The close association between astrocytes and microglia causes great difficulties to distinguish their individual roles in innate immune responses in central nervous system. Current chemical-based methods to eliminate microglia in glial cell culture introduce various molecular and functional alterations to astrocytes. Here, we describe a novel two-step approach to achieve a complete elimination of microglia without affecting the biological properties of co-cultured astrocytes by temporal treatment of histone deacetylase inhibitor trichostatin A (TSA). We verify TSA as a potent inducer for microglial-specific apoptotic cell death, which also causes comprehensive gene expression changes in astrocytes. However, withdrawal of TSA not only ensures no microglia repopulation, but also restores all the gene expression changes in terms of astrocyte functions, including neurotrophic factors, glutamate and potassium transporters, and reactive astrocyte subtypes. By contrast, withdrawal of PLX5622, the commonly used colony-stimulating factor 1 receptor inhibitor neither prevents microglia repopulation nor restores the gene expression changes mentioned above. Using this method, we are able to discriminate differential roles of microglia and astrocytes in the induced expression of antiviral and pro-inflammatory cytokines upon various pathological stimuli including the spike protein of SARS-CoV-2. This simple and efficient method can be customized for the understanding of microglia-astrocyte interaction and the development of epigenetic therapies that target over-activated microglia in neuroinflammation-related diseases.

Proteotoxic stress is a major stimulus and risk factor for the neuropathogenesis in central nervous system (CNS), which is tightly associated with neurodegenerative diseases. Here, we identify acrylamide (ACR), a type-2 alkene that is commonly detected in deep-fried starch food and used in water industry and biomedical laboratories, as a potent and universal inducer of proteotoxic stress in mouse brain, as well as in cultured mouse neural stem cells, neurons and astrocytes, three major cell types in CNS. Aggregations of ubiquitin-labeled misfolded proteins and neurodegeneration-related proteins including amyloid precursor protein and presenilin 1 were drastically induced by ACR, leading to cell-defensive aggresome formation that was able to temporarily counteract with ACR toxicity. However, a defected clearance of aggresomes eventually led to apoptotic cell death, which was largely attributed to the breakdown of cytoskeleton and impairment of macroautophagy/autophagy, as evidenced by an aggregation of filament actin and an almost complete loss of LC3-positive autophagosomes. A series of core autophagy-related genes responsible for autophagosome formation were down-regulated, indicating an ACR-induced transcriptional suppression of autophagy. In addition, FoxO1, the master transactivator of these genes, were both transcriptionally repressed and nuclear excluded by ACR. Overexpression of FoxO1 rescued ACR-induced autophagy defects and attenuated the proteotoxicity. In summary, we spotlight proteotoxic stress as a novel feature of ACR neurotoxicity in CNS, and implicate FoxO1 as a critical therapeutic target.

The close association between astrocytes and microglia causes great difficulties to distinguish their individual roles in innate immune responses in central nervous system. Current chemical-based methods to eliminate microglia in glial cell culture introduce various molecular and functional alterations to astrocytes. Here, we describe a novel two-step approach to achieve a complete elimination of microglia without affecting the biological properties of co-cultured astrocytes by temporal treatment of histone deacetylase inhibitor trichostatin A (TSA). We verify TSA as a potent inducer for microglial-specific cell death, which also causes comprehensive gene expression changes in astrocytes. However, withdrawal of TSA not only ensures no microglia repopulation, but also restores all the gene expression changes in terms of astrocyte functions, including neurotrophic factors, glutamate and potassium transporters, and reactive astrocyte subtypes. By contrast, withdrawal of PLX5622, the commonly used colony-stimulating factor 1 receptor inhibitor neither prevents microglia repopulation nor restores the gene expression changes mentioned above. Using this method, we are able to discriminate differential roles of microglia and astrocytes in the induced expression of antiviral and pro-inflammatory cytokines upon various pathological stimuli including the spike protein of SARS-CoV-2. This simple and efficient method can be customized for the understanding of microglia-astrocyte interaction and the development of epigenetic therapies that target over-activated microglia in neuroinflammation-related diseases.