Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
J Cell Sci ; 126(Pt 6): 1345-54, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23418355

RESUMO

Prion diseases are fatal neurodegenerative disorders involving the abnormal folding of a native cellular protein, named PrP(C), to a malconformed aggregation-prone state, enriched in beta sheet secondary structure, denoted PrP(Sc). Recently, autophagy has garnered considerable attention as a cellular process with the potential to counteract neurodegenerative diseases of protein aggregation such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Stimulation of autophagy by chemical compounds has also been shown to reduce PrP(Sc) in infected neuronal cells and prolong survival times in mouse models. Consistent with previous reports, we demonstrate that autophagic flux is increased in chronically infected cells. However, in contrast to recent findings we show that autophagy does not cause a reduction in scrapie burden. We report that in infected neuronal cells different compounds known to stimulate autophagy are ineffective in increasing autophagic flux and in reducing PrP(Sc). We further demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen lead to prion degradation in an autophagy-independent manner by diverting the trafficking of both PrP and cholesterol to lysosomes. Our data indicate that tamoxifen, a well-characterized, widely available pharmaceutical, may have applications in the therapy of prion diseases.


Assuntos
Lisossomos/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/tratamento farmacológico , Tamoxifeno/análogos & derivados , Animais , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Linhagem Celular , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças Priônicas/metabolismo , Transporte Proteico , Proteólise , RNA Interferente Pequeno/genética , Ratos , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico
2.
J Cell Sci ; 126(Pt 16): 3678-85, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23781027

RESUMO

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by CAG expansion in the huntingtin gene, which adds a homopolymeric tract of polyglutamine (polyQ) to the encoded protein leading to the formation of toxic aggregates. Despite rapidly accumulating evidences supporting a role for intercellular transmission of protein aggregates, little is known about whether and how huntingtin (Htt) misfolding progresses through the brain. It has been recently reported that synthetic polyQ peptides and recombinant fragments of mutant Htt are readily internalized in cell cultures and able to seed polymerization of a reporter wild-type Htt. However, there is no direct evidence of aggregate transfer between cells and the mechanism has not been explored. By expressing recombinant fragments of mutant Htt in neuronal cells and in primary neurons, we found that aggregated fragments formed within one cell spontaneously transfer to neighbors in cell culture. We demonstrate that the intercellular spreading of the aggregates requires cell-cell contact and does not occur upon aggregate secretion. Interestingly, we found that the expression of mutant, but not wild-type Htt fragments, increases the number of tunneling nanotubes, which in turn provide an efficient mechanism of transfer.


Assuntos
Nanotubos , Neurônios/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Camundongos , Neurônios/citologia , Peptídeos/genética , Transfecção
3.
Biol Cell ; 105(5): 219-233, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23398201

RESUMO

BACKGROUND INFORMATION: In animals, adipose tissue contains the main energy store as lipid droplets (LDs) composed of esterified cholesterol (CE) and triacylglycerol (TAG) enveloped in a mono-layer of phospholipid and decorated by a coat of proteins. Upon increased energy demand, dedicated lipases hydrolyse TAG stepwise into free fatty acids that are released in circulation and made available to peripheral tissue. In case of aberrant caloric load, TAGs are deposited into non-adipocyte tissues, primarily liver cells. For instance, non-alcoholic fatty liver disease (NAFLD) is a common chronic disorder characterised by an excess of TAG in the liver of patients regardless of their susceptibility to obesity, diabetes or exposure to alcohol. Several independent linkage studies have associated NAFLD with a non-synonymous variant of patatin-like phospholipase domain-containing 3 (PNPLA3/adiponutrin) encoding an isoleucine to methionine substitution at position 148 (I148M) (see Cohen et al., 2011 for review). However, the mechanism by which a variation in PNPLA3 gives susceptibility to NAFLD is not known, primarily because the physiological role of PNPLA3 still needs to be elucidated. RESULTS: We have identified PNPLA3 in a screen for genes upregulated by intracellular lipid accumulation. We investigated the sub-cellular distribution and potential function of PNPLA3 in fibroblast-like cells supplemented with lipids. We demonstrate that PNPLA3 is targetted to LDs in a process that requires an intact Brummer box domain, which is conserved in the patatin-like phospholipase family. We show that increased levels of the NAFLD-linked PNPLA3 isoform leads to larger LDs, whereas decreased levels of PNPLA3 had the opposite effect. Interestingly, however, PNPLA3 induced a reduction in LD size upon co-expression with ABDH5/CGI-58, an activator of the TAG lipase PNPLA2, which is the closest homolog of PNPLA3. By investigating LD populations according to their size and composition, we show that perturbing intracellular lipid trafficking drastically modifies LD nature. CONCLUSIONS: Taken together, our results suggest that PNPLA3 exhibits a dual function in LD metabolism, and that it participates in the restoration of lipid homeostasis upon aberrant intracellular lipid accumulation.


