Abstract
The levels of purines, essential molecules to sustain eukaryotic cell homeostasis, are regulated by the coordination of the de novo and salvage synthesis pathways. In the embryonic central nervous system (CNS), the de novo pathway is considered crucial to meet the requirements for the active proliferation of neural stem/progenitor cells (NSPCs). However, how these two pathways are balanced or separately utilized during CNS development remains poorly understood. In this study, we showed a dynamic shift in pathway utilization, with greater reliance on the de novo pathway during embryonic stages and on the salvage pathway at postnatal–adult stages. The pharmacological effects of various purine synthesis inhibitors in vitro and the expression profile of purine synthesis enzymes indicated that NSPCs in the embryonic cerebrum mainly utilize the de novo pathway. Simultaneously, NSPCs in the cerebellum require both the de novo and the salvage pathways. In vivo administration of de novo inhibitors resulted in severe hypoplasia of the forebrain cortical region, indicating a gradient of purine demand along the anteroposterior axis of the embryonic brain, with cortical areas of the dorsal forebrain having higher purine requirements than ventral or posterior areas such as the striatum and thalamus. This histological defect of the neocortex was accompanied by strong downregulation of the mechanistic target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase (S6K)/S6 signaling cascade, a crucial pathway for cell metabolism, growth, and survival. These findings indicate the importance of the spatiotemporal regulation of both purine pathways for mTORC1 signaling and proper brain development.
Significance Statement Brain development requires a balance of de novo and salvage purine synthetic pathways. However, the utilization of these pathways during brain development remains poorly understood. This study provides evidence that the spatiotemporal regulation of these two purine synthesis pathways is essential for normal brain development. We revealed that inhibition of de novo purine synthesis results in the downregulation of mammalian/mechanistic target of rapamycin (mTOR) signaling, leading to malformations in specific embryonic brain regions such as the cerebral neocortex. These results suggest a temporal and spatial gradient of purine demand during embryonic brain development. These findings could improve our understanding of neurological diseases caused by defects in purine metabolism.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Funding This work was funded by the Japan Society for the Promotion of Science grants-in-aid (KAKENHI) grant numbers 21K20701 (to S.Y.) and 19K06931 (to S.S.), Gout and Uric Acid Research Foundation 2020 (to S. S) and 2022 (to S.Y.), and Waseda University Grants for Special Research Projects 2021C-611 (to S.Y.)
The revised manuscript contains new data from several experiments, including western blotting of enzymes in specific brain regions during development, immunostaining of primary cultured apoptotic NSPCs and astrocytes, and rescue experiments following treatment with an mTOR activator in vivo and in vitro.
