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APOE polymorphisms in Alzheimer's disease
Synposium:
Epidemiology
of Alzheimer’s
Disease
s137
generates a 3 kDa C-terminal fragment (Capp3) and a 6.5 kDa amyloidogenic fragment (Capp6.5) in vitro. Endogenous Capp6.5 increases in apoptotic neurons and exhihits a precursor-product relationship with secreted amyloid ppeptide. To determine if caspase-6 could he responsible for increased amyloid @peptide in Alzheimer’a disease, we measured caspaae-6 active fragments in IO AD and 7 non-AD frontal, temporal, par&al and cerehellar brain tissues. Western blot analysis of the p10 active fragment of caspase-6 reveals increased pl0 (active fragment)/p36(pro-enzyme) ratios in the frontal lobe of AD individuals. These results suggest that caspase-6 activation is involved in neurnnal apoptosis, altered APP metabolism and AD.
S 16171ALZHEIMER’ IMPLICATIONS Takaumi
C Snide,
RIKEN
DISEASE
AS A PROTEOLYTIC
FOR DEVISING Brain
Scimre
Institute,
DISORDER
AND
THERAPY Saitama
Japan
The in vI’~.oamount of spy peptide is determined by the balance between anaholism and catabolism. Physiological hut potentially amyloidogenic peptides such as ABcould be turned into pathogenic agents by subtle alterations I” this balance over a long period of time. Cataholism/proteolysis of APin brain parenchyma can he dwided Into two major categories. One is a physiological catabolism, in which secreted wluhle APi\ fragmented under normal conditmnr. The other i? a rather pathological catabolism presumably involved in clearance of fihrillar APthat could develop into diffuse and cored plaques. Both are Important for Alzheimer reaearch because a reduction in these processes would lead to accelerated accumulation and because we may he able to prevent the.disease onset or decelerate its progression by manipulating these processes. We analyzed the mechanism of physiological catabolism by tracing multiply radiolabeled Appeptides injected into rat hippocampus. Effect of various peptidaw inhibitors and structural analysis of catabolic products suggested that a metallo-endopeptidaae similar or identical to neprilysin plays a major role. Furthermore, inhibition of this process caused hlochemical and pathological deposition of endogenous AP42. Up-regulation of Apcataholism therefore may reduce the risk of developing the disease by preventing Apaccumulation. The pathological catabolism is being studied under the assumption that matnx-degrading proteinases are likely to he capable of proteolyLing fihrillar A@. We discovered a novel matrix metalloproteinase (MMP) predominantly expressed in hrain hy cDNA cloning. This MMP i\ most ctrongly expressed in cerebellum that is known not to develop cored plaques hut only diffuse plaques. Involvement of this novel MMP and other matrix-degrading enzymes in clearance of fihrillar ADwill hc discussed.
Symposium: ETHNIC MER’S
Epidemiology VARIABHaITY
of Alzheimer’s IN
GENOTYPIC
Disease
RISKS
OF
ALZHEI-
DISEASE.
Though the asaoclation between Alaheimer’s disease (AD) and the apolipoproteiwe4 (APOE-~4) allele has been confirmed worldwide, it appears to he inconsistent among African-Americana, Hispanics and Nigerians. We have previously repotted that compared to individuals with the AP0E-e3/~3 genotype, the risk of AD wth one or more copies of the APOE-~4 allele was sigmficantly increased among Whites but not among African-Americans or Caribbean Hispanicsliving in the Washington Heights and lnwood communities of New York City. In the absence of the APOE-~4 allele, the cumulative riqk of AD to age 90 was 4 times higher for African--Americans and 2 times higher for H&panic\ compared to Whites. This variability suggests that the effects of APOE-~4 on risk in these groups may he modified by other genetic or enviromental factors or that polymorphisms in genes unrelated to APOE contribute independently to disease risk. To clarify the APOE-~4 aasoclation, we investigated flanking polymorphisms in African-Americans and Hispanics,(from upstream to downstream): a novel Kspl RFLP near the 3’ end of the TOM40gene, the -491 A/T, -427T/C and -219Gm(Thl/E47cs) polymorphisms in the APOE promoter and the Hpal RFLP in the APOCI promoter. The APOCl HpaI+ allele was associated with AD, hut only in the absence of an APOE-~4 allele. Among the promoter polymorphisms, the -219Gm T-allele in the homozygous configuration wab associated with lower risk, hut only when present with an APOE-~4 allele. Using family-based association methods, we have also examined the association between AD and the ol-2.macroglobulin polymorphism (u2m) in Caribbean Hispanics. We have found a weak, but statistically significant, association between the D/I polymorphism in u2m and AD that was stronger after adjustment for APOE-~4. No environmental factors have emerged as “genetic modifiers”, but investigation continues. Ethnic variability in genotypic risks may provide additional information regarding gene-gene and geneenvironment interaction.
ASSOCIATION FINDINGS
FROM
STRATEGIES THE
NIMH
IN
ALZHEIMER’S
GENETICS
DISEASE:
INITIATIVE
Alshrimer’s disease (AD) 1s a genetically complex disorder. whose late onwt and high prevalence complicate the application of existing genetic analytic techniques. In analyzing genome screen of approximately 500 sibling pairs and other small families collected under the auspices of the NIMH Genetics Initiative, linkage strategies point at the possible involvement of genes in multiple regions of interest, hut are unable to narrow these regions substantially. Association atrategier, aimed at identifying and characterizing posirional candidate ~uws-those identified wthin these regions based on their location nnd their potential role m AD biology-offer considerable promise in addressing this problem. Recent work in statistical genetics has greatly expanded the informativeness and flexibility of these strategies within family wnples, including those without parental data, as is typical of AD samples. Hypothesis testing procedures include a number of recently developed association tests, notably the Sibling Transmission Disequilibrium Test (S-TDT) and Sihship Di,equilihrium Tert (SDT). Procedures for effect size eqtlmation include the use of conditional logistic regression to generate odds ratios with appropriate confidence intervals. Moreover, these methods are hung adapted to adjust for genetic and environmental factors that may he involved in the disorder, to handle time-to-onset phenotypes, and to address correlated genotypes such as multiple polymorphisms in the same gene. This session will review these methods, and demonstrate their appbcability to AD data from the NIMH Genetics lnitrative sample. This will include an overwew of positive linkage findings on chromosomes 6, 12, and elsewhere, along with a more detailed dwussion of association results for several polymorphisms in alpha-2-macroglobulin and a variety of other positional candidate genes for AD.
16201APOE Marie-Chri.ytine SOB. Lille
POLYMORPHISMS
Chartier-H&in,
IN ALZHEIMER’S
Jew-Charles
Lumbrrt,
DISEASE.
Philippe
Amouyel,
INSERM
Frmce
The ~4 allele of the apohpoprotein E gene (APOE) has been associated with an increared risk of developing Alzheimer’s disease. However, it is apparent that the ~4 allele alone is not sufficient to cause the disease. Indeed APOE promoter polymorphiyms have been associated with an Increased risk of developing Alzheimer’s disease. Theae polymorphi\ma seem to control in viva and in wtro mRNA expression level( l-2). For instance, they may modulate the amount of APOE in plasma (3). The impact which the APOE expression might have on AD process, is not yet elucidated. However, in APPI717V transgenic mice, the amount of AP deposits depends directly on the copy number of the murine or human APOE gene(4,S) and we observed correlation between APOE promoter polymorphisms and AP loadmg in AD brains. So. theve data sustain the hypothesis that the APOE gene expression may he an important determinant of the etiology of AD. 1.Lamhert J-C ef al., Hum. Mol. Genet., 1998, 7,1511-6. 2. Bullido MJ et u/., Nat. &net., 1998, 18, 69-71. 3. Lamhert JC et rrl., Hum. Mol. Gene%, 2000, 9, 57-61. 4. Bales KR et al., Nat. Gem%, 1997, 17, 263-264. 5. Holtrman DM et al., .I. Clin. Invest., 1999, 103.15-21.
Epidemiology
of Alzheimer’s
Disease
s137
generates a 3 kDa C-terminal fragment (Capp3) and a 6.5 kDa amyloidogenic fragment (Capp6.5) in vitro. Endogenous Capp6.5 increases in apoptotic neurons and exhihits a precursor-product relationship with secreted amyloid ppeptide. To determine if caspase-6 could he responsible for increased amyloid @peptide in Alzheimer’a disease, we measured caspaae-6 active fragments in IO AD and 7 non-AD frontal, temporal, par&al and cerehellar brain tissues. Western blot analysis of the p10 active fragment of caspase-6 reveals increased pl0 (active fragment)/p36(pro-enzyme) ratios in the frontal lobe of AD individuals. These results suggest that caspase-6 activation is involved in neurnnal apoptosis, altered APP metabolism and AD.
S 16171ALZHEIMER’ IMPLICATIONS Takaumi
C Snide,
RIKEN
DISEASE
AS A PROTEOLYTIC
FOR DEVISING Brain
Scimre
Institute,
DISORDER
AND
THERAPY Saitama
Japan
The in vI’~.oamount of spy peptide is determined by the balance between anaholism and catabolism. Physiological hut potentially amyloidogenic peptides such as ABcould be turned into pathogenic agents by subtle alterations I” this balance over a long period of time. Cataholism/proteolysis of APin brain parenchyma can he dwided Into two major categories. One is a physiological catabolism, in which secreted wluhle APi\ fragmented under normal conditmnr. The other i? a rather pathological catabolism presumably involved in clearance of fihrillar APthat could develop into diffuse and cored plaques. Both are Important for Alzheimer reaearch because a reduction in these processes would lead to accelerated accumulation and because we may he able to prevent the.disease onset or decelerate its progression by manipulating these processes. We analyzed the mechanism of physiological catabolism by tracing multiply radiolabeled Appeptides injected into rat hippocampus. Effect of various peptidaw inhibitors and structural analysis of catabolic products suggested that a metallo-endopeptidaae similar or identical to neprilysin plays a major role. Furthermore, inhibition of this process caused hlochemical and pathological deposition of endogenous AP42. Up-regulation of Apcataholism therefore may reduce the risk of developing the disease by preventing Apaccumulation. The pathological catabolism is being studied under the assumption that matnx-degrading proteinases are likely to he capable of proteolyLing fihrillar A@. We discovered a novel matrix metalloproteinase (MMP) predominantly expressed in hrain hy cDNA cloning. This MMP i\ most ctrongly expressed in cerebellum that is known not to develop cored plaques hut only diffuse plaques. Involvement of this novel MMP and other matrix-degrading enzymes in clearance of fihrillar ADwill hc discussed.
Symposium: ETHNIC MER’S
Epidemiology VARIABHaITY
of Alzheimer’s IN
GENOTYPIC
Disease
RISKS
OF
ALZHEI-
DISEASE.
Though the asaoclation between Alaheimer’s disease (AD) and the apolipoproteiwe4 (APOE-~4) allele has been confirmed worldwide, it appears to he inconsistent among African-Americana, Hispanics and Nigerians. We have previously repotted that compared to individuals with the AP0E-e3/~3 genotype, the risk of AD wth one or more copies of the APOE-~4 allele was sigmficantly increased among Whites but not among African-Americans or Caribbean Hispanicsliving in the Washington Heights and lnwood communities of New York City. In the absence of the APOE-~4 allele, the cumulative riqk of AD to age 90 was 4 times higher for African--Americans and 2 times higher for H&panic\ compared to Whites. This variability suggests that the effects of APOE-~4 on risk in these groups may he modified by other genetic or enviromental factors or that polymorphisms in genes unrelated to APOE contribute independently to disease risk. To clarify the APOE-~4 aasoclation, we investigated flanking polymorphisms in African-Americans and Hispanics,(from upstream to downstream): a novel Kspl RFLP near the 3’ end of the TOM40gene, the -491 A/T, -427T/C and -219Gm(Thl/E47cs) polymorphisms in the APOE promoter and the Hpal RFLP in the APOCI promoter. The APOCl HpaI+ allele was associated with AD, hut only in the absence of an APOE-~4 allele. Among the promoter polymorphisms, the -219Gm T-allele in the homozygous configuration wab associated with lower risk, hut only when present with an APOE-~4 allele. Using family-based association methods, we have also examined the association between AD and the ol-2.macroglobulin polymorphism (u2m) in Caribbean Hispanics. We have found a weak, but statistically significant, association between the D/I polymorphism in u2m and AD that was stronger after adjustment for APOE-~4. No environmental factors have emerged as “genetic modifiers”, but investigation continues. Ethnic variability in genotypic risks may provide additional information regarding gene-gene and geneenvironment interaction.
ASSOCIATION FINDINGS
FROM
STRATEGIES THE
NIMH
IN
ALZHEIMER’S
GENETICS
DISEASE:
INITIATIVE
Alshrimer’s disease (AD) 1s a genetically complex disorder. whose late onwt and high prevalence complicate the application of existing genetic analytic techniques. In analyzing genome screen of approximately 500 sibling pairs and other small families collected under the auspices of the NIMH Genetics Initiative, linkage strategies point at the possible involvement of genes in multiple regions of interest, hut are unable to narrow these regions substantially. Association atrategier, aimed at identifying and characterizing posirional candidate ~uws-those identified wthin these regions based on their location nnd their potential role m AD biology-offer considerable promise in addressing this problem. Recent work in statistical genetics has greatly expanded the informativeness and flexibility of these strategies within family wnples, including those without parental data, as is typical of AD samples. Hypothesis testing procedures include a number of recently developed association tests, notably the Sibling Transmission Disequilibrium Test (S-TDT) and Sihship Di,equilihrium Tert (SDT). Procedures for effect size eqtlmation include the use of conditional logistic regression to generate odds ratios with appropriate confidence intervals. Moreover, these methods are hung adapted to adjust for genetic and environmental factors that may he involved in the disorder, to handle time-to-onset phenotypes, and to address correlated genotypes such as multiple polymorphisms in the same gene. This session will review these methods, and demonstrate their appbcability to AD data from the NIMH Genetics lnitrative sample. This will include an overwew of positive linkage findings on chromosomes 6, 12, and elsewhere, along with a more detailed dwussion of association results for several polymorphisms in alpha-2-macroglobulin and a variety of other positional candidate genes for AD.
16201APOE Marie-Chri.ytine SOB. Lille
POLYMORPHISMS
Chartier-H&in,
IN ALZHEIMER’S
Jew-Charles
Lumbrrt,
DISEASE.
Philippe
Amouyel,
INSERM
Frmce
The ~4 allele of the apohpoprotein E gene (APOE) has been associated with an increared risk of developing Alzheimer’s disease. However, it is apparent that the ~4 allele alone is not sufficient to cause the disease. Indeed APOE promoter polymorphiyms have been associated with an Increased risk of developing Alzheimer’s disease. Theae polymorphi\ma seem to control in viva and in wtro mRNA expression level( l-2). For instance, they may modulate the amount of APOE in plasma (3). The impact which the APOE expression might have on AD process, is not yet elucidated. However, in APPI717V transgenic mice, the amount of AP deposits depends directly on the copy number of the murine or human APOE gene(4,S) and we observed correlation between APOE promoter polymorphisms and AP loadmg in AD brains. So. theve data sustain the hypothesis that the APOE gene expression may he an important determinant of the etiology of AD. 1.Lamhert J-C ef al., Hum. Mol. Genet., 1998, 7,1511-6. 2. Bullido MJ et u/., Nat. &net., 1998, 18, 69-71. 3. Lamhert JC et rrl., Hum. Mol. Gene%, 2000, 9, 57-61. 4. Bales KR et al., Nat. Gem%, 1997, 17, 263-264. 5. Holtrman DM et al., .I. Clin. Invest., 1999, 103.15-21.