Cell-autonomous effects of APOE4 in restricting microglial response in brain homeostasis and Alzheimer's disease.

Microglial involvement in Alzheimer's disease (AD) pathology has emerged as a risk-determining pathogenic event. While apolipoprotein E (APOE) is known to modify AD risk, it remains unclear how microglial apoE impacts brain cognition and AD pathology. Here, using conditional mouse models expressing apoE isoforms in microglia and central nervous system-associated macrophages (CAMs), we demonstrate a cell-autonomous effect of apoE3-mediated microglial activation and function, which are negated by apoE4. Expression of apoE3 in microglia/CAMs improves cognitive function, increases microglia surrounding amyloid plaque and reduces amyloid pathology and associated toxicity, whereas apoE4 expression either compromises or has no effects on these outcomes by impairing lipid metabolism. Single-cell transcriptomic profiling reveals increased antigen presentation and interferon pathways upon apoE3 expression. In contrast, apoE4 expression downregulates complement and lysosomal pathways, and promotes stress-related responses. Moreover, in the presence of mouse endogenous apoE, microglial apoE4 exacerbates amyloid pathology. Finally, we observed a reduction in Lgals3-positive responsive microglia surrounding amyloid plaque and an increased accumulation of lipid droplets in APOE4 human brains and induced pluripotent stem cell-derived microglia. Our findings establish critical isoform-dependent effects of microglia/CAM-expressed apoE in brain function and the development of amyloid pathology, providing new insight into how apoE4 vastly increases AD risk.

APOE2 Exacerbates TDP-43 Related Toxicity in the Absence of Alzheimer Pathology.

OBJECTIVE: Recent 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. METHODS: We 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. RESULTS: We 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. INTERPRETATION: Our 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.

APOE4 exacerbates α-synuclein seeding activity and contributes to neurotoxicity in Alzheimer's disease with Lewy body pathology.

Approximately half of Alzheimer's disease (AD) brains have concomitant Lewy pathology at autopsy, suggesting that α-synuclein (α-SYN) aggregation is a regulated event in the pathogenesis of AD. Genome-wide association studies revealed that the ε4 allele of the apolipoprotein E (APOE4) gene, the strongest genetic risk factor for AD, is also the most replicated genetic risk factor for Lewy body dementia (LBD), signifying an important role of APOE4 in both amyloid-ß (Aß) and α-SYN pathogenesis. How APOE4 modulates α-SYN aggregation in AD is unclear. In this study, we aimed to determine how α-SYN is associated with AD-related pathology and how APOE4 impacts α-SYN seeding and toxicity. We measured α-SYN levels and their association with other established AD-related markers in brain samples from autopsy-confirmed AD patients (N = 469), where 54% had concomitant LB pathology (AD + LB). We found significant correlations between the levels of α-SYN and those of Aß40, Aß42, tau and APOE, particularly in insoluble fractions of AD + LB. Using a real-time quaking-induced conversion (RT-QuIC) assay, we measured the seeding activity of soluble α-SYN and found that α-SYN seeding was exacerbated by APOE4 in the AD cohort, as well as a small cohort of autopsy-confirmed LBD brains with minimal Alzheimer type pathology. We further fractionated the soluble AD brain lysates by size exclusion chromatography (SEC) ran on fast protein liquid chromatography (FPLC) and identified the α-SYN species (~ 96 kDa) that showed the strongest seeding activity. Finally, using human induced pluripotent stem cell (iPSC)-derived neurons, we showed that amplified α-SYN aggregates from AD + LB brain of patients with APOE4 were highly toxic to neurons, whereas the same amount of α-SYN monomer was not toxic. Our findings suggest that the presence of LB pathology correlates with AD-related pathologies and that APOE4 exacerbates α-SYN seeding activity and neurotoxicity, providing mechanistic insight into how APOE4 affects α-SYN pathogenesis in AD.

Impact of APOE on amyloid and tau accumulation in argyrophilic grain disease and Alzheimer's disease.

Alzheimer's disease (AD), characterized by the deposition of amyloid-ß (Aß) in senile plaques and neurofibrillary tangles of phosphorylated tau (pTau), is increasingly recognized as a complex disease with multiple pathologies. AD sometimes pathologically overlaps with age-related tauopathies such as four repeat (4R)-tau predominant argyrophilic grain disease (AGD). While AGD is often detected with AD pathology, the contribution of APOE4 to AGD risk is not clear despite its robust effects on AD pathogenesis. Specifically, how APOE genotype influences Aß and tau pathology in co-occurring AGD and AD has not been fully understood. Using postmortem brain samples (N = 353) from a neuropathologically defined cohort comprising of cases with AD and/or AGD pathology built to best represent different APOE genotypes, we measured the amounts of major AD-related molecules, including Aß40, Aß42, apolipoprotein E (apoE), total tau (tTau), and pTau181, in the temporal cortex. The presence of tau lesions characteristic of AD (AD-tau) was correlated with cognitive decline based on Mini-Mental State Examination (MMSE) scores, while the presence of AGD tau lesions (AGD-tau) was not. Interestingly, while APOE4 increased the risk of AD-tau pathology, it did not increase the risk of AGD-tau pathology. Although APOE4 was significantly associated with higher levels of insoluble Aß40, Aß42, apoE, and pTau181, the APOE4 effect was no longer detected in the presence of AGD-tau. We also found that co-occurrence of AGD with AD was associated with lower insoluble Aß42 and pTau181 levels. Overall, our findings suggest that different patterns of Aß, tau, and apoE accumulation mediate the development of AD-tau and AGD-tau pathology, which is affected by APOE genotype.

Suppression of Wnt/ß-Catenin Signaling Is Associated with Downregulation of Wnt1, PORCN, and Rspo2 in Alzheimer's Disease.

Wnt and R-spondin (Rspo) proteins are two major types of endogenous Wnt/ß-catenin signaling agonists. While Wnt/ß-catenin signaling is greatly diminished in Alzheimer's disease (AD), it remains to be elucidated whether the inhibition of this pathway is associated with dysregulation of Wnt and Rspo proteins. By analyzing temporal cortex RNA-seq data of the human postmortem brain samples, we found that WNT1 and RRPO2 were significantly downregulated in human AD brains. In addition, the expression of Wnt acyltransferase porcupine (PORCN), which is essential for Wnt maturation and secretion, was greatly deceased in these human AD brains. Interestingly, the lowest levels of WNT1, PORCN, and RSPO2 expression were found in human AD brains carrying two copies of APOE4 allele, the strongest genetic risk factor of late-onset AD. Importantly, there were positive correlations among the levels of WNT1, PORCN, and RSPO2 expression in human AD brains. Supporting observations in humans, Wnt1, PORCN, and Rspo2 were downregulated and Wnt/ß-catenin signaling was diminished in the 5xFAD amyloid model mice. In human APOE-targeted replacement mice, downregulation of WNT1, PORCN, and RSPO2 expression was positively associated with aging and APOE4 genotype. Finally, WNT1 and PORCN expression and Wnt/ß-catenin signaling were inhibited in human APOE4 iPSC-derived astrocytes when compared to the isogenic APOE3 iPSC-derived astrocytes. Altogether, our findings suggest that the dysregulations of Wnt1, PORCN, and Rspo2 could be coordinated together to diminish Wnt/ß-catenin signaling in aging- and APOE4-dependent manners in the AD brain.

APOE deficiency impacts neural differentiation and cholesterol biosynthesis in human iPSC-derived cerebral organoids.

BACKGROUND: The apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimer's disease (AD); however, how it modulates brain homeostasis is not clear. The apoE protein is a major lipid carrier in the brain transporting lipids such as cholesterol among different brain cell types. METHODS: We generated three-dimensional (3-D) cerebral organoids from human parental iPSC lines and its isogenic APOE-deficient (APOE-/-) iPSC line. To elucidate the cell-type-specific effects of APOE deficiency in the cerebral organoids, we performed scRNA-seq in the parental and APOE-/- cerebral organoids at Day 90. RESULTS: We show that APOE deficiency in human iPSC-derived cerebral organoids impacts brain lipid homeostasis by modulating multiple cellular and molecular pathways. Molecular profiling through single-cell RNA sequencing revealed that APOE deficiency leads to changes in cellular composition of isogenic cerebral organoids likely by modulating the eukaryotic initiation factor 2 (EIF2) signaling pathway as these events were alleviated by the treatment of an integrated stress response inhibitor (ISRIB). APOE deletion also leads to activation of the Wnt/ß-catenin signaling pathway with concomitant decrease of secreted frizzled-related protein 1 (SFRP1) expression in glia cells. Importantly, the critical role of apoE in cell-type-specific lipid homeostasis was observed upon APOE deletion in cerebral organoids with a specific upregulation of cholesterol biosynthesis in excitatory neurons and excessive lipid accumulation in astrocytes. Relevant to human AD, APOE4 cerebral organoids show altered neurogenesis and cholesterol metabolism compared to those with APOE3. CONCLUSIONS: Our work demonstrates critical roles of apoE in brain homeostasis and offers critical insights into the APOE4-related pathogenic mechanisms.

Trem2 H157Y increases soluble TREM2 production and reduces amyloid pathology.

BACKGROUND: The rare p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) was found to increase Alzheimer's disease (AD) risk. This mutation is located at the cleavage site of TREM2 extracellular domain. Ectopic expression of TREM2-H157Y in HEK293 cells resulted in increased TREM2 shedding. However, the physiological outcomes of the TREM2 H157Y mutation remain unknown in the absence and presence of AD related pathologies. METHODS: We generated a novel Trem2 H157Y knock-in mouse model through CRISPR/Cas9 technology and investigated the effects of Trem2 H157Y on TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathologies by conducting biochemical assays, targeted mass spectrometry analysis of TREM2, hippocampal electrophysiology, immunofluorescent staining, in vivo micro-dialysis, and cortical bulk RNA sequencing. RESULTS: Consistent with previous in vitro findings, Trem2 H157Y increases TREM2 shedding with elevated soluble TREM2 levels in the brain and serum. Moreover, Trem2 H157Y enhances synaptic plasticity without affecting microglial density and morphology, or TREM2 signaling. In the presence of amyloid pathology, Trem2 H157Y accelerates amyloid-ß (Aß) clearance and reduces amyloid burden, dystrophic neurites, and gliosis in two independent founder lines. Targeted mass spectrometry analysis of TREM2 revealed higher ratios of soluble to full-length TREM2-H157Y compared to wild-type TREM2, indicating that the H157Y mutation promotes TREM2 shedding in the presence of Aß. TREM2 signaling was further found reduced in Trem2 H157Y homozygous mice. Transcriptomic profiling revealed that Trem2 H157Y downregulates neuroinflammation-related genes and an immune module correlated with the amyloid pathology. CONCLUSION: Taken together, our findings suggest beneficial effects of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD risk, we speculate TREM2 H157Y in humans might increase AD risk through an amyloid-independent pathway, such as its effects on tauopathy and neurodegeneration which merit further investigation.

ApoE in Alzheimer's disease: pathophysiology and therapeutic strategies.

Alzheimer's disease (AD) is the most common cause of dementia worldwide, and its prevalence is rapidly increasing due to extended lifespans. Among the increasing number of genetic risk factors identified, the apolipoprotein E (APOE) gene remains the strongest and most prevalent, impacting more than half of all AD cases. While the ε4 allele of the APOE gene significantly increases AD risk, the ε2 allele is protective relative to the common ε3 allele. These gene alleles encode three apoE protein isoforms that differ at two amino acid positions. The primary physiological function of apoE is to mediate lipid transport in the brain and periphery; however, additional functions of apoE in diverse biological functions have been recognized. Pathogenically, apoE seeds amyloid-ß (Aß) plaques in the brain with apoE4 driving earlier and more abundant amyloids. ApoE isoforms also have differential effects on multiple Aß-related or Aß-independent pathways. The complexity of apoE biology and pathobiology presents challenges to designing effective apoE-targeted therapeutic strategies. This review examines the key pathobiological pathways of apoE and related targeting strategies with a specific focus on the latest technological advances and tools.

