RESUMO
Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.
Assuntos
Degeneração Hepatolenticular , Animais , Humanos , Degeneração Hepatolenticular/genética , Degeneração Hepatolenticular/tratamento farmacológico , Degeneração Hepatolenticular/metabolismo , Cobre/toxicidade , Cobre/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Hepatócitos/metabolismoRESUMO
The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.
Assuntos
Oryzias , Osteocondrodisplasias , Animais , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oryzias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Mutação , Osteocondrodisplasias/genéticaRESUMO
Gyrate atrophy of choroid and retina (GACR) is a chorioretinal degeneration caused by pathogenic variants in the gene encoding ornithine aminotransferase (OAT), an enzyme mainly expressed in liver. Affected patients have increased ornithine concentrations in blood and other body fluids and develop progressive constriction of vision fields leading to blindness. Current therapies are unsatisfactory and better treatments are highly needed. In two mouse models of OAT deficiency that recapitulates biochemical and retinal changes of GACR, we investigated the efficacy of an intravenously injected serotype 8 adeno-associated (AAV8) vector expressing OAT under the control of a hepatocyte-specific promoter. Following injections, OAT-deficient mice showed reductions of ornithine concentrations in blood and eye cups compared with control mice injected with a vector expressing green fluorescent protein. AAV-injected mice showed improved electroretinogram response and partial restoration of retinal structure up to one-year post-injection. In summary, hepatic OAT expression by AAV8 vector was effective at correction of hyperornithinemia and improved function and structure of the retina. In conclusion, this study provides proof-of-concept of efficacy of liver-directed AAV-mediated gene therapy of GACR.
Assuntos
Atrofia Girata , Degeneração Retiniana , Animais , Camundongos , Atrofia Girata/genética , Atrofia Girata/patologia , Ornitina-Oxo-Ácido Transaminase/genética , Ornitina-Oxo-Ácido Transaminase/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Ornitina/genética , Ornitina/metabolismo , Terapia Genética , Fígado/patologiaRESUMO
Cysteamine is currently the only therapy for nephropathic cystinosis. It significantly improves life expectancy and delays progression to end-stage kidney disease; however, it cannot prevent it. Unfortunately, compliance to therapy is often weak, particularly during adolescence. Therefore, finding better treatments is a priority in the field of cystinosis. Previously, we found that genistein, an isoflavone particularly enriched in soy, can revert part of the cystinotic cellular phenotype that is not sensitive to cysteamine in vitro. To test the effects of genistein in vivo, we fed 2-month-old wild-type and Ctns-/- female mice with either a control diet, a genistein-containing diet or a cysteamine-containing diet for 14 months. Genistein (160 mg/kg/day) did not affect the growth of the mice or hepatic functionality. Compared with untreated mice at 16 months, Ctns-/- mice fed with genistein had lower cystine concentrations in their kidneys, reduced formation of cystine crystals, a smaller number of LAMP1-positive structures and an overall better-preserved parenchymal architecture. Cysteamine (400 mg/kg/day) was efficient in reverting the lysosomal phenotype and in preventing the development of renal lesions. These preclinical data indicate that genistein ameliorates kidney injury resulting from cystinosis with no side effects. Genistein therapy represents a potential treatment to improve the outcome for patients with cystinosis.
Assuntos
Cistinose , Nefropatias , Animais , Feminino , Camundongos , Cisteamina/uso terapêutico , Cistina/uso terapêutico , Cistinose/tratamento farmacológico , Cistinose/genética , Modelos Animais de Doenças , Genisteína/farmacologia , Genisteína/uso terapêutico , RimRESUMO
Endosomal trafficking is essential for cellular homeostasis. At the crossroads of distinct intracellular pathways, the endolysosomal system is crucial to maintain critical functions and adapt to the environment. Alterations of endosomal compartments were observed in cells from adult individuals with Down syndrome (DS), suggesting that the dysfunction of the endosomal pathway may contribute to the pathogenesis of DS. However, the nature and the degree of impairment, as well as the timing of onset, remain elusive. Here, by applying imaging and biochemical approaches, we demonstrate that the structure and dynamics of early endosomes are altered in DS cells. Furthermore, we found that recycling trafficking is markedly compromised in these cells. Remarkably, our results in 18-20 week-old human fetal fibroblasts indicate that alterations in the endolysosomal pathway are already present early in development. In addition, we show that overexpression of the polyphosphoinositide phosphatase synaptojanin 1 (Synj1) recapitulates the alterations observed in DS cells, suggesting a role for this lipid phosphatase in the pathogenesis of DS, likely already early in disease development. Overall, these data strengthen the link between the endolysosomal pathway and DS, highlighting a dangerous liaison among Synj1, endosomal trafficking and DS.
RESUMO
Three main approaches are used to combat severe viral respiratory infections. The first is preemptive vaccination that blocks infection. Weakened or dead viral particles, as well as genetic constructs carrying viral proteins or information about them, are used as an antigen. However, the viral genome is very evolutionary labile and changes continuously. Second, chemical agents are used during infection and inhibit the function of a number of viral proteins. However, these drugs lose their effectiveness because the virus can rapidly acquire resistance to them. The third is the search for points in the host metabolism the effect on which would suppress the replication of the virus but would not have a significant effect on the metabolism of the host. Here, we consider the possibility of using the copper metabolic system as a target to reduce the severity of influenza infection. This is facilitated by the fact that, in mammals, copper status can be rapidly reduced by silver nanoparticles and restored after their cancellation.
Assuntos
Cobre/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Ceruloplasmina/fisiologia , Proteínas de Transporte de Cobre/metabolismo , ATPases Transportadoras de Cobre/fisiologia , Farmacorresistência Viral , Interações Hospedeiro-Patógeno , Humanos , Vacinas contra Influenza , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Mamíferos/metabolismo , Nanopartículas Metálicas/uso terapêutico , Chaperonas Moleculares/metabolismo , Proteínas PrPC/fisiologia , RNA Viral/fisiologia , Prata/uso terapêutico , Superóxido Dismutase-1/fisiologia , Proteínas Virais/fisiologia , Replicação ViralRESUMO
Recent studies highlight the continued growth in the identification of a variety of cellular functions that involve the Golgi apparatus. Apart from well-known membrane sorting/trafficking and glycosylation machineries, the Golgi harbors molecular platforms operating in intracellular signaling, cytoskeleton organization, and protein quality control mechanisms. One of new emerging Golgi functions consists in the regulation of copper homeostasis by coordinating the relocation and activity of copper transporters. Of these, the Cu-transporting ATPase ATP7B (known as Wilson disease protein) plays a key role in the maintenance of the Cu balance in the body via the supply of essential Cu to the systemic circulation and via elimination of excess Cu into the bile. These activities require tightly regulated shuttling of ATP7B between the Golgi and different post-Golgi compartments. Despite significant progress over recent years, a number of issues regarding ATP7B trafficking remain to be clarified. This review summarizes current views on ATP7B trafficking pathways from and to the Golgi and underscores the challenges that should be addressed to define the ATP7B trafficking routes and mechanisms in health and disease.
Assuntos
ATPases Transportadoras de Cobre/metabolismo , Complexo de Golgi/metabolismo , Animais , Humanos , Transporte ProteicoRESUMO
BACKGROUND: Geleophysic dysplasia (GPHYSD) is a disorder characterized by dysmorphic features, stiff joints and cardiac involvement due to defects of TGF-ß signaling. GPHYSD can be caused by mutations in FBN1, ADAMTLS2, and LTBP3 genes. METHODS AND RESULTS: Consistent with previous reports, we found intracellular inclusions of unknown material by electron microscopy (EM) in skin fibroblasts of two GPHYSD individuals carrying FBN1 mutations. Moreover, we found that the storage material is enclosed within lysosomes and is associated with the upregulation of several lysosomal genes. Treatment of GPHYSD fibroblasts carrying FBN1 mutations with the angiotensin II receptor type 1 inhibitor losartan that inhibits TGF-ß signaling did not reduce the storage but improved the extracellular deposition of fibrillin-1 microfibrils. CONCLUSION: Losartan is a promising candidate drug for treatment of GPHYSD due to FBN1 defects.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Fibrilina-1/genética , Fibroblastos/metabolismo , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/metabolismo , Losartan/farmacologia , Lisossomos/metabolismo , Microfibrilas/metabolismo , Pele/metabolismo , Pele/patologia , Adolescente , Doenças do Desenvolvimento Ósseo/patologia , Criança , Pré-Escolar , Matriz Extracelular , Feminino , Fibroblastos/ultraestrutura , Humanos , Lactente , Deformidades Congênitas dos Membros/patologia , Masculino , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
In humans, copper is an important micronutrient because it is a cofactor of ubiquitous and brain-specific cuproenzymes, as well as a secondary messenger. Failure of the mechanisms supporting copper balance leads to the development of neurodegenerative, oncological, and other severe disorders, whose treatment requires a detailed understanding of copper metabolism. In the body, bioavailable copper exists in two stable oxidation states, Cu(I) and Cu(II), both of which are highly toxic. The toxicity of copper ions is usually overcome by coordinating them with a wide range of ligands. These include the active cuproenzyme centers, copper-binding protein motifs to ensure the safe delivery of copper to its physiological location, and participants in the Cu(I) â Cu(II) redox cycle, in which cellular copper is stored. The use of modern experimental approaches has allowed the overall picture of copper turnover in the cells and the organism to be clarified. However, many aspects of this process remain poorly understood. Some of them can be found out using abiogenic silver ions (Ag(I)), which are isoelectronic to Cu(I). This review covers the physicochemical principles of the ability of Ag(I) to substitute for copper ions in transport proteins and cuproenzyme active sites, the effectiveness of using Ag(I) to study copper routes in the cells and the body, and the limitations associated with Ag(I) remaining stable in only one oxidation state. The use of Ag(I) to restrict copper transport to tumors and the consequences of large-scale use of silver nanoparticles for human health are also discussed.
Assuntos
Cobre/metabolismo , Íons/metabolismo , Prata/metabolismo , Transporte Biológico , Humanos , Nanopartículas MetálicasRESUMO
Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Inteínas , Retina/virologia , Trans-Splicing/genética , Animais , Vetores Genéticos/administração & dosagem , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Organoides/ultraestrutura , Organoides/virologia , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/virologia , SuínosRESUMO
Phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide with key roles in the Golgi complex, is made by Golgi-associated phosphatidylinositol-4 kinases and consumed by the 4-phosphatase Sac1 that, instead, is an ER membrane protein. Here, we show that the contact sites between the ER and the TGN (ERTGoCS) provide a spatial setting suitable for Sac1 to dephosphorylate PI4P at the TGN. The ERTGoCS, though necessary, are not sufficient for the phosphatase activity of Sac1 on TGN PI4P, since this needs the phosphatidyl-four-phosphate-adaptor-protein-1 (FAPP1). FAPP1 localizes at ERTGoCS, interacts with Sac1, and promotes its in-trans phosphatase activity in vitro. We envision that FAPP1, acting as a PI4P detector and adaptor, positions Sac1 close to TGN domains with elevated PI4P concentrations allowing PI4P consumption. Indeed, FAPP1 depletion induces an increase in TGN PI4P that leads to increased secretion of selected cargoes (e.g., ApoB100), indicating that FAPP1, by controlling PI4P levels, acts as a gatekeeper of Golgi exit.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Células HeLa , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Camundongos , Fosfatos de Fosfatidilinositol/genéticaRESUMO
BACKGROUND & AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD. METHODS: We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy. RESULTS: After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.
Assuntos
Apoptose , Autofagia/genética , ATPases Transportadoras de Cobre/genética , Hepatócitos/fisiologia , Degeneração Hepatolenticular/fisiopatologia , Fígado/fisiopatologia , Animais , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Benzilaminas/farmacologia , Sobrevivência Celular , Cobre/toxicidade , ATPases Transportadoras de Cobre/metabolismo , Feminino , Células Hep G2 , Hepatócitos/ultraestrutura , Humanos , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Transporte Proteico , Quinazolinas/farmacologia , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND & AIMS: In Wilson disease, ATP7B mutations impair copper excretion into bile. Hepatic copper accumulation may induce mild to moderate chronic liver damage or even acute liver failure. Etiologic factors for this heterogeneous phenotype remain enigmatic. Liver steatosis is a frequent finding in Wilson disease patients, suggesting that impaired copper homeostasis is linked with liver steatosis. Hepatic mitochondrial function is affected negatively both by copper overload and steatosis. Therefore, we addressed the question of whether a steatosis-promoting high-calorie diet aggravates liver damage in Wilson disease via amplified mitochondrial damage. METHODS: Control Atp7b+/- and Wilson disease Atp7b-/- rats were fed either a high-calorie diet (HCD) or a normal diet. Copper chelation using the high-affinity peptide methanobactin was used in HCD-fed Atp7b-/- rats to test for therapeutic reversal of mitochondrial copper damage. RESULTS: In comparison with a normal diet, HCD feeding of Atp7b-/- rats resulted in a markedly earlier onset of clinically apparent hepatic injury. Strongly increased mitochondrial copper accumulation was observed in HCD-fed Atp7b-/- rats, correlating with severe liver injury. Mitochondria presented with massive structural damage, increased H2O2 emergence, and dysfunctional adenosine triphosphate production. Hepatocellular injury presumably was augmented as a result of oxidative stress. Reduction of mitochondrial copper by methanobactin significantly reduced mitochondrial impairment and ameliorated liver damage. CONCLUSIONS: A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats, causing an earlier onset of the disease and enhanced disease progression.
Assuntos
Dieta , Degeneração Hepatolenticular/patologia , Fígado/patologia , Mitocôndrias/patologia , Animais , Ácidos e Sais Biliares/biossíntese , Cobre/sangue , ATPases Transportadoras de Cobre/metabolismo , Progressão da Doença , Fígado Gorduroso/patologia , Feminino , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Degeneração Hepatolenticular/sangue , Inflamação/patologia , Lipídeos/biossíntese , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Peptídeos/farmacologia , Proteoma/metabolismo , RatosRESUMO
Nephropathic cystinosis is a rare lysosomal storage disorder caused by mutations in CTNS gene leading to Fanconi syndrome. Independent studies reported defective clearance of damaged mitochondria and mitochondrial fragmentation in cystinosis. Proteins involved in the mitochondrial dynamics and the mitochondrial ultrastructure were analyzed in CTNS-/- cells treated with cysteamine, the only drug currently used in the therapy for cystinosis but ineffective to treat Fanconi syndrome. CTNS-/- cells showed an overexpression of parkin associated with deregulation of ubiquitination of mitofusin 2 and fission 1 proteins, an altered proteolytic processing of optic atrophy 1 (OPA1), and a decreased OPA1 oligomerization. According to molecular findings, the analysis of electron microscopy images showed a decrease of mitochondrial cristae number and an increase of cristae lumen and cristae junction width. Cysteamine treatment restored the fission 1 ubiquitination, the mitochondrial size, number and lumen of cristae, but had no effect on cristae junction width, making CTNS-/- tubular cells more susceptible to apoptotic stimuli.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Cisteamina/farmacologia , Cistinose/genética , Mitocôndrias/metabolismo , Células Cultivadas , Cistinose/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
H1069Q substitution represents the most frequent mutation of the copper transporter ATP7B causing Wilson disease in Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes but moves in response to copper overload to the endo-lysosomal compartment to support copper excretion via bile canaliculi. In heterologous or hepatoma-derived cell lines, overexpressed ATP7B-H1069Q is strongly retained in the ER and fails to move to the post-Golgi sites, resulting in toxic copper accumulation. However, this pathogenic mechanism has never been tested in patients' hepatocytes, while animal models recapitulating this form of WD are still lacking. To reach this goal, we have reprogrammed skin fibroblasts of homozygous ATP7B-H1069Q patients into induced pluripotent stem cells and differentiated them into hepatocyte-like cells. Surprisingly, in HLCs we found one third of ATP7B-H1069Q localized in the Golgi complex and able to move to the endo-lysosomal compartment upon copper stimulation. However, despite normal mRNA levels, the expression of the mutant protein was only 20% compared to the control because of endoplasmic reticulum-associated degradation. These results pinpoint rapid degradation as the major cause for loss of ATP7B function in H1069Q patients, and thus as the primary target for designing therapeutic strategies to rescue ATP7B-H1069Q function.
