Citations

AbstractBackgroundCerebral amyloid angiopathy (CAA), characterized by the accumulation of amyloid‐beta in the cerebrovasculature, effects blood vessel integrity leading to brain hemorrhages and an accelerated cognitive decline in Alzheimer’s disease (AD) patients. In a previous genome‐wide association study (GWAS) of 853 neuropathologically confirmed AD cases, we identified a LINC‐PINT splice variant that associated with lower CAA levels amongst individuals that did not have a copy of the APOEe4 AD risk allele (Reddy. J et al 2021). In this study, we perform GWAS to identify further genetic risk factors for CAA and AD neuropathology (Braak stage, Thal phase) in additional AD cases, and participants lacking significant AD pathology (non‐AD).MethodWe expanded the original AD cohort (N = 853) with 400 additional AD cases genotyped using the Infinium Omni2.5+Exome array. We also identified 110 AD and 502 non‐AD donors from the Mayo Clinic Brain Bank, with existing CAA scores and genome‐wide genotypes. We performed QC and imputation of these genotypes to TOPMed. We will conduct GWAS in AD only (N = 1,363), non‐AD only, as well as the combined cohort (N = 1,865). We will test imputed variant dosages for association with square root transformed CAA, Braak and Thal, using linear regression in PLINK, adjusting for relevant covariates. We will perform interaction analysis of CAA‐associated variants with APOEe4 and sex; and pursue stratified analyses. Summary statistics will be utilized to perform SNP‐based gene set enrichment analysis to identify biological pathways implicated in CAA.ResultWe expect that the increased power of this expanded dataset will lead to the identification of additional novel genetic risk factors for CAA and AD neuropathology. Genetic interaction analysis with sex and APOEe4 may further elucidate the role of these risk factors. The addition of 1,102 participants will enable assessment of the LINC‐PINT locus for association with CAA in an independent dataset.ConclusionWe expect this study will provide further insights into the genetic architecture underlying risk for CAA, both in the context of significant AD pathology, and without. Characterization of genetic variants in the context of neuropathology may lead to new avenues of research aimed at identifying biomarkers and therapies to treat CAA, and AD.

AbstractBackgroundTwo major risk factors of Alzheimer’s disease (AD) are advanced age and the APOE‐ε4 risk allele. However, despite having these risk factors, some individuals escape from developing AD or have slower cognitive decline. In this study we hypothesized that “resilient” individuals, defined as those who remain cognitively normal despite being >80 years‐old and carrying at least one copy of the APOE‐ε4 allele, have concerted changes in their blood transcriptomes that are associated with resilience to AD.MethodWe generated blood RNA‐sequence data from 105 APOE‐ε4 carriers from the Mayo Clinic Study of Aging (MCSA) who remained cognitively normal after age 80 (resilient), or who developed mild cognitive impairment (MCI) or AD (non‐resilient). Weighted gene co‐expression network analysis (WGCNA) was performed to evaluate the association of the blood co‐expression network with primary phenotypes of resilience and memory scores. To validate our findings, we constructed a consensus co‐expression network using both MCSA and a replication cohort of blood microarray data from 91 Alzheimer’s Disease Neuroimaging Initiative (ADNI) study participants who had a similar demographic profile. We annotated the consensus modules for their enrichment of cell types, biological processes, genes within previously published genome‐wide significant (GWS) AD‐associated loci, and genes that reside in consensus modules built from seven brain regions across three different autopsy cohorts. To identify genetic variants regulating blood consensus module co‐expression, we performed module quantitative trait locus (modQTL) analysis.ResultWe found co‐expression modules significantly (q<0.05) correlated with the memory phenotype, logical memory delayed recall, across both MCSA and ADNI cohorts. Some of these modules were enriched with blood cell types and biological processes, including telomere maintenance, mitochondrial functions, and immune response. We found 1 module significantly (q<0.05) enriched with genes near GWS AD‐associated loci and 4 modules with genes in brain consensus modules. Lastly, we found 8 modQTLs significantly (Bonferroni‐p<0.05) associated with the co‐expression of 5 different modules.ConclusionWe identified blood co‐expression modules, biological pathways and specific genetic variants associated with cognitive resilience in the presence of AD risk factors. This study provides novel insights into the potential role of peripheral blood in the development of and resilience to AD.

