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In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro .Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo ,beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

Centronuclear myopathies (CNMs) are a group of rare and early-onset genetic muscle disorders for which there is no curative treatment. This fatal disease is known to be caused by mutations in several genes, including myotubularin (MTM1) and amphiphysin 2 (BIN1). Mutations in the MTM1 gene cause X-linked myotubular myopathy (XLCNM), which is the most common and severe form of CNM. In addition, mutations in the BIN1 gene have been associated with autosomal dominant and recessive CNM, a late-onset myopathy that is usually less severe. Several defects at the cellular level have been observed in CNM, such as abnormal triad structure and impaired excitation-contraction coupling, which is critical for skeletal muscle contraction.In our laboratory, we generated mtm1 zebrafish knockout mutants (KO) using CRISPR/Cas9 technology. These mtm1KO mutant show progressive degeneration of fin folds, defective motor behavior, and early mortality. However, no defects in muscular ultrastructure were observed. Recent studies have shown that mutant mRNA can trigger genetic compensation. To avoid this compensation and any active transcription of the mutant mRNA, we generated mtm1 mutant that has a large deletion encompassing the entire promoter of the mtm1 gene. Surprisingly, this promoterless (PL) mutant shows a similar phenotype to the KO mutants, including the lack of a muscle phenotype. This result suggests that no compensation was induced in the KO mutant. We also generated a bin1b mutant lacking a promoter (bin1bPL), in which we found no differences from WT larvae and adult fish. These two mutants challenge the roles of mtm1 and bin1b in zebrafish muscle. Finally, we generated an mtm1-bin1b mutant lacking a double promoter (DPL). This zebrafish mutant not only shows the same features as the mtm1PL mutants, but also exhibits severe muscle defects at both the structural and functional levels that recapitulate human CNM.In summary, we have generated a zebrafish mutant that exhibits a st, Les myopathies centronucléaires (CNM) définissent un groupe de maladies musculaires génétiques rares et à apparition précoce pour lesquelles aucun traitement curatif n'est disponible actuellement. Des mutations dans plusieurs gènes, notamment la myotubularine (MTM1) et l'amphiphysine 2 (BIN1) causent ces maladies létales. Les mutations du gène MTM1 sont à l'origine de la myopathie myotubulaire liée à l'X (XLCNM), qui est la forme la plus fréquente et la plus grave de CNM. D’autre part, des mutations dans le gène BIN1 ont été associées à des CNM autosomiques dominantes et récessives qui sont généralement moins graves et se manifestent plus tardivement chez les patients. Dans la CNM, plusieurs défauts ont été observés au niveau cellulaire, comme une structure anormale des triades et un dysfonctionnement du couplage excitation-contraction, crucial pour la contraction des muscles squelettiques.Dans notre laboratoire, nous avons généré un mutant mtm1 knockout (KO) de poisson zèbre en utilisant la technologie CRISPR/Cas9. Ce mutant mtm1KO présente une dégénérescence progressive des nageoires, une diminution du comportement moteur et une mortalité précoce. Cependant, aucun défaut dans l'ultrastructure musculaire n'a été observé chez ce mutant, contrairement à ce qui était attendu. Des études récentes ont montré, dans différents modèles, que l’expression d’ARNm mutés peut déclencher une compensation génétique et une réduction de la gravité ou une absence du phénotype attendu. Pour éviter cette compensation et toute transcription d’ARNm muté, nous avons généré des mutants mtm1 portant de larges délétions englobant la totalité du promoteur du gène mtm1. De manière surprenante, ce mutant sans promoteur (« promoterless mutant », PL) présente un phénotype similaire au mutant KO, y compris par l'absence de phénotype musculaire. Ce résultat suggère qu'aucune compensation n'a été déclenchée chez le mutant KO. N, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

DNAJC3, also known as P58IPK, is an Hsp40 family member that interacts with and inhibits PKR-like ER-localized eIF2α kinase (PERK). Dnajc3 deficiency in mice causes pancreatic β-cell loss and diabetes. Loss-of-function mutations in DNAJC3 cause early-onset diabetes and multisystemic neurodegeneration. The aim of our study was to investigate the genetic cause of early-onset syndromic diabetes in two unrelated patients, and elucidate the mechanisms of β-cell failure in this syndrome., info:eu-repo/semantics/published

Deficient as well as excessive/prolonged endoplasmic reticulum (ER) stress signaling can lead to pancreatic β cell failure and the development of diabetes. Saturated free fatty acids (FFAs) such as palmitate induce lipotoxic ER stress in pancreatic β cells. One of the main ER stress response pathways is under the control of the protein kinase R-like endoplasmic reticulum kinase (PERK), leading to phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α). The antihypertensive drug guanabenz has been shown to inhibit eIF2α dephosphorylation and protect cells from ER stress. Here we examined whether guanabenz protects pancreatic β cells from lipotoxicity. Guanabenz induced β cell dysfunction in vitro and in vivo in rodents and led to impaired glucose tolerance. The drug significantly potentiated FFA-induced cell death in clonal rat β cells and in rat and human islets. Guanabenz enhanced FFA-induced eIF2α phosphorylation and expression of the downstream proapoptotic gene C/EBP homologous protein (CHOP), which mediated the sensitization to lipotoxicity. Thus, guanabenz does not protect β cells from ER stress; instead, it potentiates lipotoxic ER stress through PERK/eIF2α/CHOP signaling. These data demonstrate the crucial importance of the tight regulation of eIF2α phosphorylation for the normal function and survival of pancreatic β cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Pancreatic β-cell lipotoxicity is a central feature of the pathogenesis of type 2 diabetes. To study the mechanism by which fatty acids cause β-cell death and develop novel approaches to prevent it, a high-throughput screen on the β-cell line INS1 was carried out. The cells were exposed to palmitate to induce cell death and compounds that reversed palmitate-induced cytotoxicity were ascertained. Hits from the screen were analyzed by an increasingly more stringent testing funnel, ending with studies on primary human islets treated with palmitate. MAP4K4 inhibitors, which were not part of the screening libraries but were ascertained by a bioinformatics analysis, and the endocannabinoid anandamide were effective at inhibiting palmitate-induced apoptosis in INS1 cells as well as primary rat and human islets. These targets could serve as the starting point for the development of therapeutics for type 2 diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, andhumanβ-cells exposed to cytokines or thapsigargininduced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure ofβ-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

SCOPUS: ar.j, info:eu-repo/semantics/published