Microglia sense astrocyte dysfunction and prevent disease progression in an Alexander disease model.

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca²⁺ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca²⁺ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca²⁺ signals, with high frequency Ca²⁺ signals in both the processes and cell bodies. These microglial Ca²⁺ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y₁₂ receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2 , an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca²⁺ signals. To determine whether these P2Y₁₂ receptor-mediated Ca²⁺ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y₁₂ receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y₁₂ receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y₁₂ receptors as an increase in extracellular ATP and alter their morphology and Ca²⁺ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD. [ABSTRACT FROM AUTHOR]