AbstractBackgroundTau proteins aggregate into spatially and morphologically distinct lesions in primary and secondary tauopathies. These signature lesions result from deposition of tau proteins that are distinctly modified via missense mutation and/or post‐translational modification for each disease strain. Identifying the unique tau protein abnormalities that lead to specific neurodegenerative diseases is beneficial in their early diagnosis and treatment. This is especially true for Progressive Supranuclear Palsy a primary tauopathy with clinical symptoms mirroring Parkinsonian syndromes and frontotemporal dementias.MethodBiochemical analysis of tau aggregates from patients with a pathological diagnosis of Progressive Supranuclear Palsy (n = 60) demonstrate that the tau protein modifications observed in this disease result from tau that is specifically modified and thereby forms the basis of the distinct disease strains observed. Here data collected using the sarkosyl‐insoluble tau fractions from tissue samples of pathologically diagnosed patients with Progressive Supranuclear Palsy without comorbidities allows for the direct observation of the modifications. Most interesting is the variability of the tau proteins that actually accumulate for this disease opening up the possibility that tau strains may exist within any given tauopathy even as the clinical manifestations appear similar. This indicates that early and specific identification and detection of these strains is critical for the development of therapies for these diseases.ResultExtraction and examination of sarkosyl‐insoluble tau proteins from deceased patients pathologically diagnosed with Progressive Supranuclear Palsy indicate that multiple tau strains exist within this primary tauopathy. Western blotting of these extracts from patient tissues demonstrates a unique banding pattern of insoluble tau in a subset of the cases that exhibit extreme hyperphosphorylation as indicated by additional high molecular weight tau banding. These patients also had an earlier age of onset and age of death indicating a more aggressive disease strain. Additional data is presented comparing the structure and seeding capability of this insoluble tau and the distribution and burden of insoluble tau in these Progressive Supranuclear Palsy patients compared to those with typical onset and insoluble tau signature.ConclusionMultiple pathological tau strains exist in Progressive Supranuclear Palsy.

AbstractBackgroundMicroglia, the resident brain immune cells, play a critical role in brain homeostasis and disease progression. In neurodegenerative conditions, microglia acquire the neurodegenerative phenotype (MGnD), whose function is poorly understood. MicroRNA‐155 (miR‐155), enriched in immune cells, critically regulates MGnD. However, its role in Alzheimer’s disease (AD) pathogenesis remains unclear.MethodsWe used RNAseq and immunohistochemistry (n = 6‐8 per sex per group) to investigate the gene expression profile and AD pathology. We further utilized single‐cell RNAseq (n = 5 per group) to identify microglial clusters. Whole‐tissue proteomics (n = 4) was applied to detect the protein changes in brain milieu. Moreover, we used spontaneous alternation and forced alternation tests to evaluate the cognition (n = 33‐36).ResultWe report that microglial deletion of miR‐155 induces a pre‐MGnD activation state via interferon‐g (IFNg) signaling and blocking IFNg signaling attenuates MGnD induction and microglial phagocytosis. Single‐cell RNAseq analysis of microglia from AD mouse model identifies Stat1 and Clec2d as pre‐MGnD markers. This phenotypic transition enhances amyloid plaque compaction, reduces dystrophic neurites, attenuates plaque‐associated synaptic degradation, and improves cognition.ConclusionOur study demonstrates a novel miR‐155‐mediated regulatory mechanism of MGnD and the beneficial role of IFNg‐responsive pre‐MGnD in restricting neurodegenerative pathology and preserving cognitive function in an AD mouse model, highlighting miR‐155 and IFNg as potential therapeutic targets for AD.

