RESUMO
Niemann-Pick type C (NP-C) disease is a fatal genetic lipidosis for which there is no Food and Drug Administration (FDA)-approved therapy. Vorinostat, an FDA-approved inhibitor of histone deacetylases, ameliorates lysosomal lipid accumulation in cultured NP-C patient fibroblasts. To assess the therapeutic potential of histone deacetylase inhibition, we pursued these in vitro observations in two murine models of NP-C disease. Npc1nmf164 mice, which express a missense mutation in the Npc1 gene, were treated intraperitoneally, from weaning, with the maximum tolerated dose of vorinostat (150 mg/kg, 5 days/week). Disease progression was measured via gene expression, liver function and pathology, serum and tissue lipid levels, body weight, and life span. Transcriptome analyses of treated livers indicated multiple changes consistent with reversal of liver dysfunction that typifies NP-C disease. Significant improvements in liver pathology and function were achieved by this treatment regimen; however, NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity were not detectably altered. Vorinostat concentrations were >200 µm in the plasma compartment of treated animals but were almost 100-fold lower in brain tissue. Apolipoprotein B metabolism and the expression of key components of lipid homeostasis in primary hepatocytes from null (Npc1-/-) and missense (Npc1nmf164 ) mutant mice were altered by vorinostat treatment, consistent with a response by these cells independent of the status of the Npc1 locus. These results suggest that HDAC inhibitors have utility to treat visceral NP-C disease. However, it is clear that improved blood-brain barrier penetration will be required to alleviate the neurological symptoms of human NP-C disease.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Proteínas/genética , Animais , Apolipoproteínas B/metabolismo , Células Cultivadas , Colesterol/genética , Colesterol/metabolismo , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/farmacocinética , Homeostase/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/patologia , Doença de Niemann-Pick Tipo C/fisiopatologia , Proteínas/metabolismo , Transcriptoma/efeitos dos fármacos , VorinostatRESUMO
Niemann-Pick type C (NP-C) disease is a fatal lysosomal lipid storage disorder for which no effective therapy exists. A genome-wide, conditional synthetic lethality screen was performed using the yeast model of NP-C disease during anaerobiosis, an auxotrophic condition that requires yeast to utilize exogenous sterol. We identified 12 pathways and 13 genes as modifiers of the absence of the yeast NPC1 ortholog (NCR1) and quantified the impact of loss of these genes on sterol metabolism in ncr1Δ strains grown under viable aerobic conditions. Deletion of components of the yeast NuA4 histone acetyltransferase complex in ncr1Δ strains conferred anaerobic inviability and accumulation of multiple sterol intermediates. Thus, we hypothesize an imbalance in histone acetylation in human NP-C disease. Accordingly, we show that the majority of the 11 histone deacetylase (HDAC) genes are transcriptionally up-regulated in three genetically distinct fibroblast lines derived from patients with NP-C disease. A clinically approved HDAC inhibitor (suberoylanilide hydroxamic acid) reverses the dysregulation of the majority of the HDAC genes. Consequently, three key cellular diagnostic criteria of NP-C disease are dramatically ameliorated as follows: lysosomal accumulation of both cholesterol and sphingolipids and defective esterification of LDL-derived cholesterol. These data suggest HDAC inhibition as a candidate therapy for NP-C disease. We conclude that pathways that exacerbate lethality in a model organism can be reversed in human cells as a novel therapeutic strategy. This "exacerbate-reverse" approach can potentially be utilized in any model organism for any disease.
Assuntos
Colesterol/metabolismo , Lisossomos/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Anaerobiose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Linhagem Celular , Colesterol/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Lisossomos/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esfingolipídeos/genéticaRESUMO
The Niemann-Pick C proteins have slowly emerged as regulators of subcellular lipid transport and sterol absorption at the small intestine. A recent article in Cell Metabolism suggests that in addition to their significant structural and sequence homology, these proteins may orchestrate their functions in a previously unappreciated fashion (Voght et al., 2007).
