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.

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.

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.

The characterization of single extracellular vesicle (EV) has been an emerging tool for the early detection of various diseases despite there being challenges regarding how to interpret data with different protocols or instruments. In this work, standard EV particles were characterized for single CD9+, single CD81+ or double CD9+/CD81+ tetraspanin molecule positivity with two single EV analytic technologies in order to optimize their EV sample preparation after antibody labelling and analysis methods: NanoImager for direct stochastic optical reconstruction microscopy (dSTORM)-based EV imaging and characterization, and Flow NanoAnalyzer for flow-based EV quantification and characterization. False positives from antibody aggregates were found during dSTORM-based NanoImager imaging. Analysis of particle radius with lognormal fittings of probability density histogram enabled the removal of antibody aggregates and corrected EV quantification. Furthermore, different machine learning models were trained to differentiate antibody aggregates from EV particles and correct EV quantification with increased double CD9+/CD81+ population. With Flow NanoAnalyzer, EV samples were prepared with different dilution or fractionation methods, which increased the detection rate of CD9+/CD81+ EV population. Comparing the EV phenotype percentages measured by two instruments, differences in double positive and single positive particles existed after percentage correction, which might be due to the different detection limit of each instrument. Our study reveals that the characterization of individual EVs for tetraspanin positivity varies between two platforms-the NanoImager and the Flow NanoAnalyzer-depending on the EV sample preparation methods used after antibody labelling. Additionally, we applied machine learning models to correct for false positive particles identified in imaging-based results by fitting size distribution data.

Microglia, brain innate immune cells, participate in the spread of inflammatory signals and aggregated proteins through secretion of extracellular vesicles (EVs). Selenoprotein P (Sepp1) is a potential regulator of microglial EV secretion. Here, we investigate the effect of Sepp1 silencing on microglial transcriptomics to elucidate the Sepp1 regulatory mechanism of EV secretion and validate this effect in APPNL-G-F knockin mice. Silencing of Sepp1 significantly reduces EV secretion and CD63 loading to EVs from BV-2 microglia, as determined by single-vesicle flow cytometry and super-resolution microscopy. Sepp1 deficiency downregulates EV biogenesis machinery, accompanied by increased lysosomal activity and lipid metabolism. Silencing of Sepp1 in astrocytes but not neurons suppresses EV secretion in vitro. Finally, Sepp1 silencing reduces EV secretion from activated neurodegenerative microglia associated with amyloid plaques in APPNL-G-F mouse brains in vivo. Sepp1 is thus an emerging therapeutic target for ameliorating microglia-mediated disease spread through EV secretion in neurodegenerative disorders.

Microglia 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. Here, we report that microglial deletion of miR-155 induces a pre-MGnD activation state via interferon-γ (IFN-γ) signaling, and blocking IFN-γ signaling attenuates MGnD induction and microglial phagocytosis. Single-cell RNA-sequencing analysis of microglia from an 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. Our study demonstrates a miR-155-mediated regulatory mechanism of MGnD and the beneficial role of IFN-γ-responsive pre-MGnD in restricting neurodegenerative pathology and preserving cognitive function in an AD mouse model, highlighting miR-155 and IFN-γ as potential therapeutic targets for AD.

