Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model

Abstract Background Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer’s disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology. Methods Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote (AppNL-G-F) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC. Results Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in AppNL-G-F mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in AppNL-G-F mice, although it increased plaque burden and plaque-associated p-tau+ dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9+ EV particles, which were significantly enhanced in Mac2+ MGnD microglia compared to Mac2− homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into AppNL-G-F mice revealed encapsulation of p-tau in microglia-specific mE-CD9+ EVs as determined by super-resolution microscopy and immuno-electron microscopy. Discussion Our findings suggest that MGnD microglia hyper-secrete p-tau+ EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in AppNL-G-F mice

Correction to: Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model

Correction to: Mol Neurodegeneration 16, 18 (2021) 10.1186/s13024-021-00440-

Inhibition of colony stimulating factor 1 receptor corrects maternal inflammation-induced microglial and synaptic dysfunction and behavioral abnormalities

Abstract Maternal immune activation (MIA) disrupts the central innate immune system during a critical neurodevelopmental period. Microglia are primary innate immune cells in the brain although their direct influence on the MIA phenotype is largely unknown. Here we show that MIA alters microglial gene expression with upregulation of cellular protrusion/neuritogenic pathways, concurrently causing repetitive behavior, social deficits, and synaptic dysfunction to layer V intrinsically bursting pyramidal neurons in the prefrontal cortex of mice. MIA increases plastic dendritic spines of the intrinsically bursting neurons and their interaction with hyper-ramified microglia. Treating MIA offspring by colony stimulating factor 1 receptor inhibitors induces depletion and repopulation of microglia, and corrects protein expression of the newly identified MIA-associated neuritogenic molecules in microglia, which coalesces with correction of MIA-associated synaptic, neurophysiological, and behavioral abnormalities. Our study demonstrates that maternal immune insults perturb microglial phenotypes and influence neuronal functions throughout adulthood, and reveals a potent effect of colony stimulating factor 1 receptor inhibitors on the correction of MIA-associated microglial, synaptic, and neurobehavioral dysfunctions

P2RX7 inhibitor suppresses exosome secretion and disease phenotype in P301S tau transgenic mice

Background: Neuronal accumulation of misfolded microtubule-associated protein tau is a hallmark of neuropathology in Alzheimer’s disease, frontotemporal dementia, and other tauopathies, and has been a therapeutic target. Microglia can spread tau pathology by secreting tau-containing exosomes, although the specific molecular target is yet to be identified for the therapeutic intervention. P2X purinoceptor 7 (P2RX7) is an ATP-gated cation channel, enriched in microglia and triggers exosome secretion. The purpose of the study is to examine the therapeutic effect of an orally applicable, CNS-penetrant P2RX7 specific inhibitor on the early disease stage of a tauopathy mouse model. Methods: Three-months-old P301S tau mice were treated with P2RX7-specific inhibitor GSK1482160 or vehicle for 30 days, followed by behavioral, biochemical and immunohistochemical assessment. GSK1482160 was also tested for exosome secretion from primary cultured murine astrocytes, neurons and microglia in vitro. Results: Oral administration of GSK1482160 significantly reduced accumulation of MC1+ and Alz50+ misfolded tau in hippocampal regions, which was accompanied with reduced accumulation of Tsg101, an exosome marker, in hippocampal neurons. Proximity ligation assay demonstrated complex formation of Alz50+ tau and Tsg101 in hippocampal neurons, which was reduced by GSK1482160. On the other hand, GSK1482160 had no effect on microglial ramification or CD68 expression, which was significantly enhanced in P301S mice, or pro/anti-inflammatory cytokine gene expression. Strikingly, GSK1482160-treated P301S mice show significantly improved working and contextual memory as determined by Y-maze and fear conditioning tests. GSK1482160 also significantly increased accumulation of Tsg101 and CD81 in microglia in vivo, suggesting its suppression of P2RX7-induced exosome secretion from microglia. This effect was confirmed in vitro, as ATP-induced secretion of tau-containing exosome was significantly suppressed by GSK1482160 treatment from primary murine microglia, but not from neurons or astrocytes. Discussion: The oral administration of P2RX7 inhibition mitigates disease phenotypes in P301S mice, likely by suppressing release of microglial exosomes. P2RX7 could be a novel therapeutic target for the early stage tauopathy development

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

P2RX7 plays a critical role in extracellular vesicle‐mediated secretion of pathogenic molecules from microglia and astrocytes

Abstract Extracellular 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

Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer’s Disease Patients: A Pilot Study

Pathological hallmarks of Alzheimer’s disease (AD) are deposits of amyloid beta (Aβ) and hyper-phosphorylated tau aggregates in brain plaques. Recent studies have highlighted the importance of Aβ and tau-containing extracellular vesicles (EVs) in AD. We therefore examined EVs separated from cerebrospinal fluid (CSF) of AD, mild cognitive impairment (MCI), and control (CTRL) patient samples to profile the protein composition of CSF EV. EV fractions were separated from AD (n = 13), MCI (n = 10), and CTRL (n = 10) CSF samples using MagCapture Exosome Isolation kit. The CSF-derived EV proteins were identified and quantified by label-free and tandem mass tag (TMT)-labeled mass spectrometry. Label-free proteomics analysis identified 2546 proteins that were significantly enriched for extracellular exosome ontology by Gene Ontology analysis. Canonical Pathway Analysis revealed glia-related signaling. Quantitative proteomics analysis, moreover, showed that EVs expressed 1284 unique proteins in AD, MCI and CTRL groups. Statistical analysis identified three proteins—HSPA1A, NPEPPS, and PTGFRN—involved in AD progression. In addition, the PTGFRN showed a moderate correlation with amyloid plaque (rho = 0.404, p = 0.027) and tangle scores (rho = 0.500, p = 0.005) in AD, MCI and CTRL. Based on the CSF EV proteomics, these data indicate that three proteins, HSPA1A, NPEPPS and PTGFRN, may be used to monitor the progression of MCI to AD

Antisense oligonucleotide-based targeting of Tau-tubulin kinase 1 prevents hippocampal accumulation of phosphorylated tau in PS19 tauopathy mice

Abstract 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

Comprehensive characterization of human brain‐derived extracellular vesicles using multiple isolation methods: Implications for diagnostic and therapeutic applications

Abstract 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

Proteomic Profiling of Extracellular Vesicles Separated from Plasma of Former National Football League Players at Risk for Chronic Traumatic Encephalopathy

Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of exposure to repetitive head impacts, including National Football League (NFL) players. Extracellular vesicles (EVs) are known to carry tau in Alzheimer’s disease and other tauopathies. We examined protein profiles of EVs separated from the plasma of former NFL players at risk for CTE. EVs were separated from the plasma from former NFL players and age-matched controls using size-exclusion chromatography. Label-free quantitative proteomic analysis identified 675 proteins in plasma EVs, and 17 proteins were significantly differentially expressed between former NFL players and controls. Total tau (t-tau) and tau phosphorylated at threonie181 (p-tau181) in plasma-derived EVs were measured by ultrasensitive immunoassay. Level of t-tau and p-tau181 in EVs were significantly different, and the area under the receiver operating characteristic curve (AUC) of t-tau and p-tau181 showed 0.736 and 0.715, respectively. Machine learning analysis indicated that a combination of collagen type VI alpha 3 and 1 chain (COL6A3 and COL6A1) and reelin (RELN) can distinguish former NFL players from controls with 85% accuracy (AUC = 0.85). Based on the plasma EV proteomics, these data provide protein profiling of plasma EVs for CTE, and indicate combination of COL6A3, RELN and COL6A1 in plasma EVs may serve as the potential diagnostic biomarkers for CTE