Multi-omics analysis reveals drivers of loss of ß-cell function after newly diagnosed autoimmune type 1 diabetes: An INNODIA multicenter study.

AIMS: Heterogeneity in the rate of ß-cell loss in newly diagnosed type 1 diabetes patients is poorly understood and creates a barrier to designing and interpreting disease-modifying clinical trials. Integrative analyses of baseline multi-omics data obtained after the diagnosis of type 1 diabetes may provide mechanistic insight into the diverse rates of disease progression after type 1 diabetes diagnosis. METHODS: We collected samples in a pan-European consortium that enabled the concerted analysis of five different omics modalities in data from 97 newly diagnosed patients. In this study, we used Multi-Omics Factor Analysis to identify molecular signatures correlating with post-diagnosis decline in ß-cell mass measured as fasting C-peptide. RESULTS: Two molecular signatures were significantly correlated with fasting C-peptide levels. One signature showed a correlation to neutrophil degranulation, cytokine signalling, lymphoid and non-lymphoid cell interactions and G-protein coupled receptor signalling events that were inversely associated with a rapid decline in ß-cell function. The second signature was related to translation and viral infection was inversely associated with change in ß-cell function. In addition, the immunomics data revealed a Natural Killer cell signature associated with rapid ß-cell decline. CONCLUSIONS: Features that differ between individuals with slow and rapid decline in ß-cell mass could be valuable in staging and prediction of the rate of disease progression and thus enable smarter (shorter and smaller) trial designs for disease modifying therapies as well as offering biomarkers of therapeutic effect.

Intra-islet insulin synthesis defects are associated with endoplasmic reticulum stress and loss of beta cell identity in human diabetes.

AIMS/HYPOTHESIS: Endoplasmic reticulum (ER) stress and beta cell dedifferentiation both play leading roles in impaired insulin secretion in overt type 2 diabetes. Whether and how these factors are related in the natural history of the disease remains, however, unclear. METHODS: In this study, we analysed pancreas biopsies from a cohort of metabolically characterised living donors to identify defects in in situ insulin synthesis and intra-islet expression of ER stress and beta cell phenotype markers. RESULTS: We provide evidence that in situ altered insulin processing is closely connected to in vivo worsening of beta cell function. Further, activation of ER stress genes reflects the alteration of insulin processing in situ. Using a combination of 17 different markers, we characterised individual pancreatic islets from normal glucose tolerant, impaired glucose tolerant and type 2 diabetic participants and reconstructed disease progression. CONCLUSIONS/INTERPRETATION: Our study suggests that increased beta cell workload is accompanied by a progressive increase in ER stress with defects in insulin synthesis and loss of beta cell identity.

Angiotensin I-converting enzyme type 2 expression is increased in pancreatic islets of type 2 diabetic donors.

AIMS: Angiotensin I-converting enzyme type 2 (ACE2), a pivotal SARS-CoV-2 receptor, has been shown to be expressed in multiple cells, including human pancreatic beta-cells. A putative bidirectional relationship between SARS-CoV-2 infection and diabetes has been suggested, confirming the hypothesis that viral infection in beta-cells may lead to new-onset diabetes or worse glycometabolic control in diabetic patients. However, whether ACE2 expression levels are altered in beta-cells of diabetic patients has not yet been investigated. Here, we aimed to elucidate the in situ expression pattern of ACE2 in Type 2 diabetes (T2D) with respect to non-diabetic donors which may account for a higher susceptibility to SARS-CoV-2 infection in beta-cells. MATERIAL AND METHODS: Angiotensin I-converting enzyme type 2 immunofluorescence analysis using two antibodies alongside insulin staining was performed on formalin-fixed paraffin embedded pancreatic sections obtained from n = 20 T2D and n = 20 non-diabetic (ND) multiorgan donors. Intensity and colocalisation analyses were performed on a total of 1082 pancreatic islets. Macrophage detection was performed using anti-CD68 immunohistochemistry on serial sections from the same donors. RESULTS: Using two different antibodies, ACE2 expression was confirmed in beta-cells and in pancreas microvasculature. Angiotensin I-converting enzyme type 2 expression was increased in pancreatic islets of T2D donors in comparison to ND controls alongside with a higher colocalisation rate between ACE2 and insulin using both anti-ACE2 antibodies. CD68+ cells tended to be increased in T2D pancreata, in line with higher ACE2 expression observed in serial sections. CONCLUSIONS: Higher ACE2 expression in T2D islets might increase their susceptibility to SARS-CoV-2 infection during COVID-19 in T2D patients, thus worsening glycometabolic outcomes and disease severity.

Identification and Validation of miR-222-3p and miR-409-3p as Plasma Biomarkers in Gestational Diabetes Mellitus Sharing Validated Target Genes Involved in Metabolic Homeostasis.

Gestational diabetes mellitus (GDM) causes both maternal and fetal adverse outcomes. The deregulation of microRNAs (miRNAs) in GDM suggests their involvement in GDM pathogenesis and complications. Exosomes are extracellular vesicles (EVs) of endosomal origin, released via exocytosis into the extracellular compartment. Through EVs, miRNAs are delivered in distant target cells and are able to affect gene expression. In this study, miRNA expression was analyzed to find new miRNAs that could improve GDM classification and molecular characterization. MiRNA were profiled in total plasma and EVs in GDM patients and normal glucose tolerance (NGT) women. Samples were collected at third trimester of gestation from two diabetes centers. MiRNA expression was profiled in a discovery cohort using the multiplexed NanoString nCounter Human v3 miRNA. Validation analysis was performed in a second independent cohort using RT-qPCR. A set of miRNAs resulted to be differentially expressed (DE) in total plasma and EVs in GDM. Among them, total plasma miR-222-3p and miR-409-3p were validated in the independent cohort. MiR-222-3p levels correlated with fasting plasma glucose (FPG) (p < 0.001) and birth weight (p = 0.012), whereas miR-409-3p expression correlated with FPG (p < 0.001) and inversely with gestational age (p = 0.001). The major validated target genes of the deregulated miRNAs were consistently linked to type 2 diabetes and GDM pathophysiology. MiR-222-3p and miR-409-3p are two circulating biomarkers that could improve GDM classification power and act in the context of the molecular events leading to the metabolic alterations observed in GDM.

The Landscape of microRNAs in ßCell: Between Phenotype Maintenance and Protection.

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia mainly due to pancreatic ß cell death and/or dysfunction, caused by several types of stress such as glucotoxicity, lipotoxicity and inflammation. Different patho-physiological mechanisms driving ß cell response to these stresses are tightly regulated by microRNAs (miRNAs), a class of negative regulators of gene expression, involved in pathogenic mechanisms occurring in diabetes and in its complications. In this review, we aim to shed light on the most important miRNAs regulating the maintenance and the robustness of ß cell identity, as well as on those miRNAs involved in the pathogenesis of the two main forms of diabetes mellitus, i.e., type 1 and type 2 diabetes. Additionally, we acknowledge that the understanding of miRNAs-regulated molecular mechanisms is fundamental in order to develop specific and effective strategies based on miRNAs as therapeutic targets, employing innovative molecules.

Non-Coding RNAs: Novel Players in Insulin Resistance and Related Diseases.

The rising prevalence of metabolic diseases related to insulin resistance (IR) have stressed the urgent need of accurate and applicable tools for early diagnosis and treatment. In the last decade, non-coding RNAs (ncRNAs) have gained growing interest because of their potential role in IR modulation. NcRNAs are variable-length transcripts which are not translated into proteins but are involved in gene expression regulation. Thanks to their stability and easy detection in biological fluids, ncRNAs have been investigated as promising diagnostic and therapeutic markers in metabolic diseases, such as type 2 diabetes mellitus (T2D), obesity and non-alcoholic fatty liver disease (NAFLD). Here we review the emerging role of ncRNAs in the development of IR and related diseases such as obesity, T2D and NAFLD, and summarize current evidence concerning their potential clinical application.

Circulating microRNAs Signature for Predicting Response to GLP1-RA Therapy in Type 2 Diabetic Patients: A Pilot Study.

