Necdin Protects Embryonic Motoneurons from Programmed Cell Death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons

Caractérisation de la plasticité épigénétique du gène Necdin/NECDIN impliqué dans le syndrome de Prader-Willi et de ses conséquences fonctionnelles sur le phenotype

Le syndrome de Prader-Willi est une maladie génétique rare. Les gènes candidats au SPW, dont le gène Necdin, sont régulés par l'empreinte génomique parentale : seul l'allèle paternel de ces gènes est exprimé, l'allèle maternel étant silencieux. Notre équipe a généré un modèle murin pour lequel l'allèle paternel de Necdin a été désactivé (+m/-p) et qui présente des similarités phénotypiques avec les patients PW. Ce phénotype est plus drastique chez les animaux -/-. Nous avons alors émis l'hypothèse que l'allèle maternel puisse avoir un rôle fonctionnel dans la survie des souris (+m/-p). L'expression de l'allèle maternel de Necdin est présente dans le système nerveux des souris (+m/-p). Cette expression, bien que faible au niveau transcriptionnel, est suffisante pour produire la protéine Necdin, ce qui a des conséquences cellulaires et physiologiques qui in fine permettent une amélioration du phénotype. Cette perte de silence de l'allèle maternel est également détectée dans l'hypothalamus de patients PW. Ces résultats révèlent une plasticité épigénétique inattendue qui permet d'envisager des perspectives thérapeutiques.The Prader-Willi Syndrome (PWS) is a rare genetic disorder. Several genes, including NECDIN gene, are involved in the PWS. These genes are regulated by the genomic imprinting mechanism: only the paternal allele of these genes is expressed, their maternal allele being silenced. Our team has generated a mouse model in which the paternal allele of the Necdin gene has been deactivated (+m/-p). This model presents phenotypical similarities with PWS patients. We observed that mortality affects more -/- pups than +m/-p mice. Therefore we venture the hypothesis of a functional role of the maternal allele in mutant mice survival. We showed an expression of this allele in the nervous system of +m/-p mice. Though transcriptionnally low, that is sufficient to produce the Necdin protein and provoke cellular as well as physiological consequences that actively improve the phenotype. Importantly, a specific expression of the maternal NECDIN allele is also detected in hypothalamic brain sections of PWS patients. These results reveal an unexpected epigenetic flexibility that allow to contemplate a therapeutic pharmacological prospect

Reduction of endoplasmic reticulum- mitochondria interactions in beta cells from patients with type 2 diabetes.

Type 2 diabetes develops when beta cells are not able to fulfill insulin needs. The role of the endoplasmic reticulum-mitochondria junction in coordinating the functions of these two organelles throughout the natural history of type 2 diabetes is determinant and may explain the alterations of insulin biosynthesis. Our goal was to study endoplasmic reticulum and mitochondrial interactions in human beta cells from organ donors with type 2 diabetes. Pancreas samples were obtained via the network for pancreatic organ donors with diabetes (nPOD) based on disease status with 12 subjects with type 2 diabetes and 9 non-diabetic controls. We examined pancreatic specimens by immunofluorescence, in situ hybridization and in situ proximity ligation assay and compared the results to an in vitro model of beta-cell dysfunction. Expression of proteins that enable tethering and exchanges between endoplasmic reticulum (ER) and mitochondria and quantification of interconnection through mitochondria associated membranes (MAM) was investigated. In beta cells from type 2 diabetic cases as compared to controls, there was a significant increase in reticular expression of inositol triphosphate receptor-2 (IP3R2) both at the protein and mRNA levels, no difference in mitochondrial transit peptide receptor TOM20 and mitofusin-2 expressions, and a decrease in the expression of voltage-dependent anion channel-1 (VDAC-1). The number of IP3R2-VDAC-1 complexes identified by in situ proximity ligation assay was significantly lower in diabetic islets and in beta cells of diabetics as compared to controls. Treatment of Min6-B1 cells with palmitate altered glucose-stimulated insulin secretion, increased ER stress and significantly reduced ER-mitochondrial interactions. We can conclude that specific changes in reticular and mitochondrial beta cell proteins characterize human type 2 diabetes with reduction in organelle interactions. This finding opens new targets of intervention

Reduction of endoplasmic reticulummitochondria interactions in beta cells from patients with type 2 diabetes

International audienceType 2 diabetes develops when beta cells are not able to fulfill insulin needs. The role of the endoplasmic reticulum±mitochondria junction in coordinating the functions of these two organelles throughout the natural history of type 2 diabetes is determinant and may explain the alterations of insulin biosynthesis. Our goal was to study endoplasmic reticulum and mitochondrial interactions in human beta cells from organ donors with type 2 diabetes. Pancreas samples were obtained via the network for pancreatic organ donors with diabetes (nPOD) based on disease status with 12 subjects with type 2 diabetes and 9 nondiabetic controls. We examined pancreatic specimens by immunofluorescence, in situ hybridization and in situ proximity ligation assay and compared the results to an in vitro model of beta-cell dysfunction. Expression of proteins that enable tethering andexchanges between endoplasmic reticulum (ER) and mitochondria and quantification of interconnection through mitochondria associated membranes (MAM) was investigated. In beta cells from type 2 diabetic cases as compared to controls, there was a significant increase in reticular expression of inositol triphosphate receptor-2 (IP3R2) both at the protein and mRNA levels, no difference in mitochondrial transit peptide receptor TOM20 and mitofusin-2 expressions, and a decrease in the expression of voltage-dependent anion channel-1 (VDAC-1). The number of IP3R2-VDAC-1 complexes identified by in situ proximity ligation assay was significantly lower in diabetic islets and in beta cells of diabetics ascompared to controls. Treatment of Min6-B1 cells with palmitate altered glucose-stimulated insulin secretion, increased ER stress and significantly reduced ER-mitochondrial interactions. We can conclude that specific changes in reticular and mitochondrial beta cell proteins characterize human type 2 diabetes with reduction in organelle interactions. This finding opens new targets of intervention

Glycine contributes to the control of hepatic response to insulin by promoting mitochondrial-endoplasmic reticulum interactions

International audienceAbstract Introduction Determinants of health and diseases in humans involve complex interactions between diet, gut microbiota and host metabolism. The liver is a major organ that coordinates host adaptations to environmental factors. Mitochondria and endoplasmic reticulum tightly regulate liver nutrient sensing and metabolic adaptations, shaping its metabolic flexibility. Both organelles interact through contact points (named MAMs) in order to exchange calcium and lipids, and MAMs’ integrity settles metabolic flexibility of the liver. Disrupted MAMs’ integrity in obesity is linked to liver steatosis and insulin resistance. This occurs in association with alterations circulating amino acid profiles, among which glycine. Meta-analyses showed that the low plasma glycine concentration observed in obesity is a predictive factor for developing type 2 diabetes. We thus aimed at exploring whether glycine could participate to the regulation of the hepatic response to insulin and whether mechanisms may involve regulation of mitochondrial-ER (MAMs) interactions. Materials and Methods The study was carried out in 12 week-old male C57B16J mice fed a standard chow (n = 12/group, 36 total) in accordance with the French guidelines for the care and use of animals. Glycine (1.2g/kg) was provided for 3 days in drinking water vs. water vs. isonitrogenous placebo amino acids. Liver was collected after an overnight fast, 15 min after ip injection of saline (n = 6/group) or insulin (0.75U/kg, n = 6/group). Liver samples were fixed in glutaraldehyde/cacodylate for quantifying MAMs by transmission electronic microscopy. Fractions enriched in MAMs were isolated by differential ultracentrifugation on fresh tissue for Western Blot explorations. Insulin response was assessed through Akt phosphorylation at Ser473. Results Glycine supplementation increased the percentage of interaction between ER and mitochondria (compare to mitochondrial surface) for all spacing from 10 to 50 nm. Thus, glycine supplementation induced a 45% increase in mitochondrial-ER (MAMs) interactions compared to other groups (p < 0.001). In agreement to the improved MAMs’ integrity, insulin response was enhanced by 50% after glycine supplementation compared to other groups (p < 0.05). Finally, protein analysis of the MAMs' fraction corroborates the importance of PP2A as a key enzyme regulating MAMs’ integrity. Conclusion Glycine is therefore an interesting nutritional actor that can play a crucial role in regulating the integrity of MAMs in the liver, thus contributing to the control of the insulin response

Reduction of endoplasmic reticulummitochondria interactions in beta cells from patients with type 2 diabetes

International audienceType 2 diabetes develops when beta cells are not able to fulfill insulin needs. The role of the endoplasmic reticulum±mitochondria junction in coordinating the functions of these two organelles throughout the natural history of type 2 diabetes is determinant and may explain the alterations of insulin biosynthesis. Our goal was to study endoplasmic reticulum and mitochondrial interactions in human beta cells from organ donors with type 2 diabetes. Pancreas samples were obtained via the network for pancreatic organ donors with diabetes (nPOD) based on disease status with 12 subjects with type 2 diabetes and 9 nondiabetic controls. We examined pancreatic specimens by immunofluorescence, in situ hybridization and in situ proximity ligation assay and compared the results to an in vitro model of beta-cell dysfunction. Expression of proteins that enable tethering andexchanges between endoplasmic reticulum (ER) and mitochondria and quantification of interconnection through mitochondria associated membranes (MAM) was investigated. In beta cells from type 2 diabetic cases as compared to controls, there was a significant increase in reticular expression of inositol triphosphate receptor-2 (IP3R2) both at the protein and mRNA levels, no difference in mitochondrial transit peptide receptor TOM20 and mitofusin-2 expressions, and a decrease in the expression of voltage-dependent anion channel-1 (VDAC-1). The number of IP3R2-VDAC-1 complexes identified by in situ proximity ligation assay was significantly lower in diabetic islets and in beta cells of diabetics ascompared to controls. Treatment of Min6-B1 cells with palmitate altered glucose-stimulated insulin secretion, increased ER stress and significantly reduced ER-mitochondrial interactions. We can conclude that specific changes in reticular and mitochondrial beta cell proteins characterize human type 2 diabetes with reduction in organelle interactions. This finding opens new targets of intervention

Reduction of endoplasmic reticulummitochondria interactions in beta cells from patients with type 2 diabetes

International audienceType 2 diabetes develops when beta cells are not able to fulfill insulin needs. The role of the endoplasmic reticulum±mitochondria junction in coordinating the functions of these two organelles throughout the natural history of type 2 diabetes is determinant and may explain the alterations of insulin biosynthesis. Our goal was to study endoplasmic reticulum and mitochondrial interactions in human beta cells from organ donors with type 2 diabetes. Pancreas samples were obtained via the network for pancreatic organ donors with diabetes (nPOD) based on disease status with 12 subjects with type 2 diabetes and 9 nondiabetic controls. We examined pancreatic specimens by immunofluorescence, in situ hybridization and in situ proximity ligation assay and compared the results to an in vitro model of beta-cell dysfunction. Expression of proteins that enable tethering andexchanges between endoplasmic reticulum (ER) and mitochondria and quantification of interconnection through mitochondria associated membranes (MAM) was investigated. In beta cells from type 2 diabetic cases as compared to controls, there was a significant increase in reticular expression of inositol triphosphate receptor-2 (IP3R2) both at the protein and mRNA levels, no difference in mitochondrial transit peptide receptor TOM20 and mitofusin-2 expressions, and a decrease in the expression of voltage-dependent anion channel-1 (VDAC-1). The number of IP3R2-VDAC-1 complexes identified by in situ proximity ligation assay was significantly lower in diabetic islets and in beta cells of diabetics ascompared to controls. Treatment of Min6-B1 cells with palmitate altered glucose-stimulated insulin secretion, increased ER stress and significantly reduced ER-mitochondrial interactions. We can conclude that specific changes in reticular and mitochondrial beta cell proteins characterize human type 2 diabetes with reduction in organelle interactions. This finding opens new targets of intervention

Editorial: Metabolic Flexibility

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