The percentage of reduction of beta cell mass in patients with Type 1 diabetes (T1D) of recent onset cannot fully explain the reduction of endogenous insulin production that leads to hyperglycemia. In addition, functional defects of the beta cells may prevail from reduction of beta cell mass during Type 2 diabetes. In boty situation chronic inflammation may explain why beta cells cannot produce sufficient amounts of insulin to cope with insulin demands. The purpose of the project is to examine in situ both the mitochondria and RE-mitochondria interactions, key organelles of beta cells. Apart from being involved in the capacities of beta cells to secrete insulin upon demand, mitochondria have a central role in beta cell fate through ROS generation, capacities to transfer apoptotic signals and in the orchestration of inflammatory responses and inflammasome activation. The levels of ER-mitochondria overlap and number of ER-mitochondrial associated membranes (MAM) will be quantified using an in situ proximity ligation assay. We plan to compare the degree of RE-mitochondria interactions in inflamed vs intact islets, in beta vs non beta cells, in patients with T1D vs T2D. Expression of specific mitochondrial proteins involved in Ca2+ uptake and in the balance of mitochondria fission and fusion processes will be analyzed. Since excessive production of ROS and release of oxidized mitochondrial DNA following mitochondrial stress can be sensed by the inflammasome, levels of NLRP3 expression will be assessed both in mononuclear cells contained in inflamed islets and eventually in the beta cells. This could be an important link between innate immunity in response to viral infections and autoimmunity to beta cells. Since miRNAs can also regulate mitochondria activity, redox state and inflammatory pathways, semi-quantitative analysis of specific miRNAs will be performed by fluorescent micro-RNA in situ hybridization (FISH) technique with digoxigenin-labeled probes. Altogether, this project will give a comprehensive analysis on the role of mitochondria in the beta cell defects during the early phases of diabetes.