A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE106928 | GEO | 2019/11/06
REPOSITORIES: GEO
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Project description:Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose-loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids ( i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp-1 on the PM of sucrose-loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose-loaded fibroblasts. The inhibition of β-glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.-Samarani, M., Loberto, N., Soldà, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.
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Project description:Mutations in the gene glucosidase, beta acid 1 (GBA1) are the strongest genetic risk factor for Parkinson’s disease (PD) and associate with faster disease progression. GBA1 is expressed in all cell types of the central nervous system, with some evidence supporting higher expression in glial cells than neurons. To elucidate possible divergent functions in neurons versus glia, we differentiated human iPSCs generated from an individual with PD heterozygous for the GBA1 pathogenic null variant IVS2+1 (GBA1IVS/+),Mutations in the gene glucosidase, beta acid 1 (GBA1) are the strongest genetic risk factor for Parkinson’s disease (PD) and associate with faster disease progression. GBA1 is expressed in all cell types of the central nervous system, with some evidence supporting higher expression in glial cells than neurons. To elucidate possible divergent functions in neurons versus glia, we generated human iPSCs from fibroblasts from an individual with PD carrying the GBA1 c.115+1 G>A, also known as IVS2+1 G>A variant (GBA1IVS/+). This intronic GBA1 variant is associated with severe neuronopathic Gaucher’s disease and interrupts the canonical splice site following exon 2, leading to nonsense mediated decay and is therefore considered a null allele. An isogenic GBA1+/+ Rev control was generated by CRISPR/Cas9 genome editing to revert the GBA1 IVS2+1 G>A variant back to wildtype, and in the genome editing process, an iPSC clone homozygous for GBA1 IVS2+1 G>A was also generated (GBA1IVS/IVS). A matched control iPSC clone that remained unedited after CRISPR/Cas9 genome editing, maintaining heterozygosity for GBA1 IVS2+1 G>A, was also isolated (GBA1IVS/+ A4).Transcriptomic analysis of GBA1-deficient neurons and astrocytes revealed cell-type specific profiles. GBA1deficiency in neurons downregulated genes associated with immune response and upregulation of cholesterol synthesis, while GBA1 deficiency in astrocytes downregulated genes associated with translation and upregulated genes involved in extracellular matrix biogenesis, suggestive of gliosis. The transcriptomic profile of GBA1-deficient astrocytes was consistent with neurotoxic reactive astrocytes. Together, these findings indicate that GBA1deficiency has cell type-specific effects, where neurons have increased vulnerability to alterations in endolysosomal trafficking leading to α-synucleinopathy while GBA1 deficiency in astrocytes leads to increased neurotoxic reactivity but not impairment in endolysosomal trafficking and development of a-synuclein pathology.
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Project description:A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest
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Project description:The GBA1 gene encodes the lysosomal enzyme acid-β-glucocerebrosidase (GCase). GBA1 mutations cause the lysosomal storage disorder Gaucher disease (GD) and are a genetic risk factor for Parkinson´s disease (PD). Many GBA1 mutations cause aberrant GCase folding and processing but how these impinge on PD pathogenesis remains unclear. We addressed GCase functions in human dopaminergic (DA) neurons derived from engineered GBA1-deficient (GBA1-/-) embryonic stem cells (hESCs) and GBA1N370S PD patient-derived induced pluripotent cells (hiPSCs). GBA1-mutant DA neurons displayed aberrant morphologies and gene expression that were rescued by recombinant GCase treatment. GBA1-/- neurons have hyperactive Calcineurin (CaN) and nuclear localization of the Transcription Factor EB (TFEB). Expression of TFEB targets and lysosomal CLEAR network components were increased in GBA1-/- DA neurons and rescued by GCase replacement and CaN inhibition. Our findings reveal a link between increased CaN activity and loss of GCase, providing a mechanism for the disturbed lysosomal/autophagy pathways in GBA1-associated PD.
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Project description:Previous research has shown that glutamine and sucrose treatment of excised poplar stems induces bark storage protein (BSP) gene expression. The objective of this research is to identify changes in gene expression associated with metabolic regulation of nitrogen storage and cycling and use this information to identify potential regulatory genes. Significant, differentially expressed genes were identified in excised poplar stems incubated in solutions of glutamine, sucrose, glycine, glutamine+glucose, and glutamine+sucrose compared to incubation in a water control.
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Project description:Lysosomal Storage Disorders (LSDs), which share common phenotypes, including enlarged lysosomes and defective lysosomal storage, are caused by mutations in lysosome-related genes. Although gene therapies and enzyme replacement therapies have been explored, there are currently no effective routine therapies against LSDs. During lysosome reformation, which occurs when the functional lysosome pool is reduced, lysosomal lipids and proteins are recycled to restore lysosome functions. Here we report that the sorting nexin protein SNX8 promotes lysosome tubulation, a process that is required for lysosome reformation, and that loss of SNX8 leads to phenotypes characteristic of LSDs in human cells. SNX8 overexpression rescued features of LSDs in cells, and AAV-based delivery of SNX8 to the brain rescued LSD phenotypes in mice. Importantly, by screening a natural compound library, we identified three small molecules that enhanced SNX8-lysosome binding and reversed LSD phenotypes in human cells and in mice. Altogether, our results provide a potential solution for the treatment of LSDs.
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Project description:Research in recent years has shown that sphingolipids are essential signalling molecules for the proper biological and structural functioning of cells. Long-term studies on the metabolism of sphingolipids have provided evidence for their role in the pathogenesis of a number of diseases. As many inflammatory diseases, such as lysosomal storage disorders and some dermatologic diseases, including psoriasis, atopic dermatitis and ichthyoses, are associated with the altered composition and metabolism of sphingolipids, more studies precisely determining the responsibilities of these compounds for disease states are required to develop novel pharmacological treatment opportunities. It is worth emphasizing that knowledge from the study of inflammatory metabolic diseases and especially the possibility of their treatment may lead to insight into related metabolic pathways, including those involved in the formation of the epidermal barrier and providing new approaches towards workable therapies.
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Project description:Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of GLUT4 (glucose transporter isoform 4) to the PM (plasma membrane) from specialized GSVs (GLUT4 storage vesicles). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. In the present study, we investigated the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs and FRAP (fluorescence recovery after photobleaching) of GLUT4-GFP (green fluorescent protein) in intracellular structures. Glycosphingolipids were located in endosomal vesicles in pre-adipocytes and redistributed to the PM with decreased expression at day 2 after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. Although insulin-induced phosphorylation of IRS (insulin receptor substrate) and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4-GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2 (vesicle-associated membrane protein 2), but not the transferrin receptor or cellubrevin, indicating that the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and, thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycaemic control and enhances insulin sensitivity in animal models of Type 2 diabetes.
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Project description:Gene expression signature was explored to identify gene targets regulate by dysfunctional glucocerebrosidase function. We found 131 upregulated genes and 63 downregulated genes. Some of the most upregulated (PON3, PLOD1) and downregulated (gpx-1b and complemewnt c9) genes were analyzed by q-PCR demonstrating the correct differential gene expression signature among control and morpholino-microinjected fish. Knockdown of GBA1 function was measured in 4 biological replicates at 2 days post fertilization after microinjection of GBA1 morpholino and control morpholino at one -cell stage embryos.
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