Assuntos
Lipase/fisiologia , Metabolismo dos Lipídeos , Proteínas de Membrana/fisiologia , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Androstenos/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Cricetinae , Expressão Gênica , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/fisiologia , Lipase/química , Lipase/metabolismo , Proteínas de Membrana/química , Organelas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico
4.
Neurobiol Dis ; 47(1): 126-34, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22516235

RESUMO

The retromer complex plays an important role in intracellular transport, is highly expressed in the hippocampus, and has been implicated in the trafficking of the amyloid precursor protein (APP). Nevertheless, the trafficking routes of the neuronal retromer and the role it plays in APP transport in neuronal processes remain unknown. Here we use hippocampal neuronal cultures to address these issues. Using fluorescence microscopy, we find that Vps35, the core element of the retromer complex, is in dendrites and axons, is enriched in endosomes and trans-Golgi network, and is found in APP-positive vesicles. Next, to identify the role the neuronal retromer plays in cargo transport, we infected hippocampal neurons with a lentivirus expressing shRNA to silence Vps35. By live fluorescence imaging, Vps35 deficiency was found to reduce the frequency, but not the kinetics, of long-range APP transport within neuronal processes. Supporting the interpretation that retromer promotes long-range transport, Vps35 deficiency led to increased APP in the early endosomes, in processes but not the soma. Finally, Vps35 deficiency was associated with increased levels of Aß, a cleaved product of APP, increased colocalization of APP with its cleaving enzyme BACE1 in processes, and caused an enlargement of early endosomes. Taken together, our studies clarify the function of the neuronal retromer, and suggest specific mechanisms for how retromer dysfunction observed in Alzheimer's disease affects APP transport and processing.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Hipocampo/metabolismo , Humanos , Transporte Proteico
5.
Nat Commun ; 4: 2250, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23907271

RESUMO

Defects in endosomal sorting have been implicated in Alzheimer's disease. Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate, a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that phosphatidylinositol-3-phosphate is selectively deficient in brain tissue from humans with Alzheimer's disease and Alzheimer's disease mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein in these organelles and reduces amyloid precursor protein sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway, including the phosphatidylinositol-3-phosphate effector Hrs and Tsg101. Amyloid precursor protein is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of amyloid precursor protein to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing phosphatidylinositol-3-phosphate deficiency as a contributing factor in Alzheimer's disease, these results clarify the mechanisms of amyloid precursor protein trafficking through the endosomal system in normal and pathological states.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Endossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Processamento de Proteína Pós-Traducional , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/ultraestrutura , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Transporte Proteico , Frações Subcelulares/metabolismo , Ubiquitinação
6.
J Biol Chem ; 283(41): 27871-27880, 2008 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-18644787

RESUMO

Most cell types acquire cholesterol by endocytosis of circulating low density lipoprotein, but little is known about the mechanisms of intra-endosomal cholesterol transport and about the primary cause of its aberrant accumulation in the cholesterol storage disorder Niemann-Pick type C (NPC). Here we report that lysobisphosphatidic acid (LBPA), an unconventional phospholipid that is only detected in late endosomes, regulates endosomal cholesterol levels under the control of Alix/AlP1, which is an LBPA-interacting protein involved in sorting into multivesicular endosomes. We find that Alix down-expression decreases both LBPA levels and the lumenal vesicle content of late endosomes. Cellular cholesterol levels are also decreased, presumably because the storage capacity of endosomes is affected and thus cholesterol clearance accelerated. Both lumenal membranes and cholesterol can be restored in Alix knockdown cells by exogenously added LBPA. Conversely, we also find that LBPA becomes limiting upon pathological cholesterol accumulation in NPC cells, because the addition of exogenous LBPA, but not of LBPA isoforms or analogues, partially reverts the NPC phenotype. We conclude that LBPA controls the cholesterol capacity of endosomes.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Colesterol/metabolismo , Endocitose , Endossomos/metabolismo , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Colesterol/genética , Cricetinae , Endocitose/efeitos dos fármacos , Endocitose/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/genética , Endossomos/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Lisofosfolipídeos/farmacologia , Monoglicerídeos/farmacologia , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA