Seizures are defined as symptoms and signs due to aberrant neuronal hyperexcitability and hyper-synchronization in the brain. Seizures induce neuronal death by glutamate-mediated excitotoxicity and neuroinflammation, which are relevant to excessive hydrogen peroxide (H2O2) and nitric oxide (NO) generation (For review, Ambrogini et al., 2019). These oxidative and nitrosative stresses consume glutathione (GSH) that is the most important redox-regulating non-enzymatic thiol to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Zängerle et al., 1992; Asahi et al., 1995; Clark and Debnam, 1988). GSH biosynthesis is regulated by various enzymes and transporters via several steps. Glutamate cysteine ligase (GCLC) is the rate-limiting enzyme that converts glutamate and cysteine (mostly derived from cystine; the oxidized dimer form of cysteine) to γ-glutamylcysteine (γGluCys). Thereafter, GSH synthetase (GSHS) adds glycine to γGluCys for generating GSH. In addition, alanine-serine-cysteine transporter 1 and 2 (ASCT1 and 2, also known as solute carrier 1 A4 and A5, respectively) and cystine/glutamate transporter (xCT or SLC7a11) import cysteine and cystine, respectively, that are substrates for GSH (Kanai and Hediger, 2003; Lu, 2009; Lewerenz et al., 2012). Glutamate-glutamine cycle supplies glutamate for GSH synthesis and affects GSHS level, which are regulated by glutaminase (GLS) and glutamine synthase (GS) (Venoji et al., 2015). Since astrocytes contain higher GSH levels than neurons and support other brain cell populations in defense against ROS and RNS, a compromised astroglial GSH system leads to an impaired capacity of the antioxidative defense in the brain (Dringen et al., 2000; Vinokurov et al., 2021). Therefore, the maintenance of appropriate GSH biosynthesis in astrocytes is one of the important therapeutic goals to protect neurons from detrimental stress and prevent the secondary lesions.

On the other hand, heat shock protein 25 (HSP25, also known as HSPB1 and HSP27 in human) has a potent ability to increase cell viability via its chaperone activity. This inducible HSP prevents denaturation and aggregation of other proteins in response to various harmful stimuli including oxidative and nitrosative stresses (van der Weerd et al., 2010; Kirbach and Golenhofen, 2011). HSP25 also regulates GSH level and the protective activity of HSP25 is GSH-dependent (Mehlen et al., 1996; Préville et al., 1999; Baek et al., 2000; Paul and Arrigo, 2000). Thus, it is likely that HSP25 functions and GSH level may be reciprocally regulated by each other. However, the administration of kainic acid (KA) induces HSP25 expression in astrocytes, but not in neurons (Plumier et al., 1996; Che et al., 2001), while it decreases total GSH level in the hippocampus (Si et al., 2016). Therefore, it is necessary to elucidate the underlying mechanisms of the reciprocal regulation between HSP25 and GSH in response to seizures, which are largely unknown.

P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels. Morphologically, P2X7R mainly expresses in microglia within the adult brain. However, pharmacological and physiological data demonstrate the presence of this receptor in neurons and astrocytes (Anderson and Nedergaard, 2006; Sperlágh et al., 2006). Therefore, P2X7R modulates various brain functions, such as neuronal excitability, oxidative stress and neuroinflammation (Panenka et al., 2001; Sim et al., 2004; Sperlágh et al., 2006; Kim and Kang, 2011). In addition, P2X7R regulates glutamate uptake, GS activity and ASCT2 functionality in astrocytes (Lo et al., 2008; Pan et al., 2015; Roth et al., 2014). Indeed, P2X7R deletion decreases the basal GSH level and N-acetylcysteine (NAC, a GSH precursor) uptake in coupled with upregulations in GS and ASCT2 level in the mouse hippocampus without affecting GCLC, GSS, GLS and xCT levels (Park and Kim, 2020; Lee and Kim, 2022). In contrast, P2X7R augments lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) level (Sperlágh et al., 1998; Hu et al., 1998; Choi et al., 2007). NO reacts with reactive cysteine thiols of target proteins forming S-nitrosylated (SNO)-proteins (referred as S-nitrosylation) (Qu et al., 2014). Under nitrosative stress, S-nitrosylation of Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1) relieves nuclear factor-erythroid 2-related factor 2 (Nrf2, a redox-sensitive transcription factor) and facilitates nuclear Nrf2 translocation to increase antioxidant-response element (ARE)-dependent gene transactivations including GSH synthetic enzymes (Itoh et al., 1999; McMahon et al., 2003). Of note, P2X7R ablation ameliorates LPS-induced S-nitrosylation of GS and ASCT2 and diminishment of GSH content by inhibiting Nrf2 downregulation in microglia and astrocytes (Lee and Kim, 2022). Therefore, it is likely that P2X7R may increase the demand of GSH for maintenance of the redox homeostasis under physiological condition, and its activation may lead to the higher GSH consumption in response to KA. Furthermore, P2X7R ablation and its antagonists lead to rapid induction and prolonged HSP25 upregulation in astrocytes following KA treatment through extracellular regulated kinase 1/2 (ERK1/2)-specificity protein 1 (SP1)-mediated signaling pathway (Kim et al., 2018; Lee and Kim, 2020). In the present study, therefore we investigated whether P2X7R is involved in the reciprocal regulation between HSP25 and GSH and nitrosative stress in response to KA.

Here, we demonstrate that following KA injection P2X7R deletion facilitated nuclear Nrf2 translocation in microglia and HSP25 induction in astrocytes, respectively. P2X7R deletion also ameliorated KA-induced iNOS upregulation and S-nitrosylated-cysteine (SNO-Cys) production in astrocytes concomitant with the maintenance of nuclear Nrf2 translocation. Furthermore, P2X7R ablation attenuated the decreased GSH content and S-nitrosylations of GS and ASCT2 in the hippocampus in response to KA. These effects of P2X7R deletion were abrogated by HSP25 knockdown. Therefore, these findings indicate that P2X7R deletion-mediated HSP25 upregulation may decrease GSH consumption and maintain its biosynthesis by mitigating iNOS-induced nitrosative stress in astrocytes following KA treatment.