冨永 薫

ABSTRACT Recent evidence indicates that the TCA cycle metabolite fumarate plays a specific role in modulating signaling pathways in immune cells. We have previously shown that dimethyl fumarate (DMF) reduces glutathione (GSH) activity and causes the accumulation of cellular reactive oxygen species (ROS), thereby compromising effector immune responses and metabolic activities in activated T‐cells. However, the precise mechanism by which DMF modulates T‐cell signaling pathways remains to be elucidated. This study demonstrates that DMF inhibits T‐cell proliferation, independent of T‐cell receptor (TCR) engagement, and this response is fully reversible by replenishing GSH. Immunoblot analysis showed that DMF had different impacts on TCR downstream signaling by decreasing MYC expression while promoting the phosphorylation of Akt and Erk1/2. Cell cycle analysis demonstrated that exposure to DMF led to negative regulation of cell cycle‐related proteins and induced T‐cells into G0/G1 arrest, which was also rescued by antioxidants. Several genes related to GSH synthesis were upregulated at the same time, suggesting that a potential compensatory response may occur to reduce oxidative burst following DMF treatment. Our results suggest that DMF‐mediated oxidative stress alters a range of cell signaling pathways, including MYC, leading to cell cycle arrest and a defective proliferative response of T‐cells during activation.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro.

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

Gliomas with Isocitrate dehydrogenase 1 (IDH1) mutation have alterations in several enzyme activities, resulting in various metabolic changes. The aim of this study was to determine a mechanism for the better prognosis of gliomas with IDH mutation by performing metabolomic analysis. To understand the metabolic state of human gliomas, we analyzed clinical samples obtained from surgical resection of glioma patients (grades II-IV) with or without the IDH1 mutation, and compared the results with U87 glioblastoma cells overexpressing IDH1 or IDH1R132H. In clinical samples of gliomas with IDH1 mutation, levels of D-2-hydroxyglutarate (D-2HG) were increased significantly compared with gliomas without IDH mutation. Gliomas with IDH mutation also showed decreased intermediates in the tricarboxylic acid cycle and pathways involved in the production of energy, amino acids, and nucleic acids. The marked difference in the metabolic profile in IDH mutant clinical glioma samples compared with that of mutant IDH expressing cells includes a decrease in β-oxidation due to acyl-carnitine and carnitine deficiencies. These metabolic changes may explain the lower cell division rate observed in IDH mutant gliomas and may provide a better prognosis in IDH mutant gliomas.

Splicing can be epigenetically regulated and involved in cellular differentiation in somatic cells, but the interplay of epigenetic factors and the splicing machinery during spermatogenesis remains unclear. To study these interactions in vivo, we generated a germline deletion of MORF-related gene on chromosome 15 (MRG15), a multifunctional chromatin organizer that binds to methylated histone H3 lysine 36 (H3K36) in introns of transcriptionally active genes and has been implicated in regulation of histone acetylation, homology-directed DNA repair, and alternative splicing in somatic cells. Conditional KO (cKO) males lacking MRG15 in the germline are sterile secondary to spermatogenic arrest at the round spermatid stage. There were no significant alterations in meiotic division and histone acetylation. Specific mRNA sequences disappeared from 66 germ cell-expressed genes in the absence of MRG15, and specific intronic sequences were retained in mRNAs of 4 genes in the MRG15 cKO testes. In particular, introns were retained in mRNAs encoding the transition proteins that replace histones during sperm chromatin condensation. In round spermatids, MRG15 colocalizes with splicing factors PTBP1 and PTBP2 at H3K36me3 sites between the exons and single intron of transition nuclear protein 2 (Tnp2). Thus, our results reveal that MRG15 is essential for pre-mRNA splicing during spermatogenesis and that epigenetic regulation of pre-mRNA splicing by histone modification could be useful to understand not only spermatogenesis but also, epigenetic disorders underlying male infertile patients.

Cellular senescence is a state of permanent growth arrest and is thought to play a pivotal role in tumor suppression. Cellular senescence may play an important role in tumor suppression, wound healing, and protection against tissue fibrosis in physiological conditions in vivo. However, accumulating evidence that senescent cells may have harmful effects in vivo and may contribute to tissue remodeling, organismal aging, and many age-related diseases also exists. Cellular senescence can be induced by various intrinsic and extrinsic factors. Both p53/p21 and p16/RB pathways are important for irreversible growth arrest in senescent cells. Senescent cells secret numerous biologically active factors. This specific secretion phenotype by senescent cells may largely contribute to physiological and pathological consequences in organisms. Here I review the molecular basis of cell cycle arrest and the specific secretion phenotype in cellular senescence. I also summarize the current knowledge of the role of cellular senescence in vivo in physiological and pathological settings.