Assuntos
ATPases Transportadoras de Cobre/genética , Hepatócitos/patologia , Degeneração Hepatolenticular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Polimorfismo de Nucleotídeo Único , Linhagem Celular , Reprogramação Celular , Cobre/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Fibroblastos/citologia , Complexo de Golgi/metabolismo , Degeneração Hepatolenticular/patologia , Humanos , Proteínas Mutantes/metabolismo , Pele/citologiaRESUMO
Pompe disease (PD) is a metabolic myopathy due to acid alpha-glucosidase deficiency and characterized by extensive glycogen storage and impaired autophagy. We previously showed that modulation of autophagy and lysosomal exocytosis by overexpression of the transcription factor EB (TFEB) gene was effective in improving muscle pathology in PD mice injected intramuscularly with an AAV-TFEB vector. Here we have evaluated the effects of TFEB systemic delivery on muscle pathology and on functional performance, a primary measure of efficacy in a disorder like PD. We treated 1-month-old PD mice with an AAV2.9-MCK-TFEB vector. An animal cohort was analyzed at 3 months for muscle and heart pathology. A second cohort was followed at different timepoints for functional analysis. In muscles from TFEB-treated mice we observed reduced PAS staining and improved ultrastructure, with reduced number and increased translucency of lysosomes, while total glycogen content remained unchanged. We also observed statistically significant improvements in rotarod performance in treated animals compared to AAV2.9-MCK-eGFP-treated mice at 5 and 8 months. Cardiac echography showed significant reduction in left-ventricular diameters. These results show that TFEB overexpression and modulation of autophagy result in improvements of muscle pathology and of functional performance in the PD murine model, with delayed disease progression.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Doença de Depósito de Glicogênio Tipo II/terapia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/fisiopatologia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Camundongos , Atividade Motora/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologiaRESUMO
Alterations in mitochondrial activity and morphology have been demonstrated in human cells and tissues from individuals with Down syndrome (DS), as well as in DS mouse models. An impaired activity of the transcriptional coactivator PGC-1α/PPARGC1A due to the overexpression of chromosome 21 genes, such as NRIP1/RIP140, has emerged as an underlying cause of mitochondrial dysfunction in DS. We tested the hypothesis that the activation of the PGC-1α pathway might indeed reverse this mitochondrial dysfunction. To this end, we investigated the effects of metformin, a PGC-1α-activating drug, on mitochondrial morphology and function in DS foetal fibroblasts. Metformin induced both the expression of PGC-1α and an augmentation of its activity, as demonstrated by the increased expression of target genes, strongly promoting mitochondrial biogenesis. Furthermore, metformin enhanced oxygen consumption, ATP production, and overall mitochondrial activity. Most interestingly, this treatment reversed the fragmentation of mitochondria observed in DS and induced the formation of a mitochondrial network with a branched and elongated tubular morphology. Concomitantly, cristae remodelling occurred and the alterations observed by electron microscopy were significantly reduced. We finally demonstrated that the expression of genes of the fission/fusion machinery, namely OPA1 and MFN2, was reduced in trisomic cells and increased by metformin treatment. These results indicate that metformin promotes the formation of a mitochondrial network and corrects the mitochondrial dysfunction in DS cells. We speculate that alterations in the mitochondrial dynamics can be relevant in the pathogenesis of DS and that metformin can efficiently counteract these alterations, thus exerting protective effects against DS-associated pathologies.