Opposing effects of apoE2 and apoE4 on microglial activation and lipid metabolism in response to demyelination.

BACKGROUND: Abnormal lipid accumulation has been recognized as a key element of immune dysregulation in microglia whose dysfunction contributes to neurodegenerative diseases. Microglia play essential roles in the clearance of lipid-rich cellular debris upon myelin damage or demyelination, a common pathogenic event in neuronal disorders. Apolipoprotein E (apoE) plays a pivotal role in brain lipid homeostasis; however, the apoE isoform-dependent mechanisms regulating microglial response upon demyelination remain unclear. METHODS: To determine how apoE isoforms impact microglial response to myelin damage, 2-month-old apoE2-, apoE3-, and apoE4-targeted replacement (TR) mice were fed with normal diet (CTL) or 0.2% cuprizone (CPZ) diet for four weeks to induce demyelination in the brain. To examine the effects on subsequent remyelination, the cuprizone diet was switched back to regular chow for an additional two weeks. After treatment, brains were collected and subjected to immunohistochemical and biochemical analyses to assess the myelination status, microglial responses, and their capacity for myelin debris clearance. Bulk RNA sequencing was performed on the corpus callosum (CC) to address the molecular mechanisms underpinning apoE-mediated microglial activation upon demyelination. RESULTS: We demonstrate dramatic isoform-dependent differences in the activation and function of microglia upon cuprizone-induced demyelination. ApoE2 microglia were hyperactive and more efficient in clearing lipid-rich myelin debris, whereas apoE4 microglia displayed a less activated phenotype with reduced clearance efficiency, compared with apoE3 microglia. Transcriptomic profiling revealed that key molecules known to modulate microglial functions had differential expression patterns in an apoE isoform-dependent manner. Importantly, apoE4 microglia had excessive buildup of lipid droplets, consistent with an impairment in lipid metabolism, whereas apoE2 microglia displayed a superior ability to metabolize myelin enriched lipids. Further, apoE2-TR mice had a greater extent of remyelination; whereas remyelination was compromised in apoE4-TR mice. CONCLUSIONS: Our findings provide critical mechanistic insights into how apoE isoforms differentially regulate microglial function and the maintenance of myelin dynamics, which may inform novel therapeutic avenues for targeting microglial dysfunctions in neurodegenerative diseases.

Elevating microglia TREM2 reduces amyloid seeding and suppresses disease-associated microglia.

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.

APOE3-Jacksonville (V236E) variant reduces self-aggregation and risk of dementia.

Apolipoprotein E (APOE) genetic variants have been shown to modify Alzheimer's disease (AD) risk. We previously identified an APOE3 variant (APOE3-V236E), named APOE3-Jacksonville (APOE3-Jac), associated with healthy brain aging and reduced risk for AD and dementia with Lewy bodies (DLB). Herein, we resolved the functional mechanism by which APOE3-Jac reduces APOE aggregation and enhances its lipidation in human brains, as well as in cellular and biochemical assays. Compared to APOE3, expression of APOE3-Jac in astrocytes increases several classes of lipids in the brain including phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, and sulfatide, critical for synaptic functions. Mice expressing APOE3-Jac have reduced amyloid pathology, plaque-associated immune responses, and neuritic dystrophy. The V236E substitution is also sufficient to reduce the aggregation of APOE4, whose gene allele is a major genetic risk factor for AD and DLB. These findings suggest that targeting APOE aggregation might be an effective strategy for treating a subgroup of individuals with AD and DLB.