Assuntos
Transporte Biológico , Drosophila melanogaster/metabolismo , Doenças de Niemann-Pick/metabolismo , Esteróis/farmacocinética , Absorção , Animais , Colesterol/farmacocinética , Camundongos , Modelos BiológicosRESUMO
Numerous studies have established the involvement of lysosomal and mitochondrial dysfunction in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson diseases. Building on our previous studies of the neurodegenerative lysosomal lipidosis Niemann-Pick C1 (NPC1), we have unexpectedly discovered that activation of the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) leads to the correction of the lysosomal storage phenotype in patient cells from multiple lysosomal storage disorders including NPC1. Using small compound activators specific for TRAP1, we find that activation of this chaperone leads to a generalized restoration of lysosomal and mitochondrial health. Mechanistically, we show that this process includes inhibition of oxidative phosphorylation and reduction of oxidative stress, which results in activation of AMPK and ultimately stimulates lysosome recycling. Thus, TRAP1 participates in lysosomal-mitochondrial crosstalk to maintain cellular homeostasis and could represent a potential therapeutic target for multiple disorders.
RESUMO
Niemann-Pick C1-like 1 (NPC1L1) is required for cholesterol absorption. Intestinal NPC1L1 appears to be a target of ezetimibe, a cholesterol absorption inhibitor that effectively lowers plasma LDL-cholesterol in humans. However, human liver also expresses NPC1L1. Hepatic function of NPC1L1 was previously unknown, but we recently discovered that NPC1L1 localizes to the canalicular membrane of primate hepatocytes and that NPC1L1 facilitates cholesterol uptake in hepatoma cells. Based upon these findings, we hypothesized that hepatic NPC1L1 allows the retention of biliary cholesterol by hepatocytes and that ezetimibe disrupts hepatic function of NPC1L1. To test this hypothesis, transgenic mice expressing human NPC1L1 in hepatocytes (L1-Tg mice) were created. Hepatic overexpression of NPC1L1 resulted in a 10- to 20-fold decrease in biliary cholesterol concentration, but not phospholipid and bile acid concentrations. This decrease was associated with a 30%-60% increase in plasma cholesterol, mainly because of the accumulation of apoE-rich HDL. Biliary and plasma cholesterol concentrations in these animals were virtually returned to normal with ezetimibe treatment. These findings suggest that in humans, ezetimibe may reduce plasma cholesterol by inhibiting NPC1L1 function in both intestine and liver, and hepatic NPC1L1 may have evolved to protect the body from excessive biliary loss of cholesterol.
Assuntos
Azetidinas/farmacologia , Bile/efeitos dos fármacos , Bile/metabolismo , Colesterol/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/sangue , Ácidos e Sais Biliares/metabolismo , Membrana Celular/metabolismo , Colesterol/sangue , Ezetimiba , Humanos , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Transgênicos , Fosfolipídeos/metabolismoRESUMO
BACKGROUND INFORMATION: Within the group of lysosomal storage diseases, NPC1 [NPC (Niemann-Pick type C) 1] disease is a lipidosis characterized by excessive accumulation of free cholesterol as well as gangliosides, glycosphingolipids and fatty acids in the late E/L (endosomal/lysosomal) system (Chen et al., 2005) due to a defect in late endosome lipid egress. We have previously demonstrated that expression of the small GTPase Rab9 in NPC1 cells can rescue the lipid transport block phenotype (Walter et al., 2003), albeit by an undefined mechanism. RESULTS: To investigate further the mechanism by which Rab9 facilitates lipid movement from late endosomes we sought to identify novel Rab9 binding/interacting proteins. In the present study, we report that Rab9 interacts with the intermediate filament phosphoprotein vimentin and this interaction is altered by lipid accumulation in late endosomes, which results in inhibition of PKC (protein kinase C) and hypophosphorylation of vimentin, leading to late endosome dysfunction. Intermediate filament hypophosphorylation, aggregation and entrapment of Rab9 ultimately leads to transport defects and inhibition of lipid egress from late endosomes. CONCLUSIONS: These results reveal a previously unappreciated interaction between Rab proteins and intermediate filaments in regulating intracellular lipid transport.
Assuntos
Endossomos/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Proteína Quinase C/metabolismo , Vimentina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular , Colesterol/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Humanos , Filamentos Intermediários/metabolismo , Metabolismo dos Lipídeos , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Fosforilação , Ligação Proteica , Esfingosina/farmacologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Cholesterol accumulation in the endosomes and lysosomes of Niemann-Pick C (NPC) cells is considered to be the hallmark of this disorder, so the main focus of NPC research has revolved around cholesterol and its role in disease pathogenesis. However, recent data indicate that cholesterol is not the primary culprit in this human lipidosis. I propose a new hypothesis for the potential action or function of the NPC1 protein in the endosome. In this context, the relationship of NPC2 and NPC1 is also discussed.
Assuntos
Proteínas de Transporte/fisiologia , Colesterol/farmacocinética , Glicoproteínas de Membrana/fisiologia , Doenças de Niemann-Pick/genética , Doenças de Niemann-Pick/fisiopatologia , Colesterol/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Químicos , Proteína C1 de Niemann-PickRESUMO
OBJECTIVE: Activation of liver x receptor (LXR) raises plasma HDL-cholesterol (HDL-C) in mice. Interestingly, the LXR agonist GW3965 fails to raise plasma HDL-C in mice lacking intestinal ABCA1, indicating that intestinal ABCA1 plays a predominant role in GW3965-mediated HDL production. How this is coupled to intestinal function remains elusive. Because cholesterol is essential for HDL assembly and directly regulates intestinal ABCA1 expression via activating LXR, we hypothesized that cholesterol absorption, a major function of intestine, modulates LXR-dependent HDL formation. METHODS AND RESULTS: Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (L1-KO mice), a gene that is essential for cholesterol absorption, were treated with LXR agonist T0901317 for 7 days. Intriguingly, this treatment failed to significantly raise plasma HDL-C but caused a much greater fecal cholesterol excretion in L1-KO mice. The intestinal ABCA1 mRNA level was about 4-fold lower in L1-KO versus wild-type mice, and increased 3.9-fold and 8.8-fold after T0901317 treatment in wild-type and L1-KO mice, respectively. Hepatic ABCA1 failed to respond to T0901317 in mice of both genotypes, although hepatic mRNAs for many LXR target genes were higher in the T0901317-treated versus untreated wild-type animals. CONCLUSIONS: NPC1L1 is required for an LXR agonist to increase plasma HDL-C in mice.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Benzoatos/farmacologia , Benzilaminas/farmacologia , HDL-Colesterol/efeitos dos fármacos , HDL-Colesterol/metabolismo , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Transportadores de Cassetes de Ligação de ATP/genética , Análise de Variância , Animais , Análise Química do Sangue , HDL-Colesterol/sangue , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Injeções Intraperitoneais , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Niemann-Pick Tipo C/sangue , Doença de Niemann-Pick Tipo C/genética , Receptores Nucleares Órfãos , Probabilidade , Distribuição Aleatória , Receptores Citoplasmáticos e Nucleares , Sensibilidade e EspecificidadeRESUMO
Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin-6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1-/- mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1-/- mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1-/- mouse brain.
Assuntos
Citocinas/metabolismo , Endossomos/metabolismo , Fibroblastos/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Fatores de Transcrição STAT/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Células Cultivadas , Meios de Cultura/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Interferon beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Longevidade , Camundongos , Camundongos Knockout , Neuroglia , Doença de Niemann-Pick Tipo C/patologia , Doença de Niemann-Pick Tipo C/fisiopatologiaRESUMO
Recent studies have documented the importance of Niemann-Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1-/- mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1-/- mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1-/- mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1.
Assuntos
Anticolesterolemiantes/farmacologia , Azetidinas/farmacologia , Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores Depuradores Classe B/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Anticolesterolemiantes/química , Apolipoproteína A-I/farmacologia , Azetidinas/química , Relação Dose-Resposta a Droga , Ezetimiba , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/ultraestrutura , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Micelas , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Receptores Depuradores Classe B/genéticaRESUMO
We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann-Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.
Assuntos
Anticolesterolemiantes/farmacologia , Azetidinas/farmacologia , Colesterol/metabolismo , Mucosa Intestinal , Intestinos , Proteínas de Membrana Transportadoras/metabolismo , Microvilosidades , Animais , Anticolesterolemiantes/química , Azetidinas/química , Membrana Celular/metabolismo , Ezetimiba , Mucosa Intestinal/metabolismo , Intestinos/anatomia & histologia , Intestinos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.
Assuntos
Antineoplásicos/farmacologia , Ativadores de Enzimas/farmacologia , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Ativadores de Enzimas/química , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Ligação Proteica , Proteína Fosfatase 2/química , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NPC (Niemann-Pick type C) disease is a rare lipidosis characterized by the accumulation of LDL (low-density lipoprotein)-derived non-esterified cholesterol in the E/L (endosomal/lysosomal) system. The gene products that are responsible for the two NPC complementation groups are distinct and dissimilar, yet their cellular and disease phenotypes are virtually indistinguishable. To investigate the relationship between NPC1 and NPC2 and their potential role in NPC disease pathogenesis, we have developed a method for the rapid and efficient isolation of late endocytic vesicles from mouse liver by magnetic chromatography. Late endosomes from Wt (wild-type) and NPC1 mice were found to differ not only in their cholesterol and sphingomyelin content, as expected, but also in their non-esterified ('free') fatty acid content, with NPC1 vesicles showing an approx. 7-fold increase in non-esterified fatty acid levels compared with Wt vesicles. Furthermore, we show that the NPC2 protein is in an incompletely deglycosylated form in NPC1 late endosomes by a mechanism that is specific to the NPC2 protein and not a global aberration of protein glycosylation/deglycosylation or trafficking, since NPC2 secreted from NPC1 cells is indistinguishable from that secreted from Wt cells. Also, a greater proportion of the normally soluble cellular NPC2 protein partitions with detergent-insoluble late endosomal internal membrane domains in NPC1 vesicles. In addition, we show that, although a small amount of the NPC2 protein associates with these membranes in Wt vesicles, this localization becomes much more pronounced in NPC1 vesicles. These results suggest that the function of the NPC2 protein may be compromised as well in NPC1 endosomes, which might explain the paradoxical phenotypic similarities of the two NPC disease complementation groups.
Assuntos
Endossomos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Proteínas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Glicosilação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Proteína C1 de Niemann-Pick , Doenças de Niemann-Pick/metabolismoRESUMO
Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of alpha-galactosidase A, using the lower dose of Ad2/CMVHI-alpha gal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-alpha gal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-alpha gal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-alpha gal (10(9) particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of alpha-galactosidase A in the liver.
Assuntos
Adenoviridae/genética , Doença de Fabry/terapia , Terapia Genética , Vetores Genéticos , Animais , Ácido Clodrônico/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Células de Kupffer/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Transdução Genética , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , gama-Globulinas/farmacologiaRESUMO
BACKGROUND: Congenital absence of alpha-galactosidase in Fabry disease leads eventually to renal failure. Fabry disease is an attractive candidate for gene therapy, but uptake mechanisms of the enzyme must be understood for it to be used in treating patients with Fabry disease. METHODS: Immortalized human renal epithelial cells from three regions of the tubule were grown in culture on collagen-coated Transwell filters and were incubated with recombinant alpha-galactosidase protein placed at either the luminal or basolateral side of the cells. Uptake into cells was measured, and kinetic studies were performed. Blocking experiments were done with mannose 6-phosphate. RESULTS: Uptake from the basolateral side of the filters predominated in all three cell types. Only in distal tubule cells was mannose 6-phosphate able to block uptake to any degree. The kinetic data reveal a high Km for both luminal and basolateral cell surfaces. CONCLUSIONS: These data suggest that to correct the renal phenotype in Fabry disease, high levels of the enzyme will be need to be delivered to kidney cells. This will likely best be achieved with local administration of a vector containing the transgene directly to the kidney.
Assuntos
Polaridade Celular , Túbulos Renais/citologia , Túbulos Renais/metabolismo , alfa-Galactosidase/metabolismo , Células Cultivadas , HumanosRESUMO
Gene therapy has become the next frontier in the treatment and potential cure of many disorders that are refractive to current therapies. The lysosomal storage disorders (LSDs) collectively constitute one of the largest groups of inherited metabolic disorders. Propelled by the exciting success of enzyme replacement therapies applied to LSDs without neuropathology, the development of effective gene therapy protocols for the LSDs is underway. For the LSDs with neuropathology, in particular, it has become clear that gene therapy is at present one of only a few therapeutic options with the potential for success. Studies summarised in this review indicate that gene therapy using a variety of vectors both in vivo and ex vivo have shown great promise for the treatment of these diseases. However, several problems require serious attention before it will be feasible to embark on human gene therapy trials.
Assuntos
Terapia Genética , Doenças por Armazenamento dos Lisossomos/terapia , Animais , Modelos Animais de Doenças , Terapia Enzimática , Vetores Genéticos , Humanos , Lisossomos/fisiologiaRESUMO
Activation of protein kinase C (PKC) has previously been shown to ameliorate the cholesterol transport defect in Niemann Pick Type C1 (NPC1) cells, presumably by increasing the soluble levels of one of its substrates, vimentin. This activity would then restore the vimentin cycle in these cells and allow vimentin-dependent retrograde transport to proceed. Here, we further investigate the effects of PKC activation in NPC1 cells by evaluating different isoforms for their ability to solubilize vimentin and correct the NPC1 cholesterol storage phenotype. We also examine the effects of PKC activators, including free fatty acids and the PKC-specific activator diazoxide, on the NPC1 disease phenotype. Our results indicate that PKC isoforms α, ßII, and ε have the greatest effects on vimentin solubilization. Furthermore, expression or activation of PKCε in NPC1 cells dramatically reduces the amount of stored cholesterol and restores cholesterol transport out of endocytic vesicles. These results provide further support for the contribution of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.
Assuntos
Colesterol/metabolismo , Proteína Quinase C/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Extratos Celulares , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Esterificação/efeitos dos fármacos , Ácidos Graxos/farmacologia , Doença de Niemann-Pick Tipo C/enzimologia , Fenótipo , Solubilidade , Esfingolipídeos/metabolismo , Vimentina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
MLN64 is an integral membrane protein localized to the late endosome and plasma membrane that is thought to function as a mediator of cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria. The protein consists of two distinct domains: an N-terminal membrane-spanning domain that shares homology with the MENTHO protein and a C-terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain that binds cholesterol. To further characterize the MLN64 protein, full-length and truncated proteins were overexpressed in cells and the effects on MLN64 trafficking and endosomal morphology were observed. To gain insight into MLN64 function, affinity chromatography and mass spectrometric techniques were used to identify potential MLN64 interacting partners. Of the 15 candidate proteins identified, 14-3-3 was chosen for further characterization. We show that MLN64 interacts with 14-3-3 in vitro as well as in vivo and that the strength of the interaction is dependent on the 14-3-3 isoform. Furthermore, blocking the interaction through the use of a 14-3-3 antagonist or MLN64 mutagenesis delays the trafficking of MLN64 to the late endosome and also results in the dispersal of endocytic vesicles to the cell periphery. Taken together, these studies have determined that MLN64 is a novel 14-3-3 binding protein and indicate that 14-3-3 plays a role in the endosomal trafficking of MLN64. Furthermore, these studies suggest that 14-3-3 may be the link by which MLN64 exerts its effects on the actin-mediated endosome dynamics.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas 14-3-3/genética , Animais , Sítios de Ligação , Células COS , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Espectrometria de Massas , Proteínas de Membrana/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologiaRESUMO
Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- ß-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders.
Assuntos
Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Lisossomos/metabolismo , beta-Ciclodextrinas/farmacologia , 2-Hidroxipropil-beta-Ciclodextrina , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Excipientes/farmacologia , Exossomos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Proteína C1 de Niemann-Pick , Proteínas/genética , Proteínas/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (NPC1L1(-/-)mice) exhibit a defect in intestinal absorption of cholesterol and phytosterols. However, wild-type (WT) mice do not efficiently absorb and accumulate phytosterols either. Cell-based studies show that NPC1L1 is a much weaker transporter for phytosterols than cholesterol. In this study, we examined the role of NPC1L1 in phytosterol and cholesterol trafficking in mice lacking ATP-binding cassette (ABC) transporters G5 and G8 (G5/G8(-/-) mice). G5/G8(-/-) mice develop sitosterolemia, a genetic disorder characterized by the accumulation of phytosterols in blood and tissues. We found that mice lacking ABCG5/G8 and NPC1L1 [triple knockout (TKO) mice] did not accumulate phytosterols in plasma and the liver. TKO mice, like G5/G8(-/-) mice, still had a defect in hepatobiliary cholesterol secretion, which was consistent with TKO versus NPC1L1(-/-) mice exhibiting a 52% reduction in fecal cholesterol excretion. Because fractional cholesterol absorption was reduced similarly in NPC1L1(-/-) and TKO mice, by subtracting fecal cholesterol excretion in TKO mice from NPC1L1(-/-) mice, we estimated that a 25g NPC1L1(-/-) mouse may secrete about 4 mumol of cholesterol daily via the G5/G8 pathway. In conclusion, NPC1L1 is essential for phytosterols to enter the body in mice.