Assuntos
Síndrome de Down/genética , Metformina/administração & dosagem , Mitocôndrias/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Trifosfato de Adenosina/biossíntese , Animais , Modelos Animais de Doenças , Síndrome de Down/tratamento farmacológico , Síndrome de Down/patologia , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/biossíntese , Humanos , Camundongos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossínteseRESUMO
BACKGROUND: Nephropathic cystinosis is a lysosomal storage disease that is caused by mutations in the CTNS gene encoding a cystine/proton symporter cystinosin and an isoform cystinosin-LKG which is generated by an alternative splicing of exon 12. We have investigated the physiological role of the cystinosin-LKG that is widely expressed in epithelial tissues. METHODS: We have analyzed the intracellular localization and the function of the cystinosin-LKG conjugated with DsRed (cystinosin-LKG-RFP) in Madin-Darby canine kidney cells (MDCK II) and in proximal tubular epithelial cells carrying a deletion of the CTNS gene (cystinotic PTEC), respectively. RESULTS: Cystinosin-LKG-RFP colocalized with markers of lysosomes, late endosomes and was also expressed on the apical surface of polarized MDCK II cells. Moreover, immune-electron microscopy images of MDCK II cells overexpressing cystinosin-LKG-RFP showed stacked lamellar membranes inside perinuclear lysosomal structures. To study the role of LKG-isoform, we have investigated cystine accumulation and apoptosis that have been described in cystinotic cells. Cystinosin-LKG decreased cystine levels by approximately 10-fold similarly to cystinosin-RFP. The levels of TNFα- and actinomycin D-inducted apoptosis dropped in cystinotic cells expressing LKG-isoform. This effect was also similar to the main isoform. CONCLUSION: Our results suggest that cystinosin-LKG and cystinosin move similar functional activities in cells.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cistina/metabolismo , Cistinose/metabolismo , Cistinose/patologia , Processamento Alternativo , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Apoptose , Células Cultivadas , Cistinose/genética , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Microscopia Eletrônica de Transmissão , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The lysosomal system operates as a focal point where a number of important physiological processes such as endocytosis, autophagy and nutrient sensing converge. One of the key functions of lysosomes consists of regulating the metabolism/homeostasis of metals. Metal-containing components are carried to the lysosome through incoming membrane flows, while numerous transporters allow metal ions to move across the lysosome membrane. These properties enable lysosomes to direct metal fluxes to the sites where metal ions are either used by cellular components or sequestered. Copper belongs to a group of metals that are essential for the activity of vitally important enzymes, although it is toxic when in excess. Thus, copper uptake, supply and intracellular compartmentalization have to be tightly regulated. An increasing number of publications have indicated that these processes involve lysosomes. Here we review studies that reveal the expanding role of the lysosomal system as a hub for the control of Cu homeostasis and for the regulation of key Cu-dependent processes in health and disease.
Assuntos
ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Homeostase/fisiologia , Lisossomos/metabolismo , Animais , HumanosRESUMO
Iron is an essential nutrient for many microbes. According to the "Trojan Horse Hypothesis", biological systems have difficulties distinguishing between Fe(3+) and Ga(3+), which constitutes the antimicrobial efficacy of the gallium(iii) ion. Nine novel tris(quinolono)gallium(iii) complexes and their corresponding iron(iii) analogs have been synthesized and fully characterized. Quinolone antimicrobial agents from three drug generations were used in this study: ciprofloxacin, enoxacin, fleroxacin, levofloxacin, lomefloxacin, nalidixic acid, norfloxacin, oxolinic acid, and pipemidic acid. The antimicrobial efficacy of the tris(quinolono)gallium(iii) complexes was studied against E. faecalis and S. aureus (both Gram-positive), as well as E. coli, K. pneumonia, and P. aeruginosa (all Gram-negative) in direct comparison to the tris(quinolono)iron(iii) complexes and the corresponding free quinolone ligands at various concentrations. For the tris(quinolono)gallium(iii) complexes, no combinational antimicrobial effects between Ga(3+) and the quinolone antimicrobial agents were observed.