Butovsky, Oleg

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.

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.