Pharmacological inhibition of astrocytic transglutaminase 2 facilitates the expression of a neurosupportive astrocyte reactive phenotype in association with increased histone acetylation

Abstract

AAstrocytes are the primary support cells of the central nervous system (CNS) that help maintain the energetic requirements and homeostatic environment of neurons. CNS injury causes astrocytes to take on reactive phenotypes with altered overall function that can range from supportive to harmful for recovering neurons. The characterization of reactive astrocyte populations is a rapidly developing field, and the underlying factors and signaling pathways governing which type of reactive phenotype that astrocytes take on is poorly understood. Our previous studies suggest that transglutaminase 2 (TG2) has an important role in determining the astrocytic response to injury. TG2 is upregulated in astrocytes across multiple injury models, and selectively deleting TG2 from astrocytes improves functional outcomes after CNS injury and causes widespread changes in gene regulation, which is associated with its nuclear localization. The underlying molecular mechanisms by which TG2 causes these functional changes are unknown, and its interactions in the nucleus of astrocytes has not yet been described. To begin to understand how TG2 impacts astrocytic function, we used a neuron-astrocyte co-culture paradigm to compare the effects of TG2-/- and wild type (WT) astrocytes on neurite outgrowth and synapse formation. We assayed neurons on both a growth-supportive substrate and an injury-simulating matrix comprised of inhibitory chondroitin sulfate proteoglycans (CSPGs). Compared to WT astrocytes, TG2-/- astrocytes supported neurite outgrowth to a significantly greater extent only on the CSPG matrix, while synapse formation assays showed mixed results depending on the pre- and post-synaptic markers analyzed. We hypothesize that TG2 regulates the supportive functions of astrocytes in injury conditions by modulating the expression of a wide range of genes through interactions with transcription factors and transcription complexes. Based on results of a previous yeast two-hybrid screen for TG2 interactors, we further investigated the interaction of TG2 with Zbtb7a, a ubiquitously expressed transcription factor. Co-immunoprecipitation and colocalization analyses confirmed the interaction of TG2 and Zbtb7a in the nucleus of astrocytes. Genetic overexpression or knockdown of Zbtb7a levels in TG2-/- and WT astrocytes revealed that Zbtb7a robustly influenced astrocytic morphology and the ability of astrocytes to support neuronal outgrowth, which was significantly modulated by the presence of TG2. These findings support our hypothesis that astrocytic TG2 acts as a transcriptional regulator to influence astrocytic function, with greater influence under injury conditions that increase its expression, and Zbtb7a likely contributes to the overall effects observed with astrocytic TG2 deletion.

Pharmacological inhibition of astrocytic transglutaminase 2 facilitates the expression of a neurosupportive astrocyte reactive phenotype in association with increased histone acetylation

“…There is growing evidence that TG2 plays a significant role in regulating the phenotype of reactive astrocytes (Espitia Pinzon et al 2017, van Strien et al 2011b, Elahi et al 2021, Emerson et al 2023, Monteagudo et al 2018, Delgado and Johnson 2024. In different injury models, it has been demonstrated that deletion or depletion of TG2 from astrocytes enhances their ability to support neuronal viability and recovery after injury compared to controls (Colak and Johnson 2012, Elahi et al 2021, Emerson et al 2023, Monteagudo et al 2018. Most notably, mice with astrocyte-specific TG2 deletion recover motor function significantly faster and to an overall greater extent after SCI compared to WT mice.…”

“…Nuclear fractionation has shown that TG2 is enriched in the chromatin fraction (Lesort et al 1998). IT exhibits a functionally significant interaction with the transcription factor-like protein Zbtb7a, which regulates gene expression in part by altering promoter accessibility (Emerson et al 2023, Pittol et al 2018. To interrogate how VA4 may alter the interaction of TG2 with Zbtb7a, and thus gene expression, HEK cells were transfected with V5-tagged TG2 and FLAG-tagged Zbtb7a, followed by VA4 or vehicle-only treatment and subsequent analysis of TG2-Zbtb7a interactions (Emerson et al 2023).…”

“…IT exhibits a functionally significant interaction with the transcription factor-like protein Zbtb7a, which regulates gene expression in part by altering promoter accessibility (Emerson et al 2023, Pittol et al 2018. To interrogate how VA4 may alter the interaction of TG2 with Zbtb7a, and thus gene expression, HEK cells were transfected with V5-tagged TG2 and FLAG-tagged Zbtb7a, followed by VA4 or vehicle-only treatment and subsequent analysis of TG2-Zbtb7a interactions (Emerson et al 2023). Immunoprecipitation (IP) of TG2 from the VA4-treated cells resulted in significantly less pull down of Zbtb7a compared to vehicle-only treated cells (Fig.…”

“…TG2 and Zbtb7a both interact with components of Sin3a, a transcriptional repressor complex that facilitates chromatin modifications by HDAC1 and HDAC2 (Emerson et al 2023, Laudes et al 2008. Considering this and the predominant upregulation of gene expression when TG2 is deleted (Elahi et al 2021, Monteagudo et al 2018, we hypothesized that TG2 limits the chromatin accessibility of reactive astrocytes in part through regulating histone acetylation.…”

“…Inflammatory and stress signals, whether from injury, infection, or other sources, cause astrocytes to take on a range of phenotypes from neuroprotective to neurotoxic, although the exact molecular determinants of these phenotypes are poorly understood (Anderson et al 2014, Burda et al 2022, Escartin et al 2021, Linnerbauer and Rothhammer 2020, Davies et al 1997, Guttenplan et al 2021, Liddelow et al 2017, Smith and Strunz 2005, Yiu and He 2006. Previously, our lab has implicated the protein transglutaminase 2 (TG2) as a key factor in modulating the phenotype of reactive astrocytes (Colak and Johnson 2012, Elahi et al 2021, Emerson et al 2023, Monteagudo et al 2018.…”