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Syntaxin 4 Enrichment in β-Cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice

ABSTRACT: Syntaxin 4 (STX4), a plasma membrane–localized SNARE protein, regulates human islet β-cell insulin secretion and preservation of β-cell mass. We found that human type 1 diabetes (T1D) and NOD mouse islets show reduced β-cell STX4 expression, consistent with decreased STX4 expression, as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible β-cell–specific STX4-expressing NOD mice (NOD-iβSTX4). Of NOD-iβSTX4 mice, 73% had sustained normoglycemia vs. <20% of control NOD (NOD-Ctrl) mice by 25 weeks of age. At 12 weeks of age, before diabetes conversion, NOD-iβSTX4 mice demonstrated superior whole-body glucose tolerance and β-cell glucose responsiveness than NOD-Ctrl mice. Higher β-cell mass and reduced β-cell apoptosis were also detected in NOD-iβSTX4 pancreata compared with pancreata of NOD-Ctrl mice. Single-cell RNA sequencing revealed that islets from NOD-iβSTX4 had markedly reduced interferon-γ signaling and tumor necrosis factor-α signaling via nuclear factor-κB in islet β-cells, including reduced expression of the chemokine CCL5; CD4+ regulatory T cells were also enriched in NOD-iβSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in β-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect β-cell grafts.

ORGANISM(S): Mus musculus

PROVIDER: GSE318972 | GEO | 2026/02/12

REPOSITORIES: GEO

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Project description:Type 1 diabetes (T1D) is characterized by autoimmune destruction of pancreatic β-cells, insulin insufficiency, and hyperglycemia. Receptor interacting protein kinase 1 (RIPK1) is a multifunctional regulator of cell fate with kinase and scaffolding functions. We previously identified RIPKs as regulators of beta-cell cytotoxicity in vitro, and Ripk1 expression is increased in islets from aged non-obese diabetic (NOD) mice and β-cells from T1D donors. We hypothesized that RIPK1 regulates cytokine- and autoimmune-mediated β-cell demise in a mouse model of T1D. Using NIT-1 beta-cells derived from NOD mice, we tested control (CTL) and Ripk1 gene-edited (Ripk1delta, CRISPR/Cas9 KO) beta-cells treated with TNFα+IFNγ or co-cultured with self-reactive splenocytes from diabetic NOD mice. We performed live cell imaging, RNAseq, kinome profiling, and flow cytometry, tested small molecule RIPK1 inhibitors, and evaluated resistance to beta-cell autoimmunity in vivo. We found TNF alpha +IFN gamma increase RIPK1 phosphorylation, caspase 3/7 activity, and cell death in NIT-1 CTL cells. In contrast, cytotoxicity was blocked with small molecule RIPK1 inhibition or in NIT-1 Ripk1delta cells. Co-labeling of caspase 3/7 activation and cell death in single cells revealed protection from caspase-dependent and -independent death in Ripk1Δ cells. RNAseq uncovered differential cell death-, immune-, and identity-related gene expression between genotypes, and kinome profiling identified changes in MAPK, Eph, JAK, and other kinase activity that were associated with protection from cell death. Furthermore, NIT-1 Ripk1delta cells were protected from destruction following co-culture with self-reactive splenocytes in vitro, and NIT-1 implant graft luminescence was 399-fold greater in Ripk1delta versus CTL cells 19 days post splenocyte administration in vivo. Collectively, our findings indicate RIPK1 regulates beta-cell fate in a model of autoimmune diabetes via actions on gene expression and kinase signaling. Additional studies are needed to characterize RIPK1 signaling in primary islets and human beta-cells, and to determine whether RIPK1 can be targeted to protect beta-cells in T1D. In this data set, NIT-1 cells (control and Ripk1 KO), vehicle treated or treated with IFN-gamma and TNF alpha were used for kinome profiling.

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Syntaxin 4 Enrichment in β-Cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice

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