Ruan, Zhi

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.

Abstract We have previously reported the therapeutic effect of depletion and regeneration of endogenous microglia on autism spectrum disorder-like behaviors in offspring from the dams under maternal immune activation (MIA) by dampening their neuritogenic activation. Here we show a long-lasting pathological effect by MIA, leading to abnormal behaviors in offspring mice in a sex-specific manner at 3 months of age. MIA was induced by injecting Polyinosinic:polycytidylic acid [Poly(I:C)] at 10mg/kg at E9.5 with preselected dams based on the immunoreactivity to low-dose Poly(I:C) injection. MIA offspring show impairments in sociability, repetitive behavior, and spatial working and associative memories in both sexes, whereas only male offspring show social novelty deficit. MIA has no effect on anxiety-like behavior nor sensori-motor or locomotor activity. Administering colony stimulating factor receptor (CSF1R) inhibitor in young adult MIA offspring regenerates microglia and ameliorates sociability deficits in males and spatial working and associative memory impairments in both sexes without effect on social novelty impairments. Transcriptomic analysis of prefrontal cortex and hippocampal tissues reveals MIA and sex-specific pathways and highlights potential key genes involved in the corrective effect of microglial regeneration in MIA offspring. Our results underscore the potential of the sex-specific therapeutic applications of CSF1R inhibitor for MIA-related social and cognitive disorders.

AbstractBackgroundWe previously identified the novel mechanism of pathological tau transfer via extracellular vesicles (EVs) in Alzheimer’s disease (AD). Targeting EV secretion to mitigate tau transfer is therefore a promising therapeutic approach for AD. P2X purinoreceptor 7 (P2RX7), an ATP‐gated cationic channel, regulates microvesicle shedding or secretion of multivesicular body‐derived exosomes. We aim to investigate the effect of P2rx7 deficiency on disease progression in PS19 tauopathy mouse model in vivo and by proteomic profiling of brain EVs.MethodPS19:P2rx7–/– mice at 9 months of age were tested for the fear conditioning, and pathology assessment of the brain atrophy and tau using immunofluorescence against aggregated tau (Alz50) or phosphorylated tau (AT8) and ELISA. Mice were intracranially injected with viral vectors expressing mEmerald‐CD9 in microglia and P301L tau in neurons to visualize the secretion of microglial EV and extracellular tau in vivo. Brain EV samples were subjected to proteomic profiling by data independent acquisition mass‐spectrometry.ResultPS19:P2rx7–/– mice showed significant improvement in contextual and cued memory compared to age‐matched PS19 mice, which was accompanied by preserved cortical and hippocampal volume, and significant reduction of hippocampal tau pathology and pS396 tau in sarkosyl‐insoluble fraction of brain tissues. The number of GFP+ microglial EVs were significantly reduced in P2rx7–/– compared to WT mice. Gene ontology pathway analysis of EV proteome showed significant enrichment of mitochondrial pathways in PS19 group compared to WT group, which was significantly downregulated in PS19:P2rx7–/– group.ConclusionOur study demonstrated that P2rx7 deficiency ameliorates cognitive dysfunction and tau pathology development in PS19 mice by dampening microglial EV secretion and EV‐mediated mitochondria transfer, further indicating the therapeutic potential of targeting P2RX7, an EV regulatory molecule, to ameliorate AD progression.

Abstract Extracellular vesicle (EV) secretion is mediated by purinergic receptor P2X 7 (P2RX7), an ATP‐gated cation channel highly expressed in microglia. We have previously shown that administration of GSK1482160, a P2RX7 selective inhibitor, suppresses EV secretion from murine microglia and prevents tauopathy development, leading to the recovery of the hippocampal function in PS19 mice, expressing P301S tau mutant. It is yet unknown, however, whether the effect of GSK1482160 on EV secretion from glial cells is specifically regulated through P2RX7. Here we tested GSK1482160 on primary microglia and astrocytes isolated from C57BL/6 (WT) and P2rx7 –/– mice and evaluated their EV secretion and phagocytotic activity of aggregated human tau (hTau) under ATP stimulation. GSK1482160 treatment and deletion of P2rx7 significantly reduced secretion of small and large EVs in microglia and astrocytes in both ATP stimulated or unstimulated condition as determined by nanoparticle tracking analysis, CD9 ELISA and immunoblotting of Tsg101 and Flotilin 1 using isolated EVs. GSK1482160 treatment had no effect on EV secretion from P2rx7 –/– microglia while we observed significant reduction in the secretion of small EVs from P2rx7 –/– astrocytes, suggesting its specific targeting of P2RX7 in EV secretion except small EV secretion from astrocytes. Finally, deletion of P2rx7 suppressed IL‐1β secretion and phagocytosed misfolded tau from both microglia and astrocytes. Together, these findings show that GSK1482160 suppresses EV secretion from microglia and astrocytes in P2RX7‐dependment manner, and P2RX7 critically regulates secretion of IL‐1β and misfolded hTau, demonstrating as the viable target of suppressing EV‐mediated neuroinflammation and tau propagation.

AbstractBackgroundP2X purinoceptor 7 (P2RX7), an ATP‐gated cation channel present abundantly in the microglia, induces membrane depolarization and extracellular vesicle (EV) secretion. Previous study demonstrated that administration of GSK1482160, a P2RX7 selective inhibitor, suppressed EVs secretion from murine microglia resulting in accumulation of Tsg101+ intraluminal vesicles (ILVs) in the hippocampus, and restored partial behavioral deficits in P301S tau mice. However, the specificity of GSK1482160 to P2RX7 has never been investigated in P301S tau mice. It is crucial to validate whether P2RX7 is a potential target for alleviating the tauopathy phenotype in P301S tau mice. The purpose of this study is to determine if deletion of P2RX7 is sufficient to recapiturate the therapeutic effect of GSK1482160 in tauopathy mouse model.MethodFours and nine‐months‐old P2RX7‐/‐:P301S and P301S mice were generated for the evaluation of ILV accumulation and tau pathology. Primary microglia and astrocytes from WT (C57BL/6) and P2RX7‐/‐ mice, treated with GSK1482160 and ATP for EV secretion. EVs were isolated from conditioned media via the sequential centrifugation and ultracentrifugation. Finally, EVs were quantified by nanoparticle tracking analysis and exosome specific marker CD9 ELISA. Effects of GSK1482160 on EVs secretion from astrocytes/microglia were also assessed using Western blot with EV specific markers Tsg101 and CD9.ResultExosome specific marker Tsg101, CD9, and tau expression levels were reduced in the hippocampus of nine‐months‐old P2RX7‐/‐:P301S mice compared to P301S mice. No significant difference between the two groups at 4 months of age was observed. Strikingly, primary cultured microglia and astrocytes showed reduction in EV secretion in both ATP stimulated or unstimulated condition from P2RX7‐/‐ group compared to WT group. Additionally, GSK1482160 pretreatment significantly reduced EV secretion from WT astrocytes and microglia, although it had no effects in P2RX7‐/‐ cells. These findings were validated by ELISA of EV marker CD9 and immunoblotting of Tsg101 and flotillin‐1 using isolated EVs.ConclusionResults demonstrate that P2RX7 is primarily responsible for ATP‐induced EV secretion from astrocytes and microglia, and systemic disruption of P2RX7 mimics the effect of GSK1482160 in P301S tau mice, including accumulation of ILVs in microglia and suppression of tau accumulation in the hippocampal regions.