Glutamate-induced oxidative cytotoxicity is a major driver of neuronal loss in neurodegenerative diseases, yet the upstream transcriptional regulators linking oxidative stress to neuronal death remain unclear despite the known involvement of the p53–GADD45α pathway. CREB2 (ATF4) is a stress-responsive transcription factor, but its role in microglia-mediated oxidative neurotoxicity has not been fully defined. Here, we investigated CREB2 function in oxidative glutamate toxicity using HT22 hippocampal neurons, primary mouse hippocampal cells, and a kainic acid (KA)-injected rat model. Oxidative stress was induced by glutamate, intracellular ROS levels were quantified with DCFDA, and the antioxidant N-acetylcysteine (NAC) was used to confirm oxidative dependency. Microglia-derived glutamate was assessed by stimulating BV2 cells with lipopolysaccharide (LPS) and applying glutamate-containing conditioned medium (LPS-CM) to HT22 cells. Exogenous glutamate robustly increased CREB2 expression in HT22 and primary neurons, accompanied by ROS accumulation and cell death, whereas NAC suppressed these effects. Inhibition of p53 by siRNA or pifithrin-α (PFT-α) attenuated glutamate-induced CREB2 upregulation, and CREB2 knockdown blocked GADD45α induction and protected neurons. In Vivo, KA injection caused robust CREB2 upregulation in the damaged CA3 region. Importantly, conditioned medium from LPS-activated BV2 microglia increased CREB2 expression and ROS levels in HT22 cells in an NAC-sensitive manner, supporting a glutamate-associated oxidative mechanism rather than receptor-mediated excitotoxicity. Collectively, these results suggest that CREB2 functions between upstream p53 signaling and downstream GADD45α activation as a redox-sensitive mediator of oxidative neuronal death, and may represent a potential therapeutic target in neurodegenerative diseases associated with oxidative stress and neuroinflammation.

Objective The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome has been proposed to be a downstream mediator of neuroinflammation in individuals with Parkinson’s disease (PD). However, its involvement across disease stages and related synucleinopathies, such as multiple system atrophy (MSA), remains unclear. We aimed to analyze the peripheral mRNA expression of NLRP3-related genes and cytokines across individuals with isolated REM sleep behavior disorder (iRBD), early-stage PD, late-stage PD, and MSA.Methods Peripheral blood mononuclear cells (PBMCs) were collected from 151 participants: 35 healthy controls (HCs), 31 patients with iRBD, 41 patients with early-stage PD, 21 patients with late-stage PD, and 23 patients with MSA. mRNA expression was measured using quantitative real-time polymerase chain reaction. Statistical comparisons were performed using analysis of variance (ANOVA) or Welch’s ANOVA, and associations with clinical variables were analyzed through stepwise multiple linear regression.Results NLRP3 expression was significantly lower in patients with iRBD (p=0.0263) and patients with early-stage PD (p= 0.0101) than in HCs. NIMA-related kinase 7 (NEK7) expression progressively decreased across the disease spectrum (HCs vs. patients with early-stage PD, p=0.0008; vs. patients with late-stage PD, p<0.0001). In contrast, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 were elevated in patients with PD, especially those in the late stages. Levels of patients with MSA resembled those of HCs but differed from those of patients with PD. Interleukin (IL)-1β and IL-18 levels were not significantly different. In patients with early-stage PD, NLRP3 expression was negatively correlated with disease duration, the Movement Disorder Society-Unified Parkinson’s Disease Rating Scale Part II score, and the cognitive score.Conclusion Our findings challenge the prevailing hypothesis that NLRP3 inflammasome activation directly contributes to PD pathogenesis. Instead, the observed increase in ASC and caspase-1 expression suggests the potential involvement of alternative inflammasome pathways during disease progression.

Serum Neuronal Extracellular vesicles for RT-QuIC assay to detect Pathological α-Synuclein in Synucleinopathies