Alterations in lysosomal gene expression and lipid homeostasis in patient-derived liver organoids with acid sphingomyelinase deficiency
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE236213 | GEO | 2023/07/04
REPOSITORIES: GEO
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Project description:Acid sphingomyelinase deficiency (ASMD) or Niemann-Pick disease type A (NPA), type B (NPB) and type A/B (NPA/B), is a rare lysosomal storage disease characterized by progressive accumulation of sphingomyelin (SM) in the liver, lungs, bone marrow and, in severe cases, neurons. A disease model was established by generating liver organoids from a NPB patient carrying the p.Arg610del variant in the SMPD1 gene. Liver organoids were characterized by transcriptomic and lipidomic analysis. We observed altered lipid homeostasis in the patient-derived organoids showing the predictable increase in sphingomyelin (SM), together with cholesterol esters (CE) and triacylglycerides (TAG), and a reduction in phosphatidylcholine (PC) and cardiolipins (CL). Analysis of lysosomal gene expression pointed to 24 downregulated genes, including SMPD1, and 26 upregulated genes that reflect the lysosomal stress typical of the disease. Altered genes revealed reduced expression of enzymes that could be involved in the accumulation in the hepatocytes of sphyngoglycolipids and glycoproteins, as well as upregulated genes coding for different glycosidases and cathepsins. Lipidic and transcriptome changes support the use of hepatic organoids as ideal models for ASMD investigation.
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Project description:Alterations in lysosomal gene expression and lipid homeostasis in patient-derived liver organoids with acid sphingomyelinase deficiency
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Project description:Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have contributed to its lacking treatments. Here we show that ABCA4 knockout or Stargardt patient’s induced pluripotent stem cells-derived RPE (STGD1-iRPE) differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity, indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are reduced by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Overall, our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.
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Project description:Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay among SMase activity, initial membrane properties (i.e., phase behavior and lipid lateral organization) and lipid composition, and the amount of product (Cer) generated, and how it modulates membrane properties were studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that 1), the enzyme activity and extent of hydrolysis are strongly dependent on membrane physical properties but not on substrate content, and are higher in raft-like mixtures, i.e., mixtures with liquid-disordered/liquid-ordered phase separation; and 2), Cer-induced alterations are also dependent on membrane composition, specifically the cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small ( approximately 8.5 nm) Cer/SM-gel domains. With increasing Chol, the ability of Cer to recruit SM and form gel domains strongly decreases. In the high-Chol range, a Chol-enriched/SM-depleted liquid-ordered phase predominates. Together, these data suggest that in biological membranes, Chol in particular and raft domains in general play an important role in modulating SMase activity and regulating membrane physical properties by restraining Cer-induced alterations.
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Project description:The purpose of this study was to determine whether lysosome trafficking and targeting of acid sphingomyelinase (ASMase) to this organelle contribute to the formation of lipid raft (LR) signaling platforms in the membrane of coronary arterial endothelial cells (CAECs).By measurement of fluorescent resonance energy transfer (FRET), it was found that in FasL-stimulated CAECs, membrane lamp1 (a lysosome marker protein) or Fas and GM1 (a LR marker) were trafficking together. Cofocal colocalization assay showed that ceramide was enriched in these LR platforms. Further studies demonstrated that these ceramide molecules in LR platforms were colocalized with ASMase, a ceramide producing enzyme. Fluorescence imaging of living CAECs loaded with lysosomal specific dyes demonstrated that lysosomes fused with membrane on FasL stimulation. In the presence of lysosome function inhibitors, bafilomycin (Baf) or glycyl-L-phenylalanine-beta-naphthylamide (GPN), these FasL-induced changes were abolished. Moreover, this FasL-induced formation of LR platforms was also blocked in ECs transfected with siRNA of sortilin, an intracellular transporter for targeting of ASMase to lysosomes. Functionally, FasL-induced impairment of vasodilator response was reversed by lysosomal inhibitors or sortilin gene silencing.Lysosomal trafficking and targeting of ASMase are importantly involved in LRs clustering in ECs membrane, leading to the formation of signaling platforms or signalosomes.
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Project description:The cleavage of sphingoid base phosphates by sphingosine-1-phosphate (S1P) lyase to produce phosphoethanolamine and a fatty aldehyde is the final degradative step in the sphingolipid metabolic pathway. We have studied mice with an inactive S1P lyase gene and have found that, in addition to the expected increase of sphingoid base phosphates, other sphingolipids (including sphingosine, ceramide, and sphingomyelin) were substantially elevated in the serum and /or liver of these mice. This latter increase is consistent with a reutilization of the sphingosine backbone for sphingolipid synthesis due to its inability to exit the sphingolipid metabolic pathway. Furthermore, the S1P lyase deficiency resulted in changes in the levels of serum and liver lipids not directly within the sphingolipid pathway, including phospholipids, triacyglycerol, diacylglycerol, and cholesterol. Even though lipids in serum and lipid storage were elevated in liver, adiposity was reduced in the S1P lyase-deficient mice. Microarray analysis of lipid metabolism genes in liver showed that the S1P lyase deficiency caused widespread changes in their expression pattern. These results demonstrate that S1P lyase is a key regulator of the levels of multiple sphingolipid substrates and reveal functional links between the sphingolipid metabolic pathway and other lipid metabolic pathways that may be mediated by shared lipid substrates and changes in gene expression programs. The disturbance of lipid homeostasis by altered sphingolipid levels may be relevant to metabolic diseases. Experiment Overall Design: RNA samples from liver for three sphingosine-1-phosphate lyase knock-out and three WT mice.
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Project description:The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer to regulate anabolic metabolism. However, the metabolic requirements of PKM2 in tumorigenesis have yielded contradictory results. Herein we discovered that this glycolytic enzyme regulates lipid homeostasis in cancer cell autonomous manner and at systemic levels. Transmembrane protein 33 (TMEM33) was identified as a downstream effector of PKM2, whose expression level positively correlates with plasma total cholesterol level in vivo. Loss of PKM2 leads to up-regulation of TMEM33, which recruits ring finger protein 5 (RNF5) to promote SCAP ubiquitination and degradation, thereby inhibiting Golgi translocation and activation of sterol regulatory element binding protein 1 (SREBP1). Moreover, global PKM2 knockout promoted allografted tumor growth, at least partially attributes to the elevated plasma cholesterol level caused by increased intestinal Niemann-Pick C1-Like 1 (NPC1L1) to facilitate cholesterol absorption. Collectively, PKM2-TMEM33 axis modulates cell autonomous lipid metabolism by regulating SREBP activation. PKM2 in small intestine plays critical functions in controlling systemic cholesterol level. Overall, our results underscore unappreciated functions of PKM2 in lipid homeostasis and imply that combining an allosteric activator of PKM2 with NPC1L1 inhibitor represents a therapeutic invention for cancer treatment.
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Project description:Juvenile neuronal ceroid lipofuscinoses (JNCL) is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with JNCL carry at least one allele with 1 kb deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based JNCL models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we have corrected a 1 kb deletion mutation in human induced pluripotent stem cells (iPSC) of a compound heterozygous patient (CLN3 Δ1 kb and E295K), to generate an isogenic control to be used for disease modeling along with the unedited CLN3 patient iPSCs. iPSC-derived neurons carrying this particular CLN3 mutation, CLN3 neurons, had lower levels of spike, burst and network burst activity for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis on day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis of LAMP1 expression revealed a new finding where LAMP1 was hyperglycosylated in CLN3 neurons on DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.
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