Zhao, Na

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.

AbstractBackgroundAlzheimer’s disease (AD) is neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often quantified by Thal phase and Braak stage, respectively. Aβ also frequently deposits in the cerebrovasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. These and related measures often show high variability within AD suggesting distinct underlying mechanisms, the identification of which may reveal important biological insights into AD pathophysiology. We hypothesize that, within the AD brain, neuropathology and levels of core AD‐related proteins are influenced by variations in DNA methylation (DNAm). To test this, we performed epigenome‐wide association studies (EWAS) using DNAm clusters from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathologic measures (Braak, Thal, and CAA) and brain biochemical levels of five proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodsDNAm from neuropathologically‐confirmed AD cases available through the Mayo Clinic brain bank was measured by reduced representation bisulfite sequencing (RRBS) from 471 brain samples, 200 of which had both TCX and CER measures. Neuropathology was scored by an experienced neuropathologist. TCX levels of five AD‐related proteins from three tissue fractions (buffer‐, detergent‐, and in‐soluble) were measured by ELISA (Liu 2020). CpG methylation (CpGm) was binned by a 15‐state chromatin model (Kundaje 2015) and averaged. Averaged CpGm clusters were tested for their association with each endophenotype through multi‐variable linear regression. The influence of CpGm clusters on expression levels of nearby genes was also investigated through multi‐variable regression.ResultsOur innovative binning method demonstrated expected and biologically relevant CpGm patterns. We identified multiple epigenome‐wide significant CpGm clusters unique to each AD‐related endophenotype and brain region. Comparisons between the TCX and CER further revealed brain region‐specific enrichment of distinct CpGm clusters. Significant CpGm clusters were further characterized by their influence on gene expression.ConclusionsAlthough all neuropathologic and biochemical endophenotypes tested are core to AD pathophysiology, our results suggest each has a distinct epigenetic architecture underlying their variability in the AD brain. By discovering brain region‐specific DNAm changes that contribute to each neuropathologic or biochemical outcome, we can identify core components of complex mechanisms that individually and jointly contribute to AD.

AbstractBackgroundGene expression changes have been identified in post‐mortem brain tissue of AD cases when compared with controls, implicating various genes and pathways. Such findings provide important insights into molecular dysregulation associated with this disease and nominate therapeutic targets. Here we investigate brain gene and co‐expression network changes in a cohort of AD cases to identify associations with degree of AD neuropathology including Braak stage (Tau), Thal phase (Amyloid beta) and brain tissue levels of five AD‐related proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodGene expression measures were collected from temporal cortex (TCX) tissue of 477 AD cases, available through the Mayo Clinic Brain Bank, using RNA sequencing (Illumina TruSeq mRNA) performed on the HiSeq4000. Following quality control 456 cases remained for analysis. Braak and Thal were provided by an experienced neuropathologist and TCX levels of five AD‐related proteins from three fractions (buffer‐soluble, detergent‐soluble, and insoluble) were measured by ELISA (Liu et al. 2020). Weighted gene coexpression network analysis (WGCNA) R package was used to build co‐expression networks. Genes and networks were tested for association with neuropathology and AD‐related protein levels, adjusting for relevant covariates.ResultA total of 57,353 genes were profiled, of which 19,044 were reliably expressed above background levels and formed 20 co‐expression modules. 36 genes were associated with Braak stage, 10 with Thal phase and up to 9,000 with the AD‐related protein fractions. Brain levels of pTau had the greatest number of associations, with an overrepresentation of genes involved in synaptic signalling and immune response gene ontology biological processes. 16 of the coexpression modules associated with at least one trait after Bonferroni correction for number of tests.ConclusionWe have identified a number of genes and networks associated with different aspects of AD neuropathology and associated proteins, further lending support to the involvement of synaptic and immune biology in AD. However it is not clear whether observed changes cause disease or are a consequence of the neurodegenerative process. Comparison of these findings across different proteins and fractions is expected to reveal common and distinct vulnerabilities associated with amyloid and tau, and different stages of protein aggregation.

ObjectiveRecent evidence supports a link between increased TDP‐43 burden and the presence of an APOE4 gene allele in Alzheimer's disease (AD); however, it is difficult to conclude the direct effect of APOE on TDP‐43 pathology due to the presence of mixed AD pathologies. The goal of this study is to address how APOE isoforms impact TDP‐43 pathology and related neurodegeneration in the absence of typical AD pathologies.MethodsWe overexpressed human TDP‐43 via viral transduction in humanized APOE2, APOE3, APOE4 mice, and murine Apoe‐knockout (Apoe‐KO) mice. Behavior tests were performed across ages. Animals were harvested at 11 months of age and TDP‐43 overexpression‐related neurodegeneration and gliosis were assessed. To further address the human relevance, we analyzed the association of APOE with TDP‐43 pathology in 160 postmortem brains from autopsy‐confirmed amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with motor neuron disease (FTLD‐MND) in the Mayo Clinic Brain Bank.ResultsWe found that TDP‐43 overexpression induced motor function deficits, neuronal loss, and gliosis in the motor cortex, especially in APOE2 mice, with much milder or absent effects in APOE3, APOE4, or Apoe‐KO mice. In the motor cortex of the ALS and FTLD‐MND postmortem human brains, we found that the APOE2 allele was associated with more severe TDP‐43‐positive dystrophic neurites.InterpretationOur data suggest a genotype‐specific effect of APOE on TDP‐43 proteinopathy and neurodegeneration in the absence of AD pathology, with the strongest association seen with APOE2. ANN NEUROL 2023;93:830–843

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often measured by Thal phase and Braak stage, respectively. Frequently, Aβ also deposits in the brain vasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. Assessment of these and related endophenotypes can reveal biological insights into the phenotypic variability in AD. We hypothesize that, amongst AD patients, severity of neuropathology and brain levels of AD‐related proteins are influenced by variation in DNA methylation (DNAm), the identification of which may uncover biological pathways for AD pathogenesis. To test this, we performed epigenome‐wide association studies (EWAS) using DNAm from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathology measures (Braak, Thal, and CAA) and brain levels of five proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodsDNAm from autopsy‐confirmed AD cases available through the Mayo Clinic Brain Bank was measured by reduced representation bisulfite sequencing (RRBS) from 471 brain samples, 200 of which had both TCX and CER measures. Bisulfite converted reads were sequenced on the Illumina High Seq4000 and quality filtered. Neuropathology scoring was performed by an experienced neuropathologist. TCX levels of five AD‐related proteins from three fractions (buffer‐soluble, detergent‐soluble, and insoluble) were measured by ELISA (Liu et al. 2020). Individual CpG methylation ratios were tested for association with each neuropathological and biochemical measure using the R package CpGassoc.ResultsWe identified multiple epigenome‐wide significant CpGs unique to each AD‐related measure. Comparisons between the TCX and CER revealed region specific CpG associations. The effects of sex, age at death, APOE genotype, and presence of genetic variants for each significant CpG association were also identified.ConclusionsAlthough all neuropathological and biochemical measures tested reflect core AD pathologies, our results demonstrate unique associations of epigenetic factors with these individual phenotypes, suggesting that their variability may be governed by distinct epigenetic processes. Discovery of DNAm changes that underlie specific neuropathological or biochemical outcomes can enhance our understanding of AD pathophysiology and may lead to the identification of novel gene or pathway targets for the different facets of this complex condition.

TREM2 is exclusively expressed by microglia in the brain and is strongly linked to the risk for Alzheimer’s disease (AD). As microglial responses modulated by TREM2 are central to AD pathogenesis, enhancing TREM2 signaling has been explored as an AD therapeutic strategy. However, the effective therapeutic window targeting TREM2 is unclear. Here, by using microglia-specific inducible mouse models overexpressing human wild-type TREM2 (TREM2-WT) or R47H risk variant (TREM2-R47H), we show that TREM2-WT expression reduces amyloid deposition and neuritic dystrophy only during the early amyloid seeding stage, whereas TREM2-R47H exacerbates amyloid burden during the middle amyloid rapid growth stage. Single-cell RNA sequencing reveals suppressed disease-associated microglia (DAM) signature and reduced DAM population upon TREM2-WT expression in the early stage, whereas upregulated antigen presentation pathway is detected with TREM2-R47H expression in the middle stage. Together, our findings highlight the dynamic effects of TREM2 in modulating AD pathogenesis and emphasize the beneficial effect of enhancing TREM2 function in the early stage of AD development.

Abstract Background: The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimer’s disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site and was shown to enhance shedding and impair phagocytosis in vitro by ectopic expression of TREM2-H157Y in HEK293 cells. However, the physiological and AD-related outcomes of TREM2 H157Y mutation in vivo remain unknown.Methods: We generated a novel Trem2 H157Y knock-in mouse model through CRISPR-Cas9 technology and investigated how Trem2 H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology by conducting biochemical assays, immunofluorescent staining, hippocampal electrophysiology, and in vivo micro-dialysis in awake, free-moving animals.Results: Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates Aβ clearance and reduces amyloid burden and microgliosis. Conclusion: Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

A rare APOE variant APOE3 -Jac reduces self-aggregation and promotes healthy brain aging.

Abstract Triggering receptor expressed on myeloid cell 2 (TREM2) is a surface receptor that, in the central nervous system, is exclusively expressed on microglia. TREM2 variants have been linked to increased risk for neurodegenerative diseases, but the functional effects of microglial TREM2 remain largely unknown. To this end, we investigated TAR-DNA binding protein 43 kDa (TDP-43)-related neurodegenerative disease via viral-mediated expression of human TDP-43 protein (hTDP-43) in mice or inducible expression of hTDP43 with defective nuclear localization signals in transgenic mice. We found that TREM2 deficiency impaired microglia phagocytic clearance of pathological TDP-43, and enhanced neuronal damage and motor function impairments. Mass cytometry analysis revealed that hTDP-43 induced a TREM2-dependent subpopulation of microglia with high CD11c expression and higher phagocytic ability. Using mass spectrometry, we further demonstrated an interaction between TDP-43 and TREM2, in vitro and in vivo, in hTDP-43-expressing transgenic mouse brains. We computationally identified the region within hTDP-43 that interacts with TREM2 and observed the potential interaction in ALS patient tissues. Our data reveal the novel interaction between TREM2 and TDP-43, highlighting that TDP-43 is a potential ligand for microglial TREM2 and the interaction mediates neuroprotection of microglial TREM2 in TDP-43-related neurodegeneration.