AbstractBackgroundAPOE ε4 is the strongest genetic risk factor for late‐onset Alzheimer’s disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD).MethodGeneration of CX3CR1‐CREERT2 mice crossed to APOE‐KI(E3 and E4)fl/fl: APP/PS1 and APOE‐KI(E3 and E4)fl/fl: P301S mice. Microglia and astrocytes were isolated and sequenced using either bulk RNA‐seq (MGnD‐Paradigm) or single‐cell RNA‐seq. Crosstalk between cells was determined using the NichenetR database and validated using 1) recombinant Lgals3 intracranial injection into APP/PS1 mice and 2) adoptive transfer of microglia, from APOE3‐KI, APOE4‐KI, and APOE4‐cKO mice challenged with labeled apoptotic neurons, into WT mice and isolation of astrocytes. Validation in the brain of AD donors carrying the APOE e3/3 and e3/4 alleles.ResultHere we show, in mice and in humans, a negative role of microglial APOE4 in the induction of MGnD response to neurodegeneration. Deletion of microglial APOE4 restores MGnD phenotype, associated with neuroprotection in P301S tau transgenic mice, and decreases pathology in APP/PS1 mice. MGnD‐astrocyte crosstalk associated with b‐amyloid (Ab) plaque encapsulation and clearance is mediated via Lgals3 signaling following microglial APOE4 deletion. In the brain of AD donors carrying the APOE ε4 allele, we found a sex‐dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, as compared to APOE ε3/3 carriers. Mechanistically, APOE4‐mediated induction of ITGB8‐TGFb signaling impairs MGnD response via upregulation of TGFb‐mediated microglial homeostatic checkpoints, including INPP5D. Microglial deletion of Inpp5d restores MGnD‐astrocyte crosstalk and facilitates plaque clearance in APP/PS1 mice. Genetic and pharmacological blocking of ITGB8‐TGFb signaling enhanced MGnD response associated with increased plaque clearance in AD mice.ConclusionWe identify the microglial APOE4‐ITGB8‐TGFb pathway as a negative regulator of microglia‐astrocyte response to AD pathology, and restoring MGnD phenotype via blocking ITGB8‐TGFb signaling may provide new molecular targets to modulate and restore functional microglia in AD.

AbstractBackgroundAlzheimer’s disease (AD) is a complex, multicellular disease with immunological and genetic components. Previous studies demonstrated associations between AD and the missense variants ABI3‐rs616338‐T and PLCG2‐rs72824905‐G. The perturbed expression of these genes has been demonstrated to disrupt microglial function. To date, the transcriptomic profiles of variant carriers in the context of microglial subtype and state have not been explored in human brain. This represents a knowledge gap in our understanding of AD risk and resilience. We have developed and optimized a single nucleus RNAseq (snRNAseq) approach to study the impact of ABI3 and PLCG2 variants on microglial enriched nuclei from frozen human brain.MethodDOCK8 was identified as a nuclear microglial marker based on previous brain snRNAseq data. DOCK8 based microglial enrichment using fluorescent activated nuclei sorting (FANS) was validated using RT‐qPCR and snRNAseq in human cerebellum and temporal cortex (TCX), respectively. We are applying this enrichment and snRNAseq approach to TCX tissue from a cohort of 30 donors harboring ABI3 or PLCG2 missense mutations, or neither. Standard snRNAseq alignment and quality control pipelines will be applied, and nuclei annotated based on putative microglial markers. Differential gene expression will be assessed using pseudo bulk and MAST approaches, for association with ABI3 and PLCG2 genotype, diagnosis groups, risk factors, and pathology scores.ResultDOCK8 has several commercially available antibodies, making it an ideal microglia‐selection marker. Using RT‐qPCR in microglia enriched nuclei, we demonstrated an enrichment of microglia marker genes and depletion of other brain cell type markers. This is further supported by snRNA‐seq data from DOCK8‐sorted TCX tissue that yielded a 5x increase in microglia proportion (56% vs. 11% in unselected nuclei), enabling analyses of microglia sub‐populations. Leveraging this approach, we will investigate affected genes and pathways and uncover the resilience and risk mechanisms within microglia sub‐populations in ABI3 and PLCG2 variant‐carriers.ConclusionOur study presents a novel method for enriching microglial nuclei from frozen human brains and enables transcriptomic profiling of microglial subpopulations in ABI3 and PLCG2 missense variant‐carriers. We expect these insights will reveal novel genes and pathways that may play a key role in microglial function in AD.

