Anti-SLC25A4 monoclonal antibody Made to order

Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a36-deficient colonies under trophoblast stem cell culture conditions. As a result of Slc25a36 deficiency, mtDNA of knockdown cells declined, leading to impaired mitochondria with swollen morphology, decreased mitochondrial membrane potential, and low numbers. The key transcription regulators of mitochondrial biogenesis also decreased. These results indicate that mitochondrial dysfunction leads to an inability to support the pluripotency maintenance. Moreover, down-regulated glutathione metabolism and up-regulated focal adhesion reinforced and stabilized the process of differentiation by separately enhancing OCT4 degradation and promoting cell spread. This study improves the understanding of the function of Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.

The solute carrier family 25 (SLC25) drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier SLC25A22 (also named GC1) have been identified in early epileptic encephalopathy (EEE) and migrating partial seizures in infancy (MPSI) but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an in vivo model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA) constructs using the Neon (R) Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing GC1 in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate) (NAD(P) H) formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC) was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP) in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes. Main Points: The mitochondrial respiratory chain is functional in absence of GC1 Lack of glutamate oxidation results in a lower global ATP level Lack of mitochondrial glutamate transport results in intracellular glutamate accumulation