Cognitively healthy centenarians are genetically protected against Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) prevalence increases with age, yet a small fraction of the population reaches ages > 100 years without cognitive decline. We studied the genetic factors associated with such resilience against AD. METHODS: Genome-wide association studies identified 86 single nucleotide polymorphisms (SNPs) associated with AD risk. We estimated SNP frequency in 2281 AD cases, 3165 age-matched controls, and 346 cognitively healthy centenarians. We calculated a polygenic risk score (PRS) for each individual and investigated the functional properties of SNPs enriched/depleted in centenarians. RESULTS: Cognitively healthy centenarians were enriched with the protective alleles of the SNPs associated with AD risk. The protective effect concentrated on the alleles in/near ANKH, GRN, TMEM106B, SORT1, PLCG2, RIN3, and APOE genes. This translated to >5-fold lower PRS in centenarians compared to AD cases (P = 7.69 × 10-71), and 2-fold lower compared to age-matched controls (P = 5.83 × 10-17). DISCUSSION: Maintaining cognitive health until extreme ages requires complex genetic protection against AD, which concentrates on the genes associated with the endolysosomal and immune systems. HIGHLIGHTS: Cognitively healthy cent enarians are enriched with the protective alleles of genetic variants associated with Alzheimer's disease (AD). The protective effect is concentrated on variants involved in the immune and endolysosomal systems. Combining variants into a polygenic risk score (PRS) translated to > 5-fold lower PRS in centenarians compared to AD cases, and ≈ 2-fold lower compared to middle-aged healthy controls.

Connecting dementia risk loci to the CSF proteome identifies pathophysiological leads for dementia.

Genome-wide association studies have successfully identified many genetic risk loci for dementia, but exact biological mechanisms through which genetic risk factors contribute to dementia remains unclear. Integrating CSF proteomic data with dementia risk loci could reveal intermediate molecular pathways connecting genetic variance to the development of dementia. We tested to what extent effects of known dementia risk loci can be observed in CSF levels of 665 proteins (proximity extension-based (PEA) immunoassays) in a deeply-phenotyped mixed-memory clinic cohort (n=502, mean age (sd) = 64.1 [8.7] years, 181 female [35.4%]), including patients with Alzheimer's disease (AD, n=213), dementia with Lewy bodies (DLB, n=50) and frontotemporal dementia (FTD, n=93), and controls (n=146). Validation was assessed in independent cohorts (n=99 PEA platform, n=198, MRM-targeted mass spectroscopy and multiplex assay). We performed additional analyses stratified according to diagnostic status (AD, DLB, FTD and controls separately), to explore whether associations between CSF proteins and genetic variants were specific to disease or not. We identified four AD risk loci as protein quantitative trait loci (pQTL): CR1-CR2 (rs3818361, P=1.65e-08), ZCWPW1-PILRB (rs1476679, P=2.73e-32), CTSH-CTSH (rs3784539, P=2.88e-24) and HESX1-RETN (rs186108507, P=8.39e-08), of which the first three pQTLs showed direct replication in the independent cohorts. We identified one AD-specific association between a rare genetic variant of TREM2 and CSF IL6 levels (rs75932628, P = 3.90e-7). DLB risk locus GBA showed positive trans effects on seven inter-related CSF levels in DLB patients only. No pQTLs were identified for frontotemporal dementia, either for the total sample as for analyses performed within FTD only. pQTL variants were involved in the immune system, highlighting the importance of this system in the pathophysiology of dementia. We further identified pQTLs in stratified analyses for AD and DLB, hinting at disease-specific pQTLs in dementia. Dissecting the contribution of risk loci to neurobiological processes aids in understanding disease mechanisms underlying dementia.

Functional characterization of SORL1 variants in cell-based assays to investigate variant pathogenicity.

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Cerebrospinal fluid proteomics in patients with Alzheimer's disease reveals five molecular subtypes with distinct genetic risk profiles.