AbstractBackgroundLoss of function mutations in PINK1 and PRKN are the most common causes of early‐onset Parkinson disease (PD). The encoded ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN together regulate the autophagic degradation of dysfunctional mitochondria, termed mitophagy. During this cytoprotective process, PINK1 and PRKN identify and jointly decorate damaged mitochondria with phosphorylated Ub (pS65‐Ub) that serves as the mitophagy tag. Accumulating evidence suggest a pathogenic role for impaired mitophagy in PD and related neurodegenerative conditions. This is in line with our previous finding of increased levels of the mitophagy marker pS65‐Ub in the hippocampus of postmortem Lewy body disease (LBD) brain.MethodWe performed a genome‐wide association study (GWAS) of hippocampal pS65‐Ub level to identify novel regulators of the PINK1‐PRKN‐directed mitophagy in LBD. In our two‐stage GWAS, we obtained 1,012 autopsy confirmed LBD samples (754 discovery stage, 258, replication stage) from Caucasian participants of European ancestry. Using an established, mostly automated workflow, hippocampal sections were immunostained for pS65‐Ub, scanned, and quantified with unbiased algorithms.ResultOur findings identified that APOE rs429358 (β: 0.50, 95% CI: 0.41 to 0.69, p = 8.67×10−25) was very strongly associated with pS65‐Ub levels in LBD brain. The APOE4 genotype is known to exacerbate Aβ and tau deposition. We did observe strong correlations of pS65‐Ub levels with both tau tangle and senile plaque density in the entire LBD cohort. Nevertheless, the increased pS65‐Ub levels in APOE4‐carrying LBD brains were also independent of Aβ and tau deposition in a subgroup (N = 175) with medium levels of these pathologies. To further explore APOE4 effects on mitophagy outside disease context, this observation was also confirmed in APOE‐targeted replacement mice and in induced pluripotent stem cell‐derived astrocytes.ConclusionOur findings nominate a novel mitophagy regulator in LBD brain and highlight a strong association of APOE4 with mitophagy alteration. Our results suggested that APOE4 may mediate mitophagy defects through two mechanisms, by aggravating Alzheimer’s‐type pathologies and also independent of them. With APOE4 being the strongest known risk factor for Alzheimer’s disease and dementia with Lewy bodies, our findings suggest a common mechanistic link underscoring the importance of mitochondrial quality control.

AbstractBackgroundContext‐specific networks can capture functionally important genes that may not be differentially expressed. Leveraging seven different gene expression datasets of Alzheimer’s disease (AD) and healthy control brains, we constructed a novel pipeline that includes such network analysis, augmented by in vivo validations and in silico drug repurposing to facilitate prioritization of AD therapeutic targets.MethodWe utilized publicly available gene expression data from 733 AD and 454 control brains across 7 datasets and the NetDecoder (PMID: 26975659) network biology platform to build context‐specific networks. Based on the known protein‐protein interaction and gene expression correlations, information flow from the top differentially expressed genes (DEG) to transcriptional regulator genes was calculated using a process‐guided flow algorithm. Genes with the greatest informational flow changes across AD cohorts were systematically prioritized. We next validated these genes in a medium‐throughput screen of a tau‐transgenic Drosophila model (PMID: 11408621). Further, we identified existing small‐molecule drugs to target the candidate genes utilizing curated databases.ResultsA total of 14 context‐specific networks were constructed across 7 datasets. We found AD‐related genes, such as APP, to have high flow differences between AD and control networks and high connectivity. Notably, many such genes were not DEGs between AD and control, suggesting that the information flow captures novel aspects of AD pathophysiology. We nominated 224 unique genes with the greatest flow differences for functional validation. We screened 111 of the 224 candidate genes with readily available, high‐confidence orthologs in Drosophila. We identified genes that suppress (N = 68) or enhance (N = 27) tau‐related neurodegeneration in this model. We confirmed the effects of previously nominated genes, such as HDAC1, which enhances tau‐neurodegeneration if overexpressed and suppresses it if knocked down. We also identified and validated additional therapeutic targets. Lastly, we applied a network‐proximity‐based algorithm and identified small molecule drugs for the candidate genes.ConclusionWe present a novel pipeline that integrates analytic approaches, experimental validation, and drug repurposing to facilitate therapeutic discovery in AD. We applied this pipeline and nominated previously known and novel genes implicated in AD pathogenesis. Our pipeline demonstrates a cross‐species, systems‐level approach to therapeutic nomination in AD and related neurodegenerative diseases.

