Shaffer, Scott A.

Abstract The apolipoprotein E ε4 ( APOE4 ) allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD), yet the mechanistic underpinnings by which it alters human microglial function remain poorly understood. Here, we utilize CRISPR/Cas9-engineered isogenic human induced pluripotent stem cell-derived microglia-like cells (iMGs) expressing APOE3 or APOE4 to systematically interrogate genotype-dependent effects on microglial proteomes and their secreted extracellular vesicles (EVs) under basal and fibrillar amyloid-β (fAβ)-challenged conditions. Quantitative proteomics revealed that APOE4 iMGs exhibit downregulated RNA processing, impaired phagocytosis, and exacerbated pro-inflammatory signaling, particularly upon fAβ exposure. Functionally, APOE4 iMGs demonstrated increased NF-κB activation, enhanced neuronal apoptosis and tau pathology in 2D and 3D co-culture systems. Interestingly, APOE4 iMGs released greater amount of EVs enriched in proteasomal components over APOE3 iMG. Integrated proteomic analyses further highlighted APOE4 -specific selective packaging of EV cargo linked to neuron-glia communication, inflammatory signaling, and proteostasis dysregulation, suggesting a potent, non-cell-autonomous mechanism driving neuronal damage. Consistently, these APOE4 iMGEVs were preferentially internalized by neurons, leading to disrupted neuronal proteostasis, impaired neuronal firing, and increased tau phosphorylation, particularly under pathological stress. Notably, pharmacologic inhibition of NF-κB in APOE4 iMGs reduced EV cargo-loading of proteasomal molecules and mitigated iMGEV-driven neuronal dysfunction and tau pathology. These findings establish a mechanistic link between APOE4 , dysregulated microglial homeostasis, and microglial EV-mediated propagation of proteostatic dysfunction and neurodegeneration, advancing our understanding of how APOE4 contributes to AD pathogenesis.

Abstract P2x purinoreceptor 7 (P2RX7), an ATP-gated ion channel, is known to play pivotal roles in the progression of Alzheimer’s disease (AD), although its cell type-specific pathological mechanisms have yet to be elucidated. Here, we show that genetic deletion of P2rx7 mitigates brain atrophy, tau accumulation and cognitive impairment in PS19 tauopathy mice. Specific deletion of P2rx7 in microglia, but not astrocytes, significantly suppresses tau propagation from the entorhinal cortex to CA1 in the hippocampus, an early event in AD pathology. Single-cell (sc)-RNA sequencing of mouse brains revealed specific P2rx7 expression in microglia, inducing inflammatory changes accompanied by elevated extracellular vesicles (EVs) secretion in PS19 mice. Brain-derived EVs (BDEVs) proteome demonstrated that P2RX7 increases EV cargo loading of tau and mitochondrial molecules in BDEVs from PS19 mice, which was further validated by single-molecule super-resolution. Notably, following the injection of BDEVs isolated from PS19 mice with or without P2rx7 deficiency, the microglial transcriptome of recipient mice revealed enriched DNA-sensing and type II interferon signaling in response to BDEVs from PS19 mice, which was diminished in the group injected with P2rx7-deficient BDEVs. Thus, our results indicate that P2RX7 regulates EV-mediated tau and mitochondrial transfer and inflammatory activation in microglia with increased EV secretion, thereby contributing to tauopathy and neurodegeneration, highlighting the therapeutic potential of targeting the P2RX7-EV axis in AD.

AbstractBackgroundNeuron‐derived extracellular vesicles (NDEVs) are a valuable resource for understanding brain conditions and discovering neurodegenerative diseases biomarkers, notably Alzheimer’s disease (AD). Recent interest focuses on capturing neuron‐specific EVs from patient‐derived samples, characterizing their contents as a pathological reflection of the central nervous system (CNS). Our recent study identified ATPase Na+/K+ Transporting Subunit Alpha 3 (ATP1A3) as a prevalent neuron‐specific EV marker specifically expressed in brains. This study systematically analyzes neuronal EVs to assess the specificity of ATP1A3 to NDEVs and its potential as a target for NDEV pulldown from accessible biofluids for disease monitoring. Additionally, ATP1A3 is compared with other proposed NDEV markers.MethodWe applied immunoelectron microscopy to detect ATP1A3, L1 cell adhesion molecule (L1CAM) and neural cell adhesion molecule 1 (NCAM1) in EVs isolated from iPSC‐derived excitatory neurons, brain tissue, cerebrospinal fluid (CSF) and plasma. Neuronal EV enrichment was achieved through immunoaffinity isolation, utilizing anti‐ATP1A3, L1CAM and NCAM1 antibodies separately. Evaluation involved quantitative mass‐spectrometry, immunoblotting and ELISA. To compare the enrichment of neuronal markers, EVs isolated from CSF and plasma samples were analyzed using ExoView and Nanoimager. Additionally, we investigated the potential of ATP1A3+ plasma EVs as AD biomarker by comparing the value of amyloid‐beta peptide (Aβ) in ATP1A3+ plasma EVs measured by Nanoimager and other plasma AD biomarkers as determined by SIMOA.ResultATP1A3, identified as a neuron‐specific protein, exhibits substantial enrichment in NDEVs isolated from induced human neurons, brain tissue, CSF, and plasma samples, surpassing NCAM1 or L1CAM. Both single‐ and bulk‐EV analysis consistently demonstrate a higher enrichment of ATP1A3 associated EVs in human samples. Immunoprecipitation of ATP1A3+ EVs from human brain tissues reveals superior neuronal cell‐type specificity over NCAM1+ and L1CAM+ EVs by label‐free mass spectrometry. Increased Aβ levels are observed in ATP1A3+ EVs derived from CSF and plasma of AD cases by SIMOA. Moreover, Aβ+ populations in ATP1A3+ EVs from plasma can distinguish AD from mild cognitive impairment and control cases via Nanoimager compared to the conventional quantification of AD markers.ConclusionOur findings demonstrate that ATP1A3 as a promising target for isolating NDEVs from biofluids, offering potential diagnostic advancements in neurological research.

Neuron-derived extracellular vesicles (NDEVs) are potential biomarkers of neurological diseases although their reliable molecular target is not well established. Here, we demonstrate that ATPase Na + /K + transporting subunit alpha 3 (ATP1A3) is abundantly expressed in extracellular vesicles (EVs) isolated from induced human neuron, brain, cerebrospinal fluid, and plasma in comparison with the presumed NDEV markers NCAM1 and L1CAM by using super-resolution microscopy and biochemical assessments. Proteomic analysis of immunoprecipitated ATP1A3 + brain-derived EVs shows higher enrichment of synaptic markers and cargo proteins relevant to Alzheimer’s disease (AD) compared to NCAM1 + or LICAM + EVs. Single particle analysis shows the elevated amyloid-β positivity in ATP1A3 + EVs from AD plasma, providing better diagnostic prediction of AD over other plasma biomarkers. Thus, ATP1A3 is a reliable target to isolate NDEV from biofluids for diagnostic research.

AbstractAlzheimer's disease (AD) is a pervasive neurodegeneration disease with high heritability. In this study, we employed CRISPR‐Cas9‐engineered technology to investigate the effects of a rare mutation (rs144662445) in the A kinase anchoring protein 9 (AKAP9) gene, which is associated with AD in African Americans (AA), on tau pathology and the tau interactome in SH‐SY5Y P301L neuron‐like cells. The mutation significantly increased the level of phosphorylated tau, specifically at the site Ser396/Ser404. Moreover, analyses of the tau interactome measured by affinity purification‐mass spectrometry revealed that differentially expressed tau‐interacting proteins in AKAP9 mutant cells were associated with RNA translation, RNA localization and oxidative activity, recapitulating the tau interactome signature previously reported with human AD brain samples. Importantly, these results were further validated by functional studies showing a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared with wild type cells in a tau‐dependent manner, which are mirrored with pathological phenotype frequently seen in AD. Our results demonstrated specific effects of rs14462445 on mis‐processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.

Abstract In neurodegenerative diseases, extracellular vesicles (EVs) transfer pathogenic molecules and are consequently involved in disease progression. We have investigated the proteomic profiles of EVs that were isolated from four different human‐induced pluripotent stem cell‐derived neural cell types (excitatory neurons, astrocytes, microglia‐like cells, and oligodendrocyte‐like cells). Novel cell type‐specific EV protein markers were then identified for the excitatory neurons (ATP1A3, NCAM1), astrocytes (LRP1, ITGA6), microglia‐like cells (ITGAM, LCP1), and oligodendrocyte‐like cells (LAMP2, FTH1), as well as 16 pan‐EV marker candidates, including integrins and annexins. To further demonstrate how cell‐type‐specific EVs may be involved in Alzheimer's disease (AD), we performed protein co‐expression network analysis and conducted cell type assessments for the proteomes of brain‐derived EVs from the control, mild cognitive impairment, and AD cases. A protein module enriched in astrocyte‐specific EV markers was most significantly associated with the AD pathology and cognitive impairment, suggesting an important role in AD progression. The hub protein from this module, integrin‐β1 (ITGB1), was found to be significantly elevated in astrocyte‐specific EVs enriched from the total brain‐derived AD EVs and associated with the brain β‐amyloid and tau load in independent cohorts. Thus, our study provides a featured framework and rich resource for the future analyses of EV functions in neurodegenerative diseases in a cell type‐specific manner.

AbstractBackgroundAlzheimer’s disease (AD) is a pervasive neurodegeneration disease with high heritability. Whole exome sequencing study identified two rare African American (AA)‐specific variants in AKAP9, a kinase anchor protein 9, (rs144662445 and rs149979685), that increased the odds of AD more than 2.75‐fold. However, the functional impact of the AKAP9 rs144662445 (I2558M) mutation in the central nervous system and the underlying mechanism in AD remain unclear.MethodAKAP9 I2558M mutation was introduced by CRISPR‐mediated knock‐in and validated by Sanger sequence in SH‐SY5Y cells stably expressing P301L Tau (SH‐SY5Y P301L cells), which was further differentiated into neuronal cells. Tau phosphorylated level between AKAP9 WT and I2558M cells was evaluated by immunocytochemistry and ELISA assay. Moreover, we analyzed the tau interactome in AKAP9 I2558M cells by affinity purification‐mass spectrometry. In silico analysis was performed to compare the differences between AKAP9 WT and mutation in tau‐interacting proteins. The protein synthesis and oxidative activity in SH‐SY5Y P301L neurons with and without I2558M were measured by using the Click‐iT protein synthesis kit and CellRox dye, respectively.ResultNo difference in the neuronal cell morphology was observed between differentiated WT and mutant SH‐SY5Y P301L cells. The AD‐associated AKAP9 I2558M mutation resulted in a significant increase in phosphorylated tau protein at residues S396 and S404 site in a differentiated SH‐SY5Y P301L cell line. The presence of this mutation altered the composition of tau‐interacting proteins, by increasing interaction of proteins associated with translation, RNA localization and oxidative activity. Importantly, functional studies showed a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared to wild type cells.ConclusionOur results demonstrated specific effects of rs14462445 on mis‐processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.