Impaired GSH biosynthesis disrupts eye development and PAX6 function

Glutathione (GSH) is a critical endogenous antioxidant that protects against intracellular oxidative stress. As such, pathological alterations in GSH levels are linked to a myriad of diseases including cancer, neurodegeneration and cataract. The rate limiting step in GSH biosynthesis is catalyzed by the glutamate cysteine ligase catalytic subunit (GCLC). The high expression of GCLC in the lens supports the synthesis of millimolar concentrations of GSH in this tissue. Herein, we describe the morphological consequences of deleting (knocking out) Gclc from surface ectoderm-derived ocular tissues (using the Le-Cre transgene; Gclc KO) which includes an overt microphthalmia phenotype and severely disrupted formation of multiple ocular structures (i.e., cornea, iris, lens, retina). Controlling for the Le-Cre transgene revealed that the deletion of Gclc significantly exacerbated the microphthalmia phenotype in Le-Cre hemizygous mice and resulted in dysregulated gene expression that was unique to only the lenses of KO mice. We further characterized the impaired lens development by conducting an RNA-seq experiment on KO and Gclc control (CON) mouse lens at the day of birth. RNA-sequencing revealed significant differences between Gclc knockout (KO) and Gclc control (CON) lenses, including down-regulation of crystallins and lens fiber cell identity genes, and up-regulation of lens epithelial cell identity genes. In addition, genes related to the immune system (e.g., immune system process, inflammatory response, neutrophil chemotaxis) were upregulated, and genes related to eye/lens development were downregulated. TRANSFAC analysis of differentially expressed genes (DEGs) in the lens of Gclc KO mice implicated PAX6 as a key upstream regulator of Gclc KO sensitive genes. This was further supported by a strong positive correlation between the transcriptomes of the lenses of Gclc KO and Pax6 KO mice. Strikingly, the dysregulation of PAX6-regulated genes in Gclc KO mice was observed despite no change in the ocular localization of PAX6 or decrease in the expression of PAX6 in the lens. In vitro experiments demonstrated that suppression of intracellular GSH concentrations resulted in impairment of PAX6 transactivation activity. Taken together, the present results elucidate a novel mechanism wherein intracellular GSH concentrations may modulate PAX6 activity. Examination of gene expression in the lenses of Gclc KO and CON mice aged P1.谷胱甘肽（Glutathione, GSH）是一类关键的内源性抗氧化剂，可抵御细胞内氧化应激。因此，GSH水平的病理改变与多种疾病密切相关，包括癌症、神经退行性疾病及白内障。GSH生物合成的限速步骤由谷氨酸-半胱氨酸连接酶催化亚基（glutamate cysteine ligase catalytic subunit, GCLC）催化。GCLC在晶状体中的高表达，保障了该组织中毫摩尔级浓度GSH的合成。 本研究阐明了利用Le-Cre转基因（Le-Cre transgene）在表面外胚层起源的眼组织中敲除Gclc（Gclc KO）所带来的形态学影响，该操作可导致显著的小眼表型，并严重扰乱角膜、虹膜、晶状体、视网膜等多种眼结构的发育。 通过对Le-Cre转基因进行对照分析发现，在Le-Cre半合子小鼠中敲除Gclc，可显著加重小眼表型，并引发仅存在于敲除小鼠晶状体中的特异性基因表达失调。 我们进一步通过在出生当天对Gclc敲除（KO）及Gclc对照（CON）小鼠的晶状体开展RNA测序（RNA-seq）实验，解析了受损的晶状体发育过程。RNA测序结果显示，Gclc敲除晶状体与对照晶状体间存在显著差异：包括晶状体蛋白及晶状体纤维细胞特征基因的下调，以及晶状体上皮细胞特征基因的上调。此外，与免疫系统相关的基因（如免疫过程、炎症反应、中性粒细胞趋化相关基因）表达上调，而与眼/晶状体发育相关的基因表达则出现下调。 对Gclc敲除小鼠晶状体中差异表达基因（differentially expressed genes, DEGs）的TRANSFAC分析显示，PAX6是Gclc敲除敏感基因的关键上游调控因子。这一结论进一步得到了Gclc敲除小鼠与Pax6敲除小鼠晶状体转录组间强正相关关系的支持。 值得注意的是，尽管PAX6在眼组织中的定位未发生改变，且晶状体中PAX6的表达水平未出现下降，但Gclc敲除小鼠中PAX6调控基因的表达失调现象依然出现。体外实验证实，细胞内GSH浓度的抑制会损害PAX6的反式激活活性。 综上，本研究结果揭示了一种全新的机制：细胞内GSH浓度可调控PAX6的活性。本研究还对出生后第1天（P1）的Gclc敲除与对照小鼠的晶状体基因表达进行了检测。