RESUMEN
Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.
Asunto(s)
Lisosomas , Redes y Vías Metabólicas , Lisosomas/metabolismo , Transducción de SeñalRESUMEN
Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid transport. Loss-of-function mutations in the NPC1 protein cause Niemann-Pick disease type C1, a lysosomal storage disorder characterized by the accumulation of cholesterol and sphingolipids within lysosomes. To investigate whether the NPC1 protein could also play a role in the maturation of the endolysosomal pathway, here, we have investigated its role in a lysosome-related organelle, the melanosome. Using a NPC1-KO melanoma cell model, we found that the cellular phenotype of Niemann-Pick disease type C1 is associated with a decreased pigmentation accompanied by low expression of the melanogenic enzyme tyrosinase. We propose that the defective processing and localization of tyrosinase, occurring in the absence of NPC1, is a major determinant of the pigmentation impairment in NPC1-KO cells. Along with tyrosinase, two other pigmentation genes, tyrosinase-related protein 1 and Dopachrome-tautomerase have lower protein levels in NPC1 deficient cells. In contrast with the decrease in pigmentation-related protein expression, we also found a significant intracellular accumulation of mature PMEL17, the structural protein of melanosomes. As opposed to the normal dendritic localization of melanosomes, the disruption of melanosome matrix generation in NPC1 deficient cells causes an accumulation of immature melanosomes adjacent to the plasma membrane. Together with the melanosomal localization of NPC1 in WT cells, these findings suggest that NPC1 is directly involved in tyrosinase transport from the trans-Golgi network to melanosomes and melanosome maturation, indicating a novel function for NPC1.
Asunto(s)
Enfermedad de Niemann-Pick Tipo C , Enfermedades de Niemann-Pick , Humanos , Melanosomas/metabolismo , Monofenol Monooxigenasa/metabolismo , Proteína Niemann-Pick C1/metabolismo , Colesterol/metabolismo , Enfermedades de Niemann-Pick/genética , Enfermedades de Niemann-Pick/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismoRESUMEN
Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic cancer derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small intestinal epithelial cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multipledonors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracted the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT.
Asunto(s)
Cólera , Humanos , Fucosa , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Ligandos , Glicoconjugados , Polisacáridos , GlicoesfingolípidosRESUMEN
The rare, fatal neurodegenerative disorder Niemann-Pick disease type C1 (NPC1) arises from lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. The timing and severity of NPC1 clinical presentation is extremely heterogeneous. This study analyzed RNA-Seq data from 42 NPC1 patient-derived, primary fibroblast cell lines to determine transcriptional changes induced by treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a compound currently under investigation in clinical trials. A total of 485 HPßCD-responsive genes were identified. Pathway enrichment analysis of these genes showed significant involvement in cholesterol and lipid biosynthesis. Furthermore, immunohistochemistry of the cerebellum as well as measurements of plasma from Npc1m1N null mice treated with HPßCD and adeno-associated virus gene therapy suggests that one of the identified genes, GPNMB, may serve as a useful biomarker of treatment response in NPC1 disease. Overall, this large NPC1 patient-derived dataset provides a comprehensive foundation for understanding the genomic response to HPßCD treatment.
Asunto(s)
Enfermedad de Niemann-Pick Tipo C , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Biomarcadores , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , TranscriptomaRESUMEN
Lysosomal storage diseases are inborn errors of metabolism that arise due to loss of function mutations in genes encoding lysosomal enzymes, protein co-factors or lysosomal membrane proteins. As a consequence of the genetic defect, lysosomal function is impaired and substrates build up in the lysosome leading to 'storage'. A sub group of these disorders are the sphingolipidoses in which sphingolipids accumulate in the lysosome. In this review, I will discuss how the study of these rare lysosomal disorders reveals unanticipated links to other rare and common human diseases using Niemann-Pick disease type C as an example.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal , Enfermedad de Niemann-Pick Tipo C , Esfingolipidosis , Humanos , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Esfingolípidos/metabolismo , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Esfingolipidosis/genética , Esfingolipidosis/metabolismo , Lisosomas/metabolismoRESUMEN
GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of ß-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1-/- mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1-/- mice die six weeks earlier than male Glb1-/- mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1-/- mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1-/- mice as seen in some patients. In addition, Glb1-/- mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1-/- mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1-/- mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder.