Alzheimer's disease (AD) is heterogenous at the molecular level. Understanding this heterogeneity is critical for AD drug development. Here we define AD molecular subtypes using mass spectrometry proteomics in cerebrospinal fluid, based on 1,058 proteins, with different levels in individuals with AD (n = 419) compared to controls (n = 187). These AD subtypes had alterations in protein levels that were associated with distinct molecular processes: subtype 1 was characterized by proteins related to neuronal hyperplasticity; subtype 2 by innate immune activation; subtype 3 by RNA dysregulation; subtype 4 by choroid plexus dysfunction; and subtype 5 by blood-brain barrier impairment. Each subtype was related to specific AD genetic risk variants, for example, subtype 1 was enriched with TREM2 R47H. Subtypes also differed in clinical outcomes, survival times and anatomical patterns of brain atrophy. These results indicate molecular heterogeneity in AD and highlight the need for personalized medicine.

Identifying Aging and Alzheimer Disease-Associated Somatic Variations in Excitatory Neurons From the Human Frontal Cortex.

Background and Objectives: With age, somatic mutations accumulated in human brain cells can lead to various neurologic disorders and brain tumors. Because the incidence rate of Alzheimer disease (AD) increases exponentially with age, investigating the association between AD and the accumulation of somatic mutation can help understand the etiology of AD. Methods: We designed a somatic mutation detection workflow by contrasting genotypes derived from whole-genome sequencing (WGS) data with genotypes derived from scRNA-seq data and applied this workflow to 76 participants from the Religious Order Study and the Rush Memory and Aging Project (ROSMAP) cohort. We focused only on excitatory neurons, the dominant cell type in the scRNA-seq data. Results: We identified 196 sites that harbored at least 1 individual with an excitatory neuron-specific somatic mutation (ENSM), and these 196 sites were mapped to 127 genes. The single base substitution (SBS) pattern of the putative ENSMs was best explained by signature SBS5 from the Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signatures, a clock-like pattern correlating with the age of the individual. The count of ENSMs per individual also showed an increasing trend with age. Among the mutated sites, we found 2 sites tend to have more mutations in older individuals (16:6899517 [RBFOX1], p = 0.04; 4:21788463 [KCNIP4], p < 0.05). In addition, 2 sites were found to have a higher odds ratio to detect a somatic mutation in AD samples (6:73374221 [KCNQ5], p = 0.01 and 13:36667102 [DCLK1], p = 0.02). Thirty-two genes that harbor somatic mutations unique to AD and the KCNQ5 and DCLK1 genes were used for gene ontology (GO)-term enrichment analysis. We found the AD-specific ENSMs enriched in the GO-term "vocalization behavior" and "intraspecies interaction between organisms." Of interest we observed both age-specific and AD-specific ENSMs enriched in the K+ channel-associated genes. Discussion: Our results show that combining scRNA-seq and WGS data can successfully detect putative somatic mutations. The putative somatic mutations detected from ROSMAP data set have provided new insights into the association of AD and aging with brain somatic mutagenesis.

The correlation between neuropathology levels and cognitive performance in centenarians.

INTRODUCTION: Neuropathological substrates associated with neurodegeneration occur in brains of the oldest old. How does this affect cognitive performance? METHODS: The 100-plus Study is an ongoing longitudinal cohort study of centenarians who self-report to be cognitively healthy; post mortem brain donation is optional. In 85 centenarian brains, we explored the correlations between the levels of 11 neuropathological substrates with ante mortem performance on 12 neuropsychological tests. RESULTS: Levels of neuropathological substrates varied: we observed levels up to Thal-amyloid beta phase 5, Braak-neurofibrillary tangle (NFT) stage V, Consortium to Establish a Registry for Alzheimer's Disease (CERAD)-neuritic plaque score 3, Thal-cerebral amyloid angiopathy stage 3, Tar-DNA binding protein 43 (TDP-43) stage 3, hippocampal sclerosis stage 1, Braak-Lewy bodies stage 6, atherosclerosis stage 3, cerebral infarcts stage 1, and cerebral atrophy stage 2. Granulovacuolar degeneration occurred in all centenarians. Some high performers had the highest neuropathology scores. DISCUSSION: Only Braak-NFT stage and limbic-predominant age-related TDP-43 encephalopathy (LATE) pathology associated significantly with performance across multiple cognitive domains. Of all cognitive tests, the clock-drawing test was particularly sensitive to levels of multiple neuropathologies.

What does heritability of Alzheimer's disease represent?

INTRODUCTION: Both late-onset Alzheimer's disease (AD) and ageing have a strong genetic component. In each case, many associated variants have been discovered, but how much missing heritability remains to be discovered is debated. Variability in the estimation of SNP-based heritability could explain the differences in reported heritability. METHODS: We compute heritability in five large independent cohorts (N = 7,396, 1,566, 803, 12,528 and 3,963) to determine whether a consensus for the AD heritability estimate can be reached. These cohorts vary by sample size, age of cases and controls and phenotype definition. We compute heritability a) for all SNPs, b) excluding APOE region, c) excluding both APOE and genome-wide association study hit regions, and d) SNPs overlapping a microglia gene-set. RESULTS: SNP-based heritability of late onset Alzheimer's disease is between 38 and 66% when age and genetic disease architecture are correctly accounted for. The heritability estimates decrease by 12% [SD = 8%] on average when the APOE region is excluded and an additional 1% [SD = 3%] when genome-wide significant regions were removed. A microglia gene-set explains 69-84% of our estimates of SNP-based heritability using only 3% of total SNPs in all cohorts. CONCLUSION: The heritability of neurodegenerative disorders cannot be represented as a single number, because it is dependent on the ages of cases and controls. Genome-wide association studies pick up a large proportion of total AD heritability when age and genetic architecture are correctly accounted for. Around 13% of SNP-based heritability can be explained by known genetic loci and the remaining heritability likely resides around microglial related genes.

Carriers of the p.P522R variant in PLCγ2 have a slightly more responsive immune system.

BACKGROUND: The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells. METHODS: We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination. RESULTS: In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination. CONCLUSION: The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers.

Cognition, function, and prevalent dementia in centenarians and near-centenarians: An individual participant data (IPD) meta-analysis of 18 studies.

INTRODUCTION: There are limited data on prevalence of dementia in centenarians and near-centenarians (C/NC), its determinants, and whether the risk of dementia continues to rise beyond 100. METHODS: Participant-level data were obtained from 18 community-based studies (N = 4427) in 11 countries that included individuals ≥95 years. A harmonization protocol was applied to cognitive and functional impairments, and a meta-analysis was performed. RESULTS: The mean age was 98.3 years (SD = 2.67); 79% were women. After adjusting for age, sex, and education, dementia prevalence was 53.2% in women and 45.5% in men, with risk continuing to increase with age. Education (OR 0.95;0.92-0.98) was protective, as was hypertension (odds ratio [OR] 0.51;0.35-0.74) in five studies. Dementia was not associated with diabetes, vision and hearing impairments, smoking, and body mass index (BMI). DISCUSSION: Among the exceptional old, dementia prevalence remains higher in the older participants. Education was protective against dementia, but other factors for dementia-free survival in C/NC remain to be understood.

Resilience and resistance to the accumulation of amyloid plaques and neurofibrillary tangles in centenarians: An age-continuous perspective.

INTRODUCTION: With increasing age, neuropathological substrates associated with Alzheimer's disease (AD) accumulate in brains of cognitively healthy individuals-are they resilient, or resistant to AD-associated neuropathologies? METHODS: In 85 centenarian brains, we correlated NIA (amyloid) stages, Braak (neurofibrillary tangle) stages, and CERAD (neuritic plaque) scores with cognitive performance close to death as determined by Mini-Mental State Examination (MMSE) scores. We assessed centenarian brains against 2131 brains from AD patients, non-AD demented, and non-demented individuals in an age continuum ranging from 16 to 100+ years. RESULTS: With age, brains from non-demented individuals reached the NIA and Braak stages observed in AD patients, while CERAD scores remained lower. In centenarians, NIA stages varied (22.4% were the highest stage 3), Braak stages rarely exceeded stage IV (5.9% were V), and CERAD scores rarely exceeded 2 (4.7% were 3); within these distributions, we observed no correlation with the MMSE (NIA: P = 0.60; Braak: P = 0.08; CERAD: P = 0.16). DISCUSSION: Cognitive health can be maintained despite the accumulation of high levels of AD-related neuropathological substrates. HIGHLIGHTS: Cognitively healthy elderly have AD neuropathology levels similar to AD patients. AD neuropathology loads do not correlate with cognitive performance in centenarians. Some centenarians are resilient to the highest levels of AD neuropathology.

