Radhakishun, Stephanie

Abstract In Alzheimer’s disease (AD), tau pathology arises in entorhinal cortex layer II (ECII) and advances through defined hippocampal circuits to CA1 and connected neocortical regions, yet the determinants of this hierarchical spread remain unclear. We previously established a circuit-defined propagation model by expressing Cre-inducible human P301L 2N4R tau selectively in Wolframin-1 (Wfs1) + ECII neurons using AAV-FLEX-Tau P301L in Wfs1-Cre mice. Here, to test how amyloid-β (Aβ) and human tau background shape propagation, we generated human MAPT knock-in Wfs1 mice and APP NL-G-F / MAPT double knock-in Wfs1 mice (T-Wfs1 and AT-Wfs1) and induced ECII-restricted Tau P301L expression. Three months after injection, phosphorylated or misfolded tau-positive neurons were enriched in proximal CA1 in Wfs1 and T-Wfs1 mice, resembling primary age-related tauopathy, whereas AT-Wfs1 mice showed preferential accumulation near the CA1/subiculum (Sub) boundary, consistent with an AD-like pattern. In T-Wfs1 and AT-Wfs1 mice, tau spread extended through Sub to neocortical regions, and phosphorylated tau accumulated predominantly in excitatory rather than inhibitory neurons. Electrophysiological analyses revealed increased spontaneous neuronal firing and impaired GABAergic transmission in the CA1/Sub boundary and neocortical areas in T-Wfs1 and AT-Wfs1 mice, indicative of impaired GABAergic input and enhanced neuronal excitability in these regions. Together, these data indicate that human MAPT and Aβ pathology shift the circuit topography of tau propagation and are associated with early network dysfunction, supporting a synergistic interaction that promotes AD-like spread and synaptic imbalance.

Abstract Background We have recently reported that wolframin‐1‐expressing (Wfs1 + ) pyramidal neurons in the entorhinal cortex layer II (ECII) that projects to the CA1 propagate phosphorylated tau (pTau) via the temporoammonic pathway, mimicking early stages of tau pathology in AD (ECII‐CA1 tau mice). We examined if amyloid pathology and human tau expression with this mouse model may change the character and distribution of tau transfer. Furthermore, using a chemogenetic approach, we aimed to determine the effect of neuronal excitability on tau transfer to the hippocampal regions in mouse brains. Method Cre‐inducible AAV expression P301L tau mutant (AAV‐Flex‐P301Ltau) was injected into the EC II region of APP NL‐G‐F human MAPT double knockin (APP NL‐G‐F : TAUKI) mice crossed with Wfs1‐Cre and Wfs1‐Cre mice at 7 months of age. Tau propagation was evaluated in the hippocampal and cortex regions by immunofluorescence using HT7 (human tau) and AT8 (pS 202 /pT 205 tau) mAbs at one‐ and 3‐months post‐injection. AAV‐Flex‐P301Ltau was co‐injected with Cre‐inducible AAV expressing hM3D(Gq) or hM4D(Gi) DREADD in the ECII in Wfs1‐Cre mice at 4 months of age, followed by subcutaneous infusion of clozapine N ‐oxide or saline via osmotic pumps for 28 days. Immunofluorescence against HT7 mAb was performed at one‐month post‐injection. Result We observed robust HT7 positivity in the CA1 and subiculum in Wfs1‐Cre mice after AAV‐Flex‐P301Ltau injection, although there was little AT8 staining at both one‐ and 3‐month. Interestingly, APP NL‐G‐F :TauKI:Wfs1‐Cre mice showed strong HT7 and AT8 positivity in the subiculum, but not in CA1 at 1‐month. Notably, p ‐tau further advanced into the visual cortex (VC) region at 3‐month in APP NL‐G‐F :TauKI:Wfs1‐Cre mice, while neither HT7 nor AT8 was positive in the VC in Wfs1‐Cre mice. Increased or suppressed neuronal excitability in the medial ECII pyramidal neurons by DREADD approach significantly induced or reduced tau propagation to the CA1 compared to the control group at one‐month post‐injection, respectively. Conclusion Our study demonstrates that APP NL‐G‐F :TAUKI:Wfs1‐Cre mice injected with P301L tau in the ECII recapitulates advanced‐stage AD pathology with amyloid beta navigating tau to the neocortical region and enhancing the maturity of tau pathology. Furthermore, ECII neuronal excitability regulates tau transfer efficiency from the ECII to CA1 region.

Abstract Background We previously identified a novel mechanism describing how pathological tau transfers 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 predominantly expressed in microglia, regulates the secretion and biogenesis of EVs. Here we investigated the effect of P2rx7 deficiency in PS19 tauopathy mouse model and by proteomic profiling of brain EVs. Method PS19: P2rx7 –/– mice at 9‐10 months of age were tested for fear conditioning and their brain sections were assessed for brain atrophy. Tau pathology was evaluated using ELISA and immunofluorescence against phosphorylated (AT8) and misfolded (Alz50) tau. Brain tissues were further assessed by bulk and single cell RNA‐seq (scRNA‐seq) and brain‐EVs were subjected to proteomic profiling by mass‐spectrometry. P2rx7 –/– mice were intracranially injected with viral vectors expressing P301L tau in neurons and mEmerald‐CD9 in microglia to visualize the secretion of microglial EVs in vivo . Mice conditional knockout (cKO) for P2rx7 in microglia, neurons and astrocytes were injected with a viral vector expressing P301L tau in entorhinal cortex for tau propagation assessment. Result PS19: 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 synapse loss and tau pathology. By weighted gene co‐expression network analysis (WGCNA) we identified a microglial and EV‐related module strongly correlated with tau pathology which was remarkably downregulated in PS19: P2rx7 –/– mice hippocampus. PS19 mice showed increased secretion of EVs containing tau and mitochondrial proteins in the mouse brain parenchyma, which was significantly downregulated in PS19: P2rx7 –/– mice. scRNA‐seq analysis showed that P2rx7 is required for microglia conversion to inflammatory phenotype with increased expression of EV genes in PS19 mice. P2rx7 deficiency reduced microglial mEmerald‐CD9 + ‐EV secretion in mice expressing P301L tau. Moreover, microglia and neuron‐specific deletion of P2rx7 inhibit tau propagation from the entorhinal cortex to the mouse hippocampus. Conclusion Our study demonstrated a microglial P2RX7‐EV axis with potential implications on neuroinflammation and neurodegeneration associated with tau pathology, further indicating the therapeutic potential of targeting P2rx7 to ameliorate AD progression.

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.