AbstractBackgroundThe coarse‐grained plaque is a biochemically and morphologically distinct amyloid‐β (Aβ) deposit that was discovered in clinically‐defined atypical Alzheimer’s disease (AD). Unlike other Aβ deposits, this plaque is not observed in cognitively normal people. This study investigates the hypothesis that the coarse‐grained plaque is distinctly associated with AD neuropathologic change and glial activation within the context of AD subtypes.MethodSixty AD cases from the FLorida Autopsied Multi‐Ethnic (FLAME) cohort were assessed. We included 20 hippocampal sparing (HpSp) AD, 20 typical AD, and 20 limbic predominant AD. Immunohistochemistry was performed on the middle frontal, inferior temporal, and inferior parietal cortex for AD neuropathology (Aβ [6F/3D], phospho‐tau [AT8], tau [GT38]) and glial activation (CD68 [KP1] and GFAP). Digital pathology was used to quantify burden. Thioflavin‐S tangle counts were performed at time of neuropathologic diagnosis. Distinct deposit types, including classic cored plaques, coarse‐grained plaques, cerebral amyloid angiopathy (CAA)‐Type 1, and CAA‐Type 2, were semi‐quantitatively scored on Aβ (Fig. 1). Correlations between demographic, clinical, and neuropathologic characteristics were assessed using Spearman rho analysis and reported if p<0.05. An independent cohort of ∼100 HpSp AD cases will be assessed to validate initial findings.ResultIn the frontal cortex of HpSp AD, higher scores of classic cored plaques correlated with older age at onset and death, non‐APOE ε4 carriership, and less CAA‐Type 1 and Type 2 (Fig. 2). On the contrary, the severity of coarse‐grained plaques correlated with a younger age at onset and death, APOE ε4 carriership, higher scores for CAA‐Type 1, CAA‐Type 2, and increased burden of Aβ, GFAP, and CD68. Classic cored plaques correlated negatively with phospho‐tau and tau, whereas coarse‐grained plaques correlated positively with phospho‐tau, tau, and tangles. The two deposit types were negatively correlated with each other. Correlations were weaker but showed similar trends in other AD subtypes (Fig. 2) and regions.ConclusionWithin HpSp AD, classic cored and coarse‐grained plaques show distinct correlations with glial activation, (phospho‐)tau, and tangles. These results suggest opposite directions of association for these deposits on AD neuropathologic change that may impact clinical symptoms. These findings are relevant to therapies targeting overall Aβ pathology.

AbstractBackgroundBrain glucose hypometabolism is among the earliest pathogenic changes in Alzheimer’s disease (AD). This metabolic dysfunction points to the personal bioenergetic capacity, defined as the ability to maintain energy homeostasis under all circumstances including deregulated glucose uptake, as a potential source of resilience to the disease. Fasting blood acylcarnitine profiles are a central readout for this capacity in the absence of dietary glucose and capture the activity and efficiency of glucose‐independent routes of mitochondrial energy metabolism.MethodWe used fasting serum acylcarnitine profiles of 1,531 participants (465 with normal cognition, 762 with mild cognitive impairment, and 304 with clinical AD) from the AD Neuroimaging Initiative to perform unsupervised subgroup identification using hierarchical clustering. Identified subgroups were investigated for differences in A/T/N biomarker profiles and cognitive status. The contributions of genetic and potentially modifiable factors defining the subgroups were quantified using analysis of explained variance. The influence of the strongest determining factors on longitudinal cognitive trajectories was estimated using linear mixed‐effects models and gene‐by‐environment interaction analysis.ResultWe found several bioenergetically distinct subgroups with significant differences in AD biomarker profiles and cognitive function. The strongest genetic contribution to this bioenergetic endophenotype seems to be specifically linked to succinylcarnitine metabolism and significantly modulates the rate of future cognitive decline. In contrast, potentially modifiable sustainment of beta‐oxidation efficiency seems to decelerate bioenergetic aging, thus creating a bioenergetic reserve that delays progression of cognitive decline. Using gene‐by‐environment interaction analysis, we demonstrate that this molecular framework identifies a subgroup of individuals that is likely to benefit significantly from personalized therapeutic, dietary or lifestyle interventions tailored to increase resilience against bioenergetic disturbances in AD.ConclusionOur study reports on a set of genetic and metabolic markers that define bioenergetically distinct subgroups with significant differences on the AD biomarker and cognitive level. Longitudinal data suggest that targeting the modifiable fraction of this endophenotype might be a promising strategy to slow down disease progression in individuals with specific allelic configurations.