Basavaraj Hooli

Alzheimer’s Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta

The transmembrane protein CD33 is a sialic acid-binding immunoglobulin-like lectin that regulates innate immunity but has no known functions in the brain. We have previously shown that the CD33 gene is a risk factor for Alzheimers disease (AD). Here, we observed increased expression of CD33 in microglial cells in AD brain. The minor allele of the CD33 SNP rs3865444, which confers protection against AD, was associated with reductions in both CD33 expression and insoluble amyloid beta 42 (Aβ42) levels in AD brain. Furthermore, the numbers of CD33-immunoreactive microglia were positively correlated with insoluble Aβ42 levels and plaque burden in AD brain. CD33 inhibited uptake and clearance of Aβ42 in microglial cell cultures. Finally, brain levels of insoluble Aβ42 as well as amyloid plaque burden were markedly reduced in APP(Swe)/PS1(ΔE9)/CD33(-/-) mice. Therefore, CD33 inactivation mitigates Aβ pathology and CD33 inhibition could represent a novel therapy for AD.

A three-dimensional human neural cell culture model of Alzheimer’s disease

Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

Genome-wide association analysis reveals putative Alzheimer's disease susceptibility loci in addition to APOE.

Alzheimers disease (AD) is a genetically complex and heterogeneous disorder. To date four genes have been established to either cause early-onset autosomal-dominant AD (APP, PSEN1, and PSEN2(1-4)) or to increase susceptibility for late-onset AD (APOE5). However, the heritability of late-onset AD is as high as 80%, (6) and much of the phenotypic variance remains unexplained to date. We performed a genome-wide association (GWA) analysis using 484,522 single-nucleotide polymorphisms (SNPs) on a large (1,376 samples from 410 families) sample of AD families of self-reported European descent. We identified five SNPs showing either significant or marginally significant genome-wide association with a multivariate phenotype combining affection status and onset age. One of these signals (p = 5.7 x 10(-14)) was elicited by SNP rs4420638 and probably reflects APOE-epsilon4, which maps 11 kb proximal (r2 = 0.78). The other four signals were tested in three additional independent AD family samples composed of nearly 2700 individuals from almost 900 families. Two of these SNPs showed significant association in the replication samples (combined p values 0.007 and 0.00002). The SNP (rs11159647, on chromosome 14q31) with the strongest association signal also showed evidence of association with the same allele in GWA data generated in an independent sample of approximately 1,400 AD cases and controls (p = 0.04). Although the precise identity of the underlying locus(i) remains elusive, our study provides compelling evidence for the existence of at least one previously undescribed AD gene that, like APOE-epsilon4, primarily acts as a modifier of onset age.

Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate α-secretase activity

ADAM10, a member of a disintegrin and metalloprotease family, is an alpha-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimers disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated alpha-secretase activity of ADAM10 (>70% decrease), and elevated Abeta levels (1.5-3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.

Assessment of Alzheimer’s disease case–control associations using family-based methods

The genetics of Alzheimer’s disease (AD) is heterogeneous and remains only ill-defined. We have recently created a freely available and continuously updated online database (AlzGene; http://www.alzgene.org) for which we collect all published genetic association studies in AD and perform systematic meta-analyses on all polymorphisms with sufficient genotype data. In this study, we tested 27 genes (ACE, BDNF, CH25H, CHRNB2, CST3, CTSD, DAPK1, GALP, hCG2039140, IL1B, LMNA, LOC439999, LOC651924, MAPT, MTHFR, MYH13, PCK1, PGBD1, PRNP, PSEN1, SORCS1, SORL1, TF, TFAM, TNK1, GWA_14q32.13, and GWA_7p15.2), all showing significant association with AD risk in the AlzGene meta-analyses, in a large collection of family-based samples comprised of 4,180 subjects from over 1,300 pedigrees. Overall, we observe significant association with risk for AD and polymorphisms in ACE, CHRNB2, TF, and an as yet uncharacterized locus on chromosome 7p15.2 [rs1859849]. For all four loci, the association was observed with the same alleles as in the AlzGene meta-analyses. The convergence of case–control and family-based findings suggests that these loci currently represent the most promising AD gene candidates. Further fine-mapping and functional analyses are warranted to elucidate the potential biochemical mechanisms and epidemiological relevance of these genes.

