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.

Tau pathology emerges early in Alzheimer's disease within entorhinal cortex layer II (ECII) and reaches hippocampal CA1, but how this circuit-level spread translates into sex-dependent vulnerability remains unclear. Using a circuit-defined model in which P301L human tau is expressed selectively in Wolframin-1 (Wfs1 + ) ECII neurons and propagates to CA1, we found that the extent and proximal-distal distribution of tau-positive CA1 neurons were comparable in males and females. Despite similar propagation, females exhibited broad hippocampal-dependent cognitive impairment (working memory, object recognition, fear acquisition, trace associative memory, and contextual fear memory), whereas males showed a selective deficit in trace associative memory. Consistent with these behavioral outcomes, CA1 pyramidal neurons in tau-propagated females displayed reduced excitability (slower action potential kinetics, reduced firing during depolarizing steps) and reduced spontaneous excitatory postsynaptic current (EPSC) amplitude, while males showed subtler intrinsic changes with altered EPSC kinetics. Bulk RNA sequencing of entorhinal cortex and CA1 revealed robust immune pathway engagement after tau propagation, with males showing a stronger Th1/Th2 and neuroinflammatory signature and CTLA4-associated signaling changes, whereas females showed prominent complement-phagosome pathway enrichment and a female-specific increase in Clec7a + microglia density in CA1. CD4 + T-cell infiltration into CA1 was detected in both sexes. Together, these results indicate that sex-specific neuroimmune programs, rather than differences in tau propagation load, shape CA1 electrophysiological dysfunction and the breadth of memory impairment following early entorhinal-to-hippocampal tau spread.

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.

Neuron-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. We 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. ATP1A3, 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. Our findings demonstrate that ATP1A3 as a promising target for isolating NDEVs from biofluids, offering potential diagnostic advancements in neurological research.

Extracellular vesicles (EVs) are critical mediators of neuronal communication and have been implicated in propagating pathological processes in neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular interactome of neuronal EVs in vivo remains poorly defined. Here, we employed TurboID-CD9-based proximity biotinylation to label and capture EV-interacting proteins in the hippocampus of wild-type (WT) and APP NLGF knock-in AD mouse models. Adeno-associated viral delivery of hSyn1 promoter-driven TurboID-CD9 enabled neuron-specific EV tagging, followed by in vivo biotinylation and affinity purification of labeled proteins. Proteomic analysis using data independent acquisition liquid chromatography - mass spectrometry identified 5,502 proteins, with enriched pathways involving synaptic transmission, vesicle trafficking, and inhibitory neurotransmission. Comparative analyses revealed robust enrichment of GABAergic signaling components, including GABAA receptor subunits (Gabrb3, Gabra1, Gabbr2), Ncam1, and chloride transporters, in both WT and APP NLGF EV interactomes, with additional disease-associated proteins (Mapt, Snca) and potassium channel enrichment observed in APP NLGF mice. Proximity ligation assays validated direct EV-associated biotinylation of Ncam1, Gabrb3, and Gad1, with Gad1 showing significant upregulation in the APP NLGF cohort. In silico HADDOCK docking supported stable interactions between CD9 and these target proteins, revealing plausible EV-protein interfaces. These findings define the in vivo neuronal EV interactome and its remodeling in amyloid pathology, implicating EV-associated GABAergic and ion channel proteins in network excitability regulation. This work establishes a proteomic and structural framework for understanding EV-mediated signaling in health and disease, providing candidate targets for therapeutic modulation of excitatory / inhibitory balance in AD.

Brain cells secrete extracellular vesicles (EVs) containing signaling and pathological proteins related to Alzheimer's disease (AD) and related disorders. Recent studies demonstrate brain-derived EVs (BDEVs) contain misfolded tau and are transmissible of tau pathology in the brain. However, the underlying mechanism regarding EV-mediated tau pathology is still largely uncharacterized. We conducted the immuno-affinity purification of tau in human brain-derived EVs (BDEVs) isolated from age and sex-matched 14 AD and 14 CTRL cases and performed tandem mass-tag mass spectrometry to profile unbiased tau interactome in BDEVs. We used Nanotemper Monolith to validate the interaction of EV-tau interactors with tau, and super-resolution microscopy (Nanoimager) to detect their colocalization in a single-EV level. We next designed siRNAs to silence EV-tau interactors in SH-SY5Y cells overexpressing human P301L tau (SH-SY5Y-P301Ltau) and assessed tau loading to EVs by Flow Nanoanalyzer. These EVs were tested for their uptake and tau seeding by human iPSC-derived neurons (iNeurons). We finally tested the inhibitors of identified molecules on BDEV-mediated tau propagation in vivo . A total of 764 proteins were identified in BDEV-associated tau interactome from CTRL and AD patients. Sixty-five proteins were significantly downregulated in AD BDEVs; whereas 5 proteins were significantly upregulated in AD BDEVs compared to CTRL BDEVs. The most enriched tau-interacting proteins were significantly positively correlated with Braak stage. We confirmed the direct binding of candidate proteins to tau with micromolar binding affinities by Monolith, and their colocalization with tau in human BDEVs using Nanoimager. Silencing of candidate molecules in human SH-SY5Y-P301Ltau cells showed reduced loading of tau in EVs, their reduced uptake by iNeurons, and tau seeding activities. Finally, intracerebroventricular injection of neutralizing antibody against the most promising target results in reduction of tau pathology in PS19 mice expressing P301S human tau mutant. Our study identified tau-interacting molecules highly enriched in AD BDEVs compared to CTRL BDVs. Silencing of candidate molecule reduced tau loading to the EVs and their uptake by iNeurons and neutralizing antibody suppressed tau dissemination, highlighting them as promising therapeutic targets to halt tau pathology in AD and related tauopathy.

Brain-derived extracellular vesicles (BDEVs) carry tau filaments and promote tau transmission in Alzheimer's disease (AD). However, how APOE ε4 allele, a key genetic risk factor for AD, may change BDEV molecular structures thereby facilitate disease progression is poorly understood. Here we report comprehensive analyses of BDEVs isolated from human E3/3 and E4/4 AD brains with a biological multi-omics approach. E4/4 BDEVs significantly enhanced tau propagation in aged human MAPT (Tau) knock-in and APP NL-G-F: Tau double knock-in mouse brains in vivo and increased neuronal uptake and excitability in induced pluripotent stem cell-derived neurons (iNeurons) compared to E3/3 BDEVs in vitro. Notably, correlation analysis of BDEV-lipidome and proteome exhibited synergistic enrichment in unsaturated free fatty acid (FFA)18:2, a precursor of inflammatory w6 FFA, and neural cell adhesion molecule 1 (NCAM1). Treatment of iNeurons with FFA 18:2 induces NCAM1 expression, recruits tau into EVs and enhance their tau seeding activity, which is blocked by NCAM1 antibody in vitro. Finally, intracerebroventricular injection of NCAM1 antibody significantly alleviated pathological tau accumulation and glial inflammation in PS19 tauopathy mouse brains, which had previously been reported to exhibit increased level of 18:2 FFA. This highlights novel pathological mechanism in tau transfer mediated by E4/4 BDEVs and emphasizes strong therapeutic potential of targeting EV molecules in AD progression.

