Role of APOE in Alzheimer's disease-like neurodegeneration

S138

)621) Lit&q

S~vmposium: Apolipoprotein

CANDIDATE EASE

GENES

ASSOCIATED

WITH

ALZHEIMER

DIS-

A Fan-w. Boston University School of Medicine, Boston, MA

The discovery of an association between APOE genotype and risk of Alzheimer disease (AD) in 1993 and the subsequent confirmation of thi5 finding in a large number of diverse populations revolutionized the thinking about the genetic basis of the disorder. Until that time, it was widely held that pathogenic mutations cause AD only in rare families segregating a form of the disorder as an autosomal dominant trait manifesting before the age of 65 years. Genetic modeling studies suggest that AD risk is governed by a combination of multiple susceptibility genes and other factors. It is estimated that APOE ~4 accounts for approximately 50% of the genetic variance for AD in Caucasian populations, but much less in other groups including African Americans. Several studies including a large meta analysis of pooled primary data indicate that the effect of ~4 varies with age and sex. For the most pan, initial reports of association between AD and other candidate susceptibility genes have not been replicated satisfactorily in independent samples. Disagreement across studies can be attributed to several factors namely diagnostic misclassification (particularly among controls), genetic heterogeneity, confounding or interaction with other factors, and population stratification between patient and control samples. Recent studies suggest that the effect of APOE genotype may be modified by polymorphisms located in the APOE promoter region. Two biologically plausible candidates, a-2.macroglobuhn and LRP receptor genes, map to a region of chromosome 12 showing linkage in multiplex families of late-onset AD, however they appear to have at most a weak connection to inherited susceptibility to AD. Hypothesis- driven approaches have focused attention on genes involved in oxidative metabolism and the vascular system. At present the most promising of these candidates is the angiotensin converting enzyme (ACE) gene. Evidence from diverse populations implicates an insertiondeletion polymorphism in ACE as a modulator of disease risk, although there is disagreement across studies about the pattern of association. These studies highlight the importance of replicating gene associations in independent samples regardless of the statistical significance obtatned from any given ample.

A FULL 1622) DISEASE.

GENOME

SCAN

Michael Owen. University of Wales Collqe Kingdom

FOR LATE

ONSET

ALZHEIMERS

of Medicine, Cardi& CFl4 4XN United

M. .I. Owen’. F. WavrantDeVrieze’. R. Crooke2, P.G. Kehor’. W. S. Wu-‘, A. My&. 1. Fenton’, F. Rice’. A. Wood’, P. Holmmw’, S. Lowstone’, N. Tunstal14, J. PerezTu?, M. Hutton’, S. Shears-‘, K. Rorhl.‘, .I.Booth.‘, J. Williams’, J.Hardy2, A. Goatc’. I) Neuropsychiatric Genetics Unit, Tenovus Building. University of Wales College of Medicine, Heath Park, Cardifi CF4 4XN, UK: Dept of Pswhologicul Medicine, University of’ Wales College of Medicine, Cardlfi S Clam, Wales; 2) Birdsall Building, Muyo Clinic. 4500 San Pablo Road, Jacksonville, FL32084. USA; 3) Department of Psych&y and Genetics, Wushington University School of Medicine, 490 Childrens Place, St Louis, MO 63110. USA: 4) Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SES BAF, UK. We have genotyped 514 affected sibling pairs (ASPS) with probable/definite Alzheimers disease (AD) according to NINCDS-ADRDA diagnostic criteria with onsets above 65 years. The genome scan comprised two stages. In the first 292 ASPS were genotyped using 237 microsatellite markers separated by an average distance of 16.3cM. 16 peaks with a multipoint lad score (MLS) greater than I either in the whole sample, the e4 positive or negative subgroups were observed on chromosomes I (two peaks) 2, 5, 6, 9 (two peaks), IO (two peaks), 12, 13, 14, 19, 21 and X (two peaks). Simulation studies revealed that these findings exceeded those expected by chance, although many are likely to be false positives. The highest lod scores on chromosomes I (MLS 2.67), 9 (MLS 2.38), IO (MLS 2.27) and 19 (MLS 1.79) fuliil Lander and Kmglyak F definition of suggestive linkage In stage two we genotyped the I6 regions showing an MLS of greater than I in an independent sample of 222 ASPS. using the same markers as in stage one. Of these regions two now yield lod scores in excess of 3 and 4 give lads between 2 and 3. These data will be presented in detail.

E and Alzheimer’s

Disease

Virtually all individuals with DS have neuropathological changes consistent with a diagnosis of AD by the time they reach 40 years of age, including deposition of P-amyloid in diffuse and neuritic plaques. While most adults with DS will develop dementia by the end of the seventh decade, the average age at onset is between 50 and 55 years, with a range from 38 to 70. Hence there is a IO-30 year discordance between the presence of neuropathology and the onset of clinical dementia. The neuropathological manifestations of AD in DS have been attributed to tripication and overexpression of the gene for beta-amyloid precursor protein (APP), located on chromosome 21, but the factors influencing age at onset of dementia are unresolved. It ha\ been proposed that clinical dementia is initiated by the transition from diffuse to neuritic plaques. Studies of factors that may influence age at onset of AD by increasing P-amyloid load or accelerating the accumulation of fibrillar aggregates of amyloid p protein and neutitic plaques will be reviewed. Factors that increase or reflect the rate of P-amyloid aggregation and deposition, such as the apolipoprotein E (APOE)-~4 allele, amyloid p protein species ApI-42, male gender. and estrogen deficiency in women-indicated by age at onset of menopause- are associated with earlier onset of dementia in DS. In contrast, the presence of an APOEallele has consistently been associated with improved survival and reduced risk for AD. Atypical karyotypes leading to reduced APP dose (e.g., mosaicism for trisomy 2l), normal APP gene number (e.g.,in partial trisomy 21 )or unusal allelic variabiltty at locus D2lSl I promote longevity without evidence of cogmtive decline. Thew findings point to the importance of amyloid a protein in the development of AD.

Symposium:

16241APOE:

Apolipoprotein

E and Alzheimer’s

Disease

BIOLOGY AND BIOCHEMISTRY

Karl H. Weisgraber, Gladstone Institute oj’Neumlogiru1 Diseuse, San Francicm

CA

A protein’s structue and biophysical properties are intimately related to function. The common isoforms of apolipoprotein (ape) E have differential effects in Alzheimer’a disease (AD) and on neutite outgrowth in cell culture. To gain insight into the basis for these effects, we have examined the structure and biophysical properties of the isoforms. X-ray crystallography and site-directed mutagenesis studies showed that in apoE4, which has arginine at position 112, Arg-61 in the amino-terminal domain interacts with Glu-255 in the carboxyl-terminal domain. This domain interaction does not occur in apoE or apoE (both have cyst&e at 112) because Arg-61 ia in a different conformation. We hypothesize that domain interaction contributes to the increased risk of AD associated with apoE4. Several species, including the mouse. contain equivalents of Arg-I I2 and Glu-255, but lack the critical Arg-6lrequired for domain interaction in human apoE4, suggesting that introducing an Arg-61 codon into the Apoe gene. might “humanize” mouse apoE to mimic apoE4. We have generated targeted Arg-61 knock-in mice, which are now being characterized at the biochemical, morphological. and behavioral level, particularly with respect to alterations observed rn AD. ApoE isofonns also differ in their stability and lipid binding properties. ApoE is the most stable and apoE% the least stable, whereas apoE is the best lipid binder, suggesting the isoforms differ in their ability to transport lipids. Denaturation studies revealed that apoE forms a stable folding intermediate, which is a prominent species at low pH. whereas apoE and apoE do so to a lesser extent. Since folding intermediates have been implicated in membrane translocation and cellular processing of proteins, it is possible that apoE is differentially processed by cells and that some may escape lysosomal degradation and tramlocate into the cytoplasm following internalim.tion. Domain interaction and differences in hpid binding or cellular processing may underlie the differential effect of the apoE isoforma on neurite outgrowth and AD.

ROLE OF APOE NEURODEGENERATION

IN

ALZHEIMER’S

DISEASE-LIKE

DOWN

David M Holtzman. Anne M Fagan, Washington University, St. Louis. MO; Kelly R. Bales, Eli Lil/y and Company, Indianapoli.~, IN: Ronald DrMattm. Washington University. St. Louis, MO: Michael C Irizmy, Mass General Hospital, Boston, MA; Karen H Ashe, Universi~ of Minnesota, Minneapolis, MN; Bradley T Hymn, Mass General Hospital, Boston, MA: Steven M. Paul, Eli Lilly and Company, Indianapolis, IN

Down syndrome(DS), defined cytogenetically by trisomy 21, is the most common chromosomal disorder associated with mental retardation. Older indivdiuals with DS show age-related changes in health and functional capacities suggestive of premature or accelerated aging. The most extensively studied aspect of aging in this population is the unique association between DS and Alzheimer’s disease(AD), and it has been suggested that DS may serve as a model for the study of pathogenic factors in AD.

The ~4 allele of apolipoprotein E (apoE) is an important genetic risk factor for Alzheimer’s disease (AD) as well as cerebral amyloid angiopathy (CAA). Increasing evidence suggests that one of the major reasons for this association is related to the ability of apoE to interact with the amylaid-! (AP) peptide and influence its concentration and structure. To determine the effect of apoE on A!3 deposition and AD as well as CAA-related pathology, we have analyzed 2 different amyloid precursor protein (APP) transgenic mice, APPsw and APPy7”‘, expressing human apoE isoforms, mouse apoE, or no apoE (apoE -/-). In both APPsw and APP”“” mice, A@ deposition was attenuated but still occurred in the absence of apoE. In

16231EPIDEMIOLOGY SYNDROME

OF

AGING

AND

DEMENTIA

IN

Nicolr Schupf NYS Institute fh- Basic Research, Staten Islund, NY

Symposium:

Apolipoprotein

E and Alzheimer’s

Disease

contrast, fibrillar A@ was never seen in the absence of apoE in the brain parenchyma of either APPsw or APPV7’7r mice through 15 months of age. CAA, which only occurs in APPsw but not APP”“” mice, was blocked by removing apoE. Importantly, despite AP deposition in the absence of apoE, neuritic degeneration (i.e. neuritic plaques) only developed when AP deposition occurred in the presence of apoE. Interestingly, the expression of human apoE &forms in the brain of both APPsw and APPv7’7” mice suppressed early AP deposition for several months. Despite this, AP deposition eventually developed in APPY7’7F, human apoE-positive rmce by 15 months of age. Further, the AP was fibrillar, and neuritic degeneration became evident. Of note, apoEA was associated with significantly greater A@ deposition and neuritic degeneration than apoE3. Our data suggest that apoE is involved in AP clearance and tibrillogenesis in viw and that it is a critical modulator of neuritic and cerebrovascular plaque formation. In viva and in vitro studies are underway to determine the bimhemical mechanisms underlying the effects of CNS apoE/lipoproteins on AP metabolism.

AS A ~THERAPEUTIC [6261 APOE OF ALZHEIMER’S DISEASE

TARGET

FOR THE TREATMENT

Judes Poiner, McGill Univrrsuy / Dou&s Hospital, Verdun. PQ Cunadu; Michrl Pmisset, McGi// Universitv. Verdun. PQ Canuda The discovery that the apolipoprotein ~4 (apoE4) allele is strongly linked to both sporadic and familial late onset Alzheimer’s disease (AD) raisea the possibility that a dysfunction of the lipid transport system could seriously affect lipid homeostasis in the brain. It has been shawn that the presence of the t4 allele is associated with reduced levels of apoE in both serum and brain tissues of AD subjects. Moreover, synaptic integrity and plasticity is markedly compromised in an ~4 allele dosedependent manner in several brain areas in AD. These observations led us to propose that the reduced concentrations of apoE observed in the brain of apoEl-AD subjects may compromise cholesterol and phospholipids transport in the CNS in a manner consistent with what has been described in apoE knockout and apoE knockin mice. This, in turn, would indirectly impair the synaptic integrity and plasticny in several key neurotransmitter systems in the CNS; particularly in the cholinergic system which relies heavily on lipids to synthesize acetylcholine. With the objective of reversing the reported apoE deficit in the brain of c4 allele carriers, we identified and characterised several apoE inducer druga from a low throughput astrocyte screening assay and characterised their effects in brain cells. Tacrine and Heptylphysostigmine were found to be potent inducers of apoE secretion in vitro. In contrast, donepesil (Aricept) and rlvastigmine (Exelon) both lack this particular activity. Another apoE inducer drug called probucol was examined in mice and rat brains as well as in humans suffenng from nuld to moderate sporadic AD. Cerebroapinal fluid (CSF) evels of apoE, beta amyloid I-40, lipid peroxides and Tau were determined before and during treatment and subsequently contrasted with clinical outcome measures (CIBIC, CDR and ADAS-Cog) at 3 and 6 months. Eleven Eubject with mild-to moderate AD were Invited to take pm in this studies. The proof-of-principle climcal trial revealed that apoE induction in the brain (CSF) indeed affects disease progressmn in an apoEconcentration dependent manner. Supported by grants from the Medical Research Council of Canada. Alzheimer Society of Canada, FRSQ, Alzheimer Group of Montreal and Nova Molecular Inc.

DIET-INDUCED HYPERLIPIDEMIA ACCELERATES AMYLOID DEPOSITION IN THE APP”“= TRANSGENIC MOUSE MODEL OF ALZHEIMER DISEASE. Kelly R. Bales, Cindy l;ishmun, Cindy DeLon~, Eli Lilly and Company, Indianapolis, IN: Yanvhmg Du, lndiurru University School of Medicine. Indianapolis. IN; Willium Jordun. Strwn M. Paul, Eli Lilly and Cornpuny, Indiunapolis, IN Several genetic and nongenetic factors are associated with an increased risk for developing Alzheimer’s disease (AD) including the inheritance of an apolipoprotein t4 allele, increased age, and hyperlipidemia. Tu investigate the role of elevated serum lipids on amyloid deposition we fed male and female APPY7”’ transgenic mice (TG) mice a high fat (21.2% fat, 0.2% cholesterol) or normal (5.5% fat, 0% cholesterol) diet for various lengths of time. At 6,12,1X, and 24 weeks APPV717F TG mice were sacrificed and serum cholesterol, triglycerides as well as plaque burden in various brain regions were quantified. After 12 weeks on a high fat diet both male and female APPV7”r TG mice had a qigmticant elevation in serum triglycerides when compared to APP”“” TG mice fed a normal diet. Serum cholesterol, however, was signiticandy elevated after only 6 weeks on a high fat diet and remained elevated throughout the course of the study (24 weeks). Amyloid burden (thioflavine-S fluorescent deposits as well as AP immunorextivity) was significantly increased (pcO.05) in both the hippocampus as well as the cerebral cortex in APP”7’7F TG mice after 12 weeks on high fat diet. Although amyloid burden increased significantly in an age-dependent fashion in male APPV7”F TG rmce fed a control diet, no further accrual of amyloid occurred when mice were on a high fat diet for a longer (24 weeks)

s139

period of time, suggesting that a high fat diet-induced hyperlipidemia may accelerate amylold deposition in these mice. Interestingly APPV717F TG mice without apolipoprotein E have markedly elevated (greater than lo-fold) levels of serum cholesterol, and yet few if any amyloid deposits. Taken together, our results support the epidemiological association between the elevated risk to develop AD and hyperlipidemia perhaps through an apoE-dependent mechanism which leads to an increase in brain amyloid.

