Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

Nova1 is a master regulator of alternative splicing in pancreatic beta cells.

Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the 'neuron-specific' Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1.

miR-296-3p, miR-298-5p and their downstream networks are causally involved in the higher resistance of mammalian pancreatic α cells to cytokine-induced apoptosis as compared to ß cells.

BACKGROUND: The molecular bases of mammalian pancreatic α cells higher resistance than ß to proinflammatory cytokines are very poorly defined. MicroRNAs are master regulators of cell networks, but only scanty data are available on their transcriptome in these cells and its alterations in diabetes mellitus. RESULTS: Through high-throughput real-time PCR, we analyzed the steady state microRNA transcriptome of murine pancreatic α (αTC1-6) and ß (ßTC1) cells: their comparison demonstrated significant differences. We also characterized the alterations of αTC1-6 cells microRNA transcriptome after treatment with proinflammatory cytokines. We focused our study on two microRNAs, miR-296-3p and miR-298-5p, which were: (1) specifically expressed at steady state in αTC1-6, but not in ßTC1 or INS-1 cells; (2) significantly downregulated in αTC1-6 cells after treatment with cytokines in comparison to untreated controls. These microRNAs share more targets than expected by chance and were co-expressed in αTC1-6 during a 6-48 h time course treatment with cytokines. The genes encoding them are physically clustered in the murine and human genome. By exploiting specific microRNA mimics, we demonstrated that experimental upregulation of miR-296-3p and miR-298-5p raised the propensity to apoptosis of transfected and cytokine-treated αTC1-6 cells with respect to αTC1-6 cells, treated with cytokines after transfection with scramble molecules. Both microRNAs control the expression of IGF1Rß, its downstream targets phospho-IRS-1 and phospho-ERK, and TNFα. Our computational analysis suggests that MAFB (a transcription factor exclusively expressed in pancreatic α cells within adult rodent islets of Langerhans) controls the expression of miR-296-3p and miR-298-5p. CONCLUSIONS: Altogether, high-throughput microRNA profiling, functional analysis with synthetic mimics and molecular characterization of modulated pathways strongly suggest that specific downregulation of miR-296-3p and miR-298-5p, coupled to upregulation of their targets as IGF1Rß and TNFα, is a major determinant of mammalian pancreatic α cells resistance to apoptosis induction by cytokines.

Chronic exposure to GLP-1 increases GLP-1 synthesis and release in a pancreatic alpha cell line (α-TC1): evidence of a direct effect of GLP-1 on pancreatic alpha cells.

AIMS/HYPOTHESIS: Incretin therapies, which are used to treat diabetic patients, cause a chronic supra-physiological increase in GLP-1 circulating levels. It is still unclear how the resulting high hormone concentrations may affect pancreatic alpha cells. The present study was designed to investigate the effects of chronic exposure to high GLP-1 levels on a cultured pancreatic alpha cell line. METHODS: α-TC1-6 cell line was cultured in the presence or absence of GLP-1 (100 nmol/l) for up to 72 h. In our model GLP-1 receptor (GLP-1R) was measured. After the cells were exposed to GLP-1 the levels of glucagon secretion were measured. Because GLP-1 acts on intracellular cAMP production, the function of GLP-1R was studied. We also investigated the effects of chronic GLP-1 exposure on the cAMP/MAPK pathway, Pax6 levels, the expression of prohormone convertases (PCs), glucagon gene (Gcg) and protein expression, glucagon and GLP-1 production. RESULTS: In our model, we were able to detect GLP-1R. After GLP-1 exposure we found a reduction in glucagon secretion. During further investigation of the function of GLP-1R, we found an activation of the cAMP/MAPK/Pax6 pathway and an increase of Gcg gene and protein expression. Furthermore we observed a significant increase in PC1/3 protein expression, GLP-1 intracellular content and GLP-1 secretion. CONCLUSIONS/INTERPRETATION: Our data indicate that the chronic exposure of pancreatic alpha cells to GLP-1 increases the ability of these cells to produce and release GLP-1. This phenomenon occurs through the stimulation of the transcription factor Pax6 and the increased expression of the protein convertase PC1/3.

Palmitate affects insulin receptor phosphorylation and intracellular insulin signal in a pancreatic alpha-cell line.

This study investigated in a pancreatic alpha-cell line the effects of chronic exposure to palmitate on the insulin and IGF-I receptor (IGF-IR) and intracellular insulin pathways. alpha-TC1-6 cells were cultured in the presence or absence of palmitate (0.5 mmol/liter) up to 48 h. Glucagon secretion, insulin and IGF-IR autophosphorylation, and insulin receptor substrate (IRS)-1, IRS-2, phosphatidylinositol kinase (PI3K) (p85 alpha), and serine-threonine protein kinase (Akt) phosphorylated (active) forms were measured. Erk 44/42 and p38 phosphorylation (P) (MAPK pathway markers) were also measured. Because MAPK can regulate Pax6, a transcription factor that controls glucagon expression, paired box gene 6 (Pax6) and glucagon gene and protein expression were also measured. Basal glucagon secretion was increased and the inhibitory effect of acute insulin exposure reduced in alpha-TC1 cells cultured with palmitate. Insulin-stimulated insulin receptor phosphorylation was greatly reduced by exposure to palmitate. Similar results were observed with IRS-1-P, PI3K (p85 alpha), and Akt-P. In contrast, with IGF-IR and IRS-2-P, the basal levels (i.e. in the absence of insulin stimulation) were higher in cells cultured with palmitate. Similar data were obtained with Erk 44/42-P and p-38-P. Pax6 and glucagon gene and protein expression were higher in cells cultured with palmitate. In these cells cultured, specifics MAPKs inhibitors were able to reduce both Pax6 and glucagon gene and protein expression. These results indicate that alpha-cells exposed to palmitate show insulin resistance of the IRS-1/PI3K/Akt pathway that likely controls glucagon secretion. In contrast, the IRS-2/MAPKs pathway is stimulated, through an activation of the IGF-IR, leading to increased Pax6 and glucagon expression. Our data support the hypothesis that the chronic elevation of fatty acids contribute to alpha-cell dysregulation frequently observed in type 2 diabetes.