A novel histone deacetylase inhibitor‐based approach to eliminate microglia and retain astrocyte properties in glial cell culture

Abstract

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.

Exploring epigenetic reprogramming during central nervous system infection

“…Clearly, neutropenia may be detrimental in the case of infection; therefore, prudent clinical judgment will be paramount for the proper use of these compounds as their availability expands. Additionally, persistent HDAC inhibition is toxic to microglia, which has been leveraged to deplete microglia from mixed glial populations in vitro to yield purified astrocytes, further supporting the potential immunological danger of these compounds 115 . The toxic effects of HDAC inhibitors have proven beneficial in oncology, showing promise in the treatment of multiple hematologic cancers 13 .…”

Acrylamide Induces Protein Aggregation in CNS through Suppression of FoxO1-mediated Autophagy

“…Primary astrocytes were extracted from postnatal day 1 mouse cortices and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% Penecillin/Streptomycin in vitro, passaged once with a modified mild trypsin method [49] and underwent 2 days of trichostatin A treatment [16] to eliminate all the microglia contamination. Primary NSCs were extracted from embryonic day 13 mouse cortices, plated onto poly-L-ornithine and fibronectin double-coated culture materials and maintained in a modified N2 media supplemented with B27.…”

“…RNA preparation, cDNA synthesis and PCR analysis were performed as previously described [16]. The PCR primers are summarized in Supplementary Table S1.…”

“…Primary astrocytes were extracted from postnatal day 1 mouse cortices and cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 1% Penecillin/Streptomycin in vitro, passaged once with a modified mild trypsin method [49] and underwent 2 days of trichostatin A treatment [16] to eliminate all the microglia contamination.…”

Roles of Embryonic Lethal Abnormal Vision-Like RNA Binding Proteins in Cancer and Beyond

“…Among these inhibitors, MS-444, dehydromutactin, okicenone, SRI-42127, AZA-9, b-40, KH-3, CMLD1 and CMLD2 are specific inhibitors of ELAVL1, while others are not. For example, eltrombopag often acts as a thrombopoietin (TPO) receptor agonist ( Bussel et al, 2019 ), AZA as a DNA methyltransferase inhibitor ( Song et al, 2022 ) and TSA as a histone deacetylase inhibitor ( He et al, 2022 ). Pyrvinium pamoate ( Faheem et al, 2022 ), Rottlerin ( Hufnagel et al, 2009 ), dihydrotanshinone-I ( Sun et al, 2022 ), quercetin ( Zaragozá et al, 2022 ) and suramin ( Zhang et al, 2022 ) all have effects on other physiological or pathological processes.…”