Extracellular vesicles (EVs) carry pathogenic molecules and play a role in the disease spread, including aggregated tau proteins. The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is responsible for the biogenesis of small EVs (exosomes), thus targeting critical ESCRT molecules can disrupt EV synthesis. We hypothesize that microglia-specific targeting of ESCRT-I molecule Tsg101 suppresses microglia-derived EV-mediated propagation of tau pathology, leading to amelioration of the disease phenotype of the tauopathy mouse model. PS19 tau transgenic mouse line was crossed with Cx3cr1 CreERT2/+ :Tsg101 fl/fl lines for tamoxifen-inducible Cx3cr1-specific deletion of Tsg101 (Tsg101 cKO) in PS19 mice to generate WT, Tsg101 cKO, PS19, and PS19:Tsg101cKO groups. The animals were treated with tamoxifen or corn oil at 2 months of age and subjected to comprehensive behavioral, neuropathological, biochemical, and molecular biological assessments at 6-7 months of age. The microglia isolated from Cx3cr1 CreERT2/+ :Tsg101 fl/fl pups were subjected to in vitro synaptosome uptake analysis with or without 4-Hydroxytamoxifen treatment. PS19 mice develop cognitive impairment as determined by Y-maze, forced alternation, novel object recognition and fear conditioning, which are reversed in PS19:Tsg101cKO mice. This is correlated with reduced Alz50 + tau accumulation, neurodegenerative microglial activation, neuroinflammation and complement pathway activation as determined by bulk RNA sequencing, ELISA and neuropathology. Primary cultured Cx3cr1 CreERT2/+ :Tsg101 fl/fl microglia with 4-Hydroxytamoxifen treatment show reduced phagocytosis of E. coli particles and synaptosome in C1q dependent manner. Tsg101 cKO microglia show reduced expression of C3aR1 and CD68, and secretion of total and Tau + EVs in vivo. Microglia-specific targeting of Tsg101 show beneficial effect for ameliorating the disease progression of tauopathy mouse model via suppression of EV secretion, microglial activation, tau accumulation and complement-dependent synaptic pruning. Microglial Tsg101 is a potential therapeutic target of Alzheimer’s disease and related tauopathy.

We 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. PS19: 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. 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 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. Our 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.

Tauopathies are a group of neurodegenerative disorders which are characterized by the accumulation of abnormal tau protein in the brain. However, the mechanistic understanding of pathogenic tau formation and spread within the brain remains elusive. Astrocytes are major immune reactive cells in the brain and have been implicated in exacerbating tau pathology by releasing extracellular vesicles (AEVs) containing pro-inflammatory cytokines and chemokines upon activation. Our prior investigation revealed a significant association between AEVs and tau pathology development, as well as cognitive function, by analyzing brain-derived EV proteins from AD patients. In this study, we explore the potential roles of AEVs in tau pathogenesis using a human induced pluripotent stem cell (iPSC) model. We obtained two male P301L tau mutant iPSC lines from a Polish family with frontotemporal dementia. By including two male control lines, these iPSCs were differentiated into astrocytes (iAs) and characterized by immunocytochemistry and subjected for bulk RNAseq. Bioinformatics analysis was conducted to compare the transcriptome profile between wild-type (WT) and P301L iAs. EVs from WT and P301L iAs were isolated by ultracentrifugation combined with size exclusion chromatography. Characterization of WT and P301L iAEVs involved nanoparticle tracking analysis, nano-flow cytometry and super-resolution microscopy. We successfully differentiated WT and P301L mutant iPSC lines into astrocytes with >99% purity. P301L iAs displayed distinctive astrocyte reactivity compared to WT cells, with elevated levels of pan-reactive astrocyte genes (e.g., GFAP, CD44) and decreased expression of neuroprotective A2 astrocyte-specific genes (e.g., TM4SF1, PTGS2). Additionally, gene enrichment set analysis of RNAseq data revealed dysregulation in the endo-lysosomal pathway and extracellular matrix in P301L iAs compared to WT iAs. The count of intraluminal vesicles marked by CD9+ were reduced in P301L iAs compared to WT cells. Moreover, we observed a significant increase in the internalization of Tau by P301L iAs compared to WT iAs following incubation with preformed Tau fibrils, resulting in an augmented release of tau-containing EVs from P301L iAs. Our findings suggest a potential alteration in EV biogenesis in P301L iAs, potentially contributing to astrocyte-mediated tau pathology. Future investigations will focus on understanding how AEVs contribute to tau propagation and accumulation.

Solid tissue-derived extracellular vesicles (ST-EVs) are extracellular vesicles (EVs) separated directly from solid tissues of both vertebrates and invertebrates. ST-EVs provide a physiologically relevant snapshot of tissue-specific molecular dynamics and can be enriched directly in situ, from tissues in their natural state, preserving the native characteristics of ST-EVs. However, their enrichment presents unique technical challenges compared to EVs derived from biofluids or cell culture media. The need for transparent reporting in ST-EV research is crucial to enhance the reproducibility, comparability, and reliability of research findings. The Solid Tissue Task Force, part of the Scientific Reproducibility Subcommittee of International Society for Extracellular Vesicles, aims to recommend reporting parameters and identify outstanding questions related to the pre-analytical and analytical handling of solid tissues, as well as ST-EV separation and characterization. These steps are essential for advancing the understanding of the biological roles of ST-EVs and their potential clinical applications.

We have previously reported the therapeutic effect of depletion and renewal 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 are induced by injecting Polyinosinic:polycytidylic acid [Poly(I:C)] at 10 mg/kg at E9.5 with preselected dams based on the immunoreactivity to low-dose Poly(I:C) injection. MIA offspring show impairments in sociability and associative memories in both sexes, whereas spatial working memory deficit was observed only in females. MIA does not affect social novelty, novel object recognition, anxiety-like behavior nor sensori-motor or locomotor activity. Administering colony-stimulating factor receptor (CSF1R) inhibitor in young adult MIA offspring renews microglia and ameliorates sociability deficits only in males, and spatial working memory only in females while associative memory impairments are reversed in males and partially restored in females. Transcriptomic analysis of prefrontal cortex and hippocampal tissues reveals synaptogenesis and cholesterol biosynthesis as male and female specific MIA pathways respectively, underpinning sex-dependent response to CSF1R inhibitor treatment. Our results reveal the sex specific therapeutic applications of CSF1R inhibitor for MIA-related social and cognitive disorders.

Extracellular vesicles (EVs) are key mediators in transferring pathological proteins associated with Alzheimer's disease (AD). The apolipoprotein E (APOE) gene, particularly the ε4 allele, is a major genetic risk factor for late-onset AD. However, the influence of APOE genotype on the biological characteristics and cargo composition of brain-derived extracellular vesicles (BDEVs) in AD remains poorly understood. In this study, BDEVs were isolated from human AD temporal cortex with APOE3/3 and APOE4/4 genotypes ( N  = 20 / group). Nanoflow cytometry and super-resolution microscopy were used to evaluate the tau load within these BDEVs. EV-mediated tau transmission was assessed in aged human Tau KI and APP NL-G-F : Tau KI mice in vivo . BDEV uptake and tau seeding were evaluated in iPSC-derived neurons through live imaging. Additionally, shotgun lipidomics and data independent acquisition proteomics were analyzed lipidome and proteome of BDEVs, respectively. Key molecules linked to APOE genotype were identified through integrated weighted gene co-expression network and trait correlation analysis. APOE4/4 BDEVs exhibited a higher proportion of phosphorylated tau (pS396) at single EV levels. Upon injection of 300pg tau of BDEVs into aged Tau KI and APP NL-G-F : Tau KI mice, APOE4/4 BDEVs significantly increased AT8 + phosphorylated tau levels, and induced neuroinflammation in vivo . Moreover, APOE4/4 BDEVs demonstrated stronger neuronal uptake and tau transfer compared to APOE3/3 BDEVs, impairing the neuronal activity of iPSC-derived neurons. Lipidomic revealed that APOE4/4 BDEVs were significantly more enriched in pro-inflammatory long-chain polyunsaturated fatty acids (PUFA) compared to APOE3/3 with Braak stage association. Furthermore, gene ontology pathway analysis using differentially expressed proteins in APOE4/4 BDEVs showed increased activity in oxidative phosphorylation and sterol-binding. Multi-omics integrated data revealed that upregulation of PUFA was significantly associated with elevated cell adhesion molecules and ATPase transporter activity with neural cell adhesion molecule as one of key molecule. Notably, neutralizing antibody against the molecule effectively reduced APOE4/4 BDEV internalization and mitigated EV-mediated tau pathology in recipient cells. Our comprehensive analysis suggested that AD APOE4/4 BDEV may enhance transmissibility of tau pathology and neuroinflammation via enriched pro-inflammatory PUFA and cell adhesion molecules. Targeting these pathways presents a promising therapeutic strategy to mitigate disease progression, particularly in APOE4 individuals.