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease.

Alzheimer's disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals-16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-ß precursor protein processing, amyloid-ß aggregation, lipid metabolism and microglial function in AD.

Distinct cell type-specific protein signatures in GRN and MAPT genetic subtypes of frontotemporal dementia.

Frontotemporal dementia is characterized by progressive atrophy of frontal and/or temporal cortices at an early age of onset. The disorder shows considerable clinical, pathological, and genetic heterogeneity. Here we investigated the proteomic signatures of frontal and temporal cortex from brains with frontotemporal dementia due to GRN and MAPT mutations to identify the key cell types and molecular pathways in their pathophysiology. We compared patients with mutations in the GRN gene (n = 9) or with mutations in the MAPT gene (n = 13) with non-demented controls (n = 11). Using quantitative proteomic analysis on laser-dissected tissues we identified brain region-specific protein signatures for both genetic subtypes. Using published single cell RNA expression data resources we deduced the involvement of major brain cell types in driving these different protein signatures. Subsequent gene ontology analysis identified distinct genetic subtype- and cell type-specific biological processes. For the GRN subtype, we observed a distinct role for immune processes related to endothelial cells and for mitochondrial dysregulation in neurons. For the MAPT subtype, we observed distinct involvement of dysregulated RNA processing, oligodendrocyte dysfunction, and axonal impairments. Comparison with an in-house protein signature of Alzheimer's disease brains indicated that the observed alterations in RNA processing and oligodendrocyte function are distinct for the frontotemporal dementia MAPT subtype. Taken together, our results indicate the involvement of different brain cell types and biological mechanisms in genetic subtypes of frontotemporal dementia. Furthermore, we demonstrate that comparison of proteomic profiles of different disease entities can separate general neurodegenerative processes from disease-specific pathways, which may aid the development of disease subtype-specific treatment strategies.

Mapping the genetic landscape of early-onset Alzheimer's disease in a cohort of 36 families.

BACKGROUND: Many families with clinical early-onset Alzheimer's disease (EOAD) remain genetically unexplained. A combination of genetic factors is not standardly investigated. In addition to monogenic causes, we evaluated the possible polygenic architecture in a large series of families, to assess if genetic testing of familial EOAD could be expanded. METHODS: Thirty-six pedigrees (77 patients) were ascertained from a larger cohort of patients, with relationships determined by genetic data (exome sequencing data and/or SNP arrays). All families included at least one AD patient with symptom onset <70 years. We evaluated segregating rare variants in known dementia-related genes, and other genes or variants if shared by multiple families. APOE was genotyped and duplications in APP were assessed by targeted test or using SNP array data. We computed polygenic risk scores (PRS) compared with a reference population-based dataset, by imputing SNP arrays or exome sequencing data. RESULTS: In eight families, we identified a pathogenic variant, including the genes APP, PSEN1, SORL1, and an unexpected GRN frameshift variant. APOE-ε4 homozygosity was present in eighteen families, showing full segregation with disease in seven families. Eight families harbored a variant of uncertain significance (VUS), of which six included APOE-ε4 homozygous carriers. PRS was not higher in the families combined compared with the population mean (beta 0.05, P = 0.21), with a maximum increase of 0.61 (OR = 1.84) in the GRN family. Subgroup analyses indicated lower PRS in six APP/PSEN1 families compared with the rest (beta -0.22 vs. 0.10; P = 0.009) and lower APOE burden in all eight families with monogenic cause (beta 0.29 vs. 1.15, P = 0.010). Nine families remained without a genetic cause or risk factor identified. CONCLUSION: Besides monogenic causes, we suspect a polygenic disease architecture in multiple families based on APOE and rare VUS. The risk conveyed by PRS is modest across the studied families. Families without any identified risk factor render suitable candidates for further in-depth genetic evaluation.

A hidden layer of structural variation in transposable elements reveals potential genetic modifiers in human disease-risk loci.