No Association between CALHM1 and Alzheimer's Disease Risk

In a recent paper in Cell, Dreses-Werringloer et al. (2008) reported the identification and functional characterization of a new Alzheimer’s disease (AD) gene, CALHM1, encoding calcium homeostasis modulator 1. CALHM1 (formerly annotated as FAM26C) represents a compelling candidate gene for late-onset AD as it is located on chromosome 10q24, a consistently replicated AD linkage region (Bertram et al., 2007), is highly expressed in the hippocampus, which is severely affected by AD-related pathology, and is involved in calcium ion homeostasis, which may be disrupted in AD. In view of these converging leads, Dreses-Werringloer et al. (2008) sequenced the open reading frame of CALHM1 in a small sample of AD patients and healthy controls and identified a nonsynonymous polymorphism (Pro86Leu, rs2986017) whose minor leucine allele showed a higher frequency in the AD patients. Followup analyses in four additional independent samples of ~3400 DNAs revealed a consistent overrepresentation of the same allele in AD cases compared to controls in each dataset. From the combined analyses the authors estimated that inheritance of the leucine allele modestly, but significantly, elevated the risk for AD by ~40% (p value = 2 × 10−10). Their genetics findings were supported by data generated in a large number of functional genomics and biochemical experiments showing evidence that the risk-associated leucine allele leads to a loss of protein function, including attenuated permeability to calcium ions and reduced cytosolic calcium ion levels, which, in turn, were associated with an increase in the pathogenic peptide, amyloid-β (Aβ). Here, we present an independent assessment of the potential association between AD and the Pro-86Leu single-nucleotide polymorphism (SNP) in CALHM1 in more than 8100 subjects from several independent datasets comprised of AD families—including those in which the original chromosome 10q24 linkage signal was identified (Bertram et al., 2000)—as well as unrelated cases and controls, and we find no evidence of a genetic association in these samples. The family-based datasets (CAG, NIA, NIMH, NCRAD) tested in this project are of self-reported European (Caucasian) ancestry collected in the US for the study of genetic factors in AD (see Table S1, available online, for a summary of sample characteristics). All samples were primarily sibships and lacked parental genotypes. With the exception of the CAG sample, the majority of pedigrees analyzed here were nuclear families ascertained on the basis of multiple affected individuals. In addition to containing at least one affected relative pair, many pedigrees also had DNA available from additional affected or unaffected individuals (mostly siblings). The diagnosis of definite, probable, or possible AD was made according to NINCDS/ADRDA criteria for affected individuals in all four samples (McKhann et al., 1984). In addition to the family samples, we genotyped ~1300 unrelated AD cases and controls, which were collected at two sites in Northern Europe, Sweden, and Finland (Table S1). All subjects were Caucasian, and AD patients fulfilled NINCDS/ADRDA criteria for probable AD. The Swedish AD patients were ascertained at the Memory Disorder Unit at Uppsala University Hospital. Healthy control subjects were recruited from the same geographic region following advertisements in local newspapers and displayed no signs of dementia upon neuropsychological testing. The Finnish subjects were gathered from Eastern Finland and were examined in the Department of Neurology of Kuopio University Hospital. Control subjects had no signs of dementia following neuropsychological testing. Finally, we assessed the CALHM1 locus in two previously published high-density genome-wide association studies (GWAS) (Li et al., 2008; Reiman et al., 2007) for which genotype data are publicly available. Together, these studies investigated 2900 unrelated AD cases and controls. Given that the rs2986017 (Pro86Leu) variant was not tested directly in either of the GWAS, we tested two SNPs showing strong and significant linkage disequilibrium with the rs2986017 (Pro86Leu) variant (rs2986030 [D′ = 0.89, r2 = 0.71] and rs1555823 [D′ = 0.89, r2 = 0.64]). Note that the power to detect the effect sizes described in the original report (Dreses-Werringloer et al., 2008) was still very high (>90%) due to the strong linkage disequilibrium between these variants (Table S2). Genotyping of the rs2986017 (Pro86Leu) variant in the US family samples and the Northern European case-control datasets was based on an individually optimized single-base extension reaction detected by high-efficiency fluorescent polarization (HEFP; described in Bertram et al., 2005; protocols available on request). Overall, genotyping efficiency was 97.6%, and the average error rate was below 0.2%. Resequencing of the Pro86Leu variant in 20 individuals revealed 100% concordance with the HEFP genotype results. None of the markers deviated significantly from Hardy-Weinberg equilibrium. To test for association with AD risk in the family-based samples we used PBAT (http://www.biostat.harvard.edu/~clange/default.htm) applying an additive model. Odds ratios (ORs) for the family samples were calculated by fitting a conditional logistic regression model to each dataset, where family defines the stratum. To test for association in the case-control samples (including the markers extracted from the GWAS) we calculated allele-based study-specific crude ORs, 95% confidence intervals, and p values for each marker (Bertram et al., 2007). To combine the effect size estimates obtained in this study with those estimated in the original publication, summary ORs across all samples were calculated using the DerSimonian and Laird random effects model, in line with the analyses routinely performed for the AlzGene database (Bertram et al., 2007). Power calculations (performed in PBAT) suggested that we had sufficient (i.e., 70% or greater) power to detect the genetic effect size estimated in the original study in each of the samples, with the exception of the CAG dataset (Table S2). As shown in Figure S1 and Table S2, none of the eight samples we investigated showed evidence for significant association between the rs2986017 (Pro86Leu) variant in CALHM1 and AD risk (p values ranging from 0.15 to 0.84). Stratification by age of AD onset (using 65 years as cutoff) or APOE e4-genotype did not appreciably change these results (data not shown; stratified analyses were not possible in the GWAS samples as no onset age or APOE e4 data were supplied). Effect size estimates indicated insignificant ORs that were opposite in direction to those reported by Dreses-Werringloer et al. (2008) in six of the eight samples (see Figure S1). Accordingly, summary ORs calculated across the newly genotyped samples in our study (labeled “This study” in Figure S1) were insignificant (OR = 0.94 [95% CI: 0.83–1.07], p = 0.4) and tended toward null when combined with the published GWAS genotype data (“All follow-up”; OR = 0.99 [95% CI: 0.91–1.09], p = 0.9). Upon combining these data with the results of the original study (“All studies”), that is, generating a meta-analysis on all ~11,700 currently available subjects, the overall summary OR became insignificant as well (OR = 1.13 [95% CI: 0.99–1.27], p = 0.06). Using rs1555823 (instead of rs2986030) as proxy for the rs2986017 (Pro86Leu) variant in the GWAS samples revealed even less pronounced and less significant overall effects (data not shown). Thus, we have independently assessed the potential association between AD risk and the rs2986017 (Pro86Leu) variant in the CALHM1 gene in a large number of independent datasets, including AD families in which the original chromosome 10q24 linkage signal was identified (Bertram et al., 2000). Despite good to excellent power to detect genetic effect sizes on the order described by Dreses-Werringloer et al. (2008), no association between CALHM1 and AD was observed, either in the individual samples or in the combined analyses of more than 8100 subjects. Based on these negative data, it is doubtful that CALHM1 represents more than a minor genetic determinant of AD risk.