Tau tubulin kinase-1 (TTBK1), a neuron-specific tau kinase, is highly expressed in the entorhinal cortex and hippocampal regions, where early tau pathology evolves in Alzheimer’s disease (AD). The protein expression level of TTBK1 is elevated in the cortex brain tissues with AD patients compared to the control subjects. We therefore hypothesized that antisense oligonucleotide (ASO) based targeting Ttbk1 could prevent the accumulation of phosphorylated tau, thereby delaying the development of tau pathology in AD. Here we show that in vivo administration of ASO targeting mouse Ttbk1 (ASO- Ttbk1 ) specifically suppressed the expression of Ttbk1 without affecting Ttbk2 expression in the temporal cortex of PS19 tau transgenic mice. Central administration of ASO- Ttbk1 in PS19 mice significantly reduced the expression level of representative phosphor-tau epitopes relevant to AD at 8 weeks post-dose, including pT231, pT181, and pS396 in the sarkosyl soluble and insoluble fractions isolated from hippocampal tissues as determined by ELISA and pS422 in soluble fractions as determined by western blotting. Immunofluorescence demonstrated that ASO- Ttbk1 significantly reduced pS422 phosphorylated tau intensity in mossy fibers region of the dentate gyrus in PS19 mice. RNA-sequence analysis of the temporal cortex tissue revealed significant enrichment of interferon-gamma and complement pathways and increased expression of antigen presenting molecules (Cd86, Cd74, and H2-Aa) in PS19 mice treated with ASO- Ttbk1 , suggesting its potential effect on microglial phenotype although neurotoxic effect was absent. These data suggest that TTBK1 is an attractive therapeutic target to suppress TTBK1 without compromising TTBK2 expression and pathological tau phosphorylation in the early stages of AD.

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.

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain-derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size-exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG-UC). Our results showed that SG-UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass-tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG-UC showing the best enrichment of common EV proteins with less contamination of non-EV proteins. In addition, SG-UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell-substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single-particle analysis using the super-resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.

Microglia, 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. We 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). We 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. Our 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.

Alzheimer'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.

Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.

Single cell RNA sequencing studies identified novel neurodegeneration-associated microglial (MGnD/DAM) subtypes activated around cerebral amyloid plaques. Micro-RNA (miR)-155 of the TREM2-APOE pathway was shown to be a key transcriptional regulator of MGnD microglial phenotype. Despite growing interest in studying manifestations of Alzheimer’s disease (AD) in the retina, a CNS organ accessible to noninvasive high-resolution imaging, to date MGnD microglia have not been studied in the AD retina. Here, we discovered the presence and increased populations of Clec7a + and Galectin-3 + MGnD microglia in retinas of transgenic APP SWE /PS1 L166P AD-model mice. Conditionally targeting MGnD microglia by miR-155 ablation via the tamoxifen-inducible Cre ERT2 system in APP SWE /PS1 L166P mice diminished retinal Clec7a + and Galectin-3 + microglial populations while increasing homeostatic P2ry12 + microglia. Retinal MGnD microglia were often adhering to microvessels; their depletion protected the inner blood-retina barrier and reduced vascular amyloidosis. Microglial miR-155 depletion further limits retinal inflammation. Mass spectrometry analysis revealed enhanced retinal PI3K-Akt signaling and predicted IL-8 and Spp1 decreases in mice with microglia-specific miR-155 knockout. Overall, this study identified MGnD microglia in APP SWE /PS1 L166P mouse retina. Transcriptional regulation of these dysfunctional microglia mitigated retinal inflammation and vasculopathy. The protective effects of microglial miR-155 ablation should shed light on potential treatments for retinal inflammation and vascular damage during AD and other ocular diseases.

P2X 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. Fours 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. Exosome 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. Results 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.

There is increasing evidence showing that microglia play a critical role in mediating synapse formation and spine growth, although the molecular mechanism remains elusive. Here, we demonstrate that the secreted morphogen WNT family member 5A (WNT5A) is the most abundant WNT expressed in microglia and that it promotes neuronal maturation. Co-culture of microglia with Thy1-YFP+ differentiated neurons significantly increased neuronal spine density and reduced dendritic spine turnover rate, which was diminished by silencing microglial Wnt5a in vitro. Co-cultured microglia increased post-synaptic marker PSD95 and synaptic density as determined by the co-localization of PSD95 with pre-synaptic marker VGLUT2 in vitro. The silencing of Wnt5a expression in microglia partially reduced both PSD95 and synaptic densities. Co-culture of differentiated neurons with microglia significantly enhanced neuronal firing rate as measured by multiple electrode array, which was significantly reduced by silencing microglial Wnt5a at 23 days differentiation in vitro. These findings demonstrate that microglia can mediate spine maturation and regulate neuronal excitability via WNT5A secretion indicating possible pathological roles of dysfunctional microglia in developmental disorders.

Autism spectrum disorder and maternal immune activation: Environmental factors during pregnancy, such as infections, maternal stress or autoimmune disorders, are closely associated with the prevalence of neurodevelopmental disorders including autism spectrum disorder (ASD), bipolar disorder and schizophrenia. It has been shown that severe infections during pregnancy cause maternal immune activation (MIA) and significantly increase the risk of ASD in the offspring although the mechanisms are poorly understood. Many rodent MIA studies support this causal link by showing that offspring of dams administered with polyinosinic:polycytidylic acid (polyI:C), a viral mimetic Toll-like receptor 3 agonist, exhibit longlasting ASD-like behavioral abnormalities such as increased repetitive behavior, impaired social interaction and communication. Interestingly, MIA alters inflammatory cytokine expressions persisting through development and adulthood in the brain of the offspring, suggesting that chronic neuroimmune dysfunction plays a role in mediating the deleterious effects of MIA on neurodevelopment (Garay et al., 2013).

Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of mild repetitive brain injury. The initial neuropathologic changes of CTE include perivascular deposition of phosphorylated microtubule-associated protein tau (p-tau). Extracellular vesicles (EVs) are known to carry pathogenic molecules, such as tau in Alzheimer's disease and CTE suggesting their contribution in pathogenesis. We therefore examined the protein composition of EVs separated from CTE and an age-matched control brain tissues by tandem mass tag -mass spectrometry. The reporter ion intensity was used to quantify the identified molecules. A total of 516 common proteins were identified among three sets of experiments. Weighted protein co-expression network analysis identified 18 unique modules of co-expressed proteins. Two modules were significantly correlated with total tau (t-tau) and p-tau protein in the isolated EVs and enriched in cellular components and biological processes for synaptic vesicle secretion and multivesicular body-plasma membrane fusion. The p-tau (Thr181) level is significantly higher in CTE EVs compared to control EVs and can distinguish the two groups with 73.6% accuracy. A combination of t-tau or p-tau (Thr181) with SNAP-25, PLXNA4 or UBA1, enhanced the accuracy to 96.3, 93.8 and 93.8%, respectively. Bioinformatic protein-protein interaction analysis revealed the functional interaction of SNAP-25 and PLXNA4 with tau, suggesting their interaction in CTE EVs. These data indicate the future application of identified EV proteins for monitoring the CTE risk assessments and understanding the EV-mediated disease progression mechanism.

Abnormally phosphorylated tau, an early neuropathologic marker of Alzheimer’s disease (AD), first occurs in the brain’s entorhinal cortex layer II (ECII) and then spreads to the CA1 field of the hippocampus. Animal models of tau propagation aiming to recapitulate this phenomenon mostly show tau transfer from ECII stellate neurons to the dentate gyrus, but tau pathology in the dentate gyrus does not appear until advanced stages of AD. Wolframin-1–expressing (Wfs1+) pyramidal neurons have been shown functionally to modulate hippocampal CA1 neurons in mice. Here, we report that Wfs1+ pyramidal neurons are conserved in the ECII of postmortem human brain tissue and that Wfs1 colocalized with abnormally phosphorylated tau in brains from individuals with early AD. Wfs1+ neuron–specific expression of human P301L mutant tau in mouse ECII resulted in transfer of tau to hippocampal CA1 pyramidal neurons, suggesting spread of tau pathology as observed in the early Braak stages of AD. In mice expressing human mutant tau specifically in the ECII brain region, electrophysiological recordings of CA1 pyramidal neurons showed reduced excitability. Multielectrode array recordings of optogenetically stimulated Wfs1+ ECII axons resulted in reduced CA1 neuronal firing. Chemogenetic activation of CA1 pyramidal neurons showed a reduction in c-fos+ cells in the CA1. Last, a fear conditioning task revealed deficits in trace and contextual memory in mice overexpressing human mutant tau in the ECII. This work demonstrates tau transfer from the ECII to CA1 in mouse brain and provides an early Braak stage preclinical model of AD.