Extracellular vesicles (EVs) are critical mediators of intercellular communication by transferring proteins, lipid and nucleic acids between cells. EVs in biofluids, particularly blood, have gathered significant interest as potential biomarkers for disease diagnosis. However, isolating EVs from blood poses a challenge due to the high concentration of plasma proteins, which obscure the detection of low abundant EV-associated proteins. Here, we optimized a simplified and efficient method for isolating plasma-derived EVs by combining size exclusion chromatography (SEC) with flow-through chromatography using Capto Core 700 beads. A brief incubation of SEC-derived EV fractions with Capto Core beads (qEV + CC) enabled us to isolate intact, high-purity EVs with reduced soluble plasma protein contamination. As a comparison, MagReSyn-based method was not compatible with elution of intact EVs after the purification and showed significant contamination of soluble plasma proteins. Data-independent acquisition-based liquid chromatography-mass spectrometry of isolated plasma-EVs using the qEV + CC approach identified over 1,000 EV-associated proteins, including an increased presence of brain derived proteins and markers linked to neurodegenerative diseases, such as amyloid precursor protein and apolipoprotein E. These findings were further validated by super-resolution microscopy at a single EV resolution. Bioinformatic pathway and network analyses revealed enrichment of pathways involved in RNA processing, cell adhesion and synaptic function, highlighting the potential of EV molecules for broad disease biomarker discovery. Our findings present an optimized method for efficient purification of plasma-derived EVs, providing a valuable tool for advancing EV-based biomarker development.

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. 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. 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. 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.

INTRODUCTION:Transgenic mice overexpressing familial Alzheimer's disease (AD) mutations (FAD) show non-physiological traits, and their immunocompetent backgrounds limit their use in AD immunotherapy research. Preclinical models that reflect human immune responses in AD are needed. METHODS:Using CRISPR-Cas9, we developed single (NA) and double (NAPS) knock-in (KI) amyloid precursor protein (APP)KM670,671NL (Swedish) and presenilin 1 (PS 1)M146VFAD mutations on an immunodeficient NOG (NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac) background. The models were confirmed by Sanger sequencing and evaluated for AD-like pathology. RESULTS:Both NA and NAPS mice developed pathology without overexpression artifacts. Mutation-induced upregulation of APP-CTF-β led to intraneuronal human amyloid beta (Aβ) (6E10) deposits and amyloid-associated microgliosis as early as 3 months, which increased with age. The addition of the PS 1M146V mutation doubled the amyloid load. The models displayed broad neuronal loss, resulting in brain atrophy in older mice. DISCUSSION:These models replicate intraneuronal amyloid pathology and, with human immune reconstitution potential, enable novel studies of human immune responses in AD. HIGHLIGHTS:A novel Alzheimer's disease (AD) knock-in (KI) mouse was developed and characterized on an immunodeficient NOG background. The model provides a platform for human immune studies and the evaluation of immunotherapies for AD. The KI mice demonstrate intraneuronal Aβ deposits and amyloid-associated microglial reactions. KI mice demonstrate extensive neuronal loss. Human immune reconstitution enables studies of infectious AD co-morbidities, such as the human immunodeficiency and herpes simplex viruses.

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. 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. 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. 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.

There are few in vitro models available to study microglial physiology in a homeostatic context. Recent approaches include the human induced pluripotent stem cell model, but these can be challenging for large-scale assays and may lead to batch variability. To advance our understanding of microglial biology while enabling scalability for high-throughput assays, we developed an inducible immortalized murine microglial cell line using a tetracycline expression system. The addition of doxycycline facilitates rapid cell proliferation, allowing for population expansion. Upon withdrawal of doxycycline, this monoclonal microglial cell line differentiates, resembling in vivo microglial physiology as demonstrated by the expression of microglial genes, innate immune responses, chemotaxis, and phagocytic abilities. We utilized live imaging and various molecular techniques to functionally characterize the clonal 2E11murine microglial cell line. Transcriptomic analysis showed that the 2E11 line exhibited characteristics of immature, proliferative microglia during doxycycline induction, and further differentiation led to a more homeostatic phenotype. Treatment with transforming growth factor-β modified the transcriptome of the 2E11 cell line, affecting cellular immune pathways. Our findings indicate that the 2E11 inducible immortalized cell line is a practical and convenient tool for studying microglial biology in vitro.

AbstractExtracellular vesicle (EV) secretion is mediated by purinergic receptor P2X7 (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.