Type 2 diabetes (T2D) represents one of the major health issues of this century. Despite the availability of an increasing number of anti-hyperglycemic drugs, a significant proportion of patients are inadequately controlled, thus highlighting the need for novel biomarkers to guide treatment selection. MicroRNAs (miRNAs) are small non-coding RNAs, proposed as useful diagnostic/prognostic markers. The aim of our study was to identify a miRNA signature occurring in responders to glucagon-like peptide 1 receptor agonists (GLP1-RA) therapy. We investigated the expression profile of eight T2D-associated circulating miRNAs in 26 prospectively evaluated diabetic patients in whom GLP1-RA was added to metformin. As expected, GLP1-RA treatment induced significant reductions of HbA1c and body weight, both after 6 and 12 months of therapy. Of note, baseline expression levels of the selected miRNAs revealed two distinct patient clusters: "high expressing" and "low expressing". Interestingly, a significantly higher percentage of patients in the high expression group reached the glycemic target after 12 months of treatment. Our findings suggest that the evaluation of miRNA expression could be used to predict the likelihood of an early treatment response to GLP1-RA and to select patients in whom to start such treatment, paving the way to a personalized medicine approach.

MicroRNA Expression in the Aqueous Humor of Patients with Diabetic Macular Edema.

We identified and compared secreted microRNA (miRNA) expression in aqueous humor (AH) and plasma samples among patients with: type 2 diabetes mellitus (T2D) complicated by non-proliferative diabetic retinopathy (DR) associated with diabetic macular edema (DME) (DME group: 12 patients); T2D patients without DR (D group: 8 patients); and non-diabetic patients (CTR group: 10 patients). Individual patient AH samples from five subjects in each group were profiled on TaqMan Low Density MicroRNA Array Cards. Differentially expressed miRNAs identified from profiling were then validated in single assay for all subjects. The miRNAs validated in AH were then evaluated in single assay in plasma. Gene Ontology (GO) analysis was conducted. From AH profiling, 119 mature miRNAs were detected: 86 in the DME group, 113 in the D group and 107 in the CTR group. miRNA underexpression in the DME group was confirmed in single assay for let-7c-5p, miR-200b-3p, miR-199a-3p and miR-365-3p. Of these four, miR-199a-3p and miR-365-3p were downregulated also in the plasma of the DME group. GO highlighted 54 validated target genes of miR-199a-3p, miR-200b-3p and miR-365-3p potentially implied in DME pathogenesis. Although more studies are needed, miR-200b-3p, let-7c-5p, miR-365-3p and miR-199a-3p represent interesting molecules in the study of DME pathogenesis.

Targeting microRNAs as a Therapeutic Strategy to Reduce Oxidative Stress in Diabetes.

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia as a consequence of pancreatic ß cell loss and/or dysfunction, also caused by oxidative stress. The molecular mechanisms involved inß cell dysfunction and in response to oxidative stress are also regulated by microRNAs (miRNAs). miRNAs are a class of negative gene regulators, which modulate pathologic mechanisms occurring in diabetes and its complications. Although several pharmacological therapies specifically targeting miRNAs have already been developed and brought to the clinic, most previous miRNA-based drug delivery methods were unable to target a specific miRNA in a single cell type or tissue, leading to important off-target effects. In order to overcome these issues, aptamers and nanoparticles have been described as non-cytotoxic vehicles for miRNA-based drug delivery. These approaches could represent an innovative way to specifically target and modulate miRNAs involved in oxidative stress in diabetes and its complications. Therefore, the aims of this review are: (i) to report the role of miRNAs involved in oxidative stress in diabetes as promising therapeutic targets; (ii) to shed light onto the new delivery strategies developed to modulate the expression of miRNAs in diseases.

MicroRNAs as Regulators of Insulin Signaling: Research Updates and Potential Therapeutic Perspectives in Type 2 Diabetes.

The insulin signaling pathway is composed of a large number of molecules that positively or negatively modulate insulin specific signal transduction following its binding to the cognate receptor. Given the importance of the final effects of insulin signal transduction, it is conceivable that many regulators are needed in order to tightly control the metabolic or proliferative functional outputs. MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively modulate gene expression through their specific binding within the 3'UTR sequence of messenger RNA (mRNA), thus causing mRNA decoy or translational inhibition. In the last decade, miRNAs have been addressed as pivotal cellular rheostats which control many fundamental signaling pathways, including insulin signal transduction. Several studies demonstrated that multiple alterations of miRNAs expression or function are relevant for the development of insulin resistance in type 2 diabetes (T2D); such alterations have been highlighted in multiple insulin target organs including liver, muscles, and adipose tissue. Indirectly, miRNAs have been identified as modulators of inflammation-derived insulin resistance, by controlling/tuning the activity of innate immune cells in insulin target tissues. Here, we review main findings on miRNA functions as modulators of insulin signaling in physiologic- or in T2D insulin resistance- status. Additionally, we report the latest hypotheses of prospective therapies involving miRNAs as potential targets for future drugs in T2D.

MicroRNA Expression Analysis of In Vitro Dedifferentiated Human Pancreatic Islet Cells Reveals the Activation of the Pluripotency-Related MicroRNA Cluster miR-302s.

ß-cell dedifferentiation has been recently suggested as an additional mechanism contributing to type-1 and to type-2 diabetes pathogenesis. Moreover, several studies demonstrated that in vitro culture of native human pancreatic islets derived from non-diabetic donors resulted in the generation of an undifferentiated cell population. Additional evidence from in vitro human ß-cell lineage tracing experiments, demonstrated that dedifferentiated cells derive from ß-cells, thus representing a potential in vitro model of ß-cell dedifferentiation. Here, we report the microRNA expression profiles analysis of in vitro dedifferentiated islet cells in comparison to mature human native pancreatic islets. We identified 13 microRNAs upregulated and 110 downregulated in islet cells upon in vitro dedifferentiation. Interestingly, among upregulated microRNAs, we observed the activation of microRNA miR-302s cluster, previously defined as pluripotency-associated. Bioinformatic analysis indicated that miR-302s are predicted to target several genes involved in the control of ß-cell/epithelial phenotype maintenance; accordingly, such genes were downregulated upon human islet in vitro dedifferentiation. Moreover, we uncovered that cell-cell contacts are needed to maintain low/null expression levels of miR-302. In conclusion, we showed that miR-302 microRNA cluster genes are involved in in vitro dedifferentiation of human pancreatic islet cells and inhibits the expression of multiple genes involved in the maintenance of ß-cell mature phenotype.

Class III phosphoinositide 3-kinase/VPS34 and dynamin are critical for apical endocytic recycling.

Recycling is a limiting step for receptor-mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan-class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP-2, causing surface down-regulation. GFP-(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002-treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin-GFP and dependence of dynamin-GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K-III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.

A set of circulating microRNAs belonging to the 14q32 chromosome locus identifies two subgroups of individuals with recent-onset type 1 diabetes.

Circulating microRNAs (miRNAs) are linked to the onset and progression of type 1 diabetes mellitus (T1DM), thus representing potential disease biomarkers. In this study, we employed a multiplatform sequencing approach to analyze circulating miRNAs in an extended cohort of prospectively evaluated recent-onset T1DM individuals from the INNODIA consortium. Our findings reveal that a set of miRNAs located within T1DM susceptibility chromosomal locus 14q32 distinguishes two subgroups of individuals. To validate our results, we conducted additional analyses on a second cohort of T1DM individuals, confirming the identification of these subgroups, which we have named cluster A and cluster B. Remarkably, cluster B T1DM individuals, who exhibit increased expression of a set of 14q32 miRNAs, show better glycemic control and display a different blood immunomics profile. Our findings suggest that this set of circulating miRNAs can identify two different T1DM subgroups with distinct blood immunomics at baseline and clinical outcomes during follow-up.

TFEB controls syncytiotrophoblast formation and hormone production in placenta.

TFEB, a bHLH-leucine zipper transcription factor belonging to the MiT/TFE family, globally modulates cell metabolism by regulating autophagy and lysosomal functions. Remarkably, loss of TFEB in mice causes embryonic lethality due to severe defects in placentation associated with aberrant vascularization and resulting hypoxia. However, the molecular mechanism underlying this phenotype has remained elusive. By integrating in vivo analyses with multi-omics approaches and functional assays, we have uncovered an unprecedented function for TFEB in promoting the formation of a functional syncytiotrophoblast in the placenta. Our findings demonstrate that constitutive loss of TFEB in knock-out mice is associated with defective formation of the syncytiotrophoblast layer. Indeed, using in vitro models of syncytialization, we demonstrated that TFEB translocates into the nucleus during syncytiotrophoblast formation and binds to the promoters of crucial placental genes, including genes encoding fusogenic proteins (Syncytin-1 and Syncytin-2) and enzymes involved in steroidogenic pathways, such as CYP19A1, the rate-limiting enzyme for the synthesis of 17ß-Estradiol (E2). Conversely, TFEB depletion impairs both syncytial fusion and endocrine properties of syncytiotrophoblast, as demonstrated by a significant decrease in the secretion of placental hormones and E2 production. Notably, restoration of TFEB expression resets syncytiotrophoblast identity. Our findings identify that TFEB controls placental development and function by orchestrating both the transcriptional program underlying trophoblast fusion and the acquisition of endocrine function, which are crucial for the bioenergetic requirements of embryonic development.

A Plasma miR-193b-365 Signature Combined With Age and Glycemic Status Predicts Response to Lactococcus lactis-Based Antigen-Specific Immunotherapy in New-Onset Type 1 Diabetes.

Immunomodulation combined with antigen therapy holds great promise to arrest autoimmune type 1 diabetes, but clinical translation is hampered by a lack of prognostic biomarkers. Low-dose anti-CD3 plus Lactococcus lactis bacteria secreting proinsulin and IL-10 reversed new-onset disease in nonobese diabetic (NOD) mice, yet some mice were resistant to the therapy. Using miRNA profiling, six miRNAs (i.e., miR-34a-5p, miR-125a-3p, miR-193b-3p, miR-328, miR-365-3p, and miR-671-3p) were identified as differentially expressed in plasma of responder versus nonresponder mice before study entry. After validation and stratification in an independent cohort, plasma miR-193b-3p and miR-365-3p, combined with age and glycemic status at study entry, had the best power to predict, with high sensitivity and specificity, poor response to the therapy. These miRNAs were highly abundant in pancreas-infiltrating neutrophils and basophils with a proinflammatory and activated phenotype. Here, a set of miRNAs and disease-associated parameters are presented as a predictive signature for the L. lactis-based immunotherapy outcome in new-onset type 1 diabetes, hence allowing targeted recruitment of trial participants and accelerated trial execution. ARTICLE HIGHLIGHTS: Low-dose anti-CD3 combined with oral gavage of genetically modified Lactococcus lactis bacteria secreting human proinsulin and IL-10 holds great promise to arrest autoimmune type 1 diabetes, but the absence of biomarkers predicting therapeutic success hampers clinical translation. A set of cell-free circulation miRNAs together with age and glycemia at baseline predicts a poor response after L. lactis-based immunotherapy in nonobese mice with new-onset diabetes. Pancreas-infiltrating neutrophils and basophils are identified as potential cellular sources of discovered miRNAs. The prognostic signature could guide targeted recruitment of patients with newly diagnosed type 1 diabetes in clinical trials with the L. lactis-based immunotherapy.

Multi-Omics Integrative Approach of Extracellular Vesicles: A Future Challenging Milestone.

In the era of multi-omic sciences, dogma on singular cause-effect in physio-pathological processes is overcome and system biology approaches have been providing new perspectives to see through. In this context, extracellular vesicles (EVs) are offering a new level of complexity, given their role in cellular communication and their activity as mediators of specific signals to target cells or tissues. Indeed, their heterogeneity in terms of content, function, origin and potentiality contribute to the cross-interaction of almost every molecular process occurring in a complex system. Such features make EVs proper biological systems being, therefore, optimal targets of omic sciences. Currently, most studies focus on dissecting EVs content in order to either characterize it or to explore its role in various pathogenic processes at transcriptomic, proteomic, metabolomic, lipidomic and genomic levels. Despite valuable results being provided by individual omic studies, the categorization of EVs biological data might represent a limit to be overcome. For this reason, a multi-omic integrative approach might contribute to explore EVs function, their tissue-specific origin and their potentiality. This review summarizes the state-of-the-art of EVs omic studies, addressing recent research on the integration of EVs multi-level biological data and challenging developments in EVs origin.

Circulating microRNAs as clinically useful biomarkers for Type 2 Diabetes Mellitus: miRNomics from bench to bedside.

