Glutathione (GSH) is a crucial redox scavenger, essential for maintaining cellular redox balance. This study explores the long-term effects of chronic GSH deficiency on lifespan, motor function, cognitive performance, redox status and inflammation. GCLM-/- mice, with a 70-90% reduction in GSH levels, were compared to GCLM+/+ controls across their lifespan (5, 10 and 20 months). We assessed lifespan, motor performance using balance and coordination tests, cognitive function through anxiety and memory tests, redox markers, and inflammation markers, particularly TNF-α and IL-6. Biochemical analyses of GSH levels in peripheral tissues and brain regions were conducted to evaluate redox state changes. GCLM-/- mice displayed extended lifespans and improved motor function at young and adult stages, with a delayed onset of motor decline with age. Cognitive function remains largely unaffected, although there are reductions in anxiety-related behaviors and minor deficits in fear-associated memory. Age-related increases in TNF-α, an inflammatory marker, are observed in both genotypes, with GCLM-/- mice showing a less pronounced increase, particularly in females. There were significant GSH reductions in peripheral tissues, with sporadic changes in brain regions. This stress likely triggers compensatory antioxidant responses, modulating inflammation and redox-sensitive pathways. The data suggests that lifelong GSH deficiency provides protective effects against inflammation and motor decline in younger animals but exacerbates these issues in older mice. The study offers insights into potential therapeutic strategies that leverage mild oxidative stress to promote healthy aging, emphasizing the importance of redox state and antioxidant defenses in the aging process.

Glutamate-cysteine ligase catalytic subunit (GCLC), previously known as gamma-glutamyl-cysteine synthetase, is an essential rate-limiting step in glutathione synthesis. Glutathione modulates multitudes of critical cellular processes and scavenges free radicals. Its deficiency is reported to cause hemolysis of variable severity and is a rare cause of neurological abnormalities such as spinocerebellar ataxia. We report a 55-year-old female patient with progressive late-onset ataxia, lower limb spasticity, and chronic hemolytic anemia found to have a GCLC pathogenic variant and low glutathione level. Magnetic resonance imaging of the head and cervical spine showed global cerebellar atrophy with widened folia and decreased diameter of the upper cervical spine. Blood workup revealed hemolytic anemia with genetic testing confirmed a homozygous variant, c.514 T>A in exon 4 of the GCLC gene, resulting in Ser172Thr (TCC>ACC). Management encompassed a multidisciplinary approach with a trial of high-dose alpha-lipoic acid, glutathione supplement, and physical therapy. GCLC deficiency manifesting with hemolysis has been reported in 12 cases worldwide from 6 independent families, with only 4 cases having additional neurological manifestations. To date, no specific GCLC gene mutation has been attributed to the reported neurological constellation of symptoms. To the best of our knowledge, this is the first case report of late-onset spinocerebellar degeneration as a manifestation of c.514T>A (p. S172T) GCLC pathological variant genetic mutation.

Oxidative stress is intimately involved in the pathogenesis of fatty liver disease (FLD). A major factor contributing to oxidative stress is the depletion of the ubiquitous antioxidant glutathione (GSH). Unexpectedly, chronic GSH deficiency renders glutamate-cysteine ligase modifier subunit (Gclm)-null mice protected from fatty liver injuries. Epigenetic regulation serves as an important cellular mechanism in modulating gene expression and disease outcome in FLD, although it is not well understood how systemic redox imbalance modifies the liver epigenome. In the current study, utilizing the Gclm-null mouse model, we aimed to elucidate redox-associated epigenomic changes and their implications in liver stress response. We performed high-throughput array-based DNA methylation profiling (MeDIP array) in 22,327 gene promoter regions (from -1300 bp to +500 bp of the Transcription Start Sites) in the liver and peripheral blood cells. Results from the MeDIP array demonstrate that, although global methylation enrichment in gene promoters did not change, low GSH resulted in prevalent demethylation at the individual promoter level. Such an effect likely attributed to a declined availability of the methyl donor S-adenosyl methionine (SAM) in Gclm-null liver. Functional enrichment analysis of liver target genes is suggestive of a potential role of epigenetic mechanisms in promoting cellular survival and lipid homeostasis in Gclm-null liver. In comparison with the liver tissue, MeDIP array in peripheral blood cells revealed a panel of 19 gene promoters that are candidate circulating biomarkers for hepatic epigenomic changes associated with chronic GSH deficiency. Collectively, our results provided new insights into the in vivo interplay between liver redox state and DNA methylation status. The current study laid the groundwork for future epigenetic/epigenomic investigations in experimental settings or human populations under conditions of liver oxidative stress induced by environmental or dietary challenges.

Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1-aged Gclcf/f;Le-CreTg/- (KO) and Gclcf/f;Le-Cre-/- control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclcw/w;Le-CreTg/- (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-l-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), α-SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.

Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are poorly characterized. Here, we find that cystine starvation of non-small-cell lung cancer cell lines induces an unexpected accumulation of γ-glutamyl-peptides, which are produced due to a non-canonical activity of glutamate-cysteine ligase catalytic subunit (GCLC). This activity is enriched in cell lines with high levels of NRF2, a key transcriptional regulator of GCLC, but is also inducible in healthy murine tissues following cysteine limitation. γ-glutamyl-peptide synthesis limits the accumulation of glutamate, thereby protecting against ferroptosis. These results indicate that GCLC has a glutathione-independent, non-canonical role in the protection against ferroptosis by maintaining glutamate homeostasis under cystine starvation.