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Senile dementia and Alzheimer's disease: A current view
Life Sciences, Vol. 27, pp. 1-14 Printed in the U.S.A.
Pergamon Press
MINIRL~VILI~: SENILE DEMENTIA AND ALZHEIMER'S DISEASE: A CURRENT VIEW Umberto De Boni* and Donald R. Crapper McLachlan Department of Physiology U n i v e r s i t y of Toronto Toronto, Ontario, M5S 1A8
Summary Senile dementia, of which the most common cause is known as Alzheimer's disease, is a neurological disorder characterized c l i n i c a l l y by memory and learning d e f i c i t s . The disease may be shown to have a prevalence rate in the United States of greater than three per i000 of the general population, and i t may be shown that at any one time 600,000 individuals are affected by advanced stages of this disease. The disease, unique to man, is relentlessly progressive and refractory to treatment. In most cases, i t is unrelated to cerebral vascular insufficiency, i t is however associated with well defined morphological and biochemical changes at the c e l l u l a r level. These include altered dendritic morphology, i n t r a c e l l u l a r changes such as granulovacuolar and n e u r o f i b r i l l a r y degeneration, and extrac e l l u l a r deposits termed neuritic plaques. Moreover, a selective dysfunction in central cholinergic mechanisms has been demonstrated. Recent results from this laboratory have indicated that significant changes in chromatin conformation may be demonstrated to occur in this disease. Specifically, a significant "heterochromatization" was found to occur in chromatin from brains of patients who had died with this disease, suggesting that a major alteration in chromatin structure may be related to the etiology of this condition. In addi t i o n , an accumulation of the environmental agent aluminum upon brain cell chromatin has been implicated to contribute to the pathophysiology of Alzheimer's disease. During the past few years i t has become possible to model several of the morphological markers of this disease in the laboratory. Specifically, the paired helical filament, representing a major i n t r a c e l l u l a r marker can now be induced in cultured neurons and a filamentous hyperplasia similar to neurof i b r i l l a r y degeneration, but not identical in morphology, may be induced in experimental animals and in neurons in v i t r o by trace amounts of aluminum. The understanding of Alzheimer's disease is in i t s infancy, however, the a v a i l a b i l i t y of model systems now permits extensive investigations into the nature and mechanisms of the agents responsible for the changes observed.
Address a l l correspondence
to U. De Boni
0024-3205/80/270001-14502.00/0 Copyright (c) 1980 Pergamon Press Ltd
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Introduction At the present time and with increasing prevalence in our f u t u r e , an i n creasingly common neurological disorder is emerging. This is the problem of dementia. Dementia is a term which denotes a state of altered brain function. Of the many causes of dementia, the most common is Alzheimer's disease, named aft e r Alois Alzheimer, who in 1906 f i r s t described t h i s condition and recognized i t as a separate disease (1). A condition c l i n i c a l l y and p a t h o l o g i c a l l y ident i c a l to senile dementia may occur in the presenile age group, as well as in a l l patients with Down's syndrome who come to autopsy aged over 40 years. These observations support the hypothesis that the degenerative changes found in the brain of the e l d e r l y may be considered to r e s u l t from a disease rather than representing pre-programmed, i n e v i t a b l e consequences of advancing chronological age. We are faced with no known cause and no cure. Therefore, work into the etiology of t h i s mind destroying disease, which a f f e c t s so many of our elderl y , must c e r t a i n l y assume a more prominent place in our research e f f o r t s than that which i t has occupied in the past. Prevalence of senile dementia Senile dementia has t r a d i t i o n a l l y been accepted as an inescapable consequence of advancing age. Moreover, the disease has commonly, and i n c o r r e c t l y , been a t t r i b u t e d s o l e l y to cerebral vascular i n s u f f i c i e n c y . However, close a t t e n t i o n to brain pathology has shown that only 20 to 30% of brains from sen i l e i n d i v i d u a l s show s u f f i c i e n t atheromatous vascular change to account sati s f a c t o r i l y for the c l i n i c a l signs of dementia. Moreover, there is evidence to indicate that the reduction in cerebral blood flow observed in some cases may be occurring secondarily to tissue atrophy. In contrast, 60 to 70% (2) of the e l d e r l y who present with c l i n i c a l signs of dementia, e x h i b i t ' t h e c l a s s i c a l signs of Alzheimer's disease. S t a t i s t i c s for the United States are a v a i l a b l e to show that approximately 4.4% of i n d i v i d u a l s aged 65 years and older, display some form of moderate to severe dementia. This leads to the estimate that at any one time 600,000 advanced cases, or a prevalence rate greater than three per 1000 of the t o t a l population, are affected by Alzheimer's disease (3). This r a t i o w i l l d r a s t i c a l l y increase over the next few decades, as the f r a c t i o n of the population aged 65 years and older increases from the present 11% to 12% by the year 2000 and to a staggering 17% by the year 2030 (4). C l i n i c a l course of Alzheimer's disease In the past, the term Alzheimer's disease had been reserved for cases of presenile dementia, that i s , f o r cases with ages of onset of less than 60 years. However, i t is now recognized that the underlying pathology in both presenile and senile dementia of t h i s type are i d e n t i c a l , and the term Alzheimer's disease is now applied regardless of age of onset. Alzheimer's disease is a global, slowly progressive, f a t a l encephalopathy, the diagnosis of which however cannot be made with c e r t a i n t y in the l i v i n g p a t i e n t s , unless brain tissue from e i t h e r biopsy or autopsy is a v a i l a b l e f o r examination. Few l o n g i t u d i n a l studies of the c l i n i c a l course of this disorder have been reported and no adequate measures of changes in sensory, motor and
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cognitive behaviour e x i s t . However, available longitudinal data suggests z,e following c l i n i c a l course (5): Early stages of this disease are characterized by loss of memory and learning a b i l i t i e s , with preservation of f e e l i n g , perception and movement. In the middle stages, a more pronounced loss of memory is coupled with the appearance of focal neurological signs, such as a l t e r a t i o n s in the acquisition and r e t r i e val of auditory and visual events. In a d d i t i o n , there are signs of spatial and temporal d i s o r i e n t a t i o n , and the a b i l i t y to use speech, language and arithmetic are impaired. In the terminal stage, which occurs on the average 8 years (range 2-19 years) following the appearance of f i r s t symptoms, a profound loss of a l l higher mental functions necessitates continuous nursing care. Histopathology Alzheimer's disease is associated with several brain changes. Gross examination of brains from affected i n d i v i d u a l s frequently reveals pronounced cort i c a l atrophy and d i l a t e d v e n t r i c l e s , i n d i c a t i n g loss of brain substance. Micra scopic examination reveals that the most commonly involved structures are the hippocampus and the more recently evolved neocortical association or polysensory regions of f r o n t a l , p a r i e t a l , temporal and o c c i p i t a l lobes. In these regions, an evident loss of neurons is associated with altered d e n d r i t i c morphology and other histopathological changes, among them granulovacuolar degeneration of hippocampal pyramidal c e l l s , n e u r i t i c (senile) plaques with or without amyloid cores and n e u r o f i b r i l l a r y degeneration (NFD). I.
Altered d e n d r i t i c morphology
Scheibel and Tomiyasu (6) have shown that the d e n d r i t i c tree of c o r t i c a l neurons in brains of senescent i n d i v i d u a l s undergoes progressive, degenerative changes. These changes include loss of d e n d r i t i c spines, dying back of dendrites and swelling of neuronal somata. Moreover, neurons from patients with presenile dementia of the Alzheimer type were characterized by the presence of haphazardly placed clusters of new d e n d r i t i c growth, at a time when the o r i g i n a l dendrites were devoid of t h e i r normal spine complement. Due to the aberrant d i s t r i b u t i o n and o r i e n t a t i o n of these newly sprouted dendrites, not coinciding with any presynaptic terminal f i e l d s , t h i s new outgrowth is referred to as"lawless". These findings are supported by the results of a more recent, similar study of morphological changes in hippocampal neurons in ageing humans (7). In addition however, these workers showed evidence to indicate the presence of " p l a s t i c i t y " in the ageing human brain. This was demonstrated by the presence of s i g n i f i c a n t increases, in non demented e l d e r l y individuals (mean age: 79.6 y r s . ) , in the number and average length of terminal segments of the d e n d r i t i c tree, compared to adult individuals (mean age =51.2 y r s . ) . In cases of senile dementia (mean age =76.0 y r s . ) , d e n d r i t i c trees were less extensive than in cont r o l , adult brain, as a r e s u l t of fewer and shorter terminal segments. II.
Granulovacuolar degeneration
This degeneration, occurring mainly in hippocampal pyramidal c e l l s and in the medial temporal gyri', was f i r s t described by Simchowicz (8) in cases of senile dementia of the Alzheimer type. I t consists of one or more unstained, spherical vacuoles, 3 to 5 u in diameter and containing at the centre an argyrop h i l i c and hematoxilinophilic granule. U l t r a s t r u c t u r a l analysis (9,10,11) reveals l i t t l e more; the vacuoles consisting of a membrane bound, electron lucent p a r t i c l e with a c e n t r a l , electron-dense core. Although the o r i g i n and functional consequences of these intraneuronal p a r t i c l e s are unknown at this time, i t
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Figure 1 A. Normal, pyramidal shaped neurons in cerebral cortex of human brain. B. Granulovacuolar degeneration (arrow head) in pyramidal neuron of hippocampus in patient with Alzheimer's disease. C. Three neuritic plaques adjacent to granule cell layer ( l e f t ) in hippocampus of patient with Alzheimer's disease. Note core in largest plaque. D. Low power electron micrograph of neuritic plaque, with electrondense inclusions. E. Higher power electron micrograph of plaque. Note paired helical filaments among electrondense debris. A, B, C: Bielchowski stain. D, E: Glutaraldehyde/osmium preparation. Magnification: A,C: 300 X. B: 700 X. D: 11250 X. E: 37500 X.
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• ay be shown that both the incidence and degree of granulovacuolar degeneration are s i g n i f i c a n t l y higher in brains of demented i n d i v i d u a l s , and especially in those with the histopathological diagnosis of Alzheimer's disease (12,13,14). Moreover, a highly s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n could be demonstrated between the severity of granulovacuolar change, in p a r t i c u l a r in the posterior hippocampus, and the severity of n e u r o f i b r i l l a r y degeneration (see below) (15). This c o r r e l a t i o n was quite d i s t i n c t from that showing the increase of granulovacuolar change as a function of age~ In the absence of an experimental model for the induction of granulovacuolar change, this c h a r a c t e r i s t i c cytological change has been l a r g e l y ignored, and at the present time no hypothesis for induction is available. III.
N e u r i t i c (senile) plaques
These terms describe an extremely common microscopic, spherical accumulation of c e l l u l a r and e x t r a c e l l u l a r debris, often containing amyloid cores and frequently surrounded by reactive c e l l s . U l t r a s t r u c t u r a l examination reveals that such plaques are composed of degenerating neuronal processes and t h e i r cytoplasmic components, including the f i b r i n o u s , aberrant protein characterist i c of n e u r o f i b r i l l a r y degeneration ~see below) (16,17). I t is of i n t e r e s t to note that n e u r i t i c plaques are seen in a v a r i e t y of organic brain diseases of man and animals, sometimes associated with v i r a l e t i o l o g i e s (18,19). This has supported the hypothesis, long held by several i n v e s t i g a t o r s , that a unique, v i r a l - l i k e agent may be involved in the pathogenesis of Alzheimer's disease. Plaques with amyloid cores, resembling, but not identical to, those in senile dementia of the Alzheimer type occur in mouse brain following the inoculation of scrapie agent and may occur in human brain in patients with Kuru and JakobCreutzfeld disease (20,21). The transmissible factors which cause these infectious, spongiform degenerative brain diseases, have not been defined. These agents are atypical in that they remain stable at temperatures up to 80°C and that i n a c t i v a t i o n may not be complete at IO0°C (21). In addition i t is noteworthy that these agents under certain conditions, may show a remarkable resistance to u l t r a v i o l e t i r r a d i a t i o n and, with ionizing radiation (neutron beam) reveal an unusually small i n a c t i v a t i o n target size (23,24,25,26,27). Recent work (28) has indicated that the agent of scrapie may be as small as 40S (S2o,W) and that i t may contain a hydrophobic surface,explaining some of the unusual features observed. The s t a b i l i t y to heating is consistent with this view, as high temperatures s t a b i l i z e hydrophobic i n t e r a c t i o n s . Moreover, the hydrophobic properties may be associated with the observed association of scrapie agent with membrane f r a c t i o n s , since hydrophobic components may r e a d i l y be inserted into membranes. I f freed from associated membranes, the i n f e c t i v i t y of the agent may be shown to be s i g n i f i c a n t l y reduced by RNAse. Moreover, a t e n - f o l d increase in binding to hydroxyapatite and a greater i n a c t i v a t i o n by DNAse may be demonstrated following the pretreatment of the agent with proteinase K. This data is considered to indicate that the scrapie agent may contain low molecular weight, single stranded DNA of approximately 2000 nucleotides. The observed heat s t a b i l i t y f u r t h e r supports the p o s s i b i l i t y that the genetic information is contained in single stranded DNA. There is no d i r e c t evidence to indicate that a transmissible agent capable of the induction of n e u r i t i c plaques exists in Alzheimer's disease. Specifica l l y , unpublished work from this laboratory over the past 7 years has f a i l e d to induce Alzheimer changes in laboratory animals. Saline extracts of Alzheimer affected brains, alone and in combination with aluminum chloride or l a c t a t e , have f a i l e d to induce human Alzheimer type pathology in monkeys (Rhesus macaqu~ Saimiri sciureus), guinea pigs, rabbits and cats. Nevertheless, while none of these observations implicate v i r u s - l i k e mechanisms in the pathogenesis of Alzheimer's disease, the evidence available should stimulate the search for existence of such an agent.
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% ,,
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Figure 2 A. Neuron ( r i g h t ) with perinuclear aggregate of argentophilic n e u r o f i b r i l l a r y d e generation. Normal neuron at l e f t . B. Ultrastructure of normal, human neuropil, with abundant microtubules in neuronal cytoplasma ( * ) . C. Ulstrastructural appearance of aggregate of paired helical filaments in cerebral cortical neuron of patient with Alzheimer's disease. Electron-dense p a r t i c l e (arrow head) is lipofuscin or "age pigment". D. Higher power micrograph of paired helical f i l a ments characteristic of Alzheimer's disease. Note regular period of approximately 800 A° . A: Bielchowski stain. B,C,D: Glutaraldehyde/osmium preparation. Magnification: A: 700 X. B: 8000 X. C: 19000 X. D: 94000 X.
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I
Figure 3 Micrographs representative of the model system employed in the study of senile dementia. A. Phase contrast micrograph of living, human neuron with abundant neurites, 25 days in vitro. B. Ultrastructural appearance of human brain cell, 28 days in vitro. Synaptic contact (arrow head) identifies cell as neuron. C. Ultrastructure of neuropil representative of the culture system employed, 28 days in vitro. Asterisks identify synaptic contacts. Note similarity to human neuropil in vivo, Fig. 2 B. D. Examples of paired helical filaments induced in cultured human neurons, closely similar to the PHF's characteristics of Alzheimer's disease, shown in Fig. 2 D. A: Unstained, living preparation. B, C, D: Glutaraldehyde/osmium preparation. Magnificiation: A: 300 X. B,C: 8000 X. D: 56000 X.
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N e u r o f i b r i l l a r y degeneration (NFD)
Alois Alzheimer f i r s t described as NFD the a r g e n t o p h i l i c aggregates of n e u r o f i b r i l s in neurons of a patient with senile dementia (1). Modern a n a l y s i s , by electronmicroscopy, of brain tissue from senile i n d i v i d u a l s has revealed that NFD represents an intraneuronal accumulation of arrays of so called "paired h e l i c a l filaments" (PHF's), structures composed of two 100 A° diameter, solid filaments forming a h e l i c a l structure with an average period of 800 A° (29,30). This structure is unique to neurons of man, and is not known to occur in animal brain diseases. I t is now however recognized that PHF's may be encountered in human cerebral neurons in several conditions, with apparently d i f f e r e n t e t i o l o gical factors. As such, NFD may be associated with repeated head trauma,resul~ ing in dementia p u g i l i s t i c a , and in the l i p i d storage diseases named a f t e r Kuf and Neiman-Pick (31). Moreover, NFD may be associated with the delayed expression of a conventional v i r a l i n f e c t i o n . In p a r t i c u l a r , NFD may occur in brain stem nuclei, s p e c i f i c a l l y in the substantia nigra, several years a f t e r epidemic encephalitis lethargica (32). In a d d i t i o n , neurons of the brainstem of patients with Parkinson's disease with a previous h i s t o r y of v i r a l encephal i t i s , may display NFD (33). Viral e n c e p h a l i t i s may also be accompanied by NFD in brain of young patients with progressive multi focal leukoencephalopathy (34) and subacute sclerosing panencephalitis (35). While the former condition appears to be related to the unsuppressed p r o l i f e r a t i o n of an otherwise benign v i r u s , subacute sclerosing panencephalitis appears to be caused by the apparent incomplete suppression of a measles-like paramyxo virus permitting slow and chronic r e p l i c a t i o n of the agent. PHF formation also occurs in spinal cord in Guam amyotrophic l a t e r a l sclerosis cases (33) and in a s i m i l a r condition found in certain regions in the Kii peninsula of Japan (36,37). Moreover, as recentl y reviewed (38), NFD may be associated with a large number of additional d i s eases. Whether these various conditions permit the expression of a class of c l o s e l y related agents to form PHF's or whether PHF's represent a non-specific morphological response of human neurons to a diverse group of i n s u l t s , is unknown at present. Various subsets of neuronal types may e x h i b i t quite d i f f e r ent s u s c e p t i b i l i t i e s to the formation of PHF's. While some neurons, such as cerebellar Purkinje c e l l s , dorsal root ganglion c e l l s and primary sensory nuclei in the brain stem remain r e f r a c t o r y , large pyramidal shaped neurons in the hippocampus and in cerebral cortex commonly e x h i b i t NFD in Alzheimer's disease. I t is remarkable that the neurons with NFD f a i l to e x h i b i t morphological evidence of a response to control the p r o l i f e r a t i o n of PHF m a t e r i a l . This appears to indicate that there may e x i s t a s u f f i c i e n t s i m i l a r i t y between normal structural proteins and PHF's so that the appropriate p r o t e o l y t i c processes f a i l to respond. A l t e r n a t i v e l y , the PHF's could accumulate as a r e s u l t of a disorder in the system regulating the production of catabolism of neuronal f i brinous proteins. The chemical and i mmunohistochemical characterization of PHF proteins of Alzheimer's disease is complicated by the f a c t that at the time of death, only i in I00 to I in 10 neurons show NFD. Therefore. contamination of the isolated f r a c t i o n s with associated c e l l u l a r debris represents a major problem. Analysis of a neuronal f r a c t i o n enriched with neurons containing PHF's (39) showed the presence of a polypeptide band of 50,000 Daltons, a band c l o s e l y resembling the peptides derived from g l i a l f i b e r s . Since a g l i o s i s often accompanies Alzheimer's disease, i t is possible that g l i a l filaments may account for the protein p r o f i l e s . These same workers have also shown that microtubules, p u r i f i e d by two cycles of polymerization, may share immunoreactive sites with certain immunoreactive s i t e s on the n e u r o f i b r i l l a r y material from Alzheimer's d i sease (41). However, recent evidence is a v a i l a b l e (40) to show that recycled tubulin may contain subunits of 100 A° intermediate filaments with a peptide
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composition s i m i l a r to the 68,000 molecular weight subunits of neurofilaments. I t is quite conceivable therefore that this f r a c t i o n might be present in the material employed as an antigen in the studies by Grundke-lqbal (41). Additional evidence, based upon immunofluorescence has recently been obtained (42) to indicate that normal neurofilaments as well as the filaments comprising the Alzheimer NFD, and the filaments induced by aluminum (see below) may be a n t i genically related. Employing r a b b i t anti-chicken-neurofilaments as antiserum, i t was possible to show thatneurofilaments in axons, the filaments of Alzheimer's disease and the aluminum induced filaments a l l stained i n t e n s i v e l y . Although the absolute p u r i t y of the o r i g i n a l antigens is uncertain, there exists considerable s p e c i f i c i t y , as demonstrated by persistence of reaction with neurofilaments, following adsorption of the antineurofilament serum with tubulin. Much work is c u r r e n t l y carried out to define the I00 A° diameter, intermediate filaments of normal c e l l s and the f i b r i n o u s proteins comprising the Alzheimer n e u r o f i b r i l l a r y tangle. However, before such d e f i n i t i o n s may be successfully concluded, methods must be developed to prepare these subcellular f r a c t i o n s free of other cytoplasmic contaminants. V.
Altered chromatin conformation
We have recently presented evidence (43) that chromatin isolated from brains of patients with senile dementia of the Alzheimer type, has undergone "heterochromatization". Chromatin f r a c t i o n s isolated by c e n t r i f u g a t i o n following disruption of brain cell nuclei yielded three f r a c t i o n s ; heavy or heterochromatin, not transcribed in v i t r o , and two l i g h t f r a c t i o n s ; intermediate euchromatin and l i g h t euchromatin. Results of a study involving 21 nuclear preparations from cerebral cortex of 12 controls aged 2 to 69 years showed that 75.2 ±5.5 per cent of the DNA was found in the euchromatin f r a c t i o n . In cont r a s t , in 25 preparations from 12 brains with advanced Alzheimer's disease, the f r a c t i o n of DNA in the euchromatin varied between 78 and 25 per cent, with an average value of 5 5 . 6 ± 1 6 . 8 per cent. This observed increase in the proportion of heterochromatin in advanced Alzheimer's disease was independent of chromatin protein content, age, g l i o s i s and post-mortem events. Moreover, the analysis of f r a c t i o n s enriched in neurons and g l i a l c e l l s r e s p e c t i v e l y showed that this "heterochromatization" occurred both in neurons and g l i a l c e l l s . Together with the results of a t r a n s c r i p t i o n study carried out on these fractions, the findings indicate that a smaller proportion of genetic information is available f o r t r a n s c r i p t i o n in brain of affected i n d i v i d u a l s , and that a major a l t e r a t i o n in protein synthesis may be associated with dementia of the Alzheimer type. VI. Biochemistry of cerebral cortex in Alzheimer's disease The analysis of the chemical pathology of senile dementia in man is fraught with d i f f i c u l t i e s a r i s i n g from secondary g l i a l reaction, changes related to age and not dementia, agonal or terminal pre-mortemevents and post-mortemchan~es. Nevertheless by employing careful handling of biopsy and autopsy samples, s i g n i f i cant biochemical changes may be i d e n t i f i e d (44,45). Results from an early study (46) employing biopsied brain from 3 patients with Alzheimer's disease showed that r e s p i r a t o r y rates and l a c t i c acid production as well as glucose u t i l i z a t i o n were within normal l i m i t s . Moreover, while total protein was reduced compared to controls, the incorporation of labelled lysine into protein of a microsomal f r a c t i o n occurred at normal rates. In contrast, another study showed(47) that the a c t i v i t i e s of 2 phosphatases in biopsied brain from patients with large numbers of neurons with NFDandneuritic plaques were sign i f i c a n t l y increased while changes measured in oxidative enzyme a c t i v i t i e s were a t t r i b u t e d to degeneration. I t is important to note that the increasedphosphatase a c t i v i t y was found associated with regions o f n e u r o f i b r i l l a r y d e g e n e r a t i o n ,
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and could not be demonstrated in unaffected neurons in this study. The original observation that choline acetyl transferase is s e l e c t i v e l y d e pressed in about two thirds of autopsy and biopsy samples from patients with pre-senile or senile dementia (48) has focused much a t t e n t i o n on the possible dysfunction of cholinergic projections in this condition. Several studies have since confirmed that the a c t i v i t i e s of choline acetyl transferase and acetyl choline esterase may be reduced over 50% in Alzheimer's disease. Moreover, i t has been possible to show that these reductions are p a r a l l e l l e d by increasing morphological changes, which in turn correlate with dementia scores (49,50,51). Moreover, available evidence indicates that the presynaptic part of the cholinergic system might be s e l e c t i v e l y affected in Alzheimer's disease. Attempts to modulate memory performance in Alzheimer's disease by raising plasma choline levels or by the use of antichol~nesterases have been met with l i t t l e success. The only anticholinesterase r e a d i l y crossing the blood brain b a r r i e r , physostigmine, has only a short duration of action, and exerts pronounced parasympathomimetic e f f e c t . Moreover,as recently reviewed (53) the use of d i e t a r t choline or i t s precursor l e c i t h i n (53) to raise plasma choline l e v e l s , has resulted in l i t t l e or no improvement in performance. Etienne et al. (54) reported no improvement in several patients with Alzheimer's disease following t h e i r t r e a t ment for i month with up to 8 g of choline bitamtrate per day, and no changes in cognitive performance occurred in ten Alzheimer patients treated daily for 2 weeks with 9 g choline b i t a r t r a t e (55). S i m i l a r l y , improvement in performance was limited to a f r a c t i o n of the individuals tested in a study employing dietary l e c i t h i n (53). In summary, i t is evident that preliminary t r i a l s of long term choline treatment of patients with senile dementia of the Alzheimer type have f a i l e d to s i g n i f i c a n t l y improve t h e i r performance or to ameliorate t h e i r condition. Possible etiology of Alzheimer's disease and model systems employed Nothing is known concerning the factors which i n i t i a t e any of the brain changes occurring in Alzheimer's disease. Available evidence indicates however that NFD may be associated with conventional v i r a l i n f e c t i o n s and that n e u r i t i c plaques may be found associated with i n f e c t i o n s by the unconventional agents causing Kuru and Jakob-Creutzfeld disease. Therefore a search for mechanisms or agents present in brain affected by Alzheimer's disease becomes mandatory. Such investigations have resulted in the i d e n t i f i c a t i o n of models suitable f o r the i n v e s t i g a t i o n of at least some of the subsets of the changes occurring in brain affected by Alzheimer's disease. While to date no experimental model f o r granulovacuolar degeneration e x i s t s , both the n e u r i t i c plaque and the PHF, as well as the p r o l i f e r a t i o n of neuronal filamentous, structural proteins, may now be manipulated in model systems in the laboratory. The primary pathogenic event may be the r e s u l t of e i t h e r endogenous gene f a i l u r e or may be i n i t i a t e d by an as of yet u n i d e n t i f i e d agent foreign to the human brain. Based upon these assumptions, three hypothesis concerning the etiology of Alzheimer's disease may be formulated, and include: I. A reduction of the amount of RNA produced, by a pathogenic event operating in the nucleus, 2. A primary event r e s u l t i n g in the assembly of PHF's with a concomi t t e n t reduction in the number of microtubules and secondarily r e s u l t i n g in a reduction in dendroplasmic transport, and 3. a defect in the blood b a r r i e r permitting the accumulation within brain parenchyma of p o t e n t i a l l y toxic environmental compounds, including trace metals.
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The f i r s t hypothesis, related to altered function w i t h i n the c e l l nucleus, is supported by the f i n d i n g of altered chromatin conformation, as described above. Therefore, future exploration of mechanisms which control and regulate gene expression and chromatin conformation w i l l y i e l d new insights into the altered brain metabolism underlying Alzheimer's disease. In evaluating the second hypothesis, and considering the large number of pathological i n s u l t s which are associated with NFD (38), the p o s s i b i l i t y must be considered that the primary pathogenic event releases a mechanisms or agent capable of assembling PHF's. We have r e c e n t l y shown that brain affected by Alzheimer's disease may contain such a f a c t o r and i t s properties are c u r r e n t l y under study (56,57). Employing several types of e x t r a c t of Alzheimer affected brain i t has been possible to r o u t i n e l y induce PHF's in v i t r o , in f e t a l human cerebral c o r t i c a l neurons, in neurons of human f e t a l spinal cord and in r a b b i t cerebral cortex in v i t r o . In a d d i t i o n , we have recently i d e n t i f i e d that the PHF assembly f a c t o r may also be present in cerebral spinal f l u i d (CSF) of each of three patients with a c l i n i c a l diagnosis of Alzheimer's disease. In cont r a s t , CSF from a p a t i e n t with a dementia associated with progressive supranuclear palsy and from 2 patients withspondylosis without evidence of dementia f a i l e d to induce PHF's in the cultured c e l l s . Since PHF's occur in neurons in many human brain diseases in a l l age groups (38) and in many older i n d i v i d u a l s without overt signs of dementia (58) a brain e x t r a c t was prepared from a 33 year old n e u r o l o g i c a l l y normal p a t i e n t , who had died acutely of myocardial disease. While no PHF's were encountered in 8 out of 9 explant cultures t r e a t ed with t h i s e x t r a c t , I single PHF was encountered in one c u l t u r e . This observation raises the p o s s i b i l i t y that the agent responsible may be present in low t i t r e in apparently normal brain. To i d e n t i f y more exactly the nature of the agent, brain extracts were also subjected to a series of s p e c i f i c t r e a t ments, and subsequently assayed f o r t h e i r a b i l i t y to induce PHF in cultured neurons (57). Information derived from these experiments to date indicates that the f a c t o r is associated with a p a r t i c l e size of 40S and above. Moreover, the f a c t o r appears s e n s i t i v e to UV l i g h t of 2 x 10 s ergs/mm2, and does not lose the a b i l i t y to induce PHF's following exposure to DNAse I and RNAse. However, extracts exposed to trypsin f a i l to induce PHF's. The r e s u l t s indicate that Alzheimer affected brain may contain a f a c t o r which is capable of inducing PHF's in cultured neurons, a factor with properties d i s t i n c t l y d i f f e r e n t from those of a conventional virus or unconventional virus of the scrapie type. I n s u f f i c i e n t evidence is a v a i l a b l e to assess whether the f a c t o r contains nucleic acid. The t h i r d a l t e r n a t i v e concerns the hypothesis that the primary i n s u l t a l ters the blood brain and cytoplasmic b a r r i e r s , and as a secondary consequence a l t e r s b a r r i e r s to p o t e n t i a l l y toxic environmental substances. Among these, accumulated evidence strongly implicates aluminum as a toxic f a c t o r in senile brain disease; aluminum has been demonstrated to occur in brain of patients with Alzheimer's disease at increased concentrations, compared to age matched controls (59,60). In these cases, the aluminum concentration in some brain regions approached that which induces an over-production of 100 A° diameter filaments in susceptible neurons of experimental animals (61,62). Moreover, at t h i s same concentration, a class of human c o r t i c a l neurons exposed to aluminum in tissue c u l t u r e , respond with a filamentous hyperplasia of s i n g l e , unpaired filaments, of 100 A° diameter (63,64). The i n t r a c e l l u l a r locus of aluminum accumulation is nuclear chromatin, and i t has been shown that aluminum is associated with the nucleus both in brain c e l l s of man with Alzheimer's disease (65,66) and in experimental animals with an aluminum induced encephalopathy (67,68). Moreover, recent evidence derived from studies employing the s e n s i t i v e probes f o r DNA damage, sisterchromatid exchanges in cultured human lymphocytes and unscheduled DNA synthesis in human
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Vol. 27, No. i, 1980
astrocytes in v i t r o , has c l e a r l y shown that chromatin bound aluminum interacts f u n c t i o n a l l y with i t s binding s i t e s , and may damage DNA (63). Work in our laboratory employing thermal denaturation and related techniques, has also established (57,64) that aluminum in v i t r o i n t e r a c t s with DNA in a complex manner, in part depending upon i n t e r a c t i o n with hydrating water molecules and dependent upon pH and concentration. The data is consistent with the existence of two forms of aluminum, forming at least two types of complexes with DNA. Aluminum does not induce the PHF's nor the typical n e u r i t i c plaque usually seen in dementia of the Alzheimer type. Nevertheless, through the filamentous hyperplasia associated with the presence of this element in brain of experimental animals, along with the associated behavioural changes (69,70), the aluminum induced encephalopathy provides a unique model of a dementia process. Moreover, based upon the established n e u r o t o x i c i t y of this element, i t s presence in the brain of man may well represent a deleterious compounding factor in the pathophysiology of Alzheimer's disease, and must therefore be considered to be of consequence. In conclusion, i t is apparent that Alzheimer's disease, which affects so many of our e l d e r l y , is poorly understood. Moreover, the factors c o n t r o l l i n g i t s expression are obscure, and there can be no doubt that the understanding and ultimate treatment of this mind destroying disease w i l l require the a p p l i cation of the most sophisticated s c i e n t i f i c thoughts and techniques. Acknowled~ement_s The work described here is supported by the Medical Research Council of Canada, the Ontario Mental Health Foundation, and the Canadian G e r i a t r i c Research Society. Special thanks are extended for the superb cooperation of the o b s t e t r i c ' s group at the Toronto Western Hospital. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
A. ALZHEIMER, Allg. Z. Psychiat. 64146-148 (1907). C.E. WELLS, Stroke 9 1 - 3 (1978). - R.D. TERRY, Arch. Neurol. 3 3 1 - 4 (1976). F. PLUM, Nature 279372-37-3-(1979). J. PEARCE, and J.M. SCHOFIELD, Clinical Aspects of Dementia, B a i l l i e r e Tindal, London (1979). A.B. SCHEIBEL, and U. TOMIYASU, Exp. Neurol. 6 0 1 - 8 (1978). S.J. BUELL, and P.D. COLEMAN, Science 206854-~56 (1979). T. SIMCHOWICZ, •Histologie und Histopathologische Arbeiten ueber die Grosshirnrinde. Ed. by F. Nissl and A. Alzheimer, pp. 267-444, Fischer Verlag, Jena #1911). W.G. ELLIS, J.R. MCULLOUGH, and C.L. CORELY, Neurology 24101-106 (1974). A. HIRANO, H.M. DEBITZER, L.T. KURLAND, and H.M. ZIMMERMAN, J.Neuropath. Exp. Neuroi~27167-182 (1968). R.D. TERRY, an~ H.M. WISNIEWSKI, Advances in Behavioral Biology, Vol. 3. "Ageing and the Brain", Ed. by C.M. Graitz, pp. 89-116, Plenum Press, New York (1972). B.E. TOMLINSON, G. BLESSED, and M. ROTH, J. Neurol. Sci. 7331-356 (1968). B.E. TOMLINSON, G. BLESSED, and M. ROTH, J. Neurol. Sci. 11205-242 (1970). M.J. BALL, Acta Neuropath. ( B e r l . ) 4_2273-80 (1978). M.J. BALL, and P. LO, J. Neuropath. and Explt. Neurol. 36474-487 (1977). R.D. TERRY, N.K. GONATOS, and M. WEISS, Am. J. Path. 44 269-297 (1964). R.D. TERRY, and H. WISNIEWSKI, Alzheimer's Disease and Related Conditions Ed. by G.E.W. Wolstenholme and M. O'Connor, pp. 145-165, Churchill, London ( 1970].
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P. ETIENNE, S. GAUTHIER, G. JOHNSON, B. COLLIER, T. MENDIS, D. DASTOOR, M. COLE, and J.F. MULLER, Lancet I, 508-509 (1978). C.M. SMITH, M. SWASE, A.N. EXTON-SMITH, M.J. PHILLIPS, P.W. OVERSTALL, M.E. PIPER, M.R. BAILEY, Lancet I I 318 (1978). U. DE BONI, and D.R. CRAPPER, Nature 271 566-568 (1978). D.R. CRAPPER, and U. DE BONI, Bio.chemi.str ~ of Dementia, Ed. by P.J. Roberts pp. 53-68, John Wiley and Sons, London (1980). H. MATSUYAMA, Alzheimer's Disease; Senile Dementia and Related Disorders (Aging Vol. 7). Ed. by R. Katzman, R.D. Terry, and K.L. Bick, pp 287-297 Raven Press, New York (1978). D.R. CRAPPER, S.S. KRISHNAN, and A.J. DALTON, Science 180 511-513, (1973). D.R. CRAPPER, S.S. KRISHNAN, and S. QUITTKAT, Brain 99 67-80 (1976). I. KLATZO, H. WISNIEWSKI, and E. STREICHER, Neuropath. Exp. Neurol. 24 187-199 (1965). D.R. CRAPPER, and A.J. DALTON, Physiol. and Behav. 10 935-945, (1973). U. DE BONI, M. SEGER, and D.R. CRAPPER-MCLACHLAN, Neu---rotoxicol. (In press) ( 1980). D.R. CRAPPER, S. KARLIK, and U. DE BONI, Alzheimer's Disease; Senile Dementia and Related Disorders (Aging, Vol. 7), Ed. by R'. Katzman, R.D. Terry, and K.L. Bick, pp 471-485, Raven Press, New York (1978). D.R. CRAPPER, and U. DE BONI, Symposium on the Agin 9 Brain and Senile Dementia, Ed. by K. Nandy, pp 229-246, Plenum Press, New York (1977). D.P. PERL, and A.R. BRODY, J. Neuropath. Exptl. Neurol. 38 335 (Abstract #102) (1979). U. DE BONI, J.W. SCOTT, and D.R.CRAPPER, Histochemistry 40 31-37 (1974). U. DE BONI, A. OTVOS, J.W. SCOTT, and D.R. CRAPPER, Acta Neuropath. (Berl.) 35 285-294 (1976). D.R. CRAPPER, and A.J. DALTON, Physiol. and Behav. 10 925-933 (1973). T.L. PETIT, G.B. BIEDERMAN, and P.A. MCMULLEN, Exper. Neurol. In Press.
Pergamon Press
MINIRL~VILI~: SENILE DEMENTIA AND ALZHEIMER'S DISEASE: A CURRENT VIEW Umberto De Boni* and Donald R. Crapper McLachlan Department of Physiology U n i v e r s i t y of Toronto Toronto, Ontario, M5S 1A8
Summary Senile dementia, of which the most common cause is known as Alzheimer's disease, is a neurological disorder characterized c l i n i c a l l y by memory and learning d e f i c i t s . The disease may be shown to have a prevalence rate in the United States of greater than three per i000 of the general population, and i t may be shown that at any one time 600,000 individuals are affected by advanced stages of this disease. The disease, unique to man, is relentlessly progressive and refractory to treatment. In most cases, i t is unrelated to cerebral vascular insufficiency, i t is however associated with well defined morphological and biochemical changes at the c e l l u l a r level. These include altered dendritic morphology, i n t r a c e l l u l a r changes such as granulovacuolar and n e u r o f i b r i l l a r y degeneration, and extrac e l l u l a r deposits termed neuritic plaques. Moreover, a selective dysfunction in central cholinergic mechanisms has been demonstrated. Recent results from this laboratory have indicated that significant changes in chromatin conformation may be demonstrated to occur in this disease. Specifically, a significant "heterochromatization" was found to occur in chromatin from brains of patients who had died with this disease, suggesting that a major alteration in chromatin structure may be related to the etiology of this condition. In addi t i o n , an accumulation of the environmental agent aluminum upon brain cell chromatin has been implicated to contribute to the pathophysiology of Alzheimer's disease. During the past few years i t has become possible to model several of the morphological markers of this disease in the laboratory. Specifically, the paired helical filament, representing a major i n t r a c e l l u l a r marker can now be induced in cultured neurons and a filamentous hyperplasia similar to neurof i b r i l l a r y degeneration, but not identical in morphology, may be induced in experimental animals and in neurons in v i t r o by trace amounts of aluminum. The understanding of Alzheimer's disease is in i t s infancy, however, the a v a i l a b i l i t y of model systems now permits extensive investigations into the nature and mechanisms of the agents responsible for the changes observed.
Address a l l correspondence
to U. De Boni
0024-3205/80/270001-14502.00/0 Copyright (c) 1980 Pergamon Press Ltd
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Introduction At the present time and with increasing prevalence in our f u t u r e , an i n creasingly common neurological disorder is emerging. This is the problem of dementia. Dementia is a term which denotes a state of altered brain function. Of the many causes of dementia, the most common is Alzheimer's disease, named aft e r Alois Alzheimer, who in 1906 f i r s t described t h i s condition and recognized i t as a separate disease (1). A condition c l i n i c a l l y and p a t h o l o g i c a l l y ident i c a l to senile dementia may occur in the presenile age group, as well as in a l l patients with Down's syndrome who come to autopsy aged over 40 years. These observations support the hypothesis that the degenerative changes found in the brain of the e l d e r l y may be considered to r e s u l t from a disease rather than representing pre-programmed, i n e v i t a b l e consequences of advancing chronological age. We are faced with no known cause and no cure. Therefore, work into the etiology of t h i s mind destroying disease, which a f f e c t s so many of our elderl y , must c e r t a i n l y assume a more prominent place in our research e f f o r t s than that which i t has occupied in the past. Prevalence of senile dementia Senile dementia has t r a d i t i o n a l l y been accepted as an inescapable consequence of advancing age. Moreover, the disease has commonly, and i n c o r r e c t l y , been a t t r i b u t e d s o l e l y to cerebral vascular i n s u f f i c i e n c y . However, close a t t e n t i o n to brain pathology has shown that only 20 to 30% of brains from sen i l e i n d i v i d u a l s show s u f f i c i e n t atheromatous vascular change to account sati s f a c t o r i l y for the c l i n i c a l signs of dementia. Moreover, there is evidence to indicate that the reduction in cerebral blood flow observed in some cases may be occurring secondarily to tissue atrophy. In contrast, 60 to 70% (2) of the e l d e r l y who present with c l i n i c a l signs of dementia, e x h i b i t ' t h e c l a s s i c a l signs of Alzheimer's disease. S t a t i s t i c s for the United States are a v a i l a b l e to show that approximately 4.4% of i n d i v i d u a l s aged 65 years and older, display some form of moderate to severe dementia. This leads to the estimate that at any one time 600,000 advanced cases, or a prevalence rate greater than three per 1000 of the t o t a l population, are affected by Alzheimer's disease (3). This r a t i o w i l l d r a s t i c a l l y increase over the next few decades, as the f r a c t i o n of the population aged 65 years and older increases from the present 11% to 12% by the year 2000 and to a staggering 17% by the year 2030 (4). C l i n i c a l course of Alzheimer's disease In the past, the term Alzheimer's disease had been reserved for cases of presenile dementia, that i s , f o r cases with ages of onset of less than 60 years. However, i t is now recognized that the underlying pathology in both presenile and senile dementia of t h i s type are i d e n t i c a l , and the term Alzheimer's disease is now applied regardless of age of onset. Alzheimer's disease is a global, slowly progressive, f a t a l encephalopathy, the diagnosis of which however cannot be made with c e r t a i n t y in the l i v i n g p a t i e n t s , unless brain tissue from e i t h e r biopsy or autopsy is a v a i l a b l e f o r examination. Few l o n g i t u d i n a l studies of the c l i n i c a l course of this disorder have been reported and no adequate measures of changes in sensory, motor and
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cognitive behaviour e x i s t . However, available longitudinal data suggests z,e following c l i n i c a l course (5): Early stages of this disease are characterized by loss of memory and learning a b i l i t i e s , with preservation of f e e l i n g , perception and movement. In the middle stages, a more pronounced loss of memory is coupled with the appearance of focal neurological signs, such as a l t e r a t i o n s in the acquisition and r e t r i e val of auditory and visual events. In a d d i t i o n , there are signs of spatial and temporal d i s o r i e n t a t i o n , and the a b i l i t y to use speech, language and arithmetic are impaired. In the terminal stage, which occurs on the average 8 years (range 2-19 years) following the appearance of f i r s t symptoms, a profound loss of a l l higher mental functions necessitates continuous nursing care. Histopathology Alzheimer's disease is associated with several brain changes. Gross examination of brains from affected i n d i v i d u a l s frequently reveals pronounced cort i c a l atrophy and d i l a t e d v e n t r i c l e s , i n d i c a t i n g loss of brain substance. Micra scopic examination reveals that the most commonly involved structures are the hippocampus and the more recently evolved neocortical association or polysensory regions of f r o n t a l , p a r i e t a l , temporal and o c c i p i t a l lobes. In these regions, an evident loss of neurons is associated with altered d e n d r i t i c morphology and other histopathological changes, among them granulovacuolar degeneration of hippocampal pyramidal c e l l s , n e u r i t i c (senile) plaques with or without amyloid cores and n e u r o f i b r i l l a r y degeneration (NFD). I.
Altered d e n d r i t i c morphology
Scheibel and Tomiyasu (6) have shown that the d e n d r i t i c tree of c o r t i c a l neurons in brains of senescent i n d i v i d u a l s undergoes progressive, degenerative changes. These changes include loss of d e n d r i t i c spines, dying back of dendrites and swelling of neuronal somata. Moreover, neurons from patients with presenile dementia of the Alzheimer type were characterized by the presence of haphazardly placed clusters of new d e n d r i t i c growth, at a time when the o r i g i n a l dendrites were devoid of t h e i r normal spine complement. Due to the aberrant d i s t r i b u t i o n and o r i e n t a t i o n of these newly sprouted dendrites, not coinciding with any presynaptic terminal f i e l d s , t h i s new outgrowth is referred to as"lawless". These findings are supported by the results of a more recent, similar study of morphological changes in hippocampal neurons in ageing humans (7). In addition however, these workers showed evidence to indicate the presence of " p l a s t i c i t y " in the ageing human brain. This was demonstrated by the presence of s i g n i f i c a n t increases, in non demented e l d e r l y individuals (mean age: 79.6 y r s . ) , in the number and average length of terminal segments of the d e n d r i t i c tree, compared to adult individuals (mean age =51.2 y r s . ) . In cases of senile dementia (mean age =76.0 y r s . ) , d e n d r i t i c trees were less extensive than in cont r o l , adult brain, as a r e s u l t of fewer and shorter terminal segments. II.
Granulovacuolar degeneration
This degeneration, occurring mainly in hippocampal pyramidal c e l l s and in the medial temporal gyri', was f i r s t described by Simchowicz (8) in cases of senile dementia of the Alzheimer type. I t consists of one or more unstained, spherical vacuoles, 3 to 5 u in diameter and containing at the centre an argyrop h i l i c and hematoxilinophilic granule. U l t r a s t r u c t u r a l analysis (9,10,11) reveals l i t t l e more; the vacuoles consisting of a membrane bound, electron lucent p a r t i c l e with a c e n t r a l , electron-dense core. Although the o r i g i n and functional consequences of these intraneuronal p a r t i c l e s are unknown at this time, i t
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~J
Figure 1 A. Normal, pyramidal shaped neurons in cerebral cortex of human brain. B. Granulovacuolar degeneration (arrow head) in pyramidal neuron of hippocampus in patient with Alzheimer's disease. C. Three neuritic plaques adjacent to granule cell layer ( l e f t ) in hippocampus of patient with Alzheimer's disease. Note core in largest plaque. D. Low power electron micrograph of neuritic plaque, with electrondense inclusions. E. Higher power electron micrograph of plaque. Note paired helical filaments among electrondense debris. A, B, C: Bielchowski stain. D, E: Glutaraldehyde/osmium preparation. Magnification: A,C: 300 X. B: 700 X. D: 11250 X. E: 37500 X.
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• ay be shown that both the incidence and degree of granulovacuolar degeneration are s i g n i f i c a n t l y higher in brains of demented i n d i v i d u a l s , and especially in those with the histopathological diagnosis of Alzheimer's disease (12,13,14). Moreover, a highly s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n could be demonstrated between the severity of granulovacuolar change, in p a r t i c u l a r in the posterior hippocampus, and the severity of n e u r o f i b r i l l a r y degeneration (see below) (15). This c o r r e l a t i o n was quite d i s t i n c t from that showing the increase of granulovacuolar change as a function of age~ In the absence of an experimental model for the induction of granulovacuolar change, this c h a r a c t e r i s t i c cytological change has been l a r g e l y ignored, and at the present time no hypothesis for induction is available. III.
N e u r i t i c (senile) plaques
These terms describe an extremely common microscopic, spherical accumulation of c e l l u l a r and e x t r a c e l l u l a r debris, often containing amyloid cores and frequently surrounded by reactive c e l l s . U l t r a s t r u c t u r a l examination reveals that such plaques are composed of degenerating neuronal processes and t h e i r cytoplasmic components, including the f i b r i n o u s , aberrant protein characterist i c of n e u r o f i b r i l l a r y degeneration ~see below) (16,17). I t is of i n t e r e s t to note that n e u r i t i c plaques are seen in a v a r i e t y of organic brain diseases of man and animals, sometimes associated with v i r a l e t i o l o g i e s (18,19). This has supported the hypothesis, long held by several i n v e s t i g a t o r s , that a unique, v i r a l - l i k e agent may be involved in the pathogenesis of Alzheimer's disease. Plaques with amyloid cores, resembling, but not identical to, those in senile dementia of the Alzheimer type occur in mouse brain following the inoculation of scrapie agent and may occur in human brain in patients with Kuru and JakobCreutzfeld disease (20,21). The transmissible factors which cause these infectious, spongiform degenerative brain diseases, have not been defined. These agents are atypical in that they remain stable at temperatures up to 80°C and that i n a c t i v a t i o n may not be complete at IO0°C (21). In addition i t is noteworthy that these agents under certain conditions, may show a remarkable resistance to u l t r a v i o l e t i r r a d i a t i o n and, with ionizing radiation (neutron beam) reveal an unusually small i n a c t i v a t i o n target size (23,24,25,26,27). Recent work (28) has indicated that the agent of scrapie may be as small as 40S (S2o,W) and that i t may contain a hydrophobic surface,explaining some of the unusual features observed. The s t a b i l i t y to heating is consistent with this view, as high temperatures s t a b i l i z e hydrophobic i n t e r a c t i o n s . Moreover, the hydrophobic properties may be associated with the observed association of scrapie agent with membrane f r a c t i o n s , since hydrophobic components may r e a d i l y be inserted into membranes. I f freed from associated membranes, the i n f e c t i v i t y of the agent may be shown to be s i g n i f i c a n t l y reduced by RNAse. Moreover, a t e n - f o l d increase in binding to hydroxyapatite and a greater i n a c t i v a t i o n by DNAse may be demonstrated following the pretreatment of the agent with proteinase K. This data is considered to indicate that the scrapie agent may contain low molecular weight, single stranded DNA of approximately 2000 nucleotides. The observed heat s t a b i l i t y f u r t h e r supports the p o s s i b i l i t y that the genetic information is contained in single stranded DNA. There is no d i r e c t evidence to indicate that a transmissible agent capable of the induction of n e u r i t i c plaques exists in Alzheimer's disease. Specifica l l y , unpublished work from this laboratory over the past 7 years has f a i l e d to induce Alzheimer changes in laboratory animals. Saline extracts of Alzheimer affected brains, alone and in combination with aluminum chloride or l a c t a t e , have f a i l e d to induce human Alzheimer type pathology in monkeys (Rhesus macaqu~ Saimiri sciureus), guinea pigs, rabbits and cats. Nevertheless, while none of these observations implicate v i r u s - l i k e mechanisms in the pathogenesis of Alzheimer's disease, the evidence available should stimulate the search for existence of such an agent.
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% ,,
i!!i~ ¸
Figure 2 A. Neuron ( r i g h t ) with perinuclear aggregate of argentophilic n e u r o f i b r i l l a r y d e generation. Normal neuron at l e f t . B. Ultrastructure of normal, human neuropil, with abundant microtubules in neuronal cytoplasma ( * ) . C. Ulstrastructural appearance of aggregate of paired helical filaments in cerebral cortical neuron of patient with Alzheimer's disease. Electron-dense p a r t i c l e (arrow head) is lipofuscin or "age pigment". D. Higher power micrograph of paired helical f i l a ments characteristic of Alzheimer's disease. Note regular period of approximately 800 A° . A: Bielchowski stain. B,C,D: Glutaraldehyde/osmium preparation. Magnification: A: 700 X. B: 8000 X. C: 19000 X. D: 94000 X.
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I
Figure 3 Micrographs representative of the model system employed in the study of senile dementia. A. Phase contrast micrograph of living, human neuron with abundant neurites, 25 days in vitro. B. Ultrastructural appearance of human brain cell, 28 days in vitro. Synaptic contact (arrow head) identifies cell as neuron. C. Ultrastructure of neuropil representative of the culture system employed, 28 days in vitro. Asterisks identify synaptic contacts. Note similarity to human neuropil in vivo, Fig. 2 B. D. Examples of paired helical filaments induced in cultured human neurons, closely similar to the PHF's characteristics of Alzheimer's disease, shown in Fig. 2 D. A: Unstained, living preparation. B, C, D: Glutaraldehyde/osmium preparation. Magnificiation: A: 300 X. B,C: 8000 X. D: 56000 X.
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N e u r o f i b r i l l a r y degeneration (NFD)
Alois Alzheimer f i r s t described as NFD the a r g e n t o p h i l i c aggregates of n e u r o f i b r i l s in neurons of a patient with senile dementia (1). Modern a n a l y s i s , by electronmicroscopy, of brain tissue from senile i n d i v i d u a l s has revealed that NFD represents an intraneuronal accumulation of arrays of so called "paired h e l i c a l filaments" (PHF's), structures composed of two 100 A° diameter, solid filaments forming a h e l i c a l structure with an average period of 800 A° (29,30). This structure is unique to neurons of man, and is not known to occur in animal brain diseases. I t is now however recognized that PHF's may be encountered in human cerebral neurons in several conditions, with apparently d i f f e r e n t e t i o l o gical factors. As such, NFD may be associated with repeated head trauma,resul~ ing in dementia p u g i l i s t i c a , and in the l i p i d storage diseases named a f t e r Kuf and Neiman-Pick (31). Moreover, NFD may be associated with the delayed expression of a conventional v i r a l i n f e c t i o n . In p a r t i c u l a r , NFD may occur in brain stem nuclei, s p e c i f i c a l l y in the substantia nigra, several years a f t e r epidemic encephalitis lethargica (32). In a d d i t i o n , neurons of the brainstem of patients with Parkinson's disease with a previous h i s t o r y of v i r a l encephal i t i s , may display NFD (33). Viral e n c e p h a l i t i s may also be accompanied by NFD in brain of young patients with progressive multi focal leukoencephalopathy (34) and subacute sclerosing panencephalitis (35). While the former condition appears to be related to the unsuppressed p r o l i f e r a t i o n of an otherwise benign v i r u s , subacute sclerosing panencephalitis appears to be caused by the apparent incomplete suppression of a measles-like paramyxo virus permitting slow and chronic r e p l i c a t i o n of the agent. PHF formation also occurs in spinal cord in Guam amyotrophic l a t e r a l sclerosis cases (33) and in a s i m i l a r condition found in certain regions in the Kii peninsula of Japan (36,37). Moreover, as recentl y reviewed (38), NFD may be associated with a large number of additional d i s eases. Whether these various conditions permit the expression of a class of c l o s e l y related agents to form PHF's or whether PHF's represent a non-specific morphological response of human neurons to a diverse group of i n s u l t s , is unknown at present. Various subsets of neuronal types may e x h i b i t quite d i f f e r ent s u s c e p t i b i l i t i e s to the formation of PHF's. While some neurons, such as cerebellar Purkinje c e l l s , dorsal root ganglion c e l l s and primary sensory nuclei in the brain stem remain r e f r a c t o r y , large pyramidal shaped neurons in the hippocampus and in cerebral cortex commonly e x h i b i t NFD in Alzheimer's disease. I t is remarkable that the neurons with NFD f a i l to e x h i b i t morphological evidence of a response to control the p r o l i f e r a t i o n of PHF m a t e r i a l . This appears to indicate that there may e x i s t a s u f f i c i e n t s i m i l a r i t y between normal structural proteins and PHF's so that the appropriate p r o t e o l y t i c processes f a i l to respond. A l t e r n a t i v e l y , the PHF's could accumulate as a r e s u l t of a disorder in the system regulating the production of catabolism of neuronal f i brinous proteins. The chemical and i mmunohistochemical characterization of PHF proteins of Alzheimer's disease is complicated by the f a c t that at the time of death, only i in I00 to I in 10 neurons show NFD. Therefore. contamination of the isolated f r a c t i o n s with associated c e l l u l a r debris represents a major problem. Analysis of a neuronal f r a c t i o n enriched with neurons containing PHF's (39) showed the presence of a polypeptide band of 50,000 Daltons, a band c l o s e l y resembling the peptides derived from g l i a l f i b e r s . Since a g l i o s i s often accompanies Alzheimer's disease, i t is possible that g l i a l filaments may account for the protein p r o f i l e s . These same workers have also shown that microtubules, p u r i f i e d by two cycles of polymerization, may share immunoreactive sites with certain immunoreactive s i t e s on the n e u r o f i b r i l l a r y material from Alzheimer's d i sease (41). However, recent evidence is a v a i l a b l e (40) to show that recycled tubulin may contain subunits of 100 A° intermediate filaments with a peptide
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composition s i m i l a r to the 68,000 molecular weight subunits of neurofilaments. I t is quite conceivable therefore that this f r a c t i o n might be present in the material employed as an antigen in the studies by Grundke-lqbal (41). Additional evidence, based upon immunofluorescence has recently been obtained (42) to indicate that normal neurofilaments as well as the filaments comprising the Alzheimer NFD, and the filaments induced by aluminum (see below) may be a n t i genically related. Employing r a b b i t anti-chicken-neurofilaments as antiserum, i t was possible to show thatneurofilaments in axons, the filaments of Alzheimer's disease and the aluminum induced filaments a l l stained i n t e n s i v e l y . Although the absolute p u r i t y of the o r i g i n a l antigens is uncertain, there exists considerable s p e c i f i c i t y , as demonstrated by persistence of reaction with neurofilaments, following adsorption of the antineurofilament serum with tubulin. Much work is c u r r e n t l y carried out to define the I00 A° diameter, intermediate filaments of normal c e l l s and the f i b r i n o u s proteins comprising the Alzheimer n e u r o f i b r i l l a r y tangle. However, before such d e f i n i t i o n s may be successfully concluded, methods must be developed to prepare these subcellular f r a c t i o n s free of other cytoplasmic contaminants. V.
Altered chromatin conformation
We have recently presented evidence (43) that chromatin isolated from brains of patients with senile dementia of the Alzheimer type, has undergone "heterochromatization". Chromatin f r a c t i o n s isolated by c e n t r i f u g a t i o n following disruption of brain cell nuclei yielded three f r a c t i o n s ; heavy or heterochromatin, not transcribed in v i t r o , and two l i g h t f r a c t i o n s ; intermediate euchromatin and l i g h t euchromatin. Results of a study involving 21 nuclear preparations from cerebral cortex of 12 controls aged 2 to 69 years showed that 75.2 ±5.5 per cent of the DNA was found in the euchromatin f r a c t i o n . In cont r a s t , in 25 preparations from 12 brains with advanced Alzheimer's disease, the f r a c t i o n of DNA in the euchromatin varied between 78 and 25 per cent, with an average value of 5 5 . 6 ± 1 6 . 8 per cent. This observed increase in the proportion of heterochromatin in advanced Alzheimer's disease was independent of chromatin protein content, age, g l i o s i s and post-mortem events. Moreover, the analysis of f r a c t i o n s enriched in neurons and g l i a l c e l l s r e s p e c t i v e l y showed that this "heterochromatization" occurred both in neurons and g l i a l c e l l s . Together with the results of a t r a n s c r i p t i o n study carried out on these fractions, the findings indicate that a smaller proportion of genetic information is available f o r t r a n s c r i p t i o n in brain of affected i n d i v i d u a l s , and that a major a l t e r a t i o n in protein synthesis may be associated with dementia of the Alzheimer type. VI. Biochemistry of cerebral cortex in Alzheimer's disease The analysis of the chemical pathology of senile dementia in man is fraught with d i f f i c u l t i e s a r i s i n g from secondary g l i a l reaction, changes related to age and not dementia, agonal or terminal pre-mortemevents and post-mortemchan~es. Nevertheless by employing careful handling of biopsy and autopsy samples, s i g n i f i cant biochemical changes may be i d e n t i f i e d (44,45). Results from an early study (46) employing biopsied brain from 3 patients with Alzheimer's disease showed that r e s p i r a t o r y rates and l a c t i c acid production as well as glucose u t i l i z a t i o n were within normal l i m i t s . Moreover, while total protein was reduced compared to controls, the incorporation of labelled lysine into protein of a microsomal f r a c t i o n occurred at normal rates. In contrast, another study showed(47) that the a c t i v i t i e s of 2 phosphatases in biopsied brain from patients with large numbers of neurons with NFDandneuritic plaques were sign i f i c a n t l y increased while changes measured in oxidative enzyme a c t i v i t i e s were a t t r i b u t e d to degeneration. I t is important to note that the increasedphosphatase a c t i v i t y was found associated with regions o f n e u r o f i b r i l l a r y d e g e n e r a t i o n ,
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and could not be demonstrated in unaffected neurons in this study. The original observation that choline acetyl transferase is s e l e c t i v e l y d e pressed in about two thirds of autopsy and biopsy samples from patients with pre-senile or senile dementia (48) has focused much a t t e n t i o n on the possible dysfunction of cholinergic projections in this condition. Several studies have since confirmed that the a c t i v i t i e s of choline acetyl transferase and acetyl choline esterase may be reduced over 50% in Alzheimer's disease. Moreover, i t has been possible to show that these reductions are p a r a l l e l l e d by increasing morphological changes, which in turn correlate with dementia scores (49,50,51). Moreover, available evidence indicates that the presynaptic part of the cholinergic system might be s e l e c t i v e l y affected in Alzheimer's disease. Attempts to modulate memory performance in Alzheimer's disease by raising plasma choline levels or by the use of antichol~nesterases have been met with l i t t l e success. The only anticholinesterase r e a d i l y crossing the blood brain b a r r i e r , physostigmine, has only a short duration of action, and exerts pronounced parasympathomimetic e f f e c t . Moreover,as recently reviewed (53) the use of d i e t a r t choline or i t s precursor l e c i t h i n (53) to raise plasma choline l e v e l s , has resulted in l i t t l e or no improvement in performance. Etienne et al. (54) reported no improvement in several patients with Alzheimer's disease following t h e i r t r e a t ment for i month with up to 8 g of choline bitamtrate per day, and no changes in cognitive performance occurred in ten Alzheimer patients treated daily for 2 weeks with 9 g choline b i t a r t r a t e (55). S i m i l a r l y , improvement in performance was limited to a f r a c t i o n of the individuals tested in a study employing dietary l e c i t h i n (53). In summary, i t is evident that preliminary t r i a l s of long term choline treatment of patients with senile dementia of the Alzheimer type have f a i l e d to s i g n i f i c a n t l y improve t h e i r performance or to ameliorate t h e i r condition. Possible etiology of Alzheimer's disease and model systems employed Nothing is known concerning the factors which i n i t i a t e any of the brain changes occurring in Alzheimer's disease. Available evidence indicates however that NFD may be associated with conventional v i r a l i n f e c t i o n s and that n e u r i t i c plaques may be found associated with i n f e c t i o n s by the unconventional agents causing Kuru and Jakob-Creutzfeld disease. Therefore a search for mechanisms or agents present in brain affected by Alzheimer's disease becomes mandatory. Such investigations have resulted in the i d e n t i f i c a t i o n of models suitable f o r the i n v e s t i g a t i o n of at least some of the subsets of the changes occurring in brain affected by Alzheimer's disease. While to date no experimental model f o r granulovacuolar degeneration e x i s t s , both the n e u r i t i c plaque and the PHF, as well as the p r o l i f e r a t i o n of neuronal filamentous, structural proteins, may now be manipulated in model systems in the laboratory. The primary pathogenic event may be the r e s u l t of e i t h e r endogenous gene f a i l u r e or may be i n i t i a t e d by an as of yet u n i d e n t i f i e d agent foreign to the human brain. Based upon these assumptions, three hypothesis concerning the etiology of Alzheimer's disease may be formulated, and include: I. A reduction of the amount of RNA produced, by a pathogenic event operating in the nucleus, 2. A primary event r e s u l t i n g in the assembly of PHF's with a concomi t t e n t reduction in the number of microtubules and secondarily r e s u l t i n g in a reduction in dendroplasmic transport, and 3. a defect in the blood b a r r i e r permitting the accumulation within brain parenchyma of p o t e n t i a l l y toxic environmental compounds, including trace metals.
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The f i r s t hypothesis, related to altered function w i t h i n the c e l l nucleus, is supported by the f i n d i n g of altered chromatin conformation, as described above. Therefore, future exploration of mechanisms which control and regulate gene expression and chromatin conformation w i l l y i e l d new insights into the altered brain metabolism underlying Alzheimer's disease. In evaluating the second hypothesis, and considering the large number of pathological i n s u l t s which are associated with NFD (38), the p o s s i b i l i t y must be considered that the primary pathogenic event releases a mechanisms or agent capable of assembling PHF's. We have r e c e n t l y shown that brain affected by Alzheimer's disease may contain such a f a c t o r and i t s properties are c u r r e n t l y under study (56,57). Employing several types of e x t r a c t of Alzheimer affected brain i t has been possible to r o u t i n e l y induce PHF's in v i t r o , in f e t a l human cerebral c o r t i c a l neurons, in neurons of human f e t a l spinal cord and in r a b b i t cerebral cortex in v i t r o . In a d d i t i o n , we have recently i d e n t i f i e d that the PHF assembly f a c t o r may also be present in cerebral spinal f l u i d (CSF) of each of three patients with a c l i n i c a l diagnosis of Alzheimer's disease. In cont r a s t , CSF from a p a t i e n t with a dementia associated with progressive supranuclear palsy and from 2 patients withspondylosis without evidence of dementia f a i l e d to induce PHF's in the cultured c e l l s . Since PHF's occur in neurons in many human brain diseases in a l l age groups (38) and in many older i n d i v i d u a l s without overt signs of dementia (58) a brain e x t r a c t was prepared from a 33 year old n e u r o l o g i c a l l y normal p a t i e n t , who had died acutely of myocardial disease. While no PHF's were encountered in 8 out of 9 explant cultures t r e a t ed with t h i s e x t r a c t , I single PHF was encountered in one c u l t u r e . This observation raises the p o s s i b i l i t y that the agent responsible may be present in low t i t r e in apparently normal brain. To i d e n t i f y more exactly the nature of the agent, brain extracts were also subjected to a series of s p e c i f i c t r e a t ments, and subsequently assayed f o r t h e i r a b i l i t y to induce PHF in cultured neurons (57). Information derived from these experiments to date indicates that the f a c t o r is associated with a p a r t i c l e size of 40S and above. Moreover, the f a c t o r appears s e n s i t i v e to UV l i g h t of 2 x 10 s ergs/mm2, and does not lose the a b i l i t y to induce PHF's following exposure to DNAse I and RNAse. However, extracts exposed to trypsin f a i l to induce PHF's. The r e s u l t s indicate that Alzheimer affected brain may contain a f a c t o r which is capable of inducing PHF's in cultured neurons, a factor with properties d i s t i n c t l y d i f f e r e n t from those of a conventional virus or unconventional virus of the scrapie type. I n s u f f i c i e n t evidence is a v a i l a b l e to assess whether the f a c t o r contains nucleic acid. The t h i r d a l t e r n a t i v e concerns the hypothesis that the primary i n s u l t a l ters the blood brain and cytoplasmic b a r r i e r s , and as a secondary consequence a l t e r s b a r r i e r s to p o t e n t i a l l y toxic environmental substances. Among these, accumulated evidence strongly implicates aluminum as a toxic f a c t o r in senile brain disease; aluminum has been demonstrated to occur in brain of patients with Alzheimer's disease at increased concentrations, compared to age matched controls (59,60). In these cases, the aluminum concentration in some brain regions approached that which induces an over-production of 100 A° diameter filaments in susceptible neurons of experimental animals (61,62). Moreover, at t h i s same concentration, a class of human c o r t i c a l neurons exposed to aluminum in tissue c u l t u r e , respond with a filamentous hyperplasia of s i n g l e , unpaired filaments, of 100 A° diameter (63,64). The i n t r a c e l l u l a r locus of aluminum accumulation is nuclear chromatin, and i t has been shown that aluminum is associated with the nucleus both in brain c e l l s of man with Alzheimer's disease (65,66) and in experimental animals with an aluminum induced encephalopathy (67,68). Moreover, recent evidence derived from studies employing the s e n s i t i v e probes f o r DNA damage, sisterchromatid exchanges in cultured human lymphocytes and unscheduled DNA synthesis in human
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astrocytes in v i t r o , has c l e a r l y shown that chromatin bound aluminum interacts f u n c t i o n a l l y with i t s binding s i t e s , and may damage DNA (63). Work in our laboratory employing thermal denaturation and related techniques, has also established (57,64) that aluminum in v i t r o i n t e r a c t s with DNA in a complex manner, in part depending upon i n t e r a c t i o n with hydrating water molecules and dependent upon pH and concentration. The data is consistent with the existence of two forms of aluminum, forming at least two types of complexes with DNA. Aluminum does not induce the PHF's nor the typical n e u r i t i c plaque usually seen in dementia of the Alzheimer type. Nevertheless, through the filamentous hyperplasia associated with the presence of this element in brain of experimental animals, along with the associated behavioural changes (69,70), the aluminum induced encephalopathy provides a unique model of a dementia process. Moreover, based upon the established n e u r o t o x i c i t y of this element, i t s presence in the brain of man may well represent a deleterious compounding factor in the pathophysiology of Alzheimer's disease, and must therefore be considered to be of consequence. In conclusion, i t is apparent that Alzheimer's disease, which affects so many of our e l d e r l y , is poorly understood. Moreover, the factors c o n t r o l l i n g i t s expression are obscure, and there can be no doubt that the understanding and ultimate treatment of this mind destroying disease w i l l require the a p p l i cation of the most sophisticated s c i e n t i f i c thoughts and techniques. Acknowled~ement_s The work described here is supported by the Medical Research Council of Canada, the Ontario Mental Health Foundation, and the Canadian G e r i a t r i c Research Society. Special thanks are extended for the superb cooperation of the o b s t e t r i c ' s group at the Toronto Western Hospital. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
A. ALZHEIMER, Allg. Z. Psychiat. 64146-148 (1907). C.E. WELLS, Stroke 9 1 - 3 (1978). - R.D. TERRY, Arch. Neurol. 3 3 1 - 4 (1976). F. PLUM, Nature 279372-37-3-(1979). J. PEARCE, and J.M. SCHOFIELD, Clinical Aspects of Dementia, B a i l l i e r e Tindal, London (1979). A.B. SCHEIBEL, and U. TOMIYASU, Exp. Neurol. 6 0 1 - 8 (1978). S.J. BUELL, and P.D. COLEMAN, Science 206854-~56 (1979). T. SIMCHOWICZ, •Histologie und Histopathologische Arbeiten ueber die Grosshirnrinde. Ed. by F. Nissl and A. Alzheimer, pp. 267-444, Fischer Verlag, Jena #1911). W.G. ELLIS, J.R. MCULLOUGH, and C.L. CORELY, Neurology 24101-106 (1974). A. HIRANO, H.M. DEBITZER, L.T. KURLAND, and H.M. ZIMMERMAN, J.Neuropath. Exp. Neuroi~27167-182 (1968). R.D. TERRY, an~ H.M. WISNIEWSKI, Advances in Behavioral Biology, Vol. 3. "Ageing and the Brain", Ed. by C.M. Graitz, pp. 89-116, Plenum Press, New York (1972). B.E. TOMLINSON, G. BLESSED, and M. ROTH, J. Neurol. Sci. 7331-356 (1968). B.E. TOMLINSON, G. BLESSED, and M. ROTH, J. Neurol. Sci. 11205-242 (1970). M.J. BALL, Acta Neuropath. ( B e r l . ) 4_2273-80 (1978). M.J. BALL, and P. LO, J. Neuropath. and Explt. Neurol. 36474-487 (1977). R.D. TERRY, N.K. GONATOS, and M. WEISS, Am. J. Path. 44 269-297 (1964). R.D. TERRY, and H. WISNIEWSKI, Alzheimer's Disease and Related Conditions Ed. by G.E.W. Wolstenholme and M. O'Connor, pp. 145-165, Churchill, London ( 1970].
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18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
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