Type 2 diabetes (T2D), a chronic metabolic disease, has attained the status of a global epidemic with steadily increasing incidence worldwide. Improved diagnosis, stratification and prognosis of T2D patients and the development of more effective treatments are needed. In this era of personalized medicine, the discovery and evaluation of innovative circulating biomarkers can be an effective tool for better stratification, prognosis and therapeutic selection/management of T2D patients. MicroRNAs (miRNAs), a class of small non-coding RNAs that modulate gene expression, have been investigated as potential circulating biomarkers in T2D. Several studies have investigated the expression of circulating miRNAs in T2D patients from various biological fluids, including plasma and serum, and have demonstrated their potential as diagnostic and prognostic biomarkers, as well as biomarkers of response to therapy. In this review, we provide an overview of the current state of knowledge, focusing on circulating miRNAs that have been consistently expressed in at least two independent studies, in order to identify a set of consistent biomarker candidates in T2D. The expression levels of miRNAs, correlation with clinical parameters, functional roles of miRNAs and their potential as biomarkers are reported. A systematic literature search and assessment of studies led to the selection and review of 10 miRNAs (miR-126-3p, miR-223-3p, miR-21-5p, miR-15a-5p, miR-24-3p, miR-34a-5p, miR-146a-5p, miR-148a-3p, miR-30d-5p and miR-30c-5p). We also present technical challenges and our thoughts on the potential validation of circulating miRNAs and their application as biomarkers in the context of T2D.

Increased Expression of Viral Sensor MDA5 in Pancreatic Islets and in Hormone-Negative Endocrine Cells in Recent Onset Type 1 Diabetic Donors.

The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic ß-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as melanoma differentiation associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of nondiabetic and T1D donors is still missing. In the present study, we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 nondiabetic autoantibody-negative, 2 nondiabetic autoantibody-positive, 8 recent-onset, and 2 long-standing T1D). In nondiabetic control donors, MDA5 was expressed both in α- and ß-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in α-cells. In T1D donors, we observed an increased colocalization rate of MDA5-glucagon with respect to MDA5-insulin in comparison to nondiabetic controls; such increase was more pronounced in recent-onset with respect to long-standing T1D donors. Of note, an increased colocalization rate of MDA5-glucagon was found in insulin-deficient-islets (IDIs) with respect to insulin-containing-islets (ICIs). Strikingly, we detected the presence of MDA5-positive/hormone-negative endocrine islet-like clusters in T1D donors, presumably due to dedifferentiation or neogenesis phenomena. These clusters were identified exclusively in donors with recent disease onset and not in autoantibody-positive nondiabetic donors or donors with long-standing T1D. In conclusion, we showed that MDA5 is preferentially expressed in α-cells, and its expression is increased in recent-onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.

Reduced miR-184-3p expression protects pancreatic ß-cells from lipotoxic and proinflammatory apoptosis in type 2 diabetes via CRTC1 upregulation.

The loss of functional ß-cell mass in type 2 diabetes (T2D) is associated with molecular events that include ß-cell apoptosis, dysfunction and/or dedifferentiation. MicroRNA miR-184-3p has been shown to be involved in several ß-cell functions, including insulin secretion, proliferation and survival. However, the downstream targets and upstream regulators of miR-184-3p have not been fully elucidated. Here, we show reduced miR-184-3p levels in human T2D pancreatic islets, whereas its direct target CREB regulated transcription coactivator 1 (CRTC1) was increased and protects ß-cells from lipotoxicity- and inflammation-induced apoptosis. Downregulation of miR-184-3p in ß-cells leads to upregulation of CRTC1 at both the mRNA and protein levels. Remarkably, the protective effect of miR-184-3p is dependent on CRTC1, as its silencing in human ß-cells abrogates the protective mechanism mediated by inhibition of miR-184-3p. Furthermore, in accordance with miR-184-3p downregulation, we also found that the ß-cell-specific transcription factor NKX6.1, DNA-binding sites of which are predicted in the promoter sequence of human and mouse MIR184 gene, is reduced in human pancreatic T2D islets. Using chromatin immunoprecipitation analysis and mRNA silencing experiments, we demonstrated that NKX6.1 directly controls both human and murine miR-184 expression. In summary, we provide evidence that the decrease in NKX6.1 expression is accompanied by a significant reduction in miR-184-3p expression and that reduction of miR-184-3p protects ß-cells from apoptosis through a CRTC1-dependent mechanism.

TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1, and Batten disease.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.