Role of common and rare APP DNA sequence variants in Alzheimer disease

Objectives: More than 30 different rare mutations, including copy number variants (CNVs), in the amyloid precursor protein gene (APP) cause early-onset familial Alzheimer disease (EOFAD), whereas the contribution of common APP variants to disease risk remains controversial. In this study we systematically assessed the role of both rare and common APP DNA variants in Alzheimer disease (AD) families. Methods: Families with EOFAD genetically linked to the APP region were screened for missense mutations and locus duplications of APP. Further, using genome-wide DNA microarray data, we examined the APP locus for CNVs in a total of 797 additional early- and late-onset AD pedigrees. Finally, 423 single nucleotide polymorphisms (SNPs) in the APP locus, including 2 promoter polymorphisms previously associated with AD risk, were tested in up to 4,200 individuals from multiplex AD families. Results: Analyses of 8 21q21-linked families revealed one family carrying a nonsynonymous mutation in exon 17 (Val717Leu) and another family with a partially penetrant 3.5-Mb locus duplication encompassing APP. CNV analysis in the APP locus revealed an additional family carrying a fully penetrant 380-kb duplication, merely spanning APP. Last, contrary to previous reports, association analyses of more than 400 different SNPs in or near APP failed to show significant effects on AD risk. Conclusion: Our study shows that APP mutations and locus duplications are a very rare cause of EOFAD and that the contribution of common APP variants to AD susceptibility is insignificant. Furthermore, duplications of APP may not be fully penetrant, possibly indicating the existence of hitherto unknown protective genetic factors.

Alzheimer's disease-associated TREM2 variants exhibit either decreased or increased ligand-dependent activation

TREM2 is a lipid‐sensing activating receptor on microglia known to be important for Alzheimers disease (AD), but whether it plays a beneficial or detrimental role in disease pathogenesis is controversial.

Rare autosomal copy number variations in early-onset familial Alzheimer's disease

Over 200 rare and fully penetrant pathogenic mutations in amyloid precursor protein (APP), presenilin 1 and 2 (PSEN1 and PSEN2) cause a subset of early-onset familial Alzheimer’s disease (EO-FAD). Of these, 21 cases of EO-FAD families carrying unique APP locus duplications remain the only pathogenic copy number variations (CNVs) identified to date in Alzheimer’s disease (AD). Using high-density DNA microarrays, we performed a comprehensive genome-wide analysis for the presence of rare CNVs in 261 EO-FAD and early/mixed-onset pedigrees. Our analysis revealed 10 novel private CNVs in 10 EO-FAD families overlapping a set of genes that includes: A2BP1, ABAT, CDH2, CRMP1, DMRT1, EPHA5, EPHA6, ERMP1, EVC, EVC2, FLJ35024 and VLDLR. In addition, CNVs encompassing two known frontotemporal dementia genes, CHMP2B and MAPT were found. To our knowledge, this is the first study reporting rare gene-rich CNVs in EO-FAD and early/mixed-onset AD that are likely to underlie pathogenicity in familial AD and perhaps related dementias.

GAB2 as an Alzheimer Disease Susceptibility Gene: Follow-up of Genomewide Association Results

BACKGROUND Genomewide association (GWA) studies have recently implicated 4 novel Alzheimer disease (AD) susceptibility loci (GAB2, GOLM1, and 2 uncharacterized loci to date on chromosomes 9p and 15q). To our knowledge, these findings have not been independently replicated. OBJECTIVE To assess these GWA findings in 4 large data sets of families affected by AD. DESIGN Follow-up of genetic association findings in previous studies. SETTING Academic research. PARTICIPANTS More than 4000 DNA samples from almost 1300 families affected with AD. MAIN OUTCOME MEASURES Genetic association analysis testing of 4 GWA signals (rs7101429 [GAB2], rs7019241 [GOLM1], rs10519262 [chromosome 15q], and rs9886784 [chromosome 9p]) using family-based methods. RESULTS In the combined analyses, only rs7101429 in GAB2 yielded significant evidence of association with the same allele as in the original GWA study (P =.002). The results are in agreement with recent meta-analyses of this and other GAB2 polymorphisms suggesting approximately a 30% decrease in risk for AD among carriers of the minor alleles. None of the other 3 tested loci showed consistent evidence for association with AD across the investigated data sets. CONCLUSIONS GAB2 contains genetic variants that may lead to a modest change in the risk for AD. Despite these promising results, more data from independent samples are needed to better evaluate the potential contribution of GAB2 to AD risk in the general population.