COGNITIVE DEFICITS 16281THEIR RELATIONSHIP

IN APOM TRANSGENIC MICE AND TO AGING, SEX, AND INCREASED

ARLEYELS lmnnart Muck<, Gladstone Institute of Neurological Disease, San Francisco, CA Deficits in spatial memory are a prominent feature of AD. Our analysis of transgenic mice expressing human apolipoprotein (ape) E &forms indicates that E4 plays a key role in the development of spatial memory impairments. In humans, E4 increases AD risk and accelerates disease manifestations compared with E3 and E2. We analyzed murine apoE-deficient (Apoe-‘-) mice in which the neuron-specific enolase (NSE) promoter directs expression of human E3 or E4 at comparable levels (PNAS 95: 10914; J. Neurosci. 19: 4867). These mice had normal basic visuomotor skills. At 3 months of age, E3 and E4 mice showed no deficits in spatial learning and memory in a water maze test compared with Apoe+‘+ or Apoe-‘-controls lacking human apoE. E3 mice performed well also at 6 and IX months. In contrast, E4 mice developed progressive impairments affecting spatial memory retention (probe trial) at 6 months and both spatial leaming and memory at I8 months. These E&associated deficits were detected in female, but not male, mice. Testosterone treatment significantly improved the performance of h-month-old female El mice (Raber et al., this meeting). Next, we tested whether human apoE &forms differentially affect spatial memory also in males if mice are challenged with AD-related copathogens. E3 and E4 mice were crossed with human amyloid precursor protein transgenic Apoe-‘~ mice expressing high levels of human Ahin neurons. In h-month-old male and female offspring, E3 prevented Ab-induced spatial memory deficits, but E4 did not. Molecular and morphological alterations that may relate to U-associated deficits will be discussed. Thus, E3 preserves cognitive function5 during aging and prevents Ab-induced neuronal deficits mere effectively than E4. In addition, E4 actively promotes age-related cognitive decline in a gender-dependent fashion and this effect can be counteracted with sex hormones. Ongoing studies focus on the development and assessment of apoE-targeted therapeutic strategies. Supported by the NIA, the Alzheimer’s Association, and The John Douglas French Alzheimer’s Foundation.

APOE AND 16291DISEASE.

ESTROGEN

IN MOUSE MODELS OF ALZHEIMER

Jonurkun D. Smrrk, Justine A. Lrvin, Ckru Lmninska, Yuri Bunimuvich, Rockefellrr Umwsiry. New York, NY ApoE is the most prevalent genetic susceptibility factor for Alzheimer disease (AD). Females are more likely to develop AD than males, and this differece can be accounted for by the increased likelihood of AD among EA/3 females compared to males. Also, women who take estrogen replacement therapy are less likely to develop AD. ! fs20Head injury is an environmental risk factor for AD, but only among apoE carriers. Thus we have studied the effects of various genetic and environmental risk factor5 in various inbred mouse models for AD. In C57BL/6 mice, estrogen induces apoE in the cortex, hippocampus, and diencephalon. We determined that the estrogen induction of apoE in the diencephalon, but not the hippocampus and cortex, is mediated by estrogen receptor a (for details, see abstract by Justine Levi”). Amyloid precursor protein (APP) transgenic and wildtype mice were ovariectomized (OVX) at 4 weeks of age and survival through 8 months of age was followed versus sham OVX and intact mice. Mortality was 60% in the OVX APP mice, compared to 0% in the sham OVX and intact APP plainmice, and 10% in the OVX wildtype mice. This increased death was not associated with increased brain levels of total AP, measured by ELISA. The effects of 6 week treatments with pharmacological doses of 17@- and l7a-estradiol on AP levels were determined in young OVX APP transgenic mice. 17p- and 17a-estradiol led to 10% and 25% decreases in total AP levels, respectively, compared to placebo. Fluid percusive brain injury was performed unilaterally to determine the effects of trauma on apoE and A@ levels in APP transgenic mice. 3 days! after injury, the apoE levels were about 4.fold higher in the injured versus control brain hemisphere, but apoE levels decreased close to baseline 6 days after injury. In contrast, AP levels in the injured hemisphere increased about 2.fold, but only 15 days after the injury, the latest time point examined thus far. The effect of apoE-deficiency on learning was examined using an a-arm radial maze, cued by odorants. We observed that the genetic background strain had an impact on the effect of apoE-deficiency, such that apoE-deficient mice on the FVB/N background learned as well as wildype FVBlN mice, while apoE-deficient C57BW6 mice had decreased learning Compared to their wildtype controls.