Aims/hypothesis Pancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. Methods Pancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5). Results We observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3–LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3–LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002). Conclusions/interpretation We provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes. Graphical abstract
Background: Translationally stalled proteins (including those aberrantly translated beyond their stop codons) pose dangers for eukaryotic cells. Results: The ubiquitin ligase Rkr1/Ltn1 targets translationally stalled ER-associated proteins for degradation. Conclusion: Cytosolic and ER-associated translationally stalled proteins are targeted for destruction by related mechanisms. Significance: Mechanisms regulating the degradation of translationally stalled ER-associated proteins may represent therapeutic targets for human disease.
Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.
Aims/hypothesisPancreatic beta cells are highly metabolic secretory cells that are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species (ROS) during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce ROS to protect against apoptosis both in vitro and in vivo. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesized that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsMouse pancreata were collected from chloroquine injected and non-injected NOR, nondiabetic NOD, and diabetic NOD mice. Age and BMI-matched pancreas tissue sections from human organ donors (n=34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). To assess autophagic flux, we injected the mice with chloroquine to inhibit the final stages of autophagy. We analyzed tissues for markers of autophagy via immunofluorescence analysis. Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), LC3A/B (autophagosome marker), Lamp1 (lysosome marker), and p62 (autophagy adaptor protein and marker for autophagic flux). Images were collected on a scanning laser confocal microscope then analyzed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes (formerly called lipofuscin bodies, residual bodies or tertiary lysosomes) were analyzed in electron micrographs of pancreatic tissue sections from human organ donors (nPOD; n=12) deposited in www.nanotomy.org/OA/nPOD. Energy Dispersive X-ray (EDX) analysis was also performed on these tissues to analyze distribution of elements such as nitrogen, phosphorus, and osmium in secondary lysosomes and telolysosomes of nondiabetic and autoantibody positive donor tissues (n=5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p=0.0077) and increased p62 in islets of both nondiabetic and diabetic NOD mice (p<0.0001 in both cases) when compared to NOR mice. There was also a significant reduction in autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to both nondiabetic NOD mice (p=0.0004) and NOR mice (p=0.0003). Chloroquine infusions elicited accumulation of autophagosomes in the islets of NOR (p=0.0029) and nondiabetic NOD mice (p<0.0001), but not in the islets of diabetic NOD mice. Chloroquine also stimulated an accumulation of the autophagy adaptor protein p62 in the islets of NOR mice (p<0.001), however this was not observed in NOD mice (regardless of diabetes status). In the human pancreata, we observed significantly reduced autophagosome:lysosome colocalization (p=0.0002) in the residual beta cells of donors with type 1 diabetes compared to nondiabetic controls. We also observed reduced colocalization of proinsulin with lysosomes in the residual beta cells of donors with type 1 diabetes compared to both nondiabetic (p<0.0001) and autoantibody positive donors (p<0.0001). Electron microscopy based analysis of human pancreas sections also revealed accumulation of telolysosomes in beta cells of autoantibody positive donors (p=0.0084), the majority of which had an nitrogen dense ring outside a phospholipid core.Conclusions/interpretationCollectively, we provide evidence of impairment in the final degradation stages of islet macroautophagy and crinophagy in human type 1 diabetes. We also document an accumulation of telolysosomes with nitrogen accumulation at their periphery in the beta cells of autoantibody positive donors. This demonstrates clear differences in the lysosome contents of autoantibody positive donors that may be associated with lysosome dysfunction prior to clinical hyperglycemia. We observe similar impairments in macroautophagy in the diabetic NOD mouse, a model of type 1 diabetes, suggesting that this mouse model can be appropriately used to study the pathogenesis of autophagy/crinophagy loss and how it relates to disease initiation and progression. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development.Research in contextWhat is already known about this subject?Autophagy confers a cytoprotective role in the beta cell.Autophagy is reduced in type 2 diabetes.Autophagy in the context of type 1 diabetes is unexplored.What is the key question?Is autophagy reduced during the pathogenesis of human type 1 diabetes?What are the new findings?We provide evidence of reduced autophagy and crinophagy in human type 1 diabetes.These data are supported by observations of reduced autophagy in a mouse model of autoimmune diabetes.How might this impact on clinical practice in the foreseeable future?This study provides evidence that autophagy is impaired in human type 1 diabetes. Prior studies have shown that loss of autophagy in the islet is associated with increased beta cell apoptosis, therefore we propose that therapeutic targeting of autophagy pathways may reduce beta cell death in type 1 diabetes development.
Objective: Adult polyglucosan body disease (APBD) is an adult-onset neurological variant of glycogen storage disease type IV. APBD is caused by recessive mutations in the glycogen branching enzyme gene, and the consequent accumulation of poorly branched glycogen aggregates called polyglucosan bodies in the nervous system. There are presently no treatments for APBD. Here, we test whether downregulation of glycogen synthesis is therapeutic in a mouse model of the disease. Methods: We characterized the effects of knocking out two proglycogenic proteins in an APBD mouse model. APBD mice were crossed with mice deficient in glycogen synthase (GYS1), or mice deficient in protein phosphatase 1 regulatory subunit 3C (PPP1R3C), a protein involved in the activation of GYS1. Phenotypic and histological parameters were analyzed and glycogen was quantified. Results: APBD mice deficient in GYS1 or PPP1R3C demonstrated improvements in life span, morphology, and behavioral assays of neuromuscular function. Histological analysis revealed a reduction in polyglucosan body accumulation and of astro-and micro-gliosis in the brains of GYS1-and PPP1R3C-deficient APBD mice. Brain glycogen quantification confirmed the reduction in abnormal glycogen accumulation. Analysis of skeletal muscle, heart, and liver found that GYS1 deficiency reduced polyglucosan body accumulation in all three tissues and PPP1R3C knockout reduced skeletal muscle polyglucosan bodies. Interpretation: GYS1 and PPP1R3C are effective therapeutic targets in the APBD mouse model. These findings represent a critical step toward the development of a treatment for APBD and potentially other glycogen storage disease type IV patients.
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