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

Abstract

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.

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

“…We further provided evidence that the neurodegenerative microglia phenotype MGnD produce and secrete abundant EVs containing p-tau, indicating a novel mechanism for how activated microglia may facilitate development and propagation of tau pathology. We previously reported the beneficial effects of microglia depletion as well as inhibition of EV synthesis and secretion in preventing tau spread in WT and P301S tau transgenic mice [ 22 , 44 ]. We witnessed the same, but even more beneficial effect of microglia depletion in a mouse model exhibiting the other pathological hallmark of AD, Aβ plaques.…”

Role of ATP in Extracellular Vesicle Biogenesis and Dynamics

“…The interest for EVs released upon ATP stimulation has increased exponentially, given that they have become vehicle of inflammatory signals ( Verderio et al, 2012 ), tumorigenic factors ( Graner, 2018 ) or misfolded proteins in neurodegenerative diseases ( Ruan et al, 2020 ), and their number is significantly augmented in the body fluids of patients affected by many neurological diseases ( Verderio et al, 2012 ; Colombo et al, 2018 ). Since the analysis of EV cargo may provide indications on the activation state of donor cells and the pathological state of the brain, EVs are currently under intense investigation for a possible employment in clinical practice as prognostic biomarkers.…”

“…In addition to large EVs, P2X7R stimulation triggers release of small EVs, also called exosomes, originating in the endocytic compartment ( Figure 1 Panel A) ( Asai et al, 2015 ; Ruan et al, 2020 ).…”

“…Another study implicated microglial EVs released upon ATP stimulation of P2X7R in the spreading of tau protein and disease progression in a tauopathy mouse model. Specifically, pharmacologic blockade of P2X7R with GSK1482160, an orally applicable and CNS-penetrant inhibitor, suppressed both secretion of small EVs (exosomes) and disease outcome in the early disease stages ( Ruan et al, 2020 ).…”

“…Panel (C): Graphic representation of morphological changes of EVs isolated from human mast cell lines, human blood serum, mouse lung, and Saccharomyces cerevisiae as imaged by Cvjetkovic and colleagues (Cvjetkovic et al, 2017) (left), and of a single EV in motion at the cell surface of microglia (Prada et al, 2016) (right). et al, 2005), synaptic activity (Antonucci et al, 2012;Gabrielli et al, 2015), axon-glial transfer of information (Prada et al, 2018), nerve regeneration (Lai and Breakefield, 2012) and myelin formation (Pusic et al, 2016;Van Niel et al, 2018;Lombardi et al, 2019); as well as disease-associated events, such as tumor progression, spreading of inflammation (Verderio et al, 2012) or dissemination of pathogenic proteins (Joshi et al, 2014;Asai et al, 2015;Eitan et al, 2016;Sardar Sinha et al, 2018;Crotti et al, 2019;Ruan et al, 2020). However, the contribution of ATPinduced EV shedding vs. constitutive EV release in brain disease pathogenesis is just emerging.…”