brain regions that are undergoing active degenerative changes, including the neocortex (McGeer et a l . , 1987; Rogers et a l . , 1988) and substantia innominata (McGeer et a l . , 1988). More recent studies have detected T c e l l subsets which could contribute regulatory substances (lymphokines) to modulate the local immune system activities (Itagaki et a l . , 1987a). The description of elements of the complement system in AD brain (McGeer et a l . , 1989) i s further evidence that the immune system is involved in the degenerative process. B. NEUROCHEMICAL CORRELATES OF AD Cholinergic Abnormalities in AD By the 1970s the search was well under way for neurochemical alterations in AD. Various groups had demonstrated the postmortem st a b i l i t y of neurotransmitter related metabolic enzymes for a variety of putative transmitter substances (McGeer and McGeer, 1975, Perry et a l . , 1977). The activity of neocortical ChAT, the specific synthetic enzyme for acetylcholine, was reported to be decreased in AD by a number of groups (Bowen et a l . , 1976; Davies and Maloney, 1976; Perry et a l . , 1977). There is currently general agreement that a loss of 50-90% of neocortical ChAT is seen in AD, relative to age matched neurologically normal persons. In both postmortem tissue, as well as tissue biopsies, the excellent correlation between neocortical tissue levels of ChAT, the occurrence of AD pathological changes (relative density of plaques and/or NFTs), and behavioral dysfunction, have a l l indicated the importance of cholinergic abnormalities in the disease (Perry et a l . , 1978; reviews by McGeer, 1984; Perry, 1986; Mountjoy, 1986; Hansen et a l . , 1988; E s i r i et a l . , 1990). The specificity and r e l i a b i l i t y of the cortical cholinergic d e f i c i t in AD has been confirmed by the results of a number of groups, who also demonstrated the general sparing of ChAT in other brain regions such as the basal ganglia and cerebellum. The exception to this specificity may be the ventral s t r i a t a l cholinergic neurons, which have been reported to be specifically lost in AD (Lehericy et a l . , 1989). If these cel l s are confirmed to be intrinsic to the striatum, and not projection neurons, (as are the nBM cholinergic neurons), this represents a unique finding. The marked loss of cortical ChAT activity in AD, relative to the much less severe decline observed in normal brain aging, clearly differentiates the disease state from the normal age-related neurochemical changes in the brain (cf. McGeer, 1976). Several studies on large numbers of AD and aged controls have indicated an important trend in the severity of cholinergic dysfunction; younger affected individuals display greater severity of ChAT loss (Rossor et a l . , 1984; McGeer et a l . , 1986). Regression analyses for neocortical ChAT as a function of age, show a negative slope for normal aging (ChAT decreases with age), but in AD cases the slope of the curve is positive, ie. ChAT levels are relatively higher in older affected individuals. These data are crucial, as they represent evidence that AD i s not a form of accelerated brain aging. The loss of cortical ChAT i s certainly a reflection of pathological changes in the nBM magnocellular cholinergic neurons. There i s considerable evidence that the cortical decrement i s due to c e l l loss, as reflected by the positive correlation between nBM c e l l counts and neocortical ChAT activity (McGeer et a l . , 1984; Arendt et a l . , 1984). Although the loss of the nBM neurons is well established, the loss of nBM ChAT activity in AD has been reported to be more variable, with some investigators reporting AD patients with normal nBM ChAT levels (Bird et a l . , 1983; Etienne et a l . , 1986). In the study of Etienne et a l . (1986) several AD cases were described which exhibited nBM c e l l loss and loss of temporal cortex ChAT, but also had normal ChAT levels in the nBM. These authors interpreted this finding as an indication that decreased terminal ChAT levels reflected an axonal transport defect. It was also suggested that the pathology in the nBM may be concomitant with cortical pathology. An alternative explanation for the heterogeneous nBM ChAT loss may l i e in regenerative processes occurring in the surviving nBM neurons. Evidence for regenerative changes in nBM neurons has been observed in Golgi preparations, as described by Arendt et a l . (1986), as well as by galanin immunohistochemistry (Chan-Palay, 1988; described below). In the Golgi study, reticular neurons of the nBM and nucleus of the diagonal band were observed to increase in cross-sectional area, dendritic arborizations, and to display perisomatic filopodia. It was suggested that these results reflect a compensatory mechanism within surviving neurons. In this case, i t is conceivable that regenerative ce l l s could exhibit neurochemical alterations which would mask a degenerative neurochemical change and thus explain the heterogeneity of nBM ChAT loss. Non-cholinergic Abnormalites in AD A variety of other chemically identified neuronal groups have been reported to be vulnerable in AD. The anatomical evidence for the involvement of the noradrenergic neurons of the LC and serotonergic cells of the raphe has been described. The tissue level of noradrenaline is decreased in the neocortex and hippocampus, to 30-80 % of the normal level (Adolfson et a l . , 1979; Mann et a l . , 1982; Cross et a l . , 1983; Arai et a l . , 1984; Palmer et a l . , 1987; Baker and Reynolds, 1989). In addition, decreased levels of serotonin and i t s primary catabolite, 5-hydroxyindoleacetic acid, have been reported in the neocortex and hippocampus of AD cases, although the extent of loss appears variable (Adolfson et a l . , 1979; Arai et a l . , 1984: Bowen et a l . , 1983; Rossor et a l . , 1984; D'Amato et a l . , 1987; Ebinger et a l . , 1987; Herregodts et a l . , 1989; Baker and Reynolds, 1989). It is of particular significance that the serotonergic neurons undergo c e l l loss 20 in AD. Most evidence suggests that this transmitter system, unlike the noradrenergic and dopaminergic neurons, is preserved in normal brain aging, thus i t s loss in AD is a further distinction between normal brain aging and the pathophysiology of AD. In many brain regions the large cortical pyramidal neurons exhibit pathological changes, and these are believed to use an excitatory amino acid (EAA) transmitter, as well as to have a high density of EAA receptors (Cotman et a l . , 1987). Hence, several studies have investigated both pre- and postsynaptic markers of EAA systems. The direct analysis of amino acid content of brain tissue i s complicated by the existence of non-transmitter amino acid pools, as well as rapid postmortem metabolism and the overlying effects of antemortem condition. It i s therefore not surprizing that poor quantitative agreement on the levels of these compounds has been observed in postmortem brain tissue studies. In one study, a marked decrease in glutamate, and a smaller loss of aspartate, was reported in AD brain tissue (Sasaki et a l . , 1986), but this was not observed using a more specific measurement technique and a larger series of patients (Ellison et a l . , 1986). Similarly, Perry et a l . (1987) did not observe any change in EAA levels in neocortex or hippocampus. The situation i s also confused with regard to the integrity of receptor sites for EAA receptors as judged by high a f f i n i t y binding. I n i t i a l reports on glutamate binding by in situ autoradiography revealed apparently decreased numbers of binding sites in both the hippocampus and neocortex in AD brains (Greenamyre et a l . , 1985; 1987). This loss was apparent for total glutamate binding, as well as for NMDA type binding sites, with decreases in the neocortex to about 40% of the normal values. However, the loss of NMDA receptors has not been observed by several other groups (Geddes et a l . , 1986; Monaghan et a l . , 1987; Cowburn et a l . , 1988), and the levels of kainic acid receptors appear unaffected in AD (Cowburn et al . , 1989). More recently s t i l l , i t has been reported that glycine modulation of the NMDA receptor complex is decreased (Procter et a l . , 1989; Steele et a l . , 1989). Although considerable effort has been taken to resolve the various subtypes of postsynaptic EAA receptors, there may s t i l l be methodological issues which must be resolved before these apparently conflicting results can be rationalized. Direct measurements of the receptor-associated proteins, or the corresponding mRNAs, w i l l define the involvement of the EAA systems in AD. The specificity of neuronal loss in AD is frequently judged in reference to the preservation of 7-aminobutyric acidergic (GABAergic) neurons in the affected area (Davies, 1979, Coyle et a l . , 1983). This represents somewhat of a di f f i c u l t y because of the marked effect of agonal state on the GABAergic markers (cf. Perry and Perry, 1983), including tissue levels of GABA and the synthetic enzyme glutamic acid decarboxylase (GAD). One study, using a GABA uptake technique of dubious u t i l i t y with postmortem tissue, reported a loss of GABAergic terminals (Hardy et a l . , 1987), but studies of K+-evoked release of GABA in biopsy samples failed to find any difference between AD and control (Smith et a l . , 1983). Taken in total, there i s good evidence that GABAergic neurons are probably not grossly affected in AD. A number of studies have also reported a loss of specific neuroactive peptides in AD brain (reviewed by Beal and Martin, 1986). Somatostatin i s the most extensively studied neuropeptide in AD, and the one for which the evidence i s strongest for a cortical loss in AD. Even here, however, reports to the contrary exist (Whitford et a l . , 1988). In the neocortex, somatostatin immunoreactivity i s localized to small int r i n s i c neurons. A feature of the neocortical neuropeptides is their colocalization with GAD (Hendry et a l . , 1984). One of the unexplained characteristics of the cortical losses of certain neuropeptides in AD (eg., somatostatin, neuropeptide Y, corticotropin releasing factor) i s the preservation of GABAergic indices in AD. It may be the case that a small number of these GABAergic neurons are peptide-containing. The most frugal explanation is a loss of the peptide without loss of the neuron. This appears to be the case for somatostatinergic neurons, which do not appear to be lost in number, although an occasional atrophic c e l l may be seen (Nakamura and Vincent, 1986). Recently, the neuropeptide galanin has been intensively studied, due to the observation that this peptide co-exists, in lower mammals with cholinergic neurons in the basal forebrain, with noradrenaline LC neurons, with serotonin raphe neurons, and with histamine neurons in the tuberomammillary system of the hypothalamus (Melander and Staines, 1986; Melander et a l . , 1986). Interestingly, the levels of galanin do not change in AD neocortex (Beal et a l . , 1988). Since the aminergic and cholinergic efferents to the cortex are known to degenerate, these results could suggest increased production of galanin within surviving afferents as a compensatory mechanism. In this regard, i t i s interesting that a recent immunohistochemical investigation of the AD basal forebrain identified galanin immunoreactive neurons making contacts with the somata and dendrites of cholinergic neurons (Chan-Palay, 1988) . These galanin containing neurons were claimed to appear to be hypertrophic and to hyperinnervate the surviving cholinergic neurons, although the available evidence i s fragmentary. Since galanin appears to be inhibitory to cholinergic neurotransmission (reviewed by Crawley and Wenk, 1989) , increased galanin activity on basal forebrain neurons could exacerbate the cortical cholinergic d e f i c i t in AD. The senile plaque has long been appreciated to have a distinctive morphological appearance, with a central core of amyloid and a surrounding halo, made up in part by dystrophic neurites. It was hoped that the transmitter specificity of 24 these neurites could yield suggestions on the biogenesis of the plaques. Thus, the observation of AChE enzyme activity (Friede, 1965) and ChAT immunoreactivity (Kitt et a l . , 1984) in dystrophic neurites suggested a relationship between cholinergic terminals and plaque formation. The observation of possible primitive, or immature, plaques on cholinergic axons in the neocortex has further strengthened this association (Tago et a l . , 1987). This association may be merely coincidental however, as there i s evidence that several other neurotransmitters are present in dystrophic neurites, including GAD (Walker et a l . , 1985) and a variety of neuropeptides (somatostatin, neuropeptide Y, substance P, vasoactive intestinal peptide, cholecystokinin, bombesin and galanin: Armstrong et a l . , 1985; Dawbarn and Emson, 1985; Kowal and Beal, 1988; Nakamura and Vincent, 1986; Struble et a l . , 1985). In consideration of the variety of neurotransmitters in the dystrophic neurites of the plaques, i t seems unlikely that there i s any direct relationship between transmitter specificity and plaque formation. C . GENETICS OF AD Investigation of a genetic etiology of AD appears to have been initiated by Sjogren et a l . (1953). As in many subsequent studies, these investigators found clear evidence for a greater than expected incidence of AD in relatives of affected individuals. The case for a genetic component was greatly advanced by the careful studies of Heston and colleagues 25 (Heston et a l . , 1981). Many subsequent studies have confirmed the increased incidence of AD in certain families. A further finding in the studies of Heston was the increased incidence of Down's syndrome (DS) within affected families, a finding supported by examination of some kindreds (Heyman et a l . , 1983), but possibly complicated by genetic heterogeneity (Marazita et a l . , 1987; Nee et a l . , 1983). The association of DS with AD was of great interest because of the clear genetic basis of DS (Trisomy 21), as well as the Alzheimer-type pathological changes found in persons with DS dying at > 35 years. By 40 years of age, these individuals invariably develop NFT and plaque accumulations indistinguishable from AD (Jervis, 1948; Burser and Vogel, 1973; Wisnewski et a l . , 1985). A more recent trend has been towards dissociation of AD into early onset familial Alzheimer's disease (familial AD), and late onset, possibly sporadic, AD. This trend has developed from the discovery of families with a clear autosomal dominant transmission (Feldman et a l . , 1963; Goudsmit et a l . , 1981; Nee et a l . , 1983). At present, i t i s not known how many AD affected individuals would be of the familial AD type, but the figure would appear to represent less than 15% of affected persons (Breakefield and Cambi, 1988; Heston et a l . , 1981). Again, the picture i s complicated by the possibility of genetic heterogeneity, an issue which is not yet settled (St. George-Hyslop et a l . , 1988; Bird et a l . , 26 1988). A variety of genetic markers have been examined for linkage analysis in familial AD. The use of panels of polymorphic human leukocyte antigens (HLA), as well as other polymorphic antigenic determinants, has not been of much u t i l i t y (Kay, 198 6). The use of restriction fragment length polymorphic (RFLP) markers should supply sufficient polymorphic l o c i to be able to detect the chromosomal location of the FAD gene, in analogy with the approach that has proven successful for a variety of other human genetic disorders (reviewed by Gusella, 1986). In the case of AD, however, the lack of large well characterized affected families, as well as the possibility of genetic heterogeneity in the expression of the disease, represent severe obstacles in the application of the RFLP linkage approach. The remarkable growth in attempts to apply RFLP analysis to AD has been catalyzed by the amino acid sequence data for the isolated cerebrovascular amyloid A4 peptide, i n i t i a l l y reported by Glenner and Wong (1984a,b). The isolation of cDNA clones corresponding to the Glenner and Wong sequence (Kang et a l . , 1987; Goldgaber et a l . , 1987; Tanzi et a l . , 1987a; Robakis et a l . , 1987) allowed identification of the mRNA and gene coding for the precursor polypeptide for the A4 peptide, referred to as the j8-amyloid precursor polypeptide, or /3-APP. A discussion of the terminology appropriate for this gene and i t s associated transcripts and polypeptides has been presented by Selkoe (1989). In the i n i t i a l studies localization of the |8-APP gene to chromosome 21 was also achieved, as was a genetic linkage with the FAD gene locus in some kindreds (Tanzi et a l . , 1987a; St. George-Hyslop et a l . , 1987). The apparent association between FAD, DS, and the histopathology of these disorders seemed to bring together many important observations into a single etiological picture. The report of increased gene dosage of the /3-APP l o c i in several cases of AD (Delabar et a l . , 1987), and the observed increased expression of the /3-APP mRNA in Down's syndrome cases (Tanzi et a l . , 1987a) appeared to supply the evidence necessary to rationalize many unknown features about AD. It was hypothesized that an increased gene dosage of the /3-APP gene could lead to overexpression of the protein, and the resultant accumulation of the protein could lead to c e l l death. However, the description of recombination between the /3-APP l o c i and the FAD gene suggests that a mutant /3-APP gene is not the cause of the disease (Tanzi et a l . , 1987b; Van Broeckhoven et a l . , 1987). Furthermore, subsequent studies of the gene dosage in both familial and sporadic AD (Tanzi et a l . , 1987b, Van Broeckhoven et a l . , 1987; Podlisny et a l . , 1988; Furuya et a l . , 1988; St. George-Hyslop et a l . , 1987b) failed to find an abnormal complement at this locus. More recently, there are reports of FAD families which do not demonstrate any detectable linkage with markers covering large portions of chromosome 21 (Schellenberg et a l . , 1988), suggesting that 28 other chromosomes may be involved. At the present time i t is unclear what proportion of families may carry a single gene mutation leading to the disease, or on what chromosome linkage may be detected within particular kindreds. In the ensuing period, a great deal of information on the /J-amyloid precursor polypeptide has been derived. The /3-APP cDNA clones allowed the sequencing and structure prediction of the j8-APP polypeptide (Kang et a l . , 1987; for a review see Selkoe, 1988). Recent results have shown that there are at least three (Ponte et a l . , 1988; Kitaguchi, et a l . , 1988; Tanzi et a l . , 1988), and possibly four (A714 mRNA, Kang and Muller-Hill, 1990) forms of the j8-APP mRNA generated by alternative splicing of the primary transcript. The best characterized mRNAs have predicted polypeptides of 695, 751 and 770 amino acid (aa) residues. The 695 residue polypeptide has a small C-terminal cytoplasmic domain of about 40 aa, a transmembranous domain of about 20 aa; the rest of the polypeptide, extending to the N-terminal, i s extracellular. Of the 28 aa in the amyloid core A4 peptide (which is common to a l l /3-APPs) , approximately half the residues are in the extracellular domain, with the other half extending into the transmembranous region. The extracellular polypeptide sequence possesses two canonical glycosylation sites, which have been shown to be functional in vivo. The two larger j8-APP polypeptides, containing inserted sequences, have a region of approximately 50 residues which are functionally homologous to the Kunitz serine proteinase inhibitor (KPI). The functional relevance of the inhibitor domain was indicated when cultured ce l l s transfected to express this portion of the /3-APP produced an active trypsin inhibitor (Kitaguchi et a l . , 1988). A l l of the available information on the features of the /3-APP suggest that this area of research offers some promise to the eventual understanding of the formation of the plaques, although the path to this i s not yet clear. Several reports of the isolation of APP cDNAs from AD brain have failed to detect alterations in the primary structure of AD APP (Zain et a l . , 1988; Vitek et a l . , 1988), confirming genetic data (see above) which suggest that a mutation of the polypeptide i s not a cause of the disorder. Elucidation of the KPI domains of the /3-APP may be of c r i t i c a l importance, as the balance between proteases, inhibitors, and target proteins is undoubtedly central to normal c e l l and tissue physiology. Recently Wagner et a l . (1989) examined the AD brain tissue levels of the protease inhibitor nexin-1 and reported a marked decrease in the uncomplexed form, with no decrease in total immunoreactivity. Immunohistochemical staining for nexin-1 determined a subset of plaques in AD brain which show increased immunoreactivity (Rosenblat et a l . , 1989). This protease inhibitor, as well as al-antichymotrysin, which is also associated with Alzheimer-type pathology (see below), are both secreted products of astrocytes. Thus, there i s currently considerable interest in the role of protease inhibitors in AD and the relationship between the KPI domain of /3-APP and processing of this amyloid precursor. It i s clear that this w i l l be an area of active research. D. GENE EXPRESSION IN AD BRAIN At the beginning of the 1980s, methods for the production, screening, and use of cloned cDNA libraries were becoming increasingly reliable. It was anticipated that the molecular cloning approach would be applied to the analysis of gene expression in the mammalian CNS, and to the human brain in various disease states. The i n i t i a l success of Sajdel-Sulkowska and Marotta (1983a) in the preparation of high molecular weight, biologically active RNA from frozen human brain was followed by the report of decreased total cellular RNA content in AD neocortex (Sajdel-Sulkowska et a l . 1984). Evidence was presented which suggested that the loss of total cellular RNA was related to the decreased activity of an inhibitor of an alkaline ribonuclease activity (Sajdel-Sulkowska and Marotta, 1984) , although this finding has been contested (Maschhoff et a l . , 1989). The reported loss of transcriptional fitness of AD nuclear extracts could also contribute to a generalized loss of cellular RNA yields in AD brain (Sajdel-Sulkowska et a l . , 1989) . Lewis et a l . (1981), reported alterations in nucleosome composition in AD brain. This observation could suggest an alteration of the transcriptional capability of chromatin. Several groups have now reported data on the yield and Table 1. RNA Content in Human Brain Tissue Brain RNA polyA RNA Level in AD Refer. Region8 (M9/9) (M9/9) (% Control) RNA polyA RNA Fr, T, P Ctx 100 2.8 55 36 1 Fr Ctx 193 n/a 103(62)b (66)b 2 Fr Ctx -400 n/a n/a n/a 3 Fr, T, P Ctx 230 3 . 0 93 53 4 P Ctx -520 n/a 77 n/a 5 Fr Ctxc 66.4 n/a 61 n/a 6 Cb Ctxc 37.6 n/a 58 n/a 6 n/a: not reported a. Regions as follows: Fr, frontal; T, temporal; P, parietal; Ctx, cortex; Cb, cerebellum. b. No difference was observed with determination by absorbance at 260 nm, but was observed by hybridization with probes for rRNA or polyA RNA (data in parentheses). c. These values refer to polysomal RNA, estimated by assuming 40/zg per absorbance unit. References: 1. Sajdel-Sulkowska and Marotta, 1984. 2. Taylor et a l . , 1986. 3. Johnson et a l . , 1986. 4. Guillemette et a l . , 1986. 5. Clark et a l . , 1989. 6. Langstrom et a l . , 1989. integrity of RNA from postmortem human brain. A summary of the available data is shown in Table 1. As can be seen from examination of these data, the absolute amount of total cellular RNA in the (normal) human brain on a tissue weight basis i s highly variable between investigators. A similar va r i a b i l i t y i s noted for the degree of loss of both total cellular RNA and polyadenylated RNA in AD brain. Careful examination of these studies suggests that the differences between them must have a methodological basis, as opposed to being a result of differences in postmortem interval, age, or sex, a l l of which are rather uniformly controlled. It i s conceivable that some of the differences could be derived from pathological c r i t e r i a or agonal status of the selected cases, but the magnitude of the differences between groups makes this appear less like l y . Because of the uncertainties in the literature about the levels of total cellular RNA levels in human brain tissue, as well as the relative amounts in AD versus control brains, i t becomes important to state the basis on which measurements of a particular mRNA are expressed, ie., as the amount per gram tissue, or per /xg of total RNA or polyA RNA. A promising approach is the use of in situ hybridization with oligo(dT) for the analysis of cellular polyadenylated RNA levels in AD and control brain regions (Harrison et a l . , 1991a). A number of investigators have undertaken the analysis of the biological fitness of AD mRNA to act as a template for translation in vitro. In the study of Sajdel-Sulkowska et a l . (1983a), i t was noted that, in a rabbit reticulocyte in vitro translation system, the yield of polypeptide produced by exogenous AD polyA RNA was reduced relative to control values. The opposite finding has been reported by Guillemette et a l . (1986), who found a nearly two fold increase in activity for AD polyA RNA in comparison to control. Langstrom et a l . (1989) have examined polysomal mRNA yields and translational fitness for AD and controls. In this study both the yield and activity of AD frontal cortex polysomal mRNA were reduced to approximately 50% of control values. In contrast, there was no significant change in either the yield or activity of polysomal mRNA from AD cerebellum. These results would suggest that, in affected brain regions, there would be a combined loss of both the amount and translational fitness of mRNA, which could be additive and lead to a profound loss of protein synthetic capacity. Recently, the investigation of gene expression in the AD brain has turned to the examination of specific genes. A summary of the findings for a variety of mRNAs in AD brain i s presented in Table 2. As indicated in the table, specific mRNAs examined have been divided into several categories, depending on whether genes are expressed primarily in g l i a l c e l l s , neurons, or have an unknown or mixed cellular localization in brain. It might be expected that astrocytes would alter their 34 Table 2. Selected mRNAs Investigated in AD Brain mRNA mRNA Type Region Level in AD Refer. Examined3 % Control G l i a l GFAP Fr 102 [1331b 1 Fr, T, P 137 ( 7 2 T 2 Hpc -20 0-300 6 Neuronal Neurofilament-L (NF-L) Fr 19 [36]> 1 Fr, T, P 27 (14)c 2 Preprosomatostatin Fr, T, P 97 (51)c 2 Fr 70 3 Preproenkephalin Fr 84 3 NGF T, P -80 4 NGF-receptor SI -115 5 decrease 7 Tau Fr no change 8 28 kdal calbindin-D SI, Hpc 10- 15 9 Neocortex 120 9 Mixed Calmodulin Fr, T, P 68 (36)c 2 j8-actin Fr, T, P 121 (64)c 2 a-tubulin Fr, T, P 65 (34)° 2 Fr 50 [47] b 1 |8-APP (total) Fr 37 [52] b 1 a. Regions as follows: Cortex- Fr, frontal; T, temporal; P, parietal; Hpc, hippocampus; SI, substantia innominata. b. The RNA was extensively degraded, the value in brackets refers to the total integral of the lane scanned densitometrically. c. The value in parentheses refer to data expressed relative to tissue weight, calculated on the basis of the reported loss of AD tissue polyA RNA to 53% of control (ref 2). References: 1. Clark et a l . , 1989. 2. Crapper McLachlan et a l . , 3. Taylor et a l . , 1986 4. Goedert et a l . , 1986. 5. Goedert et a l . , 1989a. 6. May et a l . , 1987. 1989. 7. Higgins and Mufson, 1989. 8. Goedert et a l . , 1989b. 9. Iacopino and Christakos, 1990. 35 gene expression as a function of the hypertrophy and hyperplasia which is known to occur in AD affected brain regions. The tissue levels of mRNAs coding for proteins expressed specifically in the astrocyte, such as the g l i a l f i b r i l l a r y acidic protein (GFAP), might be expected to increase in parallel with the increased tissue level of the protein. An increased level of GFAP mRNA has been reported in the neocortex of AD affected brain, although this has not been observed in a l l studies (Clark et a l . , 1989). In the few cases in which specific neurotransmitter related genes have been examined, mixed results have been reported. As an example, the levels of preprosomatostatin mRNA, expressed in relation to total cellular RNA, have been reported to be not different from control by two groups of investigators (Crapper McLachlan et a l . , 1988; Taylor et a l . , 1986); however, one of these groups reports a decrease in total cellular RNA, whereas the other does not, thereby complicating interpretation of the data. There is a similar lack of agreement between investigators concerning the level of tissue somatostatin-like immunoreactivity in AD versus control (see section IB). In an in situ hybridization study of AD and control brains, Harrison et a l . (1991b) have convincingly demonstrated an increased (270%) level of muscarinic M1 receptor transcripts in AD temporal cortex pyramidal neurons. One of the neuronal calcium-binding proteins, 2 8 kdal 36 calbindin-D, has been investigated in the AD brain (Iacopino and Christakos, 1990). Both the levels of the polypeptide and it s mRNA are markedly decreased in AD brain SI, hippocampus and dorsal raphe nucleus, but not in the neocortex and striatum. Similar brain regionally specific decrements were reported in the substantia nigra in Parkinson's disease and striatum of Huntington disease. The brain regional losses in these diseases, and reported effects of aging on the expression of this gene, support the notion that the levels of 28 kdal calbindin-D reflect neuronal loss. The normal levels of this mRNA in AD neocortex, compared to the decreased levels in the SI, are of considerable interest. The most intensively studied gene in the human brain has been that coding for the amyloid precursor polypeptides. As described in a previous section, these polypeptides arise from alternate splicing of the primary transcript to yield at least 3 forms of mature mRNAs. Early results with a cDNA probe complementary to a l l of the known mRNA forms suggested a decreased level of these mRNAs in the frontal cortex (Goedert, 1987) . A similar result has been reported more recently by Clark et a l . (1989), and has been observed anecdotally by Goldgaber and Schemkel (1990). In contrast, at least two reports have appeared which failed to find any change in the overall level of these mRNAs (Zain et a l . , 1988; Tanaka et al . , 1988). By the use of small specific oligodeoxynucleotide exon junction probes Tanaka et a l . (1988) were able to 37 differentiate the /3-APP-695, -751, and -770 mRNAs in frontal cortex polyA mRNA. In this study of 3 AD cases and 4 controls, only the /3-APP-770 mRNA was found to be differ e n t i a l l y expressed in AD versus control, increasing approximately two-fold in AD. By a similar approach, using small cRNA transcripts for junction probes, Johnson et a l . (1988, 1989) were able to estimate the levels of 0-APP-695 and -751 mRNAs. In these studies a decrease (to about 25-35% of control) in AD levels of the APP-695 mRNA, with no change in /3-APP-751 mRNA, was observed in polyA RNA samples of mixed neocortical regions (frontal, temporal and parietal cortices) and hippocampus, but not in the cerebellum. These results are in conflict with the analyses recently reported by Koo et a l . (1990), who failed to observe any differences between AD and control ratios of the mRNAs for jS-APP-695 to /3-APP-751 + /3-APP-770. These authors also note the independence of the ratios with age in humans or adult rhesus monkeys. Golde et a l . (1990) used the polymerase chain reaction amplification of cDNA samples to analyze the levels of the predominant jS-APP transcripts in many brain regions in AD and controls. The levels of /3-APP-770 and total /3-APP were markedly increased (up to 5-fold) in frontal cortex white matter. These authors argue convincingly for caution when interpreting the results obtained from the analysis of extracted tissue RNA, which i s derived from a mixed population of c e l l types. In this study i t was also found that the cerebral, but not cerebellar, meninges had decreased levels of 38 /3-APP-770 mRNA, with increased levels of the /3-APP-695 mRNA. These observations may have bearing on the issue of the source and form of /3-APP that becomes deposited in vessel walls in AD. An analysis of the c e l l types expressing the /3-APP mRNAs could be of considerable significance. The several studies which have examined the brain regional expression of these mRNAs by Northern blot analysis have not observed regional variations. In order to gain further insight into the biosynthesis of the amyloid precursor polypeptide, a number of investigators have applied in situ hybridization methods to human and animal brain tissues. Using cDNA probes which are not selective for the various transcripts, several groups of investigators have observed relatively high levels of transcripts in pyramidal cells of the neocortex and hippocampus (Goedert, 1987; Bahmanyar et a l . , 1987; Salim et a l . , 1987; Higgins et a l . , 1988), although i t i s also apparent that most other neurons, including those not susceptible to tangle formation, also express the gene. Double staining with cRNA in situ hybridization and GFAP immunohistochemistry has also demonstrated /3-APP transcripts in cerebral astrocytes (Golde et a l . , 1990). This is in f u l l agreement with immunohistochemical staining of these cells with anti-/3-APP antisera (Card et a l . , 1988). Expression of the /3-APP mRNAs has also been studied in non-cortical brain regions. In a recent study, Cohen et a l . (1988) examined the expression of the /3-APP gene in the nucleus basalis of AD and control cases. In order to examine the expression of this gene in the cholinergic c e l l population, cells were differentiated on the basis of perikaryal diameters; measurements of grain counts were done for cells with diameters > 20 jum, the majority of which would be cholinergic (McGeer et a l . , 1984). In these c e l l s , the /?-APP signal was increased almost 4-fold in AD versus control, with a 30% decrease in the hybridization signal for /3-tubulin Further evidence for /S-APP gene expression abnormalities in AD has come from Palmert et a l . (1988) , who found an approximately 2 fold increase in total /3-APP hybridization signal in both magnocellular nBM neurons and pigmented magnocellular neurons (ie. noradrenergic) of the LC. In the subiculum and basis pontis no difference was observed between AD and controls, using the same brains. In this study, the level of transcripts with the KPI domains ((3-APP-770 + |3-APP-751) was also determined by hybridization with a cRNA probe common to these domains. With this probe, no difference was observed between AD and control in any of the brain regions. Thus, the increased total /3-APP mRNA signal in the LC and nBM neurons appears to result from a selective increase in the /3-APP-695 mRNA. A further interesting observation was that, in tangle bearing neurons in both the LC and nBM, decreased hybridization signals were evident, indicating that the increased level of transcripts was limited to histopathologically "normal" neurons. The importance of this observation l i e s in the evidence i t presents that alterations in cellular gene expression precede overt histopathological changes. Alternatively, those ce l l s which have increased levels of this transcript may not be those susceptible to the pathology. The increased levels of /3-APP transcript observed in nBM neurons in AD brain i s not necessarily in conflict with the tissue levels in the AD nBM determined by Golde et a l . (1990). These authors point out the generalized (but not individually significant) increase in /3-APP transcript levels in several brain regions. The levels in the nBM were increased 2-fold (not significant), in a brain region known to undergo neuronal loss. A limited selection of cytoskeletal proteins have also been examined at the mRNA level. The levels of tau mRNA in AD and control frontal cortex have been compared (Goedert et a l . , 1989b). No difference in the amount of the message was observed in the small number of cases examined. The recent description of a number of tau isoforms derived by differential transcript splicing (Lee et a l . , 1988; Goedert et al . , 1989b), suggests the possibility that changes in specific isoforms of the mRNAs could occur, and be missed when employing probes common to the various isoforms, particularly in light of the evidence that the tau isotypes display c e l l s pecificity of expression (Goedert et a l . , 1989b). Tissue levels of the 68 kdal neurofilament polypeptide (NF-L) mRNA have been reported to be decreased in AD by two groups (Crapper McLachlan et a l . , 1988; Clark et a l . , 1989). In both of these studies a similar loss of a-tubulin was also observed, to approximately 50-60% of that seen in control groups. In the forebrain, nerve growth factor (NGF) and i t s mRNA have been found to be distributed in parallel with cholinergic nerve endings (Korsching et a l . , 1985; Shelton and Reichardt, 1986). In the basal forebrain cholinergic neurons, NGF acts as a trophic factor, being taken up at nerve endings and retrogradely transported to the c e l l bodies (Seiler and Schwab, 1984). The basal forebrain cholinergic neurons are rich in NGF receptor (Taniuchi et a l . , 1986; Hefti et a l . , 1986) and NGF receptor mRNA (Goedert et a l . , 1989a). Given that NGF may be a potential therapeutic possibility for modulation of cholinergic function, there i s considerable interest in this protein in relation to AD. The levels of NGF mRNA in AD and control neocortex have been reported to be similar by Goedert et a l . (1986) , suggesting that a decreased avai l a b i l i t y of NGF for cholinergic uptake is an unlikely explanation for cholinergic degeneration. Conversely, the loss of cholinergic afferents would not necessarily affect cortical levels of the mRNA for NGF. For example, in the rat, transection of the fimbria results in an increase in hippocampal NGF with no change in NGF mRNA (Korsching et a l . , 1986). A recent in situ hybridization analysis of NGF receptor (NGF-R) mRNA in the forebrain detailed a loss of positive c e l l s in AD forebrain (Higgins and Mufson, 1989), a result paralleled by immunohistochemical staining for NGF-R polypeptide (Mufson et a l . , 1989). Northern blotting of SI RNA for NGF-R mRNA was carried out to compare the levels in two AD and control brains by Higgins and Mufson (1989). In these AD cases there appeared to be a marked loss, although no quantitative data were given. In contrast, Goedert et a l . (1989a) have reported preservation of tissue levels of the NGF-R mRNA in the basal forebrain in AD. Given the decreased number of NGF-R-bearing cholinergic neurons in AD, the possible preservation of receptor mRNA suggests that the c e l l density of the mRNA could be higher in surviving neurons. L i t t l e information is available on the events subsequent to NGF receptor occupation, and i t would therefore be premature to discount the potential significance of this factor in future studies of AD, or as a potential therapeutic target. It is of considerable interest that the basal forebrain cholinergic neurons can apparently survive after substantial loss of neocortical target neurons, which have been assumed to be the source of trophic support (Sofroniew et a l . , 1990). As pointed out by these investigators, this suggests that loss of these cholinergic neurons in AD may not simply be a case of retrograde degeneration resulting from loss of trophic input. E. APPROACHES TO NEW DISEASE MARKERS OF AD Excellent progress has been made in understanding the structure and distribution of the AD plaques and tangles. It is unclear how this information relates to the disease state. It i s highly desirable to find further clues to direct the research on the cause of the disease. It is equally desirable, for a number of ethical, social, and financial reasons, that a premortem diagnosis of AD become available. For these reasons, several approaches have been taken to screen for new biochemical markers of the disease. One such approach uses immunochemical screening. The Alz-50 monoclonal antibody (Wolozin and Davies, 1987) was obtained by large scale immunochemical screening for AD associated epitopes. Homogenates of the basal forebrain of AD affected persons were employed as immunogens for the production of hybridomas. Screening of the hybridoma panel resulted in a useful monoclonal antibody which detected a 68 kdal epitope that was greatly enriched in AD brain tissue extracts, and was detectable by immunohistochemical methods (Hyman et a l . , 1988) . This epitope is detected in both NFT-bearing and NFT-free neurons, and appears to be preferentially present in neocortical pyramidal neurons, as well as in magnocellular neurons of the basal forebrain. Recent studies have demonstrated that the Alz-50 antibody is immunoreactive towards an epitope on tau polypeptide (Nukina et a l . , 1988; Ksiezak-Reding et a l . , 1988). The nature of the Alz-50 immunoreactivity suggests that this antibody detects a postranslationally modified epitope, or one that i s masked in the neurologically normal brain (Kosik et a l . , 1989). Miller et a l . (1987) used a less direct immunochemical screening approach, in which a panel of monoclonal antibodies to Drosophila heads were screened by immunohistochemical staining of AD and control tissue sections. Several monoclonal antibodies were described which recognize subpopulations of human cortical neurons. By the use of these monoclonal antibodies, i t was possible to stain presumptive intracortically projecting pyramidal neurons which appeared to be preferentially susceptible in AD. The nature of the antigens is not yet known. Other variants on the immunochemical screening approach have been described. The experiments of Abraham and colleagues (1988) employed an immunochemical screening of a cDNA expression library using antisera prepared to NFT preparations. In this study an immunoreactive clone was found to code for a protease inhibitor, a1-antichymotrypsin. The presence of this protein in the NFT has generated further interest in the role of proteases and their inhibitors in AD pathology, particularly in light of the descriptions of KPI domains in the amyloid precursor (see above). An approach with great potential for screening AD for biochemical markers is the use of nucleic acid hybridization methods. Specifically, this would involve the search for genes differentially expressed in the AD brain. In order to carry 45 out this approach, i t would be necessary to screen diffe r e n t i a l l y recombinant DNA libraries with mRNAs, or the corresponding cDNAs, from AD and control brain. The investigation of gene expression in the mammalian brain is complicated, relative to other organs, by the cellular diversity of the tissue. The complexity of brain gene expression has long been appreciated on the basis of the results from kinetic hybridization analyses (Kaplan et a l . , 1982; Chikarashi et a l . , 1979), as well as from clonal analyses from relatively unselected cDNA libraries (Milner and Sutcliffe, 1983). Results from these analyses indicate that the majority of sequence complexity of the brain l i e s in the moderate to low abundance mRNA class. Based on these kinds of experiments, i t has been estimated that there are some 3 0-50,000 distinct mRNA transcripts expressed in the CNS of mammals. Moreover, the average size of neural mRNAs are significantly greater than in other tissues, an average brain-specific mRNA being about 2600 bases, as opposed to 1200-1800 bases in a housekeeping gene (Sutcliffe, 1988). These results are not surprising, in light of the anatomical and biochemical specialization of neurons, and have bearing on the experimental approach to analyzing gene expression in the brain. In studies of sequence complexity of brain, the majority of work has been carried out on RNA prepared from the whole brains of rodents. It is probable that these estimates of 4 6 sequence complexity would be equally applicable for human brain gene expression. Although the relevant measurements have not been reported, i t might be expected that the complexity of gene expression would be somewhat less within specific neural c e l l groups, eg., in brain regions enriched in a particular type of neurotransmitter specific neuron. In the investigation of gene expression in AD brain an important consideration for experimental design would be the choice of brain region to be investigated. Because of the regional variation in AD pathological changes, the study of gene expression would best be carried out on a brain region which i s reliably affected by the disease process. Based on both neurochemical and anatomical evidence, the brain region of choice would contain the basal forebrain cholinergic neurons. 47 I I . STATEMENT OF THE PROBLEM TO BE ADDRESSED The cause of Alzheimer's disease remains unknown. This disease displays marked regional variation in pathology, and is accompanied by reliable neurochemical alterations. There are few biochemical markers of the disease, and the definitive diagnosis of the disease remains a postmortem finding. In order to advance the current understanding of the biology of AD, i t would be of use to obtain further biochemical markers of the disease. This thesis describes a search for genes of interest to AD research. Since the disease displays anatomical and neurochemical specificity, a brain regional cDNA library should enrich for genes of interest. To date the best understood and most reliable neurochemical alteration in AD is the loss of neocortical ChAT, the synthetic enzyme for acetylcholine. The brain region chosen for this study was therefore the basal forebrain, which contains the c e l l soma of cortical cholinergic projections. The approach was to screen the cDNA library for genes differentially expressed in AD affected tissue, and thus to identify biochemical markers which correlate with expression of the disease. Since relatively l i t t l e is known about gene expression in the brain, an anatomically and biochemically heterogeneous tissue, any genes identified as being brain specific and/or neuronal, would also be of interest. 48 III. EXPERIMENTAL The experimental approach of this thesis required production of a cDNA library of genes expressed in the human substantia innominata, followed by a search of this library for genes which exhibited altered expression in AD. A parallel goal was the characterization of brain regional and tissue specific gene expression. In order to carry out this approach i t was necessary to be able to obtain the highest quality RNA from postmortem human brain regions. The quality of RNA was considered to be of primary importance, in light of the frequent observation in the literature of badly degraded human brain RNA. It is certainly conceivable that the rate of RNA degradation is variable in the ante-mortem interval. Available evidence suggests that the rate of RNA degradation varies with different mRNAs, as demonstrated in c e l l culture studies (Brawerman, 1989) . It is possible that there may be differential rates of degradation of mRNAs between AD and control brains, or even between different brain regions. Moreover, RNA degradation not only represents a quantitative problem in terms of interpreting the results of hybridization analysis, but also represents a severe problem in the construction of high quality, representative cDNA libraries. For these reasons, a l l reasonable attempts have been taken to minimize the effects of RNA degeneration, particularly with respect to minimizing the post mortem interval, as well as carefully examining the medical histories of tissue donors. With reasonable care, human brain RNA can be reproducibly obtained in acceptable yield with excellent structural integrity. The choice of the substantia innominata as a target for the production of a brain regional cDNA library follows from the well established pathology of the cortically projecting basal forebrain neurons, as documented in the Introduction to this thesis. This i s not to suggest that these ce l l s are the only population affected by AD, nor does this choice of tissue require that the pathology seen in the SI i s the most important feature of the disease. The choice of these ce l l s was dictated by the general observation that AD patients reliably show decrements in neocortical ChAT. Experimentally, this consideration allows the biochemical characterization of AD cases for further study. Thus ChAT levels can be used as an indicator of AD pathology in concert with histological characterization. A further consideration i s that the SI represents a comparatively compact brain region which has a c e l l population enriched in CNS cholinergic neurons. Although the basal ganglia probably has a comparable number of cholinergic neurons, the density of these ce l l s in the SI i s at least 10-20 times higher. Based on the calculations of McGeer et a l . (1984), there are roughly 400-500,000 50 cholinergic neurons in each SI in the neurologically normal brain. These cel l s are present in a brain region which weighs approximately 3 00 mg. One of the great d i f f i c u l t i e s of research on the human brain is the relative paucity of available material. The experiments described in the current work were carried out using human brains collected over a period of several years. Since the starting material i s precious, particularly in consideration of the small amount of RNA that could be obtained from the dissected subcortical brain regions, attempts were made to carry out cDNA cloning in an efficient manner. Using what are now f a i r l y standard methods, the current work demonstrates the production of a human SI cDNA library with good preservation of sequence complexity, as reflected by cDNA insert size and number of independent transformants. Differential colony hybridization analysis was applied to screening the SI cDNA library. The small amount of SI cDNA available dictated that direct screening of a large number of recombinant clones was not feasible. The rationale for the adopted screening procedures was based on the assumption that any genes of interest to this study would not be abundantly expressed in the cerebellum, a region devoid of cholinergic c e l l bodies and not exhibiting marked AD pathological changes. Therefore, the SI cDNA library was prescreened in order to eliminate, as much as possible, genes expressed abundantly in 51 the cerebellum. The selected recombinants were then diffe r e n t i a l l y rescreened with SI cDNA probes in order to define AD related gene expression changes, as well as with other cDNA probes from white matter, placenta, liver and cerebellum, in order to examine cellular and tissue s p e c i f i c i t i e s . Hybridization analysis with cDNA probes for either colony or plasmid hybridization screening has inherent limitations in specificity and sensitivity. Therefore, a large number of selected cDNA clones were further investigated by Northern blot analysis using RNAs prepared from several brain regions. Application of these various screening methods identified a specific cDNA clone which was selected for detailed characterization. This cDNA clone corresponds to an unusually large mRNA, estimated to be 15.5 kilobases, whose expression appears to be limited to the brain. In the human brain, this transcript i s differentially expressed in various brain regions, and i t s absence in white matter suggests i t to be neuronal. A weakly hybridizing RNA of comparable size and tissue distribution was detected in rat brain regions, but not in non-neural tissues. An ribonuclease-protection solution hybridization assay identified the mRNA to be of moderate abundance, approximately 0.2 weight percent of polyA RNA. This assay indicated that the gene i s expressed at comparable levels in the AD and normal SI, but may be expressed at higher levels in AD temporal cortex than in the normal control. The 52 sequence analysis of the cDNA clone indicates that the 3 kilobases of cDNA so far determined appear to correspond to the 3'-untranslated region on the messenger RNA. Computer searches of the Genbank, EMBL, and NBRF databases failed to identify any known gene or protein with significant homology. 53 I I I A . PREPARATION AND CHARACTERIZATION OF HUMAN BRAIN RNA Preparation of high quality RNA remains one of the c r i t i c a l steps in the analysis of gene expression. In many circumstances, such as c e l l culture studies, the recovery of pure and intact RNA is relatively straightforward. At the beginning of this work there were several suggestions in the literature that this was not the case for human brain RNA (Gilbert et a l . , 1981; Morrison and G r i f f i n , 1981; Sajdel-Sulkowska et a l . , 1983a; Wallace and Winblad, 1986). Subsequent experience has confirmed these suggestions. Two causes of RNA degradation seem to predominate; 1) poor condition of the patient prior to death, and 2) inappropriate handling of tissue and RNA during tissue extraction and subsequent purification steps. Medical ethics governing human experimentation mean that l i t t l e can be done about the premortem status of a case to be studied. When possible, efforts should be made (and in the current study have been) to minimize the effects of postmortem degradation by establishing protocols for the prompt removal, dissection, and storage of the brain after death. For a number of reasons, i t is highly desirable to be able to prepare human brain RNA from frozen tissue. Several reports of successful RNA preparation from frozen brain (Sajdel-Sulkowska and Marotta, 1983a,b; Taylor et a l . , 1986) 54 indicated that this was feasible. In order to use frozen tissue for preparative purposes, strong protein denaturing agents have to be employed along with rapid homogenization. Successful RNA preparation depends on the rapid inactivation of endogenous ribonuclease activity, and, since freezing and thawing tissue i s likely to disturb intracellular storage vesicles containing nuclease activity (ie. lysosomes), maintaining the tissue in a frozen state u n t i l dispersion has become the accepted procedure. This approach has been followed in the current work. As described in a previous section, the reported yield of RNA from human brain has been variable (see section I.D, Table 1, page 31). The variabi l i t y could be due to preparative methods, choice or conditions of the tissue, or the presence of contaminating materials. The presence of contaminating materials in RNA preparations could cause problems in yield determinations due to the poor specificity of absorbance measurements at 260 nm, the usual means applied for the determination of RNA yield. Since relatively l i t t l e information is available on the chemical purity of RNA preparations, particularly from human brain tissue, this point was examined by a direct chemical method. To be useful for comparative purposes, a RNA preparative method must give high yields and be reproducible. In order to examine the reproducibility of the method, preparations were carried out on a number of neurologically normal control (NNC) and AD individuals. Hybridization analysis of human brain RNAs with labelled cDNA probes was conducted in order to examine structural integrity. Materials and Methods Case Selection. Human brains were obtained from cases coming to autopsy at Vancouver General Hospital and the University of British Columbia Health Science Center Hospital. Details regarding the histories of individuals were obtained under agreement of confidentiality, usually by way of medical records. In a l l cases drug histories were reviewed, and conditions of the individual at the time of death were considered. Cases exhibiting symptoms of neurological impairment suggesting an undesirable c l i n i c a l status, eg., cerebral infarction, drug induced confusion, anoxia, or unexplained motor or sensory impairment, were excluded from further study. In this discussion control cases refer to those individuals who had a well defined cause of death, lack of symptoms suggesting the possibility of neurological disease, a normal range of neocortical ChAT activity, and the absence of histological signs of AD-type pathology. With rare exceptions, the brains of control cases were thoroughly assessed for histopathology. AD cases were frequently very well defined c l i n i c a l l y , having been followed over a period of years by neurologists in the metropolitan region, and frequently within the Alzheimer's Disease c l i n i c of the hospital. A l l cases herein referred to as AD displayed a c l i n i c a l history of dementing disease and probable AD. Definitive diagnosis of AD was confirmed by histological observation by a staff neuropathologist at either of the two hospitals where autopsies were performed. The majority of the cases studied were examined by Dr. C. Dolman, formerly of the Department of Pathology, University of British Columbia. In most cases samples were submitted from frontal, occipital and temporal cortices, as well as the hippocampus. In a number of cases individuals were identified as displaying mild AD pathology combined with evidence of multi infarcts. None of the latter cases were used in the present study. Cases identified as AD showed clear decrements in neocortical ChAT levels. Several individuals were identified which had clear AD pathology, but also displayed some Parkinsonian c l i n i c a l features as well as catecholaminergic abnormalities. The neurochemical and histological features of these AD + Parkinsons Disease cases has recently been described (McGeer et a l . , 1988). Cases displaying this type of pathology were recognized, and are not included in the cases referred to as AD. Tissue Collection and Brain Dissection. Brains were removed as soon as possible after death. In a l l cases used for group comparisons, the post mortem interval was less than 8 hours and the average time to autopsy was less than 4 hours. Upon removal of the brain, the dura mater was removed, then the cerebellum and midbrain separated by cutting through at the level of the substantia nigra, to a line just above the tectum. The remainder of brain was bisected on the midline. For the determination of ChAT, samples of about 100 mg were removed from the cortex, stripped of pia mater and then frozen. Samples were taken from seven cortical regions as follows, with the Brodmann's areas identified: frontal, 10; temporal ti p , 38; Broca 1s area, 45; occipital pole, 17; precentral gyrus, 4; postcentral gyrus, 1; midtemporal gyrus, 21. The l e f t hand side of the brain was sliced in 1-2 cm slabs before immersion in fixative (0.1 M NaP04, pH=6.8/4 % paraformaldehyde) and was used for a variety of histological procedures or transported for examination by the neuropathologist. From the right hand side of the brain samples of 3-8 g were removed from the frontal, temporal and occipital cortices. A sample of about 15 g was removed from the posterior lobe of the cerebellum. At this point the brain was cut in 1-2 cm slabs, then appropriately sampled for the caudate nucleus (head and anterior portions, 2-3 g) and hippocampus (body, 0.75-1.5 g). For the dissection of the hippocampus, care was taken to remove the subicular cortex as well as the white matter associated with the fimbria. To obtain the SI, a coronal slab was obtained by sectioning at the level of the optic chiasma and on the posterior aspect at the level of the mammillary body. A block of tissue was obtained by cutting the slab laterally 1 cm off the midline and then at the junction of the parahippocampal gyrus. A 58 horizontal incision was made starting at the lateral limit of the exterior portion of the globus pallidus and continuing to the site nearest the midline. The resulting tissue block was a trapezoid weighing 200-500 mg. A discussion of tissue dissection in this brain region is presented by Hedreen et a l . (1985). The tissue samples for RNA extraction were immediately put in clean plastic containers and placed in a -75°C freezer, where they were stored unti l extraction. Freezing tissue in liquid nitrogen was not found to be necessary. Extraction and Purification of RNA. The isolation protocol is based on the method described by Kaplan (1979) and Chirgwin et a l . (1979). A l l plastic containers which came into contact with RNA had been ste r i l i z e d and decontaminated with diethylpyrocarbonate (DEPC) after siliconizing by treatment with dichlorodimethylsilane. A l l glassware was baked at 400°C for 8 hours. Solutions were made up from nuclease free stock solutions using water that was treated with 0.05 % diethylpyrocarbonate, then autoclaved twice. When necessary, solutions were f i l t e r e d using s t e r i l e plastic 0.2 jum f i l t r a t i o n devices (Nalgene) . The homogenization f l u i d was composed of 5 M guanidine isothiocyanate/ 50 mM Tris-HCl, pH=7.4/ 1 % j8-mercaptoethanol/ 10 mM EDTA/ 1 % lauryl sarcosine/ 2 % Antifoam A (Sigma). Tissue extraction was carried out by addition of 5-6 ml of the extraction f l u i d per g of frozen tissue. Tissue fragments were dropped into extraction solution just before homogenization. 59 Small pieces (less than 1 g) were homogenized in a 15 ml siliconized Corex centrifuge tube by use of a Polytron homogenizer (Brinkmann), using a PTF-10 low foam microprobe operated at a speed setting of 6 for 30 seconds. Larger pieces of tissue were broken up while frozen, then homogenized with a Sorvall Omnimixer, operated at a speed setting of 7 for 90 seconds. Following homogenization, solid CsCl was added at 0.15 g/ml of solution, which was vortex mixed u n t i l a l l of the salt went into solution. The resulting solution was centrifuged at 10,000 x g at room temperature for 5 minutes to remove insoluble materials. Supernatants were loaded onto a cushion of CsCl solution (5.7 M CsCl/ 0.1 M EDTA, pH=7.5) in a polyallomer ultracentrifuge tube. The volume of the CsCl solution was adjusted to f i l l the bottom 1/4 of the tube. One of three swinging bucket ultracentrifuge rotors was used, depending on the volume of extract; AH-629 rotor (Sorvall; 28 ml per tube), AH-641 rotor (Sorvall; 11 ml), SW-65 Ti rotor (Beckman; 3.5 ml). When necessary, light mineral o i l (Fisher) was used to bring the tube to volume. RNA was sedimented through the CsCl cushion by centrifugation at 20°C for the following times and speeds: AH-629, 30 hrs. at 27,000 rpm; AH-641, 18 hrs. at 28,000 rpm; SW-65 T i , for 16 hrs. at 36,000 rpm. After centrifugation, supernatants were aspirated to just under the dense solution interface, the tubes were carefully cut off just above the remaining solution, then the remaining liquid was rapidly decanted by inversion of the tube. While the tube was inverted, the walls were cleaned with s t e r i l e gauze and a guanidine-HCl solution was added (7 M guanidine-HC1/ 50 mM Tris-HCl, pH=7.5/ 10 mM dithiothreitol) corresponding to 1/4 the volume used for the i n i t i a l extraction. The RNA pellet was triturated, transferred to a s t e r i l e tube, and the remaining material present in the ultracentrifuge tube was removed by addition of a further volume of solution. The combined solutions were extensively vortexed with gentle heating u n t i l the RNA pellet was dissolved, after which 0.05 volume of 3 M sodium acetate, pH=5.0, was added. The solution was adjusted to 50% (v/v) of cold ethanol. The RNA containing solution was l e f t to precipitate at -20°C for 4-6 hours. The RNA was pelleted by centrifugation at -10°C for 10 min. at 10,000 x g. After decanting the supernatant, the pellet was resolubilized with guanidine solution, corresponding to one half the previous volume, and was reprecipitated from ethanol/sodium acetate. The f i n a l RNA pellet was washed with 7 0% ethanol at room temperature, dried in vacuo, then solubilized with 10 mM Tris-HCl/ 1 mM EDTA/ 0.1 % SDS, centrifuged at 10,000 x g for 15 minutes, and the supernatant transferred to a clean tube. Before further use, and for storage, this f i n a l RNA solution was reprecipitated from ethanol (66%) and 0.3 M sodium acetate. Electrophoresis of RNA, Blot Transfers and Hybridization. Gel electrophoresis of RNA was carried out in 0.7-1.0 % agarose formaldehyde gels using a morpholino-N-propanesulfonic acid/acetate running buffer, basically as described by Maniatis et a l . (1982). The separation was carried out at room temperature using a submerged gel with buffer recirculation for 3-5 hours at 40 V. Gels for blot transfer were washed twice for 10 min. in s t e r i l e water, followed by 10 minutes in 10 x SSC (1 x SSC i s 0.15 M NaCl/0.015 M Na-citrate, pH=6.5). Vacuum transfer of RNA from gels was carried out using a Vacu-Gene apparatus (Pharmacia-LKB) onto Gene Screen Plus (NEN-DuPont) charged nylon membranes. Of the procedures tested, the best transfer buffer was 10 x SSC, and the device was operated by i n i t i a t i n g transfer at 3 5 mm water pressure for 10 minutes, then increasing to 50 mm water pressure and allowing transfer to continue for at least 3 hrs. This completely removed from the gel any trace of an RNA calibration standard having a range of 9.5-0.24 kilobases (BRL). Following transfer, the membrane was rinsed with 2 x SSC, then allowed to dry at room temperature before baking at 80°C under vacuum. Hybridization of blotted membranes was carried out as follows, with specific changes depending on the required stringency. Membranes were sandwiched between nylon mesh and placed in polyethylene pouches, both as supplied for the Turbo Blot device (ABN). The membranes were wetted with 2 x SSC, and excess liquid was removed by aspiration. Prewarmed prehybridization solution [50 % deionized formamide/ 1 M NaCl/ 1% SDS/ 50 mM Tris-HCl, pH=7.6/ 1 mM EDTA/ 100 pg/ml sodium heparin sulphate/ 25 nq/ml of both poly(A) and poly(U)/ 100 jug/ml sonicated and denatured calf thymus DNA] was added to the bag, which was incubated for 2-4 hrs at 42°C in a water bath with agitation. Hybridization was initiated by removing about 5 ml of the prehybridization solution, followed by mixing with the 3 2P-labelled probe, generally present at 5-10 ng/ml with a specific activity of greater than 109 CPM//xg. Hybridization was allowed to proceed for 24 hours at 42°C, membranes were washed with agitation, using the following solutions: 2 x SSC/1 % SDS twice for 15 minutes at room temperature, 0.5 x SSC/1 % SDS twice for 15 minutes at 65°C, 0.1 x SSC/1 % SDS three times for 20 minutes at 65°C, 2 x SSC at room temperature for 2 minutes. The membranes were overlaid on f i l t e r paper moistened with 2 x SSC, wrapped in Saran wrap, placed in a cassette with intensifying screens, overlaid with film, and exposed at -75°C for appropriate periods of time. Autoradiographic films were Kodak XAR or X-Omat RP and were handled and developed as recommended by the manufacturer. Most membranes were reused several times. After the appropriate exposure, probes were stripped off the membrane by twice treating them with 94 % formamide/0.01 M Tris-HCl, pH=7.4/0.001 M EDTA/0.5 % SDS at 90°C for 10 minutes. After stripping, the membranes were washed with 2 x SSC, blotted dry and stored at -20°C un t i l rehybridization. Preparation of Plasmid DNAs and Radiolabelled Probes. Clones of recombinant bacteria were obtained by culture on appropriate antibiotic selection agar plates. Liquid cultures were initiated with isolated clones. Large scale liquid cultures were harvested in mid-logarithmic growth and harvested bacterial pellets were processed by the alkaline lysate method of Birnboim and Doly (1979) to produce cleared lysates containing plasmid DNA. This DNA was purified to apparent electrophoretic homogeneity by treatment with heat-treated pancreatic ribonuclease A (Sigma) and selective precipitation from high salt polyethylene glycol, basically as described by Sadhu and Gedamu (1988) . The /3-amyloid precursor polypeptide (/3-APP) cDNA clone 1-B2 has been described by Robakis et a l . (1987), and was generously supplied by Dr. H. Wisnewski (New York Institute for Basic Research in Developmental Disabilities, Staten Island N.Y.). cDNA inserts were released from vector with the appropriate restriction endonuclease and were separated from linearized vector on preparative electrophoresis gels using SeaKem (FMC) low melting point agarose with a Tris/acetate/EDTA buffer system (Maniatis et a l . , 1982). The target DNA was recovered from excised agarose blocks by melting at 65°C before solid phase extraction onto Elutip-d cartridges using the binding and elution conditions recommended by the manufacturer (Schleicher and Schuell). cDNA probes were radiolabelled using [32P]-dCTP (>3000 Ci/mmol, NEN-DuPont) in a random hexanucleotide priming reaction based on the method of Feinberg and Vogelstein (1984), using primers and a recombinant Klenow DNA polymerase I fragment obtained from Pharmacia. The specific a c t i v i t i e s of 9 the DNA probes were in excess of 1 x 10 cpm/|ig. Each radiolabelled DNA probe was purified on NEN-Sorb solid phase extraction cartridges (NEN/DuPont) by the manufacturer's recommended protocol modified in that the labelled DNA was eluted with 50 % ethanol (vol/vol). Chromatographic Analysis of RNA Purity. Enzymatic hydrolysis and dephosphorylation of nucleic acid samples were carried out using conditions patterned after Gerhke et a l . (1982, 1984). The RNA sample (0.1-1 /ig) was dissolved in 20 /xl of water containing 25 ng of the internal standard 8-bromoguanine (Br G) . The solution was heated to 70OC for 5 min. before c h i l l i n g on ice, then brought to 50 pl with adjustment to a buffer composition of 30 mM sodium acetate (pH 5.3)/ 1 mM ZnS04. Eight-tenths of a unit of Nuclease Pl (800 U/mg, Pharmacia) and 0.05 U of bacterial alkaline phosphatase (26 U/mg, Sigma Type III) were added. Digestion was allowed to proceed for 1 hr. at 37°C, after which 12.5 pl of 0.5 M Tris-HCl (pH 8.9) was added. The dephosphorylation reaction proceeded to completion within 1 hr. at 37°C. The reaction mixture was placed in a -75°C freezer u n t i l analysis, usually within 1 day. Various control . . Phe experiments with tRNA and calf thymus DNA confirmed that the digestion of RNA and DNA proceeded to completion, liberating the nucleosides quantitatively. Quantitation of the nucleosides was carried out by reversed-phase high pressure liquid chromatography. The instrument was composed of two M510 pumps controlled by an M680 gradient controller; samples of 50 / i l were introduced via the U6-K injection valve ( a l l from Waters Associates). Compounds of interest were monitored at 260 nm (0.25 s time constant) with the Kratos 757 UV-Vis absorbance detector (Applied Biosystems), whose signal was recorded on a M730 integrator (Waters). Resolution of the ribo- and deoxyribonucleosides was achieved on a 250 mm x 4.6 mm i.d. column of octyldecylsilane bonded-phase 5 jitm spherical s i l i c a (LC-18 DB, Supelco) which was placed in series with a 20 x 4.6 mm guard column packed with the same s i l i c a . Elution was carried out using a biphasic linear gradient of 0.05 M sodium phosphate buffer (pH = 4.0, Pump A) with methanol as the organic modifier (0.05 M NaPO4/50 % methanol for Pump B) . Instrument calibration was by area response factors generated for each compound in reference to the internal standard 8-• Phe bromo-deoxyguanme. Standard nucleotides and purified tRNA (used as a control RNA) were obtained from Sigma and were desiccated before preparation of stock solutions, the concentrations of which were checked by spectrophotometry. Miscellaneous Methods: Spectrophotometry was carried out on a Pye-Unicam SP-500 single beam instrument. Nucleic acid concentrations were calculated using unit extinction coefficients of 38 /ig/ml/absorbance unit for RNA, and 50 /xg/ml/absorbance unit for DNA, both at 260 nm. Tissue levels of ChAT were determined on sucrose homogenates using [ 1 4C]-acetyl coenzyme A incorporation into acetylcholine, with ion exchange separation of product on Dowex CG-50 X8, as previously described (McGeer et a l . , 1987b). Protein contents of tissue homogenates were determined by the method of Lowry (1954). Results The brain regions dissected to obtain the SI and hippocampus are outlined in Figure 1. Because the SI cholinergic cells are somewhat dispersed, a standardized region was dissected to contain the majority of these neurons. This region was noticeably shrunken in many AD cases, as was the hippocampus. On gross examination, most of the AD cases exhibited some indication of cortical atrophy, most apparent in sulcal widening in the frontal and temporal cortices. Brain weights were also noted to be lower in many cases, although no correction was made for loss of CSF. In Table 3 the group data on age, sex, neocortical ChAT and postmortem delays are presented for the AD and neurologically normal control (NNC) human brains collected during this study. As shown, there is a marked decrease in the neocortical tissue levels of ChAT in the AD group. These groups were not significantly different in the other measures. There was a significant decrement in neocortical ChAT levels with increasing age of the NNC individuals as shown in Figure 6 7 A Figure 1. The regions dissected to obtain the human hippocampus (Hpc, panel A) and substantia innominata (SI, panel B). The small arrow points in the medial direction, the large arrow head points dorsal. Abbreviations: LV, lateral ventricle; Sub, subiculum; Cn, caudate nucleus; Put, putamen; Gp, globus pallidus; AC, anterior commissure; OT, optic tract. 68 Table 3. Group Data for Collected Human Brains 8 Alzheimer Normal Control Number 12 24 Age (years) 75.3 (12.1) 72.6 (7.4) Sex (M/F) 5/7 13/11 Mean Neocortical ChAT 0.19 (0.07) 0.79 (0.17)b (/imol/min/100 mg protein) Postmortem Delay (hrs.) 4.0 (1.2) 4.6 (1.4) a. Values reported in means (standard deviation) b. p<0.0001, two-tailed t-test, 34 d.f. 1.4, 1.2-Neocortical ChAT in SDAT and NNC o SDAT • NNC 1. .8. .6. .4-.2. o o o o 0-40 -i 1— 50 60 70 80 Age (yrs) 90 100 — i 110 Figure 2. A plot of mean neocortical ChAT for the cases collected during this study. The seven cortical regions were assayed individually and the mean presented. In general a l l cortical divisions show the AD decrement in ChAT levels. The control cases are denoted by a f i l l e d c i r c l e and AD cases by an open c i r c l e . A least squares linear regression of the control cases detected a significant decrease of ChAT levels with age (r = 0.520, n = 24, p<0.01). 2. No effect of age on ChAT levels was detected in the AD group. Preliminary attempts to obtain human brain RNA by the guanidinium isothiocyante (GITC) hot phenol method (Ferramisco et a l . , 1982; Maniatis et a l . , 1982) gave low and variable results with frozen human cortex (< 60 yq/q tissue) or fresh rat whole brain (296 ± 138 nq/q, n=3). RNA was therefore prepared by GITC extraction and CsCl gradient centrifugation. The RNA resulting from this method was observed to have the absorbance spectrum expected for RNA. For 12 RNA preparations the ratios of absorbance (mean ± s.d.) were 2.21 ± 0.17 at 260 nm versus 280 nm and 2.34 ± 0.11 at 260 nm versus 230 nm. In order to obtain information on the degree of DNA contamination of RNA preparations, the material was digested to completion with the nonspecific nuclease PI from Penicillium citrinum. Subsequent dephosphorylation of ribonucleotides (and deoxyribonucleotides) with alkaline phosphatase was necessary to allow effective chromatographic analysis. As illustrated in Figure 3, the resulting nucleoside mixtures could be completely resolved by gradient elution reversed-phase HPLC. A sample of hippocampal RNA analyzed by this method is shown in the figure, as i s an aliquot which has been spiked with a 1 % (w/w) contamination of high molecular weight human DNA. The ready detection of low level DNA contamination in RNA preparations is apparent in this experiment. In 9 samples of human brain RNA, only 1 contained Figure 3. Chromatographic analysis of nucleosides. In the top panel the retention positions of the ribonucleosides and deoxyribonucleosides are identified, standard letter codes are used, Br G i s the internal standard 8-bromoguanine. The middle panel illustrates the composition of a sample equivalent to 500 ng of hippocampal RNA. The bottom panel shows the same sample spiked with 5 ng of DNA before enzymatic hydrolysis. The released deoxynucleosides are denoted by arrows. detectable DNA, present at 0.26 % (w/w) with an estimated lower limit of detection, under the conditions employed, of 0.15 % (w/w, S/N=3). It was also noted that under the conditions employed there were no unusual or modified nucleosides detected in the AD or control RNA samples. Electrophoresis of RNA on formaldehyde agarose gels confirmed the presence of intact rRNA, as shown in Figure 4. Comparison of RNA samples obtained from AD and NNC hippocampus revealed no apparent differences in the integrity of the rRNA bands. Similarly, comparison of the integrity of RNA in AD and control samples revealed no difference in either the temporal cortex or SI samples. Blot hybridization analysis was conducted on samples of AD and NNC temporal cortex RNA using a cDNA probe for the B-APP mRNA. This probe w i l l detect a l l reported forms of the precursor mRNAs. The membrane was subsequently stripped of the /3-APP cDNA probe and rehybridized. with a cDNA probe for the B-actin mRNA. The results of these blot analyses are shown in Figure 5. No differences in either the intensity of the signal or the integrity of the detected mRNAs were apparent with either of these probes. Quantitation of the tissue content of total cellular RNA was carried out for three brain regions in AD and control cases. These results are shown in Figure 6. The levels of RNA were not different between groups in either the SI or temporal cortex. In contrast, the hippocampal RNA content in AD (110 ± 73 Figure 4. Electrophoretic analysis of RNA from human brain using formaldehyde agarose gels stained with ethidium bromide. rRNA bands (28 S and 18 S) are apparent in these samples in comparison with rRNA markers from E.Coli. and yeast (Sac.C). Also shown i s the mobility relationship as a function of chain length. 74 Oc. Fr. Temporal Ctx. Ctx. Ctx. Figure 5. Northern blot hybridization analysis of RNA samples obtained from AD and control temporal cortex. The upper band corresponds to the hybridization signal for the 6-APP mRNAs (3.5-3.2 kb), the lower for the 6-actin mRNA. The cDNA probes and hybridization conditions are described in the text. 75 Table 4. Group Data for Selected AD and NNC Cases Used for RNA Yields SI Brain Region Examined Temp.Ctx Hippocampus AD NNC AD NNC AD NNC Number 8 10 ChAT 0.23 0.78 0.19 0.74 (0.11) (0.22) (0.13) (0.26) 0.29 0.94 (0.11) (0.49) Age (yrs) 76.5 (8.0) 79.8 (9.1) 75. 6 (9.7) 81.4 (12.2) 79.0 (12.9) 77.8 (10.2) Postmortem 3.6 4.8 3.3 5.2 Delay (hrs) (1.2) (1.6) (1.4) (1.9) 2.6 3.6 (0.7) (0.8) a. average neocortical ChAT in jumol/min./100 mg protein. Tests of significance are two-tailed t-tests: *, p < 0.10; **, p < 0.01; ***, p < 0.001. 76 400 n Oi "55 5 300 a> 5 D) O) r i < Z CC S 100 LO CO 200-• SDAT • NNC n=10 n=6 I n=6 n=7 n=5 I n=8 T. Ctx Hippocampus S.I. Figure 6. Total cellular RNA yields in NNC and AD brain regions, expressed as fxq RNA per g tissue wet weight. The group denoted by the asterix was significantly different from the control at p < 0.05 (two-tailed t-test, 10 d.f.). 29.8 ng/g, n=5) showed a significant decrease to 53% of control level (209 ± 73.0 ng/g, n=7; p < 0.05, 2-tailed t-test). Groups of AD and NNC individuals used for the RNA yield comparisons did not differ in age at death. The average postmortem interval was shorter for AD cases, and the mean neocortical ChAT levels were markedly reduced in AD groups, as shown in Table 4. Discussion The successful isolation of good quality RNA from frozen postmortem human brain has only been achieved in recent years. It i s apparent from the literature that success i s dependent on the use of brain tissue that has been acquired at short post mortem interval and extracted under strongly denaturing conditions (Sajdel-Sulkowska et a l . , 1983; Morrison et a l . , 1987; Guillemette et a l . , 1986; Wood et a l . , 1986). In the present study attempts were made to reduce the post mortem interval and to extract the tissue using a protocol which would favour structural integrity. By the c r i t e r i a of spectral characteristics and compositional analysis, RNA prepared by this method i s substantially free of,contamination. Previous attempts to investigate the purity of RNA preparations have been based on binding assays using fluorogenic intercalating agents (LePec et a l . , 1977; Guillemette et a l . , 1986) of dubious analytical specificity. The structural integrity of the RNA was indicated here by electrophoretic analysis on denaturing gels which 78 detected the presence of discrete rRNA bands. Similarly, Northern blot hybridization analysis of temporal cortex RNA samples with two cDNA probes failed to detect overwhelming degradation of the RNA, in comparison with previous studies in the literature. No differences in the quality of the RNAs prepared from AD and control cases were noted. Thus, the current observations are in conflict with the observed loss of structural integrity of AD total cellular RNA reported by Wallace and Winblad (1985). It is dangerous to overinterpret the degree of integrity of RNA on the basis of a small number of probes. Variable integrity of different mRNAs has been noted in human brain RNA preparations by Zain et a l . (1988), who observed the partial degradation of the predominantly neuronal /3-APP mRNAs, in comparison to the g l i a l GFAP mRNA. In the present study, there was no marked difference in the amount of detected /S-APP mRNA, in agreement with results of some, but not a l l studies (see section I.D). As described in Section I.D of this thesis, various values have been reported for yields of RNA from post mortem human brain. In normal human brain I found regional differences in the amount of RNA that can be obtained, with the highest being observed in the cerebellum (646 ± 103 pq/q, n=3), followed by the temporal cortex, SI, then hippocampus. Similar brain regional differences have been noted by others (Morrison et a l . , 1986). The values observed here in the temporal cortex are in the same range, or a l i t t l e higher, 79 than those reported for neocortex by other investigators. It is not clear what the basis is for the variable yields reported in the literature. It i s of interest that there i s a wide discrepancy between the levels of tissue RNA determined by chemical methods, such as the orcinol colorimetric reaction, which yields values approaching 1000 nq/q tissue, in comparison with the values determined for high molecular weight purified RNA, which are at most \ this value (eg. Guillemette et a l . , 1986). This difference might be due to either incomplete recovery of RNA (eg., such as tRNAs), or to poor speci f i c i t y of the orcinal assay method. Within group va r i a b i l i t y of RNA yields was reasonable, with the standard deviation being about 25 % of the means (range 12-35 % ) , indicating that between group comparisons were feasible. It should be noted that care was taken in this study to exclude individuals with certain preclinical conditions. This can be quite important, as previously suggested in the literature (Taylor et a l . , 1986), and anecdotal evidence can be misleading. In the current work, for example, one case was i n i t i a l l y classified as a normal control but was later found to have died 5 days after surgery and to have been treated for an opportunistic infection with cyclophosphamide (a microtubule inhibitor with RNA polymerase inhibitory properties); the case was excluded as a normal control. Determination of the hippocampal RNA content for this case yielded the lowest value observed in this study, 28 nq/q 80 tissue. In well characterized, age and post mortem delay matched groups of AD and control cases, there was no significant difference in RNA yield from the temporal cortex or SI. The similar yields of neocortical RNA from AD and NNC i s in agreement with some, but not a l l investigators (see Section ID, Table 1, p. 31). In the AD hippocampus RNA yield was 53% that of the control group. The decreased yield of hippocampal RNA in AD has not previously been reported to my knowledge. The decreased levels of hippocampal RNA in AD may be related to the loss of hippocampal pyramidal neuron RNA in AD, as reported by Doebler et a l . (1987). This study used microspectrophotometric measurements of the RNA-azure B complex in tissue sections in order to determine the individual neuron RNA content. In pyramidal neurons, RNA was reduced in a l l of the hippocampal sectors examined, with an even greater decrement in the subiculum. In seria l adjacent tissue sections, the density of NFT and neuron c e l l counts were examined. No correlation was observed between the RNA content in neurons in a particular sector of the hippocampus and the density of NFT or neuron c e l l loss, a l l of which were observed to vary within the hippocampal subdivisions. This latter observation suggests that the changes in RNA content might occur independent of the NFT formation or c e l l death. A further suggestion in the literature, that the observed loss of cellular RNA may be physiologically relevant, has come 81 from observations of a decreased neuronal nucleolar volume in AD brain (Mann et a l . , 1981a, 1981b,). In a recent study, Mann et a l . (1988a) report that the nucleolar volume decrease in AD cortical pyramidal ce l l s occurs relatively early, being detectable in biopsy samples, and i s decreased even further at autopsy. The interpretation of this finding has been that the shrinkage of nucleoli reflects a decreased assembly of rRNA into ribosomes. The observed decrement in hippocampal RNA yield would be compatible with these morphometric findings; however, no decrease in neocortical RNA yield was observed in the present study. There are a number of complications in interpreting tissue loss of total cellular RNA in AD, as reported here for the hippocampus, and for the neocortex by others (see Section ID), or of decreased polyA RNA, as reported elsewhere (Crapper MacLachlan et a l . , 1988; Sajdel-Sulkowska and Marotta et a l . , 1984) . It i s clear that in AD there are changes occurring in the cellular composition of a particular brain region, for example, g l i a l hypertrophy and hyperplasia, the possible invasion of lymphocytes, etc. It might be suggested that the decreased RNA levels could be a reflection of these cellular changes, particularly the loss of pyramidal neurons. Since i t is possible that g l i a l cells may have different RNA content than neurons, an increase in total c e l l numbers (as in gliosis) might occur with a net decrease, increase, or even no change in the tissue RNA content. In the present study, 3 82 brain regions which are known to be sites of AD pathology were examined, only one of which had alterations in RNA levels. Since the cellular composition of each tissue may change in different ways, with differing time courses, the observation that only one brain region shows a loss i s not surprising. A major observation from this work is that high quality RNA can be reproducibly obtained from postmortem human brains, including those affected by AD. Sufficient material can be obtained to carry out subsequent investigations on the expression of specific genes in AD brain. 83 I I I B . PREPARATION OF A SUBSTANTIA INNOMINATA CDNA LIBRARY As previously described in detail, the substantia innominata (SI) i s a brain region enriched in cortically projecting cholinergic neurons, and one which i s particularly damaged in AD. A cDNA library for the SI would be of use for investigating genes specifically expressed by the cholinergic neuron, and to supply relevant probes to investigate gene expression in AD affected brain. An appropriate SI cDNA library should have the desired features of large insert size ( f u l l length representations of mRNA), high complexity (number of independent recombinants), and production in a cloning vector which would lend i t s e l f readily to the screening scheme. For the screening scheme undertaken in this thesis, the use of plasmid vectors, as compared to bacteriophage, was the most appropriate choice. The selection of plasmid cloning was based on two features; 1) negative selection primary screening is straightforward; and, 2) the recovery of large numbers of purified recombinant DNAs is rapid and easily achieved. One of the disadvantages of using plasmids, low transformation efficiency, has been overcome by the use of electroporation transformation. In recent years a number of plasmid vectors have been 84 produced which lend themselves readily to cDNA cloning. Many contemporary plasmid vectors are based on the pUC series originated by Messing and colleagues (for a review, see Messing, 1983). The pUC series grew out of work on the M13mp series of filamentous single stranded DNA bacteriophages and share the synthetic multiple restriction endonuclease polylinker, or multiple cloning site (MCS). These plasmids permit identification of recombinants by insertional inactivation of the a-complementation of jS-galactosidase (/3-gal, recombinants lose this enzymatic activity) and allow for antibiotic selection of transformed bacteria via the plasmid-linked /3-lactamase gene. In addition, the pUC plasmids are propagated with high copy numbers, possessing a modified pBR322 replication origin. Recent further refinements have included the addition of bacteriophage RNA polymerase promoters for the preparation of synthetic RNA transcripts (Melton et a l . , 1984), and the addition of the M13 intergenic region for the preparation of single stranded DNA plasmid copies (Zagursky, 1985). The vector used in the present work, pT7T3-18U (see Figure 7), i s a pUC derivative which has a l l of these various functions. The cDNA synthetic approach in the current work has been modified from previous protocols (Gubler and Hoffman, 1983; Krug and Berger, 1987) in order to allow the handling of small amounts of cDNA. An overriding fact in the examination of the human SI is the limited amount of material available. The 85 human SI weighs about 200-500 mg per hemisphere. The maximum amount of RNA that can be obtained from a neurologically normal SI i s 75-150 /tig, corresponding to 1-3 pq of polyA RNA. In order to reduce the loss of the small amounts of material, manipulations of the cDNA prior to vector ligation and bacterial transformation have been minimized wherever possible. A key step in the construction of recombinant DNA i s the joining of target cDNA sequences to linearized vector. The ends of the linear insert DNA must be compatible with those of the vector in order to ligate the two molecules successfully. The generation of compatible ends can be accomplished by blunt ending the DNAs, by trimming away single stranded overhang with exonuclease or by f i l l i n g i t in with a DNA polymerase, by the generation of cohesive homopolymer tracts (tailing) with terminal deoxynucleotidyl transferase, or by generating an appropriately compatible cohesive end during synthesis of the insert DNA. An increasingly used method for the generation of compatible ends is the use of synthetic oligodeoxynucleotide (oligonucleotide) linkers or adaptors. These synthetic DNAs can be placed at the termini of insert DNA by ligation and can either directly generate a cohesive protruding end (adaptor), or generate a compatible end after digestion with the appropriate restriction endonuclease (linker). The rationale for the use of these reagents is to take advantage of their protruding cohesive ends, which are more efficient substrates 86 for ligation than are blunt ends. Other advantages of insertion with linker and adaptor molecules derive from the ab i l i t y to insert into a predictable, known sequence of the vector, and particularly to do so in a known orientation relative to a sequencing primer on the vector. In a negative selection library screening procedure, such as that intended for the analysis of this SI cDNA library, i t becomes extremely important to minimize the level of non-recombinant "background" clones. Although a variety of sophisticated adaptor schemes for cloning are available (for examples see Coleclough, 1987), many generate an unacceptable level of background transformants. The approach taken in this work has been to use a f i r s t strand cDNA primer-adaptor, not for the integration site for ligation to vector, but rather as a site for identification of the 3'-end of recombinants produced by direct double stranded adaptor cloning. Materials and Methods Preparation of SI polyadenylated RNA. In order to isolate SI polyA RNA from the small amounts of total cellular RNA available, a batch adsorption procedure onto oligo(dT)-cellulose was carried out. This method seems to reduce the losses involved with chromatographic methods, and is carried out as follows: oligo(dT)-cellulose (type 7, Pharmacia) is stored cold and in the dark as a 50% (hydrated volume) slurry in 0.1 M NaOH/ 10 mM EDTA; 500 jul of the slurry was placed in a 1.5 ml microcentrifuge tube and collected by 87 centrifugation at 1000 x g for 2 minutes; the matrix was washed twice by resuspension in 1 ml of water, followed by washing twice with 500 mM NaCl/ 25 mM Tris-HCl (pH 7.6)/ 1 mM EDTA (HS buffer) ; 30-150 pq of heat denatured RNA in 50 jul of water, 500 jul HS buffer, and 15 jLtl of 10% SDS was placed in the tube containing the matrix and mixed continuously for 20 minutes; the matrix, with bound RNA, was washed three times with l ml of HS buffer, then once with 1 ml of HS buffer that had been diluted 1:1 with water. The polyA RNA was eluted with three 200 j u l aliquots of water at 37°C for 5 minutes each. The volume of the RNA solution was reduced to approximately 100 /il on a Savant evaporator, adjusted to 0.3 M NaOAc and mixed with two volumes of ethanol; the material was l e f t to precipitate for at least 16 hours at -20°C. Source and purification of synthetic oligonucleotides. The primer-adaptor and adaptor oligodeoxynucleotides (see Figure 7) were prepared by automated synthesis using phosphoramidite chemistry and were obtained from the following sources: the 13-mer and 9-mer Eco RI adaptor pair from Pharmacia, the 27-mer 1st strand primer-adaptor was from Dr. T Atkinson, Dept. of Biochemistry, U.B.C. (Vancouver, B.C.). The 1st strand primer-adaptor was purified by HPLC before use, and was 5•-phosphorylated with PNK. The Eco RI adaptor was phosphorylated on the 5'-end of the 13-mer before annealing with the complementary 9-mer. 88 cDNA synthesis. The f i r s t strand of cDNA was synthesized by the action of AMV reverse transcriptase (AMV RT-XL, Life Sciences), and was primed with the 27-mer primer-adaptor. The SI polyA RNA was denatured by heating to 70°C for 3 minutes, then rapidly cooled on ice. The RNA was added to a synthetic mixture which had the f i n a l concentrations: 50 mM Tris-HCl (pH 8.3 at 41°C)/ 50 mM KCl/ 8 mM MgCl2/ 1 mM EDTA/ 5 mM dithiothreitol/ 1 mM of each dNTP/ 1.5 mM sodium pyrophosphate/ 0.5 mM spermidine/ 100 /xg/ml acetylated BSA/ 250 units/ml placental RNAase inhibitor (BRL) / 100 jug/ml 27-mer primer-adaptor/ 500 units/ml reverse transcriptase/ 10 jtiCi [a-32P]-dCTP (3000 Ci/mmol, NEN) , a l l in a f i n a l volume of 25 /nl. The reaction was allowed to s i t on ice for 5 minutes, then was transferred to a bath at 41°C for 100 minutes. At the end of this incubation, the mixture was placed on ice. The second strand synthesis was carried out by the addition of various reagents to the tube containing the f i r s t strand products. The f i n a l reaction conditions were: 25 mM Tris-HCl (pH 7.8 at 15°C)/ 100 mM KCl/ 5 mM MgCl2/ 5 mM dithiothreitol/ 0.2 mM each dNTP/ 10 units/ml E. c o l i RNAase H (BRL)/ 250 units/ml E. c o l i DNA polymerase I (BRL). The reaction had a f i n a l volume of 200 /xl, and the components were kept on ice during the various additions. For analytical purposes, a 10 /xl portion of the reaction was incubated in 32 parallel with 25 /LICI of dried [a- P]-dCTP. The reaction was 89 incubated for 1 hour at 14 °C, then the temperature was raised slowly to 22°C and the reaction allowed to proceed for a further hour, after which i t was terminated by the addition of EDTA to 5 mM. Protein was removed from the cDNA by a single extraction with phenol/chloroform/isoamyl alcohol (phenol-chloroform; 25:24:1), the organic phase was back extracted with one volume of 10 mM Tris-HCl (pH=8.0), 1 mM EDTA (TE [8.0]), the aqueous phases pooled, made to 2.5 M with NH4OAc, and two volumes of ethanol was added. The ethanolic precipitation was carried out at -20°C for 12 hours, followed by centrifugation in a SW-65 Ti rotor at 150,000 x g for 1 hour. The pellet from the centrifugation was washed with 70% ethanol, dried, resuspended in 50 fxl of water, and then reprecipitated from NH4OAc and ethanol. The double strand cDNA (approximately 450 ng) was rendered blunt end by treatment with the Klenow fragment of DNA polymerase (5 units, BRL) in 50 mM Tris-HCl (pH 7.8)/ 8 mM MgCl2/ 10 mM dithiothreitol/ 0.1 mM of each dNTP; a l l in a fi n a l volume of 20 jul, for 4 hours at 17°C. The reaction was terminated by heating to 65°C for 15 minutes, then c h i l l i n g on ice. The cDNA was ligated to Eco RI adaptor (5 jLtg) in a fi n a l volume of 100 /i l in the presence of 3 0 mM Tris-HCl (pH 7.8)/ 10 mM MgCl2/ 4 mM jS-mercaptoethanol/ 0.4 mM ATP and 2 units of T4 DNA ligase (BRL). The ligation reaction was carried out at 12°C for 14 hours, after which i t was terminated by incubation at 65°C for 10 minutes, followed by cooling in an ice bath. The adapted cDNA was phosphorylated by adding dithiothreitol to 10 mM, ATP to 1 mM, spermidine to 0.5 mM, and 10 units of polynucleotide kinase (PNK, Pharmacia). The mixture was incubated at 37°C for 30 minutes, after which a further 10 units of PNK was added, and the incubation continued for a further 30 minutes. The phosphorylation was terminated by the addition of EDTA to 10 mM, and the solution extracted with 100 jLtl of phenol-chloroform, the organic phase back extracted with 50 jtil of TE, and the two aqueous phases pooled. In order to clean up the adapted cDNA before f i n a l ligation to vector, and particularly to eliminate excess adaptor, size exclusion chromatography using Sephacryl S-400 HR was carried out. The cDNA was loaded on a 15 x 1 cm column, which was eluted by gravity at a flow rate of about 2 ml/hour with 25 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA. This chromatographic procedure was quite fast, requiring about 3 hours for the complete elution of materials, and provided a highly efficient separation of cDNA (void volume fraction 2.5-2.9 ml) from unincorporated nucleotide and other extraneous materials, including adaptors (apparent included volume of 5.8-6.4 ml). Based on model experiments with restriction fragments of lambda DNA, the exclusion limit for this matrix is 400-600 bp. The cDNA fraction was reduced in volume to 150 jul on a Savant evaporator, adjusted to 0.15 M NaOAc, and two volumes of ethanol was added. The f i n a l size selected cDNA was allowed to precipitate at -20°C for 6 hours, then recovered by centrifugation at 200,000 x g for 1 hour at 2°C. Finally, the DNA was washed with 70% ethanol by centrifugation at 100,000 x g for 10 minutes at 2°C, before drying under vacuum. The cDNA yield after size exclusion chromatography and ethanol precipitation was 140 ng. Ligation of cDNA to vector and transformation of E. c o l i . In order to define transformation potential and to reduce the level of non-recombinants, the cDNA was i n i t i a l l y tested on small scale ligations with varying amounts of the linearized and phosphorylated plasmid vector DNA. Based on these t r i a l s , the large scale ligations were carried out at a mass ratio of vector to cDNA of 3.5 to 1, corresponding to a molar ratio of about 2.5 to 1. One half of the cDNA preparation, estimated to be 70 ng, was ligated to 250 ng of Eco RI digested, dephosphorylated pT7T3-18U, in a volume of 200 jul, using the previously described ligation buffer and 1.5 units of T4 ligase for 20 hours at 12°C. The ligation mixture was precipitated out of NH4OAc/ethanol by centrifugation at 200,000 x g for 1 hour at 2°C, followed by washing with 70% ethanol and centrifugation at 100,000 x g for 5 minutes. The ligation products were resuspended in 75 jul of TE (8.0). Transformation of E. c o l i K12 strain DH5a was carried out using the electroporation protocol of Dower et a l . (1988), with the Gene Pulser apparatus (Bio Rad). Bacteria were prepared for transformation by growth in 1 l i t r e of LB broth 92 (Maniatis, 1982) to an optical density of 0.50 at 600 nm. The bacteria were collected by centrifugation at 1500 x g for 4 min. at 4°C, washed sequentially with 1 1 ice cold water, 500 ml water, and 20 ml of 15% glycerol, and then resuspended in 3 ml of 15% glycerol at a f i n a l c e l l density of approximately 3 x 1010 colony forming units (cfu)/ml. The cel l s were briefly chilled on ice, frozen in a dry ice/ethanol slurry, and stored at -75°C until use. Just before transformation, bacteria were allowed to thaw at room temperature, and 40 /xl aliquots were put in the bottom of microcentrifuge tubes, which were placed in an ice bath. The ligated DNA was heated to 65°C for 5 minutes and chilled on ice; then approximately 12 ng of DNA (vector + insert) in 3 /xl was added to an aliquot of bacteria, and the mixture returned to the ice bath. After 1 minute, the mixture of DNA and bacteria was transferred to a 0.2 cm gap parallel plate cuvette, and subjected to a 2500 volt pulse with the 200 ohm series resistor (an observed time constant of 4.6 ms). Immediately after electroporation, the contents of the cuvette were washed out with 1 ml of prewarmed SOC media (Hanahan, 1985) and transferred to a Falcon 2059 tube. The bacteria were allowed to recover by incubation at 37°C for 1 hour at 225 rpm in a gyrorotary water bath (Lab-Line). Twenty-five discrete transformation reactions were carried out for each half of the ligated cDNA/vector mixtures. Storage of primary transformants and preparation of an  amplified SI cDNA library. 93 Two of the transformation mixtures from the f i r s t half of the library construct (pSI3a) were plated out directly on 80 HATF nitrocellulose f i l t e r s (82 mm dia., Millipore Corp.), which had been laid on top of LB agar plates (containing 100 /xg/ml ampicillin [Amp] and 50 nq/ml 5-bromo-4-chloroindolyl-j8-D-galactopyranoside [X-gal]), at a density of approximately 500 colony forming units (cfu) per membrane. The primary transformation plates were incubated overnight at 37°C, and then were stored at 4°C unti l further processing. The remaining portion of pSI3a, corresponding to about 5 x 105 cfu, was diluted 10-fold in LB-Amp medium and incubated for 1 hour (corresponding to two doubling times), harvested by centrifugation at 1500 x g for 5 minutes, resuspended in SOC/10% glycerol freezing medium, and frozen in 1 ml aliquots. A portion of this material, corresponding to 50,000 cfu, was amplified in LB-Amp selection media overnight. This material was harvested by centrifugation, the bacteria washed with TE, recentrifuged and the resulting pellet frozen for plasmid extraction. The second ligation reaction, pSI3b, was used to transform bacteria (as described above). After the 1 hour recovery period in SOC, the cultures were pooled and plated out onto 42 nitrocellulose membranes (88 mm squares, Schleicher and Schuell Ltd.) overlaid on LB-Amp agarose plates (at a density of 14,000 cfu/membrane). The bacteria were grown at 37°C u n t i l the colony size was approximately 0.2 mm in diameter. This amplified cDNA library, pSI3b, was recovered from the nitrocellulose plates by scraping each membrane with a rubber policeman into 4 ml of SOB/10% glycerol medium (Hanahan, 1985). The bacteria were pooled and 1 ml aliquots were frozen in dry ice/ethanol. The t i t r e of the library, 9 after thawing, was 4 x 10 cfu/ml. Plating of a sample on X-gal indicator plates revealed 3.3% strongly blue colonies. The portion of pSI3a that was plated directly onto nitrocellulose membranes was replicated on Colony/Plaque Screen membranes (82 mm dia., Dupont) when the colony size was approximately 0.5 mm. Both the replica (on LB-Amp) and nitrocellulose master membranes (on LB-Amp/5% glycerol) were returned to the incubator to allow growth before storage at 4°C. The replicas were harvested when the colonies were about 0.7 mm. For storage, master membranes were sandwiched with a second Colony/Plaque Screen membrane and the sandwich frozen at -70°C (Hanahan and Meselson, 1985). Miscellaneous. Plasmid 'miniprep' DNAs were prepared by the method of Birnboim (1983). Restriction enzymes were obtained from various commercial suppliers, and were used as recommended by the supplier. The cloning vector pT7T3-18U was obtained from Pharmacia; this vector was linearized with Eco RI and dephosphorylated with bacterial alkaline phosphatase. Transformation experiments with this vector gave less than 5 x 4 . . . . . 10 cfu//xg of ligated vector under conditions in which supercoiled pUC18 had a transformation efficiency of 4 x 10 cfu//xg. The bacterial host, E. c o l i K12 DH5a (BRL) , has the genotype: F-<p80 dlacZAM15 A(lacZYA-argF)U169 recAl endAl hsdR17(rK", mK+) supE44 X" thi-1 gyrA relAl (Hanahan, 1987). Native agarose gel electrophoresis was carried out using Tris/acetate/EDTA buffer and cDNA was analyzed on denaturing alkaline gels (Maniatis et a l . , 1982). Results Figures 7 and 8 illustr a t e the reagents used, and the cloning protocol developed for the preparation of the human SI cDNA library pSI3. Yield and Characterization of the cDNA Products. The reaction conditions employed for the f i r s t strand cDNA synthesis were determined by optimizing the quality and quantity of cDNA products using a synthetic polyA RNA standard from BRL as template. This material has a range of transcription competent RNAs from 0.24 kilobases (kb) to 9.49 kb. In addition, a 2.3 kb polyA RNA was used in order to evaluate possible "snapback" cDNA synthesis. The addition of 1 mM Na pyrophosphate was observed to inhibit the snapback cDNA synthesis, and, at higher concentrations, to be generally inhibitory. When the cDNA synthesis was carried out with ImM Na pyrophosphate, no detectable 4.6 kb product was observed from the 2.3 kb template; this compound was therefore included in the standard reaction mixture. Conditions which gave a high yield of f u l l length cDNA product for the 2.3 kb or the 0.24-96 U. Seq. T3 RNA Pol pUC18 T7 RNA Pol i Primer promoter M.C.S. promoter | Primer-Adaptor 1st strand, 27-mer 5'-CTAGAGTGGACATTTTTTTTTTTTTTT-3' Sal I Adaptor Oligo #1, 13-mer 5' - A AT TCGGCACG AG - 3' Adaptor Oligo #2, 9-mer 5'-CTCGTGCCG-3' Alignment of Eco RI Adaptor for Ligation to cDNA: 5' - AAT TCGGCACG AG - 3' 3'-GCCGTGCCGp-5' Figure 7. Reagents used for SI cDNA cloning. A map of the cloning vector pT7T3-18U i s shown at the top, and the sequences and orientations of the oligonucleotides used for cloning are shown on the bottom of the page. cDNA cloning protocol 97 S. I. polyA+ RNA 1 i AAAAAA-3' 1. Reverse transcriptase 2. RNAase H/DNA Pol I 3. Klenow ds-cDNA 1. Ligation to Eco R1 Adaptor 2. Phosphorylation with P N K 5'-pe ip-5' 1. Size selection (> 350 bp) on S-400HR 2. Ligation to Eco R1 cut and dephosphorylated pT7T3 18U w Transformation of DH5a Figure 8. A summary of the sequence of enzymatic and preparative steps used during the production of the SI cDNA library. The f i r s t strand synthetic primer sequence i s shown in Figure 7. 98 9.49 kb polyA RNA preparations were also observed to give a good product for cerebellar polyA RNA and, as w i l l be shown, for SI polyA RNA. For quality control purposes, a reaction with the synthetic standard was always run in parallel with brain cDNA syntheses. Generally, the yield of f i r s t strand cDNA from the standards was higher than polyA RNA prepared from brain. On some occasions, the standard RNA had yields of up to 65 % cDNA, whereas the highest ever obtained with brain poly A RNA was 38 %, obtained with a cerebellar polyA RNA preparation. Generally, a yield of 15-30 % was obtained from human brain polyA RNA which had been twice selected by oligo(dT)-cellulose chromatography. The SI cDNA library was constructed from one half of the undetermined mass of poly A RNA obtained from 150 ng of total cellular RNA. This total cellular RNA was pooled from two neurologically normal donors (MC-187 and MC-213), in order to minimize the possible effect of dissection error. As detailed in Methods, the SI poly A+RNA was selected by an oligo(dT)-cellulose batch absorption. This preparation gave a f i r s t 32 strand yield of 315 ng of cDNA, based on P-dCTP incorporation into trichloroacetic acid precipitable material. The yield of the standard RNA, carried out in parallel, was 45 %. In order to assess the quality of cDNA, the products were analyzed by alkaline agarose gel electrophoresis. Figure 9 presents the electrophoretic analysis of the cDNA products, with standard and SI polyA RNAs for templates. The fitness of the reverse transcription i s readily indicated by the discrete cDNA products produced from the standard RNA, with significant amounts of the higher molecular weight species, including 7.5 kb cDNA. Only a small amount of 9.4 kb cDNA was produced. The SI cDNA products had a heterogeneous distribution extending up to at least 8-10 kb, with the greatest mass appearing in the 1-3 kb region. The second strand cDNA synthetic reaction was carried out by a modification of the method of Gubler and Hoffman (1983). The reaction was simplified by the dilution of the f i r s t strand reaction mixture, and the subsequent adjustment of the conditions for the action of RNAase H and DNA polymerase I. Under these conditions, quantitative production of the second strand was observed (104 % yield for standard), the yield for the SI cDNA was 327 ng (103 % ) . The electrophoretic analysis of the cDNA second strand is presented in Figure 9. The second strand reaction conditions yield products with a distribution similar to the f i r s t strand cDNAs, and are therefore presumably nearly f u l l length. Two methodological considerations are important in the recovery of the cDNA after the synthetic reactions. First, in agreement with Gubler (1987) , i t was found necessary to use an ammonium acetate/ethanol precipitation of the product out of the second strand reaction mixture. The use of sodium acetate 100 Figure 9. Electrophoretic analysis of cDNA products with SI polyA RNA (XI, X2; 1st and 2nd strands) and the BRL standard RNA (SI, S2) as templates. Alkaline agarose gel electrophoresis was carried out on a 1.4% gel. The products were visualized by autoradiography. Chain lengths of the A-DNA standards (M) are indicated on the l e f t of the photograph in kilobases. The standard RNA was composed of synthetic polyA RNAs with chain lengths of 9.49, 7.46, 4.40, 2.37, 1.37, and 0.24 kb. 101 results in the co-precipitation of a large amount of extraneous material, including a large amount of unincorporated dNTPs. In subsequent enzymatic manipulations of the cDNA, these materials have a deleterious effect and do not permit evaluation of product yields. Secondly, in order to obtain a reasonable recovery of the small amount of cDNA, i t is necessary to allow a sufficient period of time for ethanol precipitation (at least 6 hours), and then to use high speed centrifugation to sediment the DNA. Using this procedure, the loss of TCA precipitable counts was reduced to approximately 30% during the recovery and reprecipitation of the cDNA. Characterization of Transformants The optimal conditions for the ligation of cDNA to dephosphorylated vector was determined to be 3.5 mass vector to unit mass insert. This resulted in a yield of 0.9 x 106 transf ormants per jug of ligated cDNA. Assuming there was 1 jug of starting mRNA, this yielded an efficiency in the range of 1 x 106 transformants/jug poly A RNA. The level of nonrecombinants in the SI library was estimated by plating transf ormants on indicator plates for j3-gal, using X-gal as the chromogen. This procedure w i l l detect insertional inactivation of the j3-gal a-complementation at the lac Z1 locus. With the pSI3a transformations, the number of blue colonies, representing nonrecombinants, was 3.1 ± 0.1% (mean ± s.d., n=3). It should be noted that this was determined as the number of colonies which gave a clearly blue colour reaction within 12 hours after removal from the incubator. After the plates were l e f t for another day at 4°C, a further 2-3% of the colonies developed light blue centers. In this, and previous work, the light blue colonies yield plasmids which harbor small inserts (up to 900 bp). The size of cloned insert was determined by resection at the flanking adaptor sites with Eco Rl endonuclease. A series of randomly chosen primary transformant colonies which were negative for j8-gal were grown in small scale culture for plasmid 'minipreps'. The plasmid DNA was digested with Eco Rl, and the size of insert cDNA was determined by analytical agarose gel electrophoresis, an example of which is shown in Figure 10. A l l of the clones which underwent this analysis had detectable inserts. Many clones had more than one insert fragment released by Eco Rl, and were presumed to have insert cDNAs with internal Eco Rl restriction sites. Subsequent analyses of several such clones have confirmed this interpretation. The mean insert size for the 56 clones analyzed was 1.40 kbp. A plot of the frequency distribution of insert size for this series of randomly chosen transformants is presented in Figure 11. The distribution appears as a bimodal function, with the main peak in the range of 1.0 kbp, and a secondary maxima at 2.5-3.0 kbp. The smallest cDNA insert in this sample was 0.34 kbp and the largest was 4.85 kbp. To confirm the size distribution of cloned insert cDNAs, 103 F i g u r e 10. A n a l y t i c a l agarose g e l e l e c t r o p h o r e t i c d e t e r m i n a t i o n o f cDNA i n s e r t s i z e f o r a number o f c l o n e s from p S I 3 a . The p l a s m i d DNAs were d i g e s t e d t o c o m p l e t i o n w i t h Eco R I , and the r e s u l t i n g fragments a n a l y z e d on a 1.2% agarose g e l , which was s t a i n e d w i t h e t h i d i u m bromide . The i n s e r t l e n g t h s were c a l c u l a t e d i n r e f e r e n c e t o t h e s t a n d a r d DNA fragments , which have c h a i n l e n g t h s as i n d i c a t e d on the r i g h t o f the p h o t o g r a p h . The 2.9 kbp v e c t o r band i s denoted by V . 104 INSERT SIZE REPRESENTATION IN pSI3a Percent O c c u r r e n c e 25.0 -i 20.0-0.5 1 1.5 2 2.5 3 3.5 >3.5 Bin Range of Insert in K i lobase Figure 11. Non-cumulative frequency histogram of the size of cDNA insert for library construct pSI3a. The data are plotted with a bin width of 0.25 kbp, and were normalized for the 56 randomly selected clones which underwent this analysis. The size of Eco Rl released fragments was determined by gel electrophoresis, as described in Figure 3. Several clones released more than one insert fragment indicating internal restriction sites; in these cases, the total length of insert was calculated as the sum of released fragments. The average insert was 1.40 kbp. 105 a portion of the primary transformants, corresponding to 50,000 recombinants, was amplified in liquid culture overnight. The mixture of purified plasmid DNAs was then analyzed directly, or subjected to Kpn I restriction enzyme digestion. This enzyme has a single vector recognition site in the multiple cloning site and, given that i t i s a low frequency 6-cutter, should fragment the insert DNA at internal sites only rarely. This experiment indicated an approximate average insert size of 1.5 kbp (on the gel running in the region of 4.5 kbp = 2.9 kbp vector + 1.4 kbp insert). Inserts of up to 6-8 kbp could be found in this mixture of recombinants (corresponding to linearized vector + insert of 9-11 kbp). Discussion Despite starting with a small amount of RNA, sufficient polyA RNA could be acquired to generate a large cDNA library. The cloning protocol was highly efficient, yielding a large number of independent transformants from a small amount of polyA RNA (about 0.5-1 x 106 per u.q) , resulted in a low background of non-recombinants, and contained cDNA inserts with a size distribution similar to values reported for cultured cells or animal tissues. The use of a primer-adapter for the f i r s t strand synthesis, and of the assymetically phosphorylated Eco RI adapter yielded a library which could be conveniently analyzed. Starting with larger amounts of brain tissues, a number of others have reported similar s t a t i s t i c s 106 for the construction of cDNA libraries using lambda phage cloning vectors (Zain et a l . , 1988; Octave et a l . , 1988; Vitek et a l . , 1988). The high efficiency of library construction i s due to both efficient cDNA synthesis and the improved transformation yield obtained with the electroporation method. The SI cDNA library appears to be a good representation of gene expression in the human SI at the time of death, and is an appropriate source of clones for the analysis of differential gene expression. 107 III C. SCREENING THE SUBSTANTIA INNOMINATA CDNA LIBRARY A DNA library screening strategy requires either pre-existing structural information about a target gene, or a biological observation relating to the prevalence of the target within a population of nucleic acids. As an example of the latter, a library can be screened by differential hybridization, using labelled cDNAs produced from two different sources of polyA RNA. A comparison of the hybridization patterns for the two assemblies of clones may indicate some difference in the relative abundance of cDNAs within one probe population (from polyA RNA), relative to the other. Generally, this approach has only been successful for screening relatively small numbers of recombinant clones, at low density. The genes chosen as targets in this study of the SI cDNA library are those which are either expressed at different abundances in the SI of AD and NNC, or expressed in a brain region specific manner. Genes which would f i t both c r i t e r i a would, of course, be of the greatest interest. Although differential screening of the SI cDNA library should be capable of identifying genes of both sorts, certain experimental limitations are well known: 1) a direct differential cDNA screening of a reasonable number of clones 108 requires a large amount of cDNA; 2) colony hybridizations are subject to significant background problems, generating false positives; 3) relatively large quantitative differences are needed to generate an apparent differential signal; and 4) much of the great sequence complexity of gene expression in the brain is represented by low abundance mRNAs. The library screening scheme was, as in the case for cDNA cloning, designed to minimize the ut i l i z a t i o n of SI cDNA. The biological observation which underlies the library screening scheme is that the cerebellum does not contain c e l l bodies of cholinergic neurons, nor i s i t a site of marked AD-type pathology. The cerebellum is a brain region which expresses much of the sequence complexity of the CNS (Travis and Sutcliffe, 1988), but which should not express the AD-affected genes sought in this study. Thus this tissue was used in the SI cDNA library screening scheme as a negative, that i s , a source of polyA RNA to generate cDNA for the detection of clones to be eliminated from consideration as SI or disease specific markers. Elimination of such clones was necessary in order to have a smaller pool of clones enriched in the target sequences. Differential cDNA screening of a smaller pool of clones reduced the required amount of SI cDNA probe, which was available in limited quantity. Materials and Methods Preparation of cDNA Probes Radiolabelled cDNA probes were produced by a two-step 109 procedure. First, an unlabelled cDNA was produced by the f i r s t strand cDNA synthetic reaction described in Section III.B, and phenol-chloroform extracted and precipitated with ammonium acetate and ethanol. This single stranded DNA was employed for production of a radiolabeled cDNA probe, just before use, by the random hexanucleotide reaction described in Section III.A. Calculation of second strand synthesis, based on incorporation 32 . . . . . of [ PjdCTP, confirmed the essentially quantitative yield (>95%). Radiolabelled cDNA probes were isolated from the labelling reaction by adsorption and elution on NEN-Sorb cartridges, as previously described. The two-step probe production was developed in order to improve the specific activity of radiolabelled cDNA (> 109 cpm/jug DNA input) , as well as to allow storage of unlabelled single strand cDNA, without the usual concern for radiolytic decay of high specific activity single strand cDNA probes. This allowed more efficient use of the limited amount of SI polyA RNA. Colony screening with cDNA Keyed (orientation holes), replicate membranes bearing approximately 500 primary transformants per nylon membrane (pSI3a, see section III.B, p. 93) were allowed to thaw, placed on LB/Amp/X-gal indicator agar plates, then transferred to a 37°C incubator. The colonies were grown to 1.5 mm in diameter, then placed at 4°C for at least 6 hours before further processing. At this point non-recombinant transformants were readily detectable as blue colonies (galactosidase positive). 110 Bacterial colonies were lysed and immobilized by sequential placement on f i l t e r papers saturated with 0.5 M NaOH held at 45°C for 1 minute, then at room temperature for 2 minutes; the f i l t e r s was treated twice at room temperature with 2 M Tris-HCl, pH=7.5, for 5 minutes. These processed f i l t e r s were allowed to dry at room temperature overnight. The matching replica colony-bearing nitrocellulose membranes were placed on LB-AMP plates, and growth was established at 37°C for 2-4 hours; the plates were then transferred to a 4°C refrigerator for storage u n t i l the hybridization analysis was completed. The dried nylon membranes were transferred to Vacu-Blot (ABN, Inc.) pouches (8 membranes per 20 x 20 cm pouch), and were treated for 6 hours with 100 ml of 1 % SDS/ 1 M NaCl at 60°C with agitation. The washing f l u i d was removed by aspiration before addition of 50 ml of prehybridization f l u i d , composed of the following: 50 mM Tris-HCl, pH=7.5/ 1 M NaCl/ 5 mM EDTA/ 1 % SDS/ 10 % dextran sulfate/ 100 ug/ml sonicated and denatured calf thymus DNA/ 200 ug/ml heparin sulfate. The membranes were prehybridized at 65°C for about 12 hours. Hybridization was carried out for 24 hours at 65°C in 50 ml of fresh prehybridization f l u i d which contained radiolabelled cerebellum cDNA probe (1 ng/ml, 106 cpm/ml) and 25 ug/ml of both polyA and polyU (high molecular weight, Sigma). Following hybridizations, the membranes were sequentially washed as follows: 2 x SSC/1 % SDS, 10 min. with two changes, at room temperature; 0.2 x SSC/ 1 % SDS, 3 0 min. with two changes, at I l l 60°C; 1 x SSC/ 1 % SDS for 10 min. at room temperature. In another series of experiments, colony hybridizations were carried out with both the prehybridization and hybridization fluids containing 50% formamide/ 5 x SSPE (1 x SSPE is 0.18 M NaCl/ 0.01 M sodium phosphate/ 0.001 M d i -sodium EDTA, pH=8.0)/ 0.25 % SDS/ 10 % dextran sulfate/ 100 /ig/ml calf thymus DNA/ 200 jug/ml heparin sulfate/ 25 jiig/ml each polyA and polyU. These hybridizations were carried out at 42°C. In these experiments labelled cDNA was present at 5 ng/ml (5 x 106 cpm/ml) , and the hybridization was allowed to proceed for 24 hours. Post-hybridization washes were carried out as described above with a f i n a l stringency wash of 0.1 x SSC/ 1 % SDS with two changes for 1 hour at 60°C. Negative Selection with Cerebellum cDNA Probe Colony hybridization with cerebellum cDNA was carried out at 65°C (in the absence of formamide), as described above. After washing and autoradiographic exposure for varying times, the membranes were stripped of probe by treatment with 95 % formamide/ 1 % SDS/ 0.01 M Tris-HCl, pH= 7.5/ 0.001 M EDTA at 90°C for 10 min. with two changes of f l u i d . These membranes were then processed for the detection of plasmid DNA by hybridization with 10 ng/ml (107 cpm//xg DNA) of labelled plasmid DNA, and washed as above. Comparison of colony replica membranes with the autoradiograms generated with plasmid hybridization indicated that a l l colonies were being fai t h f u l l y detected. The matched pairs of films from the 112 plasmid and cerebellum cDNA hybridizations were aligned for the determination of colonies which did not generate a signal with the cerebellum cDNA probe. White colonies which were cerebellum cDNA negative were sampled with s t e r i l e toothpicks for inoculation of tubes containing nutrient medium. The selected clones were grown to stationary phase overnight, a sample removed and diluted 1:1 with 20 % glycerol-LB medium, and then placed in the wells of microtitre plates (Flow Labs) for storage at -70°C until use. Secondary selection of the cerebellum cDNA negative clones was carried out by transferring a 5 jLtl inoculum of the culture to form arrays" of colonies on nylon membranes, which had been overlaid on LB-Amp plates. These banks of clones were grown to 1 mm colony size, then processed for hybridization analysis as described above (65°C, no formamide). In this case, the hybridization was carried out with a 10-fold increase in probe input, 10 ng/ml (107 cpm/ml) of hybridization f l u i d . Slot Blot Hybridizations Following selection, the desired recombinants were recovered from microtitre plate banks, then grown to stationary phase as 2 ml of liquid culture in LB-Amp medium. One m i l l i l i t r e of the culture was pelleted in a microcentrifuge tube, and plasmid was extracted by alkaline lysis (Birnboim, 1983). The cleared lysate was treated with RNAase, phenol-chloroform extracted, and precipitated from 113 ammonium acetate and ethanol. The f i n a l plasmid DNA was dissolved in 20 jul of TE (8.0), and the DNA concentration was determined by spectrofluorometric assay using the dye bisbenzamide (Hoescht 33258, Calbiochem), essentially as described (Brunk et a l . , 1979)., Fluorescent intensities were determined on a modified Aminco-Bowman SPF-500 instrument using an excitation wavelength of 360 nm, an emission wavelength of 450 nm, an entrance s l i t width of 2 mm, and exit s l i t width of 0.5 mm. Yields of plasmids ranged from 0.8 /xg to 23 /xg. Isolated plasmid DNA was f i l t e r e d onto Gene-Screen Plus membranes using the Slot Blot II apparatus of Schleicher and Scheull, Inc. This was carried out by denaturing 350 ng of plasmid DNA in 200 fj.1 of 0.2 M NaOH/0.01 M EDTA, for 20 min. at room temperature. This solution was neutralized by the addition of 200 p i of 3 M ammonium acetate, and immediately loaded onto the membrane with gentle suction. The sample wells were washed with 0.5 ml of 1 M ammonium acetate, sucked dry, and the membranes were allowed to dry at room temperature before baking at 80°C in a vacuum oven. Following preparation of several replicate membranes in the above manner, hybridization analysis with cDNA probes was carried out at 65°C, as described above for colony hybridizations. In experiments which varied the tissue source of cDNA, care was taken to match the specific activities of hybridization solutions. A l l such experiments were carried out in parallel, 114 in order to assure accurate matching of the hybridization, washing, and film exposure conditions. Differential Southern Blot Hybridizations Samples of plasmid DNAs were treated with Eco Rl endonuclease to release the cDNA insert, then electrophoresed through 1 % agarose gels using the TAE buffer system. DNA was transferred to Gene-Screen Plus membranes using the Vacu-Blot apparatus under alkaline conditions, as recommended by the manufacturer (LKB-Pharmacia). These membranes were processed for differential cDNA screening by hybridization at 65°C, using a cDNA concentration of 10 ng/ml (107 cpm/ml) , and a f i n a l stringency wash of 0.1 x SSC/1 % SDS at 60°C for 45 min. Following autoradiographic exposure of the membranes, higher stringency washes were examined in order to evaluate specificity of the signals. Northern Blot Hybridizations Detection of RNA transcripts was carried out using the electrophoresis, blot transfer, and hybridization conditions described in Section IIIA. The f i n a l stringency wash was 0.1 x SSC/1 % SDS at 65°C with two changes, for a total of 1 hour. Membranes were stripped of probe and reused, as described above. The analyses of cDNA clones by Northern blotting were preferentially carried out using polyA RNAs, in order to reduce autoradiographic exposure times. PolyA RNA samples were prepared by oligo(dT)-cellulose column chromatography, as described by Maniatis et a l . (1982), with the exception of SI 115 smaples, which were prepared as described on p.86. Estimates of the size of detected transcripts were gained by electrophoresing and blotting a sample of BRL high molecular weight RNA standards on the same gel as the target polyA RNA samples. Following blot transfer, the lane containing the standards was cut from the membrane, then the positions of the bands were identified by hybridization analysis using radiolabelled bacteriophage lambda DNA. The relationship between mobility versus log chain length was fi t t e d by linear regression and used to calculate transcript length. Transcripts which had mobilities outside of the range of the standards (9.4-0.6 kilobases), were estimated by extrapolation of the fi t t e d curve. Miscellaneous Human liver was generously made available by Dr. A. Churg of the U.B.C. Hospital Pathology Department. Human term placenta was generously supplied by Dr. C.P.K. Leung of Vancouver General Hospital. Results Selection of Clones by cDNA Hybridization Analysis The various screening strategies applied to the SI cDNA library are summarized in Figure 12. Due to the short supply of SI polyA RNA, negative selection colony hybridizations were carried out on 50,000 clones using cerebellum cDNA. One hundred membranes at a density of 500 colonies each were 2 . . . . processed (c. 8 cfu/cm membrane). In this i n i t i a l screen, low Figure 12. Screening strategies for the SI cDNA library. cDNA LIBRARY FROM NORMAL SI POLY(A) RNA NEGATIVE COLONY SELECTION: HYBRIDIZATION WITH CEREBELLAR cDNA AT LOW CONCENTRATION "pSI3a Series" Negative Colony Selection: Cerebellum cDNA At High Concentration "pSI3ac Series" Differential Colony Hybridization: cDNA Probes From NNC SI, AD SI, Cerebellum, Corpus Callosum Differential Slot Blot Hybridizations: Plasmid DNAs Hybridized with cDNAs from NNC SI. AD SI, Cerebellum, Corpus Callosum Differential Southern Blot Hybridizations: Plasmid Insert DNAs Hybridized with cDNAs from NNC SI, AD SI, Cerebellum, Corpus Callosum NORTHERN BLOT HYBRIDIZATIONS: RNA SAMPLES OF BRAIN REGIONS, LIVER, AND PLACENTA PROBED WITH LABELLED cDNA INSERTS OF SELECTED CLONES input of probe was used in an attempt to limit false positives, which were generated, in part, by the spread of signal generated from abundantly represented cDNAs. In the analysis of cerebellum negative clones, the small number of background non-recombinants (c. 3 %) served the useful purpose of being a negative control in the hybridization analysis. The non-recombinant clones were readily eliminated by the blue colour generated on X-gal plates. In the f i r s t cerebellum negative screen 3970 clones were selected, representing 8 % of the primary transformants. The efficiency with which the primary cerebellum selection reduced the number of cerebellum positive clones is illustrated by hybridization analysis of slot blotted plasmid DNAs, using both selected and unselected clones, as shown in Figure 13. It is clear from this autoradiogram that the level of strongly cerebellum positive clones has been reduced by the selection regime, as indicated by the much stronger signals generated by most of the unselected clones. It is also clear that the negative selection protocol has not eliminated cerebellum positive clones, but rather has selected against them on the basis of apparent abundance in the cerebellum cDNA probe population. Thus, this single negative selection with cerebellum cDNA appears to have eliminated most cDNA clones abundantly represented in that region. Two approaches were brought to bear for further screening the selected clones. In one approach, direct diff e r e n t i a l 118 ftlltiffeill 0 n Oi i 1111 11 i i 0 0 Vector F i g u r e 13. Cerebellum cDNA probe h y b r i d i z a t i o n a n a l y s i s of SI cDNA c l o n e s . Both n e g a t i v e s e l e c t e d c l o n e s , as w e l l as a number of u n s e l e c t e d c l o n e s (hatched box) were examined. In t h i s experiment 350 ng of plasmid DNAs were immobilized, then h y b r i d i z e d w i t h 10 ng/ml of cDNA probe (1 x 10 cpm/ml). A ne g a t i v e c o n t r o l lane, c o n t a i n i n g p l a s m i d v e c t o r DNA, i s c i r c l e d and l a b e l l e d " v e c t o r " . The oth e r c i r c l e d areas i n d i c a t e c e rebellum cDNA-negative SI recombinants. 119 colony hybridization screening was attempted on 500 clones. The plasmid DNA from the 28 resulting selected clones underwent differential screening on Southern blots, using SI cDNAs from AD and NNC cases, and white matter cDNA. This approach resulted in six clones which were examined on Northern blots. In the other screening approach, another round of cerebellum cDNA screening was carried out on the primary selected clones, at a higher probe input (10 ng/ml), and lower 2 colony density (1.5 cfu/cm of membrane). From this second screen 998 clones, 2 % of the original population, were selected as cerebellum negative. Of these clones, 600 cultures were grown for plasmid DNA extraction. These plasmid DNAs were purified, immobilized on membranes in slot blots, then differentially screened with SI cDNAs from AD and NNC cases, and with cDNAs from white matter and cerebellum. Examples of the differential slot blot screens are shown in Figure 14. Many of the clones which were examined (c. 30%) did not generate a signal above background, (which was evaluated as that generated by the vector DNA), with any of the cDNA probes. Those clones which appeared to have authentic signals above background were evaluated for apparent differential signal between probes. The c r i t e r i a used for selection was differential signal in AD versus control and/or between normal SI versus other tissues. Using this approach, 14 clones were selected for further investigation by Northern blot analysis. 120 Figure 14. D i f f e r e n t i a l cDNA hybridization analysis of selected SI cDNA clones. The r e p l i c a t e arrays of plasmid DNAs were hybridized with r a d i o l a b e l e d cDNAs prepared to RNA from the NNC SI (NSI), AD SI (ADSI) or cerebellum (CBM). Several clones exhibited d i f f e r e n t i a l signals when probed with cDNA, as indicated by the arrowheads. The l e t t e r s r e f e r to clones i d e n t i f i e d i n Table 5 (p. 124); A) pSI3a-24, B) pSI3a-36, C) pSI3a-50, D) pSI3a-60. See text for d e t a i l s . 121 With these combined cDNA screening approaches, no high or moderate abundance genes were detected which were markedly specific to, or enriched in the SI. Similarly, these methods did not detect genes of these abundances which were obviously differentially expressed in the AD versus NNC SI. Recombinants were therefore selected on the basis of exhibiting weakly differential signals between AD and NNC SI or between SI and cerebellum, and not generating a signal with white matter cDNA. In total 20 clones were selected for further analysis on the basis of the cDNA hybridization analyses. Northern Blot Analysis of Selected Clones Insert cDNAs were released from cloning vector, isolated by agarose gel electrophoresis, labelled to high specific activity, and then applied to hybridization analysis of RNA blot membranes. Examples of the autoradiograms resulting from the Northern blot analysis are shown in Figure 15. The characteristics of the transcripts detected by these cDNA inserts, and the corresponding sizes of the inserts are summarized in Table 5. Half of the clones examined (10/20) detected transcripts in the liver, and therefore were not judged to be of interest with regards to tissue specificity. One such clone was pSI3a-197, a clone which, although detected with WM and cerebellum cDNA on differential cDNA screening, appeared to have a stronger signal with AD SI cDNA relative to NNC SI cDNA. Subsequent analysis of Northern blots detected a transcript in 122 Figure 15 (over, p. 123). Northern blot hybridization analysis of selected SI cDNA clones. Four examples of autoradiograms resulting from the analysis of each cDNA insert are presented. Each lane used 2 jug of polyA RNA from the indicated human tissues or brain regions. Liv, liver: Pl, placenta: Cbm, cerebellar cortex: WM, white matter (corpus callosum): SI, substantia innominata: Tex, temporal cortex. Numbers to the l e f t of each autoradiogram refer to the transcript size in kilobases of RNA. Liv PI Cbm SI Tex Liv PI Cbm Tex 1.6 pSI3a-36 Cbm WM SI Liv pSI3a-197 Liv SI WM Cbm 4.3 pSI3ac-18 pSI3a-203 124 Table 5. Summary of the Northern Blot Analyses of cDNA Clones Signal Detection Clone Insert Number S i z e(kbp) Cbm Liv Pl SI Transcript (bases) A. Strategy 1 pSI3a-9 1.9 + - - + 2100 (WM+) pSI3a-24 1.7 ++ ++ ++ nd 1320 pSI3a-30 1.2 + + ++ nd 1900 pSI3a-36 0.8 ++ - + + 4100 pSI3a-50 1.3 ++ + + nd 1600 pSI3a-60 1.8 +/- +/- +/- nd smear pSI3a-64 1.6 - - - +/- smear pSI3a-99 2.1 - - - - no detection pSI3a-156 1.9 - - - nd no detection pSI3a-164 0.6 + + + nd 950, 2150 pSI3a-182 0.8 - - - - no detection pSI3a-190 2.0 +/" + + nd 2250 pSI3a-197 1.1 + + ++ + 1600 (WM+) pSI3a-203 0.9 ++ ++ + + 4300 (WM+) B. Strategy 2 pSI3ac-2 0.8 + - + ++ 1500 (WM+) pSI3ac-7 1.4 ++ + nd ++ 4800 pSI3ac-16 0.6 ++ + + ++ 5 bands pSI3ac-18 1.0 + +/" nd + 3300 (WM+) pSI3ac-22 0.8 ++ ++ nd ++ 1400 (WM+) pSI3ac-24 3 . 0 + - - ++ 15500 - not detectable, +/- weak signal, + clearly detectable, ++ strong signal, nd not determined, Cbm cerebellum, Liv liver, Pl placenta, SI substantia innominata, WM corpus callosum. Transcript size is estimated bases of RNA, determined in comparison to BRL standards. 125 a l l tissues examined (see Figure 15). Comparison of SI RNA samples from 3 AD and 4 NNC cases failed to find a difference in the expression of this gene, so further analysis of this clone was not pursued. The majority, 14 of 20, of the selected clones, which were selected through successive cerebellum negative screening protocols, clearly detected transcripts in cerebellum RNA samples. Two such clones appeared at much higher abundance in cerebellum than in other tissues tested; in fact, one of these, pSI3ac-18, appears to be greatly enriched in the cerebellum (see Figure 15). The remaining clones, 6 of 20, failed to detect discrete RNA transcripts in cerebellum after extended autoradiographic exposure of Northern blots (2-3 weeks at -70°C with 2 intensifying screens). Of these, 5 did not generate any discrete signal with any of the RNA samples (no signal or a smear), and 1 was cerebellum negative but found in the liver and placenta. Restriction enzyme analysis of a l l cDNA clones confirmed that they possessed authentic DNA inserts with identifiable unique fragments. Presumably, these clones represent RNAs of very low abundance, below the limit of detection on these Northern blots. One clone, pSI3a-64, generated a very weak signal with SI polyA RNA, but not with polyA RNA from other tissues examined. This signal was a smear from c. 5000 bases to 1500 bases. This finding was reproduced on a separate blot, with no improvement in intensity or appearance of the signal. No further hybridization analysis 126 was carried out on this clone because attempts to characterize the transcript did not appear promising. One clone examined, pIS3ac-24, had two of the sought after properties on Northern blot analysis; higher apparent abundance in the SI versus the cerebellum, and organ specificity, not being detectable in the liver, placenta or white matter. This clone also had the unusual property of detecting a very large transcript, estimated to be 15.5 kilobases. A Northern blot demonstrating some of these features is shown in Figure 16. The original source of this clone was from the differential colony hybridization analysis, from which i t was selected on the basis of a weakly greater signal in the AD SI cDNA, in comparison to the NNC SI cDNA. Discussion Negative selection with cerebellar cDNA and subsequent differential screening of a relatively large number of SI recombinants resulted in the selection of 20 candidate clones for further analysis. During the differential screening, for both brain regional and disease specificity, no genes of interest in the high or moderately high abundance class were found. From previous studies of others, i t was anticipated that such genes would need to be present on the order of 0.1 % relative abundance in the cDNA probe population in order to be detectable by differential hybridization methods (Dworkin and Dawid, 1980). It appears, on the basis of the differential cDNA screening, that the expression of many high to moderate 127 Figure 16. Northern blot characterization of clone pSIac-24. Samples of polyA RNA (2 jug) from liver (L.), placenta (PL.), cerebellum (CBM.), SI (SI.), and temporal cortex (TCX.) were hybridized with radiolabelled cDNA insert from clone pSIac-24. The transcript detected by this cDNA was estimated to be 15.5 kilobases, by extrapolation of the mobility of BRL high molecular weight standards. 128 abundance genes is not altered in the AD SI in comparison to NNC SI . To the present time, limited work has been directed to the analysis of differential expression of uncharacterized genes in AD and NNC. Recently, May et a l . (1988, 1990) d i f f e r -entially screening a hippocampal cDNA library with cDNAs prepared to AD and control hippocampal RNAs. By screening 50,000 clones, the recombinant pADHC-9 was selected which detected an RNA with a 2-fold increased expression in AD. The detected transcript has subsequently been defined as the human sulfated glycoprotein-2 (SGP-2). In the current work, this gene would have been eliminated as a clone of interest, based on i t s wide CNS distribution and presence in nonneural tissues. Several other clones isolated by Mays et a l . detected an RNA whose expression was increased 2 to 3-fold in AD brain. These clones were later found to repesent cDNA for g l i a l f i b r i l l a r y acidic protein mRNA. In this thesis, cDNA clones corresponding to RNAs of this type, eg. GFAP and other abundant g l i a l mRNAs, would likely be eliminated by the negative selection scheme. The cDNA screening protocol employed did not recover SI specific cDNAs, although several clones with regional preferences were obtained. This suggests a low frequency of brain region specific cDNA clones in the SI cDNA library. Travis et a l . (1987) screened 25,000 monkey cortex cDNA clones for cortex specific genes using a combination of subtractive 129 hybridization (cortex - cerebellum) and differential hybridization schemes. From these clones, 5 clones were identified as being cortex specific, a l l of which were independent isolates of the same gene. These investigators recently described several further monkey cortex positive, cerebellum negative, clones from a larger cDNA library (Travis and Sutcliffe, 1988). These clones were estimated to be present at, or below, 0.001% abundance in polyA RNA. Such cDNAs would be far below the limit of detection by the differential screening methods described in this thesis. The reliable detection of these very rare cDNAs would require screening a much larger number of clones (millions) than that attempted in this work. Direct screening using SI cDNA probes would be very d i f f i c u l t , due to the limiting amount of mRNA. Subtractive hybridization methods could be suggested as an alternative method to pre-enrich cDNA probes for large-scale screening of unselected libraries for AD brain regional genes of interest. A similar approach has been used by Duguid et a l . (1988) to investigate gene expression in scrapie infected rodent brain. In fact, one gene of interest in AD has been identified by these investigators (Duguid et a l . , 1989), which has been identified to be identical to the human SGP-2 cDNA identified by Mays et a l . (1990). In the current instance, direct use of subtracted cDNA hybridization probes for screening the SI cDNA library would not seem feasible for the recovery of low abundance clones from the SI library, on account of the small amount of available RNA for subtraction. At present, i t appears that screening brain regional cDNA libraries for completely region specific clones, and the use of this approach to identify genes relevant to AD research remains a technical challenge. The described screening protocol resulted in the recovery of several clones of interest. One such clone, pSI3ac-24, detected a transcript which exhibited several properties of interest, including, differential brain regional expression, organ specificity, and remarkable size. The further characterization of this gene was therefore undertaken, as described in the next section of this thesis. 131 I I I D . INVESTIGATION OF A BRAIN CDNA CLONE FOR A LARGE mRNA The preceding section of this thesis described the process of selection leading to the human cDNA clone pSIac-24. Northern blot analysis of human RNAs, using pSIac-24 cDNA insert as probe, demonstrated that the corresponding mRNA is expressed in brain, but not in liver or placenta. Differential cDNA hybridization analysis suggested that the mRNA was present in higher abundance in the AD SI than in the normal SI, but the r e l i a b i l i t y of the measurement is questionable. A further interesting feature of this mRNA is i t s remarkable size, estimated to be 15.5 kilobases. Because of i t s large size, and for lack of a more informative name, this mRNA w i l l be referred to as immensin. To characterize immensin mRNA further, and to learn more about i t s potential relevance to the study of AD, the expression of the gene was investigated by hybridization methods. The regional distribution of the expression of the mRNA was investigated in human brain, as well as in rat brain and other tissues. Due to the large size of the mRNA, and the resulting d i f f i c u l t i e s in interpreting Northern blot analyses, a nuclease protection assay was developed to allow estimation of the pSIac-24 mRNA levels in total cellular RNA samples. The 132 abundance of the mRNA in several brain regions was determined. The levels of this transcript in SI and temporal cortex samples from AD and NNC cases were compared. The structure of immensin mRNA was investigated by analysis of the cloned cDNA. Southern blot hybridization analysis was conducted to examine the representation of the cDNA in human genomic DNA. The sequence of the 3 kbp cDNA insert was determined and analyzed by computer searches for structural motifs and for sequence similarities to previously described genes. Materials and Methods Preparation of DNA and RNA. RNA was extracted from frozen human and rat tissues as described in section IIIA. Plasmid DNA was prepared from alkaline lysates using the PEG precipitation method, and cDNA insert, or i t s fragments, were isolated by preparative gel electrophoresis, as described in section IIIA. Human genomic DNA was obtained from heparinized blood obtained from a healthy volunteer. White cells were prepared by lysis of red blood cells using the method of Herrmann and Frischauf (1987). The ce l l s from 25 ml of blood were suspended in 25 ml of TEN solution (0.05 M Tris-HCl/0.1 M NaCl/0.001 M EDTA, pH= 7.5), to which was added 2.5 mg of predigested proteinase K (IBI) and SDS to a fi n a l concentration of 0.5 % (wt/vol). The cells were digested for 4 hours at 55°C, a further 1.25 mg of proteinase was added, and the solution was digested for a 133 further 4 hours. The resulting solution was extracted 2 times at room temperature with 25 ml of phenol/chloroform solution, and the aqueous phase recovered. DNA was obtained by addition of sodium acetate to 0.2 M and ethanol to 65 %, followed by spooling the DNA onto a glass rod. While s t i l l on the rod, DNA was rinsed by repeated immersion in 70 % ethanol and allowed to dry partially. The DNA was dissolved in 10 ml of 0.01 M Tris-HCl/ 0.001 M EDTA (pH 8.0; TE 8.0), digested with 0.5 mg of heat-treated RNAase A for 2 hours at 37°C, digested with proteinase K, extracted with phenol/chloroform, and spooled onto a glass rod, as before. The f i n a l material was partially dissolved in 1.5 ml of TE 8.0, and completely resolubilized by dialysis against this buffer for 2 days at 4°C. Spectrophotometry established an absorbance ratio (260/280 nm) of 1.79. Southern Blot Hybridization Samples of DNA (10 pq) were digested for 2 hours at 37°C with 50-75 units of restriction enzyme using buffers supplied by the manufacturer (BRL or Pharmacia). Enzymatic digestion was terminated by addition of 1/5 volume of electrophoresis loading solution (20 % Ficoll/0.3 % bromophenol blue/0.3 % xylene cyanole/0.2 M EDTA), followed by brief heating and c h i l l i n g on ice. Just before electrophoresis, samples were diluted 1:1 with TAE electrophoresis buffer (section IIIA), and loaded into the wells of a 15 x 20 cm 0.7 % agarose gel. Electrophoresis proceeded at 25 volts for 16 hours. The gel 134 was stained with ethidium bromide, photographed, and washed repeatedly with water. The gel was treated with 0.25 M HCl for 10 min., follwed by two washings with 0.5 M NaOH/1 M NaCl for 15 minutes each. Transfer of DNA onto Gene Screen Plus (NEN) was effected by vacuum transfer using 10 x SSC for 4 hours. The membrane was rinsed twice with 1.0 M Tris-HCl/1.5 M NaCl, dried at room temperature, and baked at 80°C under vacuum for 2 hours. The membranes were prehybridized and hybridized using the conditions described in section IIIA, with 50 % formamide at 42°C and 5 ng/ml probe for 18 hours. The probe was a randomly 32 9 primed, P-labelled (1 x 10 cpm//xg) whole cDNA insert of pSI3ac-24. The hybridized membrane was washed as described previously to a f i n a l stringency of 0.1 x SSC/1 % SDS at 65°C for 1 hour. Autoradiographic exposure of the membrane was carried out at -75°C with an intensifying screen. Northern Blot Hybridizations Methods for electrophoresis in formaldehyde gels and blot transfer to membranes were as described in section IIIA. Several variations on the vacuum blot transfer method were examined to improve the transfer of the high molecular weight mRNA detected by the cloned cDNA. None of these variations noticeably improved detection of the mRNA. Northern blot analysis of rat total cellular RNA was conducted using either the labelled 1 or 2 kbp Eco RI released fragments, and in one case both fragments. Both prehybridization and hybridization 135 were carried out at 37°C in 50 % formamide, followed by washing the membranes to increasing stringency, with intervening autoradiographic exposure of the membranes. This procedure was necessary because of the apparent poor homology of the probe for the rat mRNA. For human Northern blot analyses, membranes were stripped and rehybridized with /3-actin probe in order to assure RNA transfer. Samples of human RNA were also examined by Northern blot analysis using cRNA probes (probes are described below). In this case, blots were prehybridized and hybridized at 55°C using the standard 50 % formamide hybridization conditions, and 5 ng/ml of cRNA probe. Post-hybridizations washes of the membrane were carried out sequentially in 0.1 x SSC/1 % SDS at 55°C (three times for 30 minutes each), 2 x SSC/ 1% SDS (two times at 55°C for 15 minutes each). The membranes were washed in 0.4 M NaCl/25 mM Tris-HCl/1 mM EDTA (pH =7.5) at room temperature for 20 minutes, and then digested in this buffer with RNAase A (10 /zg/ml) and BSA (100 /xg/ml) for 20 minutes at 37°C. The RNAase digestion was terminated by the addition of 1 x SSC/1 % SDS, and heating to 37°C for 10 minutes; the membrane was washed for a further 3 0 minutes in 2 x SSC/1 % SDS at 22°C. Autoradiographic exposure of the blot took place at -75°C with an intensifying screen. Ribonuclease Protection Assay Levels of immensin mRNA were determined by solution hybridization using a single strand cRNA probe, followed by 136 digestion of excess probe by single strand specific RNAases (RNAases A and TI), based on methods described by Lee and Costlow (1987). The single strand cRNA probes were synthesized by either T7 (antisense) or T3 (sense) RNA polymerases using a pT7T3-18U construct containing an 585 bp Pst I/Eco RI fragment excised from the 1 kbp Eco RI fragment of pSI3ac-24. The probes were synthesized with a specific activity of about 2-4 x 108 cpm//xg RNA using [a-32P]UTP at 25 MM f i n a l concentration (20 JUM unlabelled UTP) . Before use, cRNA probes were isolated by DNAase I digestion, phenol/chloroform extraction, and twice precipitating from 2.5 M ammonium acetate/70 % ethanol. The integrity of the cRNA probe was checked by denaturing gel electrophoresis through 6 % polyacrylamide/7 M urea gels (see below) . Samples of total cellular RNA (2-40 /xg) were mixed with purified yeast tRNA, to yield a total RNA mass of 50 /xg. The appropriate mass of the cRNA probe, usually 0.25ng, was added to each sample, and the RNA was precipitated by addition of ammonium acetate and ethanol. The resulting pellet was dried under vacuum, and then dissolved in 10 /xl of purified formamide and 6 /xl of water. Following complete dissolution of the sample, 4/il of 5-fold concentrated hybridization buffer was added (2 M NaCl/0.125 M piperazinediethanesulfonic acid/0.005 M EDTA, pH = 6.8). The samples were overlaid with light mineral o i l (Fisher), briefly centrifuged, capped and heat denatured at 85°C for 10 minutes. Hybridization was carried out in a water bath at 55°C for 20-24 hours. The 137 calculated Tm for the probe sequence under the conditions employed as an RNA-DNA sequence is about 70-75°C (52.8 % G+C, 50 % formamide, 0.4 M Na+; Calzone, et a l . , 1987). The samples were transferred to an ice bath, and excess (unprotected) probe digested by adding to each sample 3 00 nl of 0.4 M NaCl/0.075 M Tris-HCl/5 mM EDTA (pH = 8.0) containing 20 jug of RNAase A (Pharmacia) and 450 units of RNAase Tl (BRL). The samples were mixed, centrifuged, digested at 37°C for 1 hour, and then placed on ice. To quantify the RNAase protected probe, 300 ng of yeast tRNA was added, and the samples were precipitated by addition of 400 / i l of 10 % TCA/0.002 M sodium pyrophosphate, followed by incubation on ice for 15 minutes. TCA precipitable material was collected on Whatman GF-C f i l t e r s in a Millipore 12 well sample manifold. Radioisotope 32 . counting was carried out in the P channel of a Ph i l l i p s liquid s c i n t i l l a t i o n spectrometer using Packard PicoFluor s c i n t i l l a n t solution. Samples for gel analysis were processed by adding SDS to 0.5 % and proteinase K to 0.25 mg/ml, followed by digested at 37°C for 1 hour. To each tube was added 20 ng of tRNA, the sample was extracted with phenol/chloroform, and RNA was precipitated overnight after addition of ethanol. Following centrifugation, the RNA pellet was washed with 70 % ethanol, dried, and then brought into solution using 12 jul of gel sample solution (80 % formamide/20 mM EDTA (pH = 8.0)/0.05 % bromophemol blue/0.05 % xylene cyanol). Samples were heat denatured at 90°C before 138 electrophoresis. Electrophoretic separations were carried out on 16 x 16 cm, 0.75 mm thick 6 % polyacrylamide/7 M urea gels using the BioRad Protean II apparatus, which was maintained at 50°C with a circulating water bath. These gels were prepared and run in 0.5 x TBE at 750 volts for about 3 hours (Maniatis et a l . , 1982). They were fixed in 10 % methanol/10 % acetic acid, dried under vacuum and subjected to autoradiography. Appropriate amounts of each ribonuclease for the digestion of excess probe were established by following the degree of digestion of probe and carrier, in the absence of human RNA. The minimum amount of enzyme resulting in a plateau of digestion was used. The degree of digestion was about 95 % for RNAase A, and about 85-90% for RNAase TI. The combined action of these enzymes removed greater than 99 % of the input probe, generating a background signal of about 1000-2000 counts per minute (CPM). This background value was determined in duplicate for each sample set and subtracted for calculation of the RNAase resistant transcript recovery. Titration of a serial diluted sample set resulted in a regression line which extrapolated at zero target input to a value within 5 % of the measured background value. Sample determinations were routinely carried out in duplicate. The values reported were corrected to the mass of 15.5 kilobase mRNA, based on a value of 585 nts of cRNA for the probe. This calculation assumed the ratio of UMP to total nucleoside content was 0.25. 139 Sequence Determination and Analysis DNA sequence was obtained using pseudo-virus packaged single strand plasmid DNA template obtained by superinfection of recombinant bacteria with M13K07 helper phage. The desired insert in pT7T3 18U or 19U was propagated in the male E. c o l i strain DH5aF* (BRL). An overnight culture of the bacteria was diluted 1/100 in 2 x YT medium (YT medium is 0.8 % Bacto-tryptone/0.5 % yeast extract/0.5 % NaCl), and then grown to an absorbance of 0.4-0.5 at 600 nm. Helper phage M13K07 (Viera and Messing, 1987) was added to a multiplicity of infection of 10-20, and the mixture incubated at 37°C in a gyrotary bath for 1 hour at 350 rpm. A sample of 400 /xl of the culture was removed to 9.6 ml of 2 x YT medium, and kanamycin was added to 70 /ig/ml. Virus was propagated by incubation at 37°C for 14-16 hours. Virus particles were obtained from cleared culture supernatant using PEG precipitation from a solution made 3.5 M in ammonium acetate. Template DNA was obtained from virus particles as follows: to the pellet of virus 100 / i l of 1 % SDS was added, and the solution heated at 37°C for 10 minutes; the solution was made 2.5 M in ammonium acetate, placed on ice for 10 minutes, and then centrifuged for 5 minutes to remove insoluble material; DNA was recovered by centrifugation after addition of 2 volumes of ethanol. The template DNA was brought to f i n a l purity by extraction and recovery on NEN-Sorb cartridges, as previously described (section IIIA). The yield of template DNA from 10 ml of culture was about 20 /ig. 140 Sequencing reactions were carried out by dideoxy-nucleotide chain termination, using either the Klenow fragment of DNA Polymerase I (Sanger et a l . , 1983; BRL) or T7 DNA polymerase (Tabor and Richardson, 1987; Pharmacia). Both reactions employed a preliminary chain extension followed by the chain termination reactions, and used the reaction mixes and protocols supplied in kits obtained from the enzyme supplier. A l l reactions were carried out using thio-[a-35S]dATP (1000 Ci/mmol, NEN/DuPont). In a small number of cases, additional reactions were carried out with T7 DNA polymerase using deaza-dideoxyGTP (Pharmacia). Gel analyses of the sequencing reactions were carried out on the BRL S2 apparatus using 6 % polyacrylamide/8M urea gels with the Tris/borate/EDTA buffer system (pH = 8.3, BRL sequencing manual) at 50 W constant power. Following electrophoresis, gels were fixed with 10 % methanol/10 % acetic acid, and then dried under vacuum. Autoradiographic exposure of the gels was at room temperature with Kodax X-RP film. Sequencing gels were analyzed using a Biorad sonic digitizer connected to a personal computer. Sequence data from contiguous gels was compiled using the ASSEMGEL program supplied in the PCGENE programs from Intellegenetics. Directed deletion subclones of pSI3ac-24 and the Eco RI fragment subclones of pSI3ac-24 were obtained by use of the Exonuclease III/S1 nuclease methods described by Henikoff (1984). These subclones were sequenced using the M13 universal 141 primer, obtained from Pharmacia. Additional sequencing reactions were carried out with specific oligodeoxynucleotide sequencing primers designed to be complementary to available sequence data, according to rules described by Barnes (1987). Vector homology with primers was examined by probing M13mpl2 and pUC18 sequences using the FASTA program in PCGENE. Sequence homology searches of databases were carried out on both cDNA strands (and fragments thereof) using the FASTA program (Pearson and Lipman, 1988), with a word length of 5. The searches were mounted either on a Sun workstation (through Bionet, Intellegenetics Inc.) or a VAX 6600 computer (DuPont Experimental Station, Wilmington, DE). The nucleic acid data bases searches included the composite EMBL and Genbank databases (GENEMBL), as well as the NBRF nucleic acid data base. A l l other searches and sequence analysis methods were carried out using the programs in PCGENE. Miscellaneous Sequencing primers were synthesized by Dr. T. Atkinson at the Dept. of Biochemistry at U.B.C, and were purified on SepPak cartridges before use (section IIIB). cDNA probes were radiolabelled and purified as previously described (section IIIA). Results Basic Features of the Clone and i t s Complement in Genomic DNA. A partial restriction map of clone pSI3ac-24 i s presented in Figure 17. The cDNA insert was released from the clone on 142 treatment with Eco RI endonuclease, while generating fragments of 1 and 2 kbp. Thus the insert was 3 kbp long with a single internal Eco RI site. Southern blot analysis of human genomic DNA suggested that the immensin cDNA was detecting a single copy gene, as reflected by the small number of discrete fragments detected in genomic DNA digested with a number of restriction enzymes (Figure 18). The restriction fragments identified by Southern blot analysis could be related to the restriction fragment map of the cDNA insert. Cutting the cDNA with Hind III generated an internal fragment of about 450 bp which was also identified on the Southern blot. Similarly, the other enzymes, which a l l had 1 or two fragments on Southern blot analysis, were "~ observed to have the appropriate sites in the cDNA. This finding suggests that there is not a large intervening sequence present in the genomic DNA which is absent in the cloned cDNA for that region of the gene represented by the cDNA. Brain Regional and Tissue Expression of Immensin mRNA. Northern blot analysis of human RNA, using the cDNA insert of pSI3ac-24 as probe, detected a high molecular weight polyA RNA which has been named immensin. Calibration of agarose gels using the highest available molecular weight marker set resulted in an estimate of 15.5 kilobases for the size of this RNA. Determination of the chain length of this transcript, using denatured DNA standards, yielded a value of 143 0.5 kbp I 1 Sph I Hind III Hind ' •;,""jr;— ^4^"~r™s-^W. ,,, „ MHIii.. , . „ , _ Pst I Xba I Eco Rl Pst I Eco Rl •2.0 kbp 1 1.0 kbp 1 Figure 17. A partial restriction map of clone pSI3ac-24a. The relative position of restriction sites in the cDNA insert are presented as determined electrophoretically. Figure 18 (next page, 144). Southern blot hybridization analysis of human genomic DNA. The position of molecular weight standards is shown on the right of the autoradiogram in kbp of DNA. The membrane was hybridized with the whole cDNA insert. 144 Eco Hind Bam Pst Xho Sst ^ 23.1 4 9.4 4 6.6 4 4.4 kbp DNA 4 2.3 4 2.0 4 0.6 145 14.8 kilobases. As was presented in Figure 16 (section IIIC, p. 122), this transcript was not detected in human liver or placenta RNA samples, but was detected in several human brain regions. The widespread distribution of the immensin mRNA in human brain regions was demonstrated by Northern blot analysis, as shown in Figure 19. From this blot, i t appears that the highest levels of the transcript are present in hippocampus, caudate nucleus, and SI, whereas lower levels appear in the cerebellum. Comparison between brain regions of the signal generated by probing the same membrane with a B-actin probe suggests that the highest relative levels of immensin mRNA may be found in the hippocampus. However, the relatively high va r i a b i l i t y in sample detection by Northern blot analysis did not allow precise determination of the relative amounts of the transcript in the brain regions examined, particularly when samples from a series of brains were compared. In order to examine the gross cellular localization of the transcript, Northern blot analysis was applied to RNA prepared from human corpus callosum, a white matter rich region. Figure 20 shows the hybridization pattern obtained on on Northern blot of a white matter rich RNA sample. Although the corpus callosum lane had a weak and diffuse low molecular weight smear, the high molecular weight immensin transcript was not detected. It should be noted that this RNA sample did not show any indistinct banding when the membrane was probed 146 Temp Occ Hpc CP Cbm I S I 15.5 kb> mmm m 1.8 k b » F i g u r e 19. Northern b l o t h y b r i d i z a t i o n of human b r a i n r e g i o n RNA samples. Samples of RNA (20 /xg) were r e s o l v e d on a 0.7 % agarose g e l , t r a n s f e r r e d t o the membrane and probed w i t h the r a d i o l a b e l l e d 3 kbp cDNA i n s e r t (upper p a n e l ) . F o l l o w i n g a u t o r a d i o g r a p h i c exposure of the membrane, i t was s t r i p p e d of probe and r e a n a l y z e d with a /3-actin cDNA probe (lower panel) . 147 CBM. WM F i g u r e 20. Northern b l o t h y b r i d i z a t i o n a n a l y s i s of human b r a i n r e g i o n a l RNA samples. Samples of polyA RNA (2 /xg) from corpus cal l o s u m (lane WM), cerebellum (CBM) and SI were on the membrane. Note the absence of a hig h m o l e c u l a r weight t r a n s c r i p t i n the corpus callosum RNA sample. The i n t e g r i t y of the RNA was proven by r e p r o b i n g the membrane w i t h / 3-actin and j8-APP cDNA probes. 148 with other cDNAs, indicating that the signal on this blot did not result from degradation of high molcular transcript. Similar tissue distribution patterns were obtained on Northern blots using either the whole pSI3ac-24 cDNA insert, or the 1 and 2 kbp fragments of the insert. Northern blot hybridizations were carried out with both polyA RNA as well as total cellular RNAs. Detection of the transcript in polyA RNA samples was only modestly increased (c. 3-4 fold) relative to the signal generated by a similar amount of total cellular RNA, possibly because of preferential loss of high molecular weight polyA RNAs on oligo(dT)-cellulose. Reliable detection of the transcript in either total cellular RNA or polyA RNArequired very good quality RNA and well controlled blot transfer conditions, both of which reflect the d i f f i c u l t i e s encountered with a very high molecular weight transcript. Detection of an Immensin mRNA in Rat A variety of hybridization conditions, including the use of cDNA insert fragment probes, were applied to Northern blot analysis of rat brain regional and organ RNA samples. The best success was obtained using low stringency hybridization (37°C in 50 % formamide) and washing conditions (42°C, 0.5 x SSC/ 1% SDS), and a probe prepared from the 1 kbp Eco Rl fragment. As shown in Figure 21, a high molecular weight rat RNA showed weak hybridization to this probe, against a considerable background. The intense band on the blot comigrated with rat 149 F i g u r e 21. Northern b l o t h y b r i d i z a t i o n of r a t t i s s u e RNA. Rat RNA samples (20 jug) were h y b r i d i z e d w i t h the s m a l l Eco R l fragment of c l o n e pSI3ac-24. The 15.5 kb t r a n s c r i p t was de t e c t e d i n cerebellum (CBM), c e r e b r a l c o r t e x (CTX), caudate-putamen (CP) and ad r e n a l gland (ADR) samples. A b b r e v i a t i o n s : SC, s p i n a l c o r d ; HRT, h e a r t ; SKM, s k e l e t a l muscle; KID, kidney; SPL, s p l e e n ; LIV, l i v e r . The i n t e n s e lower mo l e c u l a r weight (smeary) s i g n a l generated i n the ADR lane was found t o be an a r t e f a c t on the membrane, as i t was not p r e s e n t when the experiment was r e r u n . 150 28S rRNA. Reducing the stringency of the post-hybridization washes increased the signal from the putative rat 28S and 18S rRNAs. Blots of human total cellular RNA samples that had been washed at low stringency, similarly exhibited a 28S band. Since this effect was reduced in human polyA RNA, this low stringency hybridization signal was ascribed to detection of human 28S rRNA. It is likely that a similar cross-hybridization occured with rat 28S rRNA. The band identified as a possible rat immensin mRNA was observed in several brain regions and in the adrenal gland, but was not observed in the other non-neural tissues examined. The poor signal detection precluded quantification of the relative abundance in various rat brain regions. Measurement of Immensin mRNA in Control and AD Brain Regions A ribonuclease protection assay was applied to the measurement of the transcript levels in human brain. cRNA probes were synthesized by bacteriophage RNA polymerase synthesis initiated at the promoters on pT7T3-18U. Northern blot hybridization of human cortex and cerebellum RNA was conducted using both sense and antisense cRNA probes. Only the antisense cRNA probe generated a hybridization signal. Recovery of labelled antisense cRNA probe (RNAase resistant TCA precipitable counts) increased linearly with addition of unlabelled in vitro synthesized "sense" cRNA (Figure 22a), or with human cerebellar total cellular RNA (Figure 22b). Denaturing polyacrylamide gel electrophoretic analysis 151 detected a RNAase resistant (protected) RNA fragment in human cerebellar RNA which had a higher mobility than the input probe cRNA, as shown in Figure 23. A similar result was obtained for a temporal cortex and SI RNA samples. Since the probe sequence contains 16 bases of vector derived sequence, the higher relative mobility of the hybrid-protected band was expected. On this gel, i t was also noted that a small amount of undigested "background" probe was present in the samples. The amount of this material is similar between lanes, and was also present in samples which did not contain human RNA. It is assumed that this material represents the background "undigested" TCA-precipitable radioactivtity. The level of background radioactivty was the same in various negative control samples, including rat RNA, liver RNA, or carrier RNA (yeast tRNA). Close examination of the autoradiogram also showed that a number of lower molecular weight, possibly protected RNA fragments, were present. Possible sources for these species include truncated cRNA probe species (which were variably present in cRNA probe preparations), fragments generated from partially degraded human RNA samples, non-specific enzymatic fragmentation of hybrids, hybridization from closely related human mRNAs, or a combination of these various sources. Taken together, these bands appear to represent 5-15 % of the radioactivity present in the sample. The solution hybridization employed probe in greater than 10-fold molar excess, at a concentration sufficient to ensure 152 Nuclease Protection Assay for the 1 5.5 kb Transcript Sense cRNA Standard 10 15 , 20 Mass of cRNA (pg) 30 Nuclease Protection Assay for the 15.5 kb Transcript Human Cerebellum RNA 10000 9000 <5 8000 15 20 25 30 Cerebellum RNA i/ig) 45 Figure 22. In the upper panel 0.5 ng of probe was used for the tit r a t i o n of T3 RNA polymerase synthesized unlabelled sense cRNA. The results are presented uncorrected for background. The predicted cRNA levels were within 1 % of the input cRNA. The lower panel shows a tit r a t i o n of human cerebellar RNA using the nuclease protection assay (0.25 ng probe/sample). The results are presented corrected for background (no human RNA), determined to be 1602 CPM. 153 Figure 23 (over, p. 154). Denaturing polyacrylamide gel analysis of the RNAase resistant products of the nuclease protection assay. Following hybridization and RNAase digestion, samples were ethanol precipitated and subjected to electrophoresis on a 6 % polyacrylamide/7 M urea gel. The mobilities of the probe and protected RNAs were confirmed on a separate gel. In this experiment, the input probe was 0.25 ng in the 2 0 pl hybridization volume. Legends: Human, human cerebellum RNA; Rat, rat cerebellum RNA. 154 155 complete hybridization of target within 12-16 hours (greater than 98%; Calzone et a l . , 1987). Since the specific activity of the cRNA probe was known with reasonable accuracy, (based on tracer radiolabel incorporation), i t was possible to estimate the copy number of target entering hybrid. Thus, the levels of immensin mRNA (as a 15.5 kilobase transcript) were estimated for two cerebellum samples to be 7.9 and 13.4 pg/pg total RNA. Based on an estimate that 2 % of total cellular RNA is polyA RNA, the 15.5 kilobase message represents about 0.05 % mass of polyA RNA in cerebellum. The levels of immensin transcript were measured in temporal cortex and SI samples obtained from several AD cases and NNC individuals (Figure 24). In the temporal cortex the levels of the transcript were increased 29 % in AD relative to controls (p<0.05; 2-tailed t-test). The transcript levels in the AD SI were slightly increased (11 %) relative to controls, but not significantly different. In the normal aged human brain, the levels of transcript were higher in SI (40.0 pg/ng) than temporal cortex (28.6 pg/pg) or cerebellum (11.2 pg/M<?) • Sequence Analysis of pSI3ac-24 The sequencing strategy employed for the clone i s summarized in Figure 25. Each of the Eco Rl fragments was subcloned in both orientations relative to the M13 universal sequencing primer sites of pT7T3-18U. Subclones were generated for the small Eco Rl fragment at unique Pst I and Hind III sites. Further subclones were generated in both directions 156 Immensin Levels in AD and NNC Brain SJ. (n=6) Temp. Cortex (n=5) Figure 24. Determination of the levels of the 15.5 kb transcript in SI and temporal cortex RNA samples from AD and NNC cases. Determinations were carried out with duplicate 10 /zg RNA samples using the numbers of cases indicated. These data are presented as means with standard error of the means, n represents the number per group. In the temporal cortex the levels of transcript in AD were significantly increased compared to NNC (p<0.05, 2-tailed t-test, 8 d.f.), while in the SI there was no significant difference between groups. The group means were not significantly different in age at death or postmortem interval, but were in the levels of neocortical ChAT. The c r i t e r i a for diagnosis of AD were met in a l l members of that group (section IIIA). along the large Eco RI fragment, and in one direction in the small Eco RI fragment, using Exonuclease III/S1 nuclease deletions. Complete sequencing of both strands of the cDNA required a series of synthetic oligonucleotide primers, which are identified in the figure at the site of use. Sequence determinations were carried out by dideoxynucleotide termination using the Klenow fragment of DNA polymerase I, which was replaced in favour of the T7 DNA polymerase. The complete 2963 bp of the cDNA is presented as Figure 26 on pages 159-160. The 3' end of the corresponding mRNA was identified by a stretch of 20 bases of polyA, which were preceded by an 8 base dT tract. An atypical polyadenylation site, -AAUAA-, was located 60 bases 5' from the f i r s t adenylate base; no AAAUAA polyadenylation signal was found in the sequence. The f i r s t strand reverse transcriptase primer and the Eco RI adaptor sequences were also identified. A search of the sequence for direct and inverted repeats identified a purine rich sequence which was directly repeated; 1414-AAGGGAAGGGAAGGGAA-1430, 2195-AGGAAGGGAAGGGGAGA-2211. A similarly purine rich sequence was also noted; 2779-GGAAAGGAAGGTGAAA-2794. No other highly repeated sequences were identified. The sequence was examined for the presence of protein coding regions. From the 5' end of the reverse-transcribed mRNA, open reading frames (ORFs) of 108 bp (frame 3) and 324 bp (frame 2) were identified. Both putative ORFs were 158 200 bp OlB EL2 2d1 E oic 1d13 1d16 " olG 1d28 1 d 2 1 -1d7 EL1 H6 D6 ES1 E H P E I I 2d5 OIH 2d9 olD-olE Sd22 d25 2d11 , FRg. 2d16 sd5 olF olA sd8 ' sd13 2016 bp 1 947 bp I 1 Figure 25. DNA sequence strategy for clone pSI3ac-24. The Eco Rl fragments were subcloned on both orientations relative to the universal primer (1 kbp fragment, ESI and ES2; 2 kbp fragment ELI and EL2). Additional clones were generated by forced cloning Eco RI/Hind III and Eco Rl/Pst I fragments of ESI, and by deletions of ES2 (sd series), ELI (Id series), and EL2 (2d series). Sequences obtained using specific primers are labelled olA through olH. 1 5 9 F i g u r e 26. DNA sequence of c l o n e p S I 3 a c - 2 4 . The p u t a t i v e p o l y a d e n y l a t i o n s i g n a l and the p u r i n e r i c h r e p e a t sequences a r e u n d e r l i n e d . 10 20 30 40 50 i i i i i 1 6GACAAT66T CAGTTCAGAG AGGGTGAGGG CAGCAAACGC TTCAGAGGAC 51 ACAGAAGCCA GAGGACCCCC CCCCGCCCCA CAGCTGGGTC AGCCTGGAAA 101 ATCCATCTAT TAGGGACTTT TTGGCAGCCA GATGGCAGCA ATAGCCCATT 151 AGGTCTCATC CCGAGTTCCA AGTCTTGGCT GCAAATGAGC CTCAGTTCGC 2 01 CTTACTGGAG AGCACCCCAG ATTCCTGGGC ACAGTTCATT TCCAGCCCTT 251 TCTAGATCTG ATCTTTTAGG GGGAAAGACA GCTTAAAATG TTCTTTTCAT 3 01 TTTAAAGAAA ATTATTCTGT CTGCTTAAGT TGGAGGCTAC TTACTCTTTC 351 ACCTGCATTT TCTTTCCTTT TATTCTTCCA GATCAGGAAT GAAATTTCCA 4 01 TGCTGCTCAT AAAGATAATA TTATTGTACT AATTATTTTT ATTACCATTG 451 TAATTATGAT CATTATGTTG ATATTTTAGT CAGGGTTTTA AATGCACATT 501 TATTCCAAGT ATCTTTGTGT TTTCTCTTTA ATATTTAAAC TTATTCTCTC 551 TGTGAGTATA TAAGTAGACT GGAGGGACAT CCAGATGTCC AGTTTTGTCA 601 GGCAAAAAAA AAAGGAAAGA CTTAGGAAGT AGGAAAATTG TTTCTGTCAT 651 CTCTATCCCA ACAAGAGACG TCAAGAAAGA TCCACCACAG AACAAAAGTT 701 TAAAGAAGAA TCAAAGCCTT GATTGGGCTT CTGACAACAT GGTCACCATC 751 AAGGTTGTCA TTTTCTAGAT CCAGAGGCCT GGGATGCGAC GTCAGGTGGC 801 ATCTCATGGG CTCGGGGAAT GTCGAGTCAC TGACTGTCCA GCCCTTAGCC 851 AGCTTCTCTC CCACATCCTC AGAGCTCTCC TGTGCTTCTG AAATCTGTTA 901 ACTAAATCTT TGGCTTGCCT CTGGTATTTA AGCAAGAAAA ATTCCCTCCC 951 GAGGTGACCC CATCCGCTTC CCCACAATCC ATCCTTTTGC CATCGGGCAC 1001 CTGGGGCGTG GCTTAGGTTC TTCAATGCAG GGACATTTGC CCCCTCCCAG 1051 AAAGCTGCTG GGCACAGTGA GGTGGCGTAA GAGTGACTGG CAGGTGGTAC 1101 CTTCCCCAGG AAATTTCACC ACACCACCCA GTTCCTCAGC CTGCCCCCTC 1151 CCCCTGTGAT GCATGCCCCC AGCACCCAAT TCTAGCCAGC TGGAAGTGGG 12 01 TGGAGGGACA GCAGGAGGCC AGAGAAACCC TGAACAAAGC TGGGCGGCTG 1251 CTCAGGCATC ACAGGCTGCA CCCCCTCTGA AAGCATCCCC ACTGGGCTCC 1301 GGCCACATCT TCAGTGCACT GTGCTGTGTG CGCTGGGTGC TCACACGCTG 1351 TCCCCAGACC CACAAAGTGC TAGGCCCCAG TTGAAGAAAG GGGTGAAATA 1401 GCCAGCTTCA CCGAAGGGAA GGGAAGGGAA GTATTGGGCG ATGCCAGCCC 1451 CACAGACGCT CAGCAAACAT TAGTGCACAT TCTCCTAGTC CTCACCCAAT 1501 GGCCTCCTCT ACCCCCATGC ATGGAGCTGC CACATCAGAA GCCCCAAGAG 1551 AAGCTCCCTG CAGGAGAGGC CAGCTCCCTG GATGCCCAAT TGCATACCTG 1601 GCCGAATCTG CCATTGAGTC ACCTTAGCAA ATAGGCTGCT GTCACTAGGA 1651 CCAAGCTCTG AAGCACAGGG ATGCCAACCT AGTCCTTACT TAGCCCACGA 1701 ATCATCTAGA GCATTCTCTA GTCTTTTGTG GGCTCCTCTT CCATTTGAAG 1751 AGACATTGTT CAGAGGAAGA GGGGAAGATT TGAATGTCAG GCTCAGGAGG 1801 AGTGTTTCAA TGGAGCCTGG TGAACCGCAG GCAATTTGCT TCTGCTCACT 1851 GGGTTCTGAC TGGCCCGTCT GGACGTGGGC CCCCATGTCT CTGTGCTTAG 1901 GGCCTCTTCA TGATGTTTTG GATGTTTCCA AGGGAAGTGG GTGAGCAGAT 1951 CAAGGGGTGG GAGAGTCGAG GCTTGATGCC AGTTAATACT GTGAAGTGGA 2001 GCGTGCGGTC AGTGGGAATT CAGAGGAAAA AGAAGGGTTG GAGCAAAGCG 2051 GCATTCATCT CCTGGACTGT TAGCCTTTCT AGTCTTCCTG GTGGCTGAGG 2101 TGTTTACGGG CTGGGGGAGC CAGCTGACCT TTGTCCTCTT CAACCTAGAA continued next page... 160 F i g u r e 2 6 c o n t i n u e d . . . 2151 GACTCAGCCC 2201 GGAAGGGGAG 2251 TTGGGAATGG 2301 GGCCGTGTGA 2351 GAGGGAGGAG 2401 GAGAGAGCGA 2451 CTTTGTCTGA 2501 GGCCTGTCAA 2551 GGTGCTCTCA 2 601 CAGGCCAGCC 2 651 AAGACATTCC 2 701 TTTTCCTTCT 2751 AGCAGGCCAG 2801 CTTTCTTTAG 2851 AATGGCCTTC 2 9 01 GCAATTCTAT 2951 AAAAAAAAAA GCCCAGACAC ATTAGCCCAA CAAACACTCA GCGTGTGTGA ATGAGGAGGC CGTGGGGACC TGCTGAGGGC GTAGACCCTA ACTAGCAGAG TGGGACTCCC AGCCCCAGGG GGTGTGTTTT AGATGTCCTT CCAAAGAACC CCTTCGTTCC TTGTCTTTCT AAA CAACGTGTGA CTGCTGCAGA TATTGGAACA GTGAGTGGGA TTCGGGAAGT CAGCTCGCCC AGGGTGGGGT GGACAGAAAA AGAATTGAGG ACAGCCGCCC ACTTTGCAGG ACAGACTTCT ACCAAATTGG CTTCTCAAAG TTTCCAGGCA. CTTTCCTCTC GACGGATGGA ACGATTTCCT AGCTTGGGGT ACAAACTTTC ATTACTGATG CAGCTTTTGT GTGGGACCAC TGGAAAGAAG AGAGGTAAGG AGCAGGAGTG CTTCATTCCC GATGGGAAGC AAAGGAAGGT ACGCCTCCAG ATAATGACAT TGTCCTTTTT CATCAGGAAG TGGTTGGACC GGAAGATTTA TTGGAAACTG GCTCATGGTT CCCAGGTTCT CACTCTTGTT GAAATGGCTC GTTCCTTCTG ACTTGGCCAC TGTCTGTGTC TTCAAACTTG GAAACTGTTC AAATGGACAA CATTAGTGAT TTTAAAAAAA T o t a l number o f bases i s : 2963. DNA sequence c o m p o s i t i o n : 736 A ; 726 C ; 749 G ; 752 T ; 161 predicted to be non-coding on examination of the sequence for protein coding regions using Ficketts method (Fickett, 1982; COD_FICK program in PCGENE). Translation of these sequences resulted in polypeptides that lacked any recognizable structural motifs, when examined by various secondary structure predicting computer programs. Neither sequence scored significantly for overall hydrophobicity, or possessed remarkable charge distribution, although the longest predicted polypeptide (108 a.a. residues), was distinguished by an unusual frequency of serine residues (17/108 a.a.), and a very high isoelectric point, 11.81. Although a variety of potential splice sites could be constructed, these did not reveal ORFs of r e a l i s t i c lengths or compositions; the best cases were 450-500 bp in length. The translation products predicted from this analysis did not yield products with striking properties. Given the assumption of a fu l l y processed cDNA, the putative donor sites that were identified are of questionable significance. Searches of the EMBL (release 22.0), GENBANK (63.0) and NBRF Nucleic Acid (36.0) data bases found no sequences with significant homology to the cDNA sequence. The polypeptides predicted from the 51 ORFs and several of the larger internal ORFs were translated, and used to search the NBRF (24.0) and Swiss Prot (14.0) protein data bases. No corresponding sequences with significant homology were identified. 162 Discussion Some of the features of a novel human brain mRNA have been determined in the experiments described. This gene was studied because of the brain regional expression pattern, based i n i t i a l l y on colony hybridizations, and later confirmed by Northern blotting. Due to the the variable Northern blot detection of this high molecular weight transcript, i t was necessary to use a nuclease protection assay. The brain regional expression pattern observed on Northern blots was further confirmed by nuclease protection, which indicated a 4-fold higher level of the transcript in SI compared to cerebellum. In both human and rat, no nonneural tissue was observed to express the gene, although the detection of the transcript in rat was, at best, poor quality. This gene was found to be expressed in a l l human brain regions examined, with the exception of the white matter rich corpus callosum. Since this region is rich in g l i a l c e l l s , (particularly oligodendrocytes and astrocytes), and is greatly depleted in neurons, the results support a predominantly neuronal localization of the transcript. Recently, there has been some success at resolving the issue of cellular localization of the transcript by in situ hybridization (Walker, Boyes, McGeer and McGeer [1990], unpublished observations). In these experiments, hybridization signal was observed over neurons in the human cerebellar cortex. Thus, available information to date indicates that this gene is expressed in a tissue and 163 cell-type specific manner. Although the original intent of the screening protocols was to obtain region specific genes whose expression was altered in AD, the gene studied was not specific to the SI. The brain regional distribution observed for the expression of this gene clearly eliminates i t s specificity for CNS cholinergic neurons. Further experiments w i l l be necessary to examine the possibility that the transcript is expressed in neurochemically or cytologically distinct neurons. The brain regional variations in transcript levels can not yet be related to specific neuronal populations. Based on the d i f f i c u l t i e s encountered in obtaining sufficiently fresh human brain for in situ hybridization analysis, as well as the limited resolution of the method, i t is lik e l y that a definitive cellular localization w i l l only be derived from immunochemical staining for the corresponding protein product of this gene. Although in situ hybridization analysis of the rat would be technically simpler than human experiments, the apparent poor sequence homology between the rat and human gene, (in the available cDNA sequence), renders this analysis impossible. The results of the Southern blot analysis of human genomic DNA using pSI3ac-24 cDNA are consistent with the interpretation that this is a single copy gene. Although a polyadenylate tract of 20 bases was present, no cannonical polyadenylation signal was present in the cDNA. An AAUAAU 164 sequence, which has been r e p o r t e d t o be a f u n c t i o n a l p o l y a d e n y l a t i o n s i t e ( B i r n s t i e l e t a l . , 1985), was found 60 bases 5' t o the p o l y a d e n y l a t e t r a c t . T h i s i s a c o n s i d e r a b l y g r e a t e r d i s t a n c e than the 15-3 0 bases of i n t e r v e n e i n g sequence u s u a l l y found between an AAUAAA and the p o l y a d e n y l a t e t r a c t . Computer searches of sequence databases d i d not i d e n t i f y any homolog f o r t h i s gene. Since the sequence of the cDNA suggests t h a t the cl o n e encompasses 3' u n t r a n s l a t e d r e g i o n , and s i n c e many e n t r i e s i n the databases are p a r t i a l cDNA sequences f o r p r o t e i n coding r e g i o n s , i t i s not p o s s i b l e t o r u l e out t h a t a p r o t e i n c o r r e s p o n d i n g t o t h i s mRNA has a l r e a d y been d i s c o v e r e d . W i t h i n the CNS, s e v e r a l l a r g e mRNAs (> 10 k i l o b a s e s ) have been r e c e n t l y d e s c r i b e d . These i n c l u d e some of the m i c r o t u b u l e a s s o c i a t e d p r o t e i n mRNAs (MAPs, see f o l l o w i n g d i s c u s s i o n ) , t r a n s c r i p t s f o r c a l c i u m - i o n t r a n s l o c a t i o n channel s u b u n i t s t h a t b i n d d i h y d r o p y r i d i n e s (Mikami e t a l . , 1989) or rhyanodine (Takesima e t a l . , 1989), the 1 , 4 , 5 - i n o s i t o l t r i p h o s p h a t e - b i n d i n g p r o t e i n ( F u r u i c h i e t a l . , 1989; Mignery et a l . , 1989; a l s o r e f e r r e d t o as the P A O 0 p r o t e i n ) , and the mRNA f o r the e r y t h r o i d a - s p e c t r i n ( a - f o d r i n , 9 kb; Moon and McMahon, 1990). Based on what i s known of these genes, on l y the MAPs mRNAs appear t o be expressed a t l e v e l s of abundance comparable t o immensin mRNA, which was determined t o be pre s e n t a t about 0.05-0.2 % of polyA RNA mass. Most o f the other l a r g e t r a n s c r i p t s which have been s t u d i e d a re expressed a t lower or much lower abundance. Although the 1 , 4 , 5 - i n o s i t o l 165 triphosphate-binding protein mRNA is rather abundant in the cerebellum, this transcript is different than immensin mRNA on the basis of both i t s brain regional expression and presence in the liver. The size of the immensin transcript, as well as i t s moderately high abundance, suggests that this gene may correspond to a protein which has previously been described in the CNS. Given that the MAPs are present at comparable abundance and have similarly large transcripts, i t seems possible that immensin could be a structural protein, perhaps with a cytoskeletal function. Certain similarities arise between the MAPs genes and immensin when the tissue specificity is considered; some of the MAP mRNAs are expressed exclusively in the CNS, and some forms appear to be neuronal (Matus, 1988). There is current interest in the MAP proteins in AD studies, and the tissue levels of the MAPI proteins, (and tau) have been reported to be decreased in AD neocortex, with preservation of the MAP2 proteins (Nieto et a l . , 1989). The MAPs are known to be represented by a diverse collection of transcripts, with each of the two major groups, MAPls and MAP2s, having subtypes generated by alternative splicing, and probably varying in polyadenylation sites. Although no sequence data is available on MAPI genes, recent cloning of MAP1B cDNA identified a brain transcript of greater than 10 kb (Safaei and Fischer, 1989). Several of the MAP2 genes have been cloned, and some have been sequenced. Rat 166 brain MAP2b and MAP2c are generated by alternate splicing of a single gene to yield transcripts of 9 and 6 kb (Papandrikopoulou et a l . , 1989; Kindler et a l . , 1990). The available 31 sequence of the rat MAP2 mRNAs show no homologies to immensin, nor does the ubiquitously expressed MAP-U (bovine sequence, Aizawa et a l . , 1990). Unfortunately for further sequence comparisons, only the internal protein-coding regions of human MAPs mRNAs have been reported, and only the 3' non-coding region of immensin is known. From careful inspection of the Northern blots, the pattern of immensin hybridization was always observed to be a thick band, with a smear to lower molecular weight.' The smearing of the band was expected, given the frequently observed partial degradation of post mortem human brain RNA. The width of the high molecular weight band suggests that alternate forms of the transcript may be present. This has recently been confirmed to be the case. In an attempt to gain cDNA sequence 5' to the pSI3ac-24 clone, D. Walker (personal communication, 1990) recovered a 4.7 kbp cDNA clone from the SI3a library. This clone extended 300 bp in the 5' direction, and 1.4 kbp in the 3' direction. A polyadenylation signal sequence has been determined on the 3' end of this cDNA. Thus, the immensin gene has been shown be expressed in at least 2 forms, differing in alternative polyadenylation sites. The 4.7 kb immensin 3* untranslated region represents the longest known for brain mRNAs (Sutcliffe, 1988), which have a tendency 167 to be longer than in other tissues, averaging about 1-1.2 kb. Since l i t t l e is known about the functional role, of mRNA 3* untranslated regions, i t is d i f f i c u l t to interpret the biological significance of the large 3' UTR of immensin. It is also possible that further forms of immensin transcript exist, generated either at other polyadenylation sites, and/or at alternative splicing sites. The generation of multiple transcripts by alternative splicing is very common in genes expressed in the CNS (Sutcliffe, 1988). Comparison of the levels of immensin transcript in AD and NNC brain regions yielded significant results. Some concern might be raised on the specificity of the measurements. The probe employed detected a single major protected species in the nuclease protection assay, although a number of minor bands were also present on denaturing gels. The source of these bands has not been determined. It is possible that the minor bands could contribute a non-specific signal, although the rather high stringency conditions employed for the assay and the highly specific Northern blot signal detection make this appear unlikely. Further p o s s i b i l i t i e s for the source of these lower molecular weight bands are that they are derived from alternatively spliced forms of the immensin mRNA, or that there are closely related mRNAs. It has not been determined whether hybridization to such specicies, i f they exist, would materially change the relative measurements observed between AD and NNC groups. Nevertheless, within the limitations of 168 these measurements, a significant increase in the level of the transcript was detected in the temporal cortex, but not SI, in AD compared to controls. Since this gene does not appear to be expressed in g l i a l c e l l s , the increased expression is likely occurring in neurons. Neuron c e l l death is a well known feature of AD pathology, although the quantitative aspects are s t i l l controversial. In the SI, the evidence for c e l l loss of magnocellular neurons has been presented in the Introduction to this Thesis (section IA and IB). Loss of neocortical neurons in AD has been reported in many studies. Morphometric analyses of AD neocortex suggest a decrease of 15-50 % of temporal cortex neurons in comparison to controls, with a reported tendency for larger diameter (pyramidal) neurons to be lost (Hansen et a l . , 1988; Mann et a l . , 1985; see also Coleman and Flood, 1987 and associated editorial responses). Thus, the increased levels of immensin mRNA in the AD temporal cortex are counter to the expectation that the tissue levels of the transcript to reflect overall c e l l numbers. This could suggest that the transcript is present in cortical neurons that are spared in AD (ie. small diameter), or, alternatively, that the expression is up-regulated in surviving pyramidal neurons. Similarly, the preservation, or even slight increase in the levels of the transcript in the SI might not be expected for a neuronal transcript that is relatively more abundant in SI cholinergic neurons, and similar reasoning 169 would prevail. In order to distinguish between these alternatives, a microscopic evaluation would be necessary. Studies of neuronal morphology in AD affected brain regions have concluded that regenerative processes occur in concert with neurodegeneration. Evidence for regenerative processes in AD has come from Golgi staining and electron microscopic studies of AD neocortex (Scheibel and Tomiyasu, 1978; Paula-Barbosa et a l . , 1980; Ferrer et a l . , 1983) and SI (Arendt et a l . , 1986), which have reported dendritic sprouting. In some cases the fine structure of dendrites have been described as resembling growth cones. Similarly, immunocytochemical studies of galanin in the nBM has suggested a dense proliferation of terminals in close proximity to cholinergic neurons (Chan-Palay, 1988). Immunohistochemical staining of AD brain for cytoskeletal proteins detects a marked rearrangement of tau and MAP2 immunoreactivities, to a state resembling that observed in developing neurites (McKee et a l . , 1989, Ihara, 1988). Thus, morphological studies of AD affected tissue supply considerable evidence for growth processes overlapping the neurodegenerative changes. The regenerative processes described morphologically in AD brain also have been observed at the molecular level. The modified tau epitope recognized by the Alz-50 antibody, which is readily detected in AD brain and is present at high levels in early development, is a l l but undetectable in normal adult brain (Wolozin et a l . , 1988). Geddes at a l . (1990) have 1 7 0 applied in situ hybridization to the measurement of the levels of the developmentally regulated human bal a-tubulin transcript in AD affected brain. In these experiments, the bal embryonal form of a-tubulin was observed to be at higher levels in hippocampal neurons of AD than adult control brain. Since this gene i s expressed at much higher levels in early brain development, and codes for a cytoskeletal protein, this result suggests that neurons may use growth-related proteins to build cytoskeletal structures to accompany dendritic growth. If the immensin transcript does code for a neuronal cytoskeletal element, the observed increased expression in AD temporal cortex could be rationalized in a similar manner. Although the level of increase of the transcript i s modest, when determined in extracted RNA, the levels in surviving neurons could be considerably different. A similar reasoning could be applied to interpreting the levels of the transcript observed in the SI samples. As is the case with any study dealing with measurement of transcript levels in tissue extract RNA, caution should be exercised in interpreting the physiological meaning of a change in abundance. Given the known and potential complexity of the immensin gene, considerable further effort w i l l be required to describe i t f u l l y . This w i l l be a prerequisite to further interpreting the measured change in transcript abundance in AD brain. 171 I V . CONCLUSION During the course of the studies described in this thesis, the direction of research on AD has expanded from the neurochemical and anatomical perspectives to include molecular genetic and molecular biological approaches. Thus, considerable information has been obtained on the structure and expression of the /3-APP gene, and there i s hope that the processes leading to the deposition of amyloid in AD brain w i l l be untangled. To the present time, the expression of only a small number of other genes has been examined in the AD brain. By the combination of molecular biological and protein chemical studies, some of the protein constituents of the NFTs have been determined, with a reasonable degree of certainty. The relationship between the biogenesis of amyloid and NFTs, and the pathological significance of these protein accumulations w i l l probably be understood in a few years. The progress made in the genetic analysis of the disease is much less certain, probably reflecting the d i f f i c u l t i e s of carrying out linkage analysis of small groups, a problem magnified by the imperfect premortem diagnosis of AD. As larger collaborative efforts define a greater number of FAD affected families, the localization of the genetic l o c i involved should become clearer. It appears that many AD 172 affected individuals are not of the familial AD-type. Unfortunately, the definitive diagnosis of AD remains a postmortem finding. The early hopes of immunochemical detection of AD via the ALZ-50 antigen or /3-APP antigen have not yet materialized. No novel candidate disease markers of predictive value have appeared. Clearly, there i s a considerable need for such markers. The approach advocated in this thesis was to use the well established anatomical/neurochemical observation of SI cholinergic c e l l loss in AD brain with a relatively wide range screening method to define disease-related gene expression changes. Similar studies, examining human cortex gene expression in AD, have been described recently by others (May et a l . , 1988, 1990; Travis and Sutcliffe, 1988). It was hoped that this would rapidly lead to new biochemical markers of AD. Limited success has been achieved to date. The novel brain mRNA described in this work represents the product of such an approach, in this case, concentrating on the SI. As defined experimentally, the gene described represents a novel brain mRNA with some interesting structural properties. The available information suggests that the further study of this gene may have some relevance to AD. Clearly, many questions remain unresolved on the biology and biochemistry of the putative gene product corresponding to the described cDNA clone. Due to the extreme size of the mRNA, as well the possible specificity to human tissue, i t may be anticipated 173 that considerable effort w i l l be required to f u l l y characterize the gene and gene product(s). Nevertheless, isolation of the cDNA clone w i l l provide a means to further investigate the structure, function, and eventually, the relevance of this gene to AD. From the outset of this work, the greatest experimental limitation was in acquiring sufficient human brain regional mRNA to investigate AD-specific changes in gene expression. This is a limitation inherent in the use of c l i n i c a l l y and histopathologically well defined specimens, and was exacerbated by the small size of the target tissue. Current developments in molecular biological methods appear to be obviating this limitation. Specifically, the use of the polymerase chain reaction (PCR) w i l l permit experiments of the type undertaken in this work to be carried out with a much decreased demand on tissue-derived mRNA. Examples of this include the recent use of PCR amplified cDNAs for library construction starting with very small amounts of RNA (Akowitz and Manuelidis, 1989). The combination of PCR amplification and subtractive hybridization regimes has recently been used to define brain gene expression changes resulting from scrapie infection (Duguid et a l . , 1989a,b). Analogously, cDNA libraries could be differentially screened using PCR amplified cDNA probes. Although this approach has not yet been reported for investigation of the problem addressed in this thesis, i t seems that this would be a possible solution to the technical 174 l i m i t a t i o n s of the cDNA l i b r a r y screening methods employed i n t h i s work. 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