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Find video protocols related to scientific articles indexed in Pubmed.
Amyotrