RESUMEN
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
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
BACKGROUND: Alteration in glycosphingolipids (GSLs) in Parkinson's disease (PD) still needs to be determined. OBJECTIVES: We evaluated if PD subjects show abnormal GSLs levels compared to healthy controls (HC) and if GSLs correlate with clinical features. METHODS: We analyzed GSLs and glucosylceramide (GlcCer) in plasma using two normal-phase high-performance liquid chromatography assays; clinico-demographic data were extracted. RESULTS: Eighty PD subjects and 25 HCs were analyzed. Levels of GlcCer, GD1b, Gb4, GalNAcGA1, and b-series were higher in PD patients than in HCs; total GSLs, GT1b, GM1a, GM3, GM2, and a-series levels were lower in PD patients than in HCs. Changes in GSLs were present in PD subjects, with GlcCer levels similar to those in HCs. The results were similar after excluding certain GBA1 mutation carriers. Movement Disorder Society Unified Parkinson's Disease Rating Scale, Part III, correlated with Gb4 and Montreal Cognitive Assessment with GD1b levels. CONCLUSIONS: Multiple GSL abnormalities in plasma were detected in patients with and without GlcCer changes, indicating a broader shift in lipid homeostasis. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.
Asunto(s)
Enfermedad de Parkinson , Glucosilceramidas , Glicoesfingolípidos/análisis , Glicoesfingolípidos/química , Humanos , Pruebas de Estado Mental y Demencia , Enfermedad de Parkinson/genética , Plasma/químicaRESUMEN
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
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
Lysosomal storage diseases are inherited monogenic disorders in which lysosome function is compromised. Although individually very rare, they occur at a collective frequency of approximately one in five thousand live births and usually have catastrophic consequences for health. The lysosomal storage diseases Niemann-Pick disease type C (NPC) is caused by mutations predominantly in the lysosomal integral membrane protein NPC1 and clinically presents as a progressive neurodegenerative disorder. In this article we review data that demonstrate significant dysregulation of innate immunity in NPC, which occurs both in peripheral organs and the CNS. In particular pro-inflammatory responses promote disease progression and anti-inflammatory drugs provide benefit in animal models of the disease and are an attractive target for clinical intervention in this disorder. Niemann-Pick disease type C is a rare, devastating, inherited lysosomal storage disease with a unique cellular phenotype characterized by lysosomal accumulation of sphingosine, various glycosphingolipids and cholesterol and a reduction in lysosomal calcium. In this review we highlight the impact of the disease on innate immune activities in both the central nervous system (CNS) and peripheral tissues and discuss their contributions to pathology and the underlying mechanisms.
Asunto(s)
Inmunidad Celular/inmunología , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Enfermedad de Niemann-Pick Tipo C/inmunología , Animales , Humanos , Células Asesinas Naturales/inmunologíaRESUMEN
OBJECTIVES: Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterised by the storage of multiple lipids, reduced lysosomal calcium levels, impaired late endosome:lysosome fusion and neuroinflammation. NPC is caused by mutations in either of the two genes, NPC1 or NPC2, which are believed to function in a common cellular pathway, the function of which remains unclear. The complexity of the pathogenic cascade in NPC disease provides a number of potential clinical intervention points. To date, drugs that target pivotal stages in the pathogenic cascade have been tested as monotherapies or in combination with a second agent, showing additive or synergistic benefit. In this study, we have investigated whether we can achieve greater therapeutic benefit in the Npc1(-/-) mouse by combining three therapies that each targets unique aspects of the pathogenic cascade. METHODS: We have treated Npc1(-/-) mice with miglustat that targets sphingolipid synthesis and storage, curcumin that compensates for the lysosomal calcium defect by elevating cytosolic calcium, and the non-steroidal anti-inflammatory drug ibuprofen to reduce central nervous system inflammation. RESULTS/INTERPRETATION: We have found that triple combination therapy has a greater neuroprotective benefit compared with single and dual therapies, increasing the time period that Npc1(-/-) mice maintained body weight and motor function and maximally delaying the onset of Purkinje cell loss. In addition, ibuprofen selectively reduced microglial activation, while curcumin had no anti-inflammatory effects, indicating differential mechanisms of action for these two therapies. When taken together, these results demonstrate that targeting multiple unique steps in the pathogenic cascade maximises the clinical benefit in a mouse model of NPC1 disease.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Curcumina/uso terapéutico , Ibuprofeno/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , 1-Desoxinojirimicina/uso terapéutico , Animales , Cerebelo/efectos de los fármacos , Cerebelo/patología , Quimioterapia Combinada , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Niemann-Pick C1 , Proteínas/genéticaRESUMEN
Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal disorder characterized by sphingolipid and cholesterol storage in the late endocytic system. In common with other neurodegenerative diseases, activation of the innate immune system occurs in the brain resulting in neuro-inflammation. Targeting inflammation in the brain therefore represents a potential clinical intervention strategy that aims to slow the rate of disease progression and improve quality of life. We evaluated non-steroidal anti-inflammatory drugs (NSAIDs) and an anti-oxidant to determine whether these agents are disease modifying in an acute mouse model of NPC1. NSAIDs significantly prolonged the lifespan of NPC1 mice and slowed the onset of clinical signs. However, anti-oxidant therapy was of no significant benefit. Combining NSAID therapy with substrate reduction therapy (SRT) resulted in additive benefit. These data suggest that anti-inflammatory therapy may be a useful adjunctive treatment in the clinical management of NPC1, alone or combined with SRT.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Modelos Animales de Enfermedad , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/patología , Animales , Antiinflamatorios no Esteroideos/farmacología , Ácido Ascórbico/farmacología , Ácido Ascórbico/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/patología , Inflamación/tratamiento farmacológico , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
Lysosomal storage diseases (LSDs) often manifest with severe systemic and central nervous system (CNS) symptoms. The existing treatment options are limited and have no or only modest efficacy against neurological manifestations of disease. We demonstrate that recombinant human heat shock protein 70 (HSP70) improves the binding of several sphingolipid-degrading enzymes to their essential cofactor bis(monoacyl)glycerophosphate in vitro. HSP70 treatment reversed lysosomal pathology in primary fibroblasts from 14 patients with eight different LSDs. HSP70 penetrated effectively into murine tissues including the CNS and inhibited glycosphingolipid accumulation in murine models of Fabry disease (Gla(-/-)), Sandhoff disease (Hexb(-/-)), and Niemann-Pick disease type C (Npc1(-/-)) and attenuated a wide spectrum of disease-associated neurological symptoms in Hexb(-/-) and Npc1(-/-) mice. Oral administration of arimoclomol, a small-molecule coinducer of HSPs that is currently in clinical trials for Niemann-Pick disease type C (NPC), recapitulated the effects of recombinant human HSP70, suggesting that heat shock protein-based therapies merit clinical evaluation for treating LSDs.
Asunto(s)
Proteínas de Choque Térmico/uso terapéutico , Esfingolipidosis/tratamiento farmacológico , Administración Intravenosa , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Proteínas Morfogenéticas Óseas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Glicoesfingolípidos/metabolismo , Proteínas de Choque Térmico/farmacología , Humanos , Hidroxilaminas/farmacología , Hidroxilaminas/uso terapéutico , Inyecciones Intraperitoneales , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/efectos de los fármacos , Lisosomas/patología , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Proteínas/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Esfingolipidosis/patología , Distribución TisularRESUMEN
Although water-soluble polymers are finding increasing use as polymer therapeutics, there has been little consideration of the effect of polymer stereochemistry on their physico-chemical and biological properties. The aim of this study was to investigate these properties using polymethacrylic acids (PMAs) of similar molecular weights with a difference in syndiotacticity of about 20% of rr triad content. Experiments to characterize the solution behaviour were conducted at pHs encountered during the transport, endocytic uptake and intracellular trafficking (7.4-3.0). These showed that with increasing rr triads, the polymer become less hydrophobic, a stronger acid, displayed a locally ordered solution conformation at pH 5.5, and interacted more strongly with dodecyl trimethylammonium bromide (DTAB) micelles. Preliminary cytotoxicity experiments using B16F10 melanoma cells showed lower toxicity in the concentration range of 1-100 µg/mL with increased rr triads. These observations indicate that the higher content of rr triads could drive a chain organization that minimize the influence of negative charges and so underline the importance of further, systematic studies to investigate the effect of tacticity on the behaviour of polymers in respect of their pharmacokinetics, toxicity and efficacy.
Asunto(s)
Química Física , Portadores de Fármacos/química , Ácidos Polimetacrílicos/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromatografía en Gel , Portadores de Fármacos/toxicidad , Endocitosis , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Ratones , Estructura Molecular , Peso Molecular , Ácidos Polimetacrílicos/síntesis química , Ácidos Polimetacrílicos/toxicidad , Soluciones , Estereoisomerismo , Tensión Superficial , ViscosidadRESUMEN
Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.