Genome-wide association studies (GWAS) have been highly informative in discovering disease-associated loci but are not designed to capture all structural variations in the human genome. Using long-read sequencing data, we discovered widespread structural variation within SINE-VNTR-Alu (SVA) elements, a class of great ape-specific transposable elements with gene-regulatory roles, which represents a major source of structural variability in the human population. We highlight the presence of structurally variable SVAs (SV-SVAs) in neurological disease-associated loci, and we further associate SV-SVAs to disease-associated SNPs and differential gene expression using luciferase assays and expression quantitative trait loci data. Finally, we genetically deleted SV-SVAs in the BIN1 and CD2AP Alzheimer's disease-associated risk loci and in the BCKDK Parkinson's disease-associated risk locus and assessed multiple aspects of their gene-regulatory influence in a human neuronal context. Together, this study reveals a novel layer of genetic variation in transposable elements that may contribute to identification of the structural variants that are the actual drivers of disease associations of GWAS loci.

Risk of dementia in APOE ε4 carriers is mitigated by a polygenic risk score.

INTRODUCTION: We investigated relationships among genetic determinants of Alzheimer's disease (AD), amyloid/tau/neurodegenaration (ATN) biomarkers, and risk of dementia. METHODS: We studied cognitively normal individuals with subjective cognitive decline (SCD) from the Amsterdam Dementia Cohort and SCIENCe project. We examined associations between genetic variants and ATN biomarkers, and evaluated their predictive value for incident dementia. A polygenic risk score (PRS) was calculated based on 39 genetic variants. The APOE gene was not included in the PRS and was analyzed separately. RESULTS: The PRS and APOE ε4 were associated with amyloid-positive ATN profiles, and APOE ε4 additionally with isolated increased tau (A-T+N-). A high PRS and APOE ε4 separately predicted AD dementia. Combined, a high PRS increased while a low PRS attenuated the risk associated with ε4 carriers. DISCUSSION: Genetic variants beyond APOE are clinically relevant and contribute to the pathophysiology of AD. In the future, a PRS might be used in individualized risk profiling.

Genome-wide association study of frontotemporal dementia identifies a C9ORF72 haplotype with a median of 12-G4C2 repeats that predisposes to pathological repeat expansions.

Genetic factors play a major role in frontotemporal dementia (FTD). The majority of FTD cannot be genetically explained yet and it is likely that there are still FTD risk loci to be discovered. Common variants have been identified with genome-wide association studies (GWAS), but these studies have not systematically searched for rare variants. To identify rare and new common variant FTD risk loci and provide more insight into the heritability of C9ORF72-related FTD, we performed a GWAS consisting of 354 FTD patients (including and excluding N = 28 pathological repeat carriers) and 4209 control subjects. The Haplotype Reference Consortium was used as reference panel, allowing for the imputation of rare genetic variants. Two rare genetic variants nearby C9ORF72 were strongly associated with FTD in the discovery (rs147211831: OR = 4.8, P = 9.2 × 10-9, rs117204439: OR = 4.9, P = 6.0 × 10-9) and replication analysis (P < 1.1 × 10-3). These variants also significantly associated with amyotrophic lateral sclerosis in a publicly available dataset. Using haplotype analyses in 1200 individuals, we showed that these variants tag a sub-haplotype of the founder haplotype of the repeat expansion that was previously found to be present in virtually all pathological C9ORF72 G4C2 repeat lengths. This new risk haplotype was 10 times more likely to contain a C9ORF72 pathological repeat length compared to founder haplotypes without one of the two risk variants (~22% versus ~2%; P = 7.70 × 10-58). In haplotypes without a pathologic expansion, the founder risk haplotype had a higher number of repeats (median = 12 repeats) compared to the founder haplotype without the risk variants (median = 8 repeats) (P = 2.05 × 10-260). In conclusion, the identified risk haplotype, which is carried by ~4% of all individuals, is a major risk factor for pathological repeat lengths of C9ORF72 G4C2. These findings strongly indicate that longer C9ORF72 repeats are unstable and more likely to convert to germline pathological C9ORF72 repeat expansions.