Asunto(s)
Gangliosidosis GM1 , Enfermedades por Almacenamiento Lisosomal , Masculino , Femenino , Animales , Ratones , Gangliosidosis GM1/genética , Ratones Noqueados , beta-Galactosidasa/genética , Enfermedades por Almacenamiento Lisosomal/genética , ExonesRESUMEN
Identification of genetic modulators of lysosomal enzyme activities and glycosphingolipids (GSLs) may facilitate the development of therapeutics for diseases in which they participate, including Lysosomal Storage Disorders (LSDs). To this end, we used a systems genetics approach: we measured 11 hepatic lysosomal enzymes and many of their natural substrates (GSLs), followed by modifier gene mapping by GWAS and transcriptomics associations in a panel of inbred strains. Unexpectedly, most GSLs showed no association between their levels and the enzyme activity that catabolizes them. Genomic mapping identified 30 shared predicted modifier genes between the enzymes and GSLs, which are clustered in three pathways and are associated with other diseases. Surprisingly, they are regulated by ten common transcription factors, and their majority by miRNA-340p. In conclusion, we have identified novel regulators of GSL metabolism, which may serve as therapeutic targets for LSDs and may suggest the involvement of GSL metabolism in other pathologies.
Asunto(s)
Glicoesfingolípidos , Enfermedades por Almacenamiento Lisosomal , Animales , Ratones , Glicoesfingolípidos/metabolismo , Enfermedades por Almacenamiento Lisosomal/metabolismo , Hidrolasas/metabolismo , Lisosomas/metabolismoRESUMEN
Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme ß-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.
Asunto(s)
Enfermedad de Gaucher/terapia , Terapia Genética , Neuronas/metabolismo , Sinapsinas/genética , Animales , Astrocitos/metabolismo , Astrocitos/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/patología , Humanos , Ratones , Neuronas/patología , Regiones Promotoras Genéticas/genética , Sinapsinas/uso terapéuticoRESUMEN
It is well established that lysosomal glucocerebrosidase gene (GBA) variants are a risk factor for Parkinson's disease (PD), with increasing evidence suggesting a loss of function mechanism. One question raised by this genetic association is whether variants of genes involved in other aspects of sphingolipid metabolism are also associated with PD. Recent studies in sporadic PD have identified variants in multiple genes linked to diseases of glycosphingolipid (GSL) metabolism to be associated with PD. GSL biosynthesis is a complex pathway involving the coordinated action of multiple enzymes in the Golgi apparatus. GSL catabolism takes place in the lysosome and is dependent on the action of multiple acid hydrolases specific for certain substrates and glycan linkages. The finding that variants in multiple GSL catabolic genes are over-represented in PD in a heterozygous state highlights the importance of GSLs in the healthy brain and how lipid imbalances and lysosomal dysfunction are associated with normal ageing and neurodegenerative diseases. In this article we will explore the link between lysosomal storage disorders and PD, the GSL changes seen in both normal ageing, lysosomal storage disorders (LSDs) and PD and the mechanisms by which these changes can affect neurodegeneration.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal , Enfermedad de Parkinson , Envejecimiento , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Glicoesfingolípidos/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismoRESUMEN
CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)-dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosides-namely monosialoganglioside GM3 and disialoganglioside GD3-as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells.
Asunto(s)
Ceramidas/metabolismo , Gangliósidos/metabolismo , Células T Asesinas Naturales/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Antígenos CD1d/metabolismo , Células de la Médula Ósea/metabolismo , Células Dendríticas/metabolismo , Gangliósido G(M3)/metabolismo , Glicoesfingolípidos/metabolismo , Masculino , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Estrés del Retículo Endoplásmico/inmunología , Activación de Linfocitos , Células T Asesinas Naturales/inmunología , Animales , Presentación de Antígeno , Antígenos CD1d/biosíntesis , Antígenos CD1d/inmunología , Autoantígenos/inmunología , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Citoesqueleto/ultraestructura , Endosomas/inmunología , Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Lípidos/inmunología , Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Células THP-1 , Tapsigargina/farmacología , Respuesta de Proteína Desplegada/inmunología , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/fisiologíaRESUMEN
Most cells acquire cholesterol by endocytosis of circulating low-density lipoproteins (LDLs). After cholesteryl ester de-esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA-approved compounds in a high-content, image-based screen of the endosomal lipids, lysobisphosphatidic acid and LDL-derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann-Pick type C (NPC), as well as in liver of Npc1-/- mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.
Asunto(s)
Colesterol/metabolismo , Endosomas/efectos de los fármacos , Lisofosfolípidos/metabolismo , Monoglicéridos/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Piperidinas/farmacología , Animales , Células Cultivadas , Endosomas/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.
Asunto(s)
Adenoviridae/genética , Proteínas Portadoras/administración & dosificación , Modelos Animales de Enfermedad , Trastornos Neurológicos de la Marcha/prevención & control , Terapia Genética , Longevidad/genética , Glicoproteínas de Membrana/administración & dosificación , Enfermedad de Niemann-Pick Tipo C/prevención & control , Animales , Proteínas Portadoras/fisiología , Trastornos Neurológicos de la Marcha/genética , Trastornos Neurológicos de la Marcha/patología , Humanos , Inflamación/genética , Inflamación/patología , Inflamación/prevención & control , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Transgénicos , Mutación , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patologíaRESUMEN
Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.
Asunto(s)
Toxina del Cólera/toxicidad , Gangliósido G(M1)/fisiología , Animales , Células Cultivadas , Gangliósido G(M1)/metabolismo , Glicosilación , Células HL-60 , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/metabolismo , Ratas , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Niemann-Pick disease type C (NPC) and Tangier disease are genetically and clinically distinct rare inborn errors of metabolism. NPC is caused by defects in either NPC1 or NPC2; whereas Tangier disease is caused by a defect in ABCA1. Tangier disease is currently without therapy, whereas NPC can be treated with miglustat, a small molecule inhibitor of glycosphingolipid biosynthesis that slows the neurological course of the disease. When a Tangier disease patient was misdiagnosed with NPC and treated with miglustat, her symptoms improved. This prompted us to consider whether there is mechanistic convergence between these two apparently unrelated rare inherited metabolic diseases. In this study, we found that when ABCA1 is defective (Tangier disease) there is secondary inhibition of the NPC disease pathway, linking these two diseases at the level of cellular pathophysiology. In addition, this study further supports the hypothesis that miglustat, as well as other substrate reduction therapies, may be potential therapeutic agents for treating Tangier disease as fibroblasts from multiple Tangier patients were corrected by miglustat treatment.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Transportador 1 de Casete de Unión a ATP/genética , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , 1-Desoxinojirimicina/uso terapéutico , Adulto , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Persona de Mediana Edad , Proteína Niemann-Pick C1 , Resultado del TratamientoRESUMEN
Lysosomal storage diseases are inborn errors of metabolism, the hallmark of which is the accumulation, or storage, of macromolecules in the late endocytic system. They are monogenic disorders that occur at a collective frequency of 1 in 5,000 live births and are caused by inherited defects in genes that mainly encode lysosomal proteins, most commonly lysosomal enzymes. A subgroup of these diseases involves the lysosomal storage of glycosphingolipids. Through our understanding of the genetics, biochemistry and, more recently, cellular aspects of sphingolipid storage disorders, we have gained insights into fundamental aspects of cell biology that would otherwise have remained opaque. In addition, study of these disorders has led to significant progress in the development of therapies, several of which are now in routine clinical use. Emerging mechanistic links with more common diseases suggest we need to rethink our current concept of disease boundaries.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal/metabolismo , Esfingolípidos/metabolismo , Animales , Vías Biosintéticas , Muerte Celular , Glicoesfingolípidos/biosíntesis , Glicoesfingolípidos/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Enfermedades por Almacenamiento Lisosomal/terapia , Lisosomas/metabolismo , Enfermedad de Parkinson/metabolismo , Esfingolípidos/biosíntesisRESUMEN
Lysosomal storage disorders (LSDs) are predominantly very rare recessive autosomal neurodegenerative diseases.Sphingolipidoses, a sub-group of LSDs, result from defects in lysosomal enzymes involved in sphingolipid catabolism, and feature disrupted storage systems which trigger complex pathogenic cascades with other organelles collaterally affected. This process leads to cell dysfunction and death, particularly in the central nervous system. One valuable approach to gaining insights into the global impact of lysosomal dysfunction is through metabolomics, which represents a discovery tool for investigating disease-induced modifications in the patterns of large numbers of simultaneously-analysed metabolites, which also features the identification of biomarkers Here, the scope and applications of metabolomics strategies to the investigation of sphingolipidoses is explored in order to facilitate our understanding of the biomolecular basis of these conditions. This review therefore surveys the benefits of applying 'state-of-the-art' metabolomics strategies, both univariate and multivariate, to sphingolipidoses, particularly Niemann-Pick type C disease. Relevant limitations of these techniques are also discussed, along with the latest advances and developments. We conclude that metabolomics strategies are highly valuable, distinctive bioanalytical techniques for probing LSDs, most especially for the detection and validation of potential biomarkers. They also show much promise for monitoring disease progression and the evaluation of therapeutic strategies and targets.
Asunto(s)
Metabolómica/métodos , Enfermedad de Niemann-Pick Tipo C/metabolismo , Biomarcadores/análisis , Humanos , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Metabolómica/normas , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , Esfingolípidos/análisis , Esfingolípidos/metabolismoRESUMEN
The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.
Asunto(s)
Canales de Calcio/genética , Calcio/metabolismo , NADP/análogos & derivados , Animales , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Células Cultivadas , Potenciales Evocados/efectos de los fármacos , Expresión Génica/fisiología , Concentración de Iones de Hidrógeno , Lisosomas/efectos de los fármacos , Lisosomas/fisiología , Ratones , Ratones Noqueados , NADP/metabolismo , NADP/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.
Asunto(s)
Glucosilceramidas/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Transporte Biológico , Línea Celular , Globósidos/biosíntesis , Globósidos/química , Globósidos/metabolismo , Glucosilceramidas/química , Glicoesfingolípidos/biosíntesis , Glicoesfingolípidos/química , Glicoesfingolípidos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Fosfatos de Fosfatidilinositol/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 or NPC2 with decreased functions leading to lysosomal accumulation of cholesterol and sphingolipids. FTY720/fingolimod, used for treatment of multiple sclerosis, is phosphorylated by nuclear sphingosine kinase 2, and its active phosphorylated form (FTY720-P) is an inhibitor of class I histone deacetylases. In this study, administration of clinically relevant doses of FTY720 to mice increased expression of NPC1 and -2 in brain and liver and decreased cholesterol in an SphK2-dependent manner. FTY720 greatly increased expression of NPC1 and -2 in human NPC1 mutant fibroblasts that correlated with formation of FTY720-P and significantly reduced the accumulation of cholesterol and glycosphingolipids. In agreement with this finding, FTY720 pretreatment of human NPC1 mutant fibroblasts restored transport of the cholera toxin B subunit, which binds ganglioside GM1, to the Golgi apparatus. Together, these findings suggest that FTY720 administration can ameliorate cholesterol and sphingolipid storage and trafficking defects in NPC1 mutant fibroblasts. Because neurodegeneration is the main clinical feature of NPC disease, and FTY720 accumulates in the CNS and has several advantages over available histone deacetylase inhibitors now in clinical trials, our work provides a potential opportunity for treatment of this incurable disease.-Newton, J., Hait, N. C., Maceyka, M., Colaco, A., Maczis, M., Wassif, C. A., Cougnoux, A., Porter, F. D., Milstien, S., Platt, N., Platt, F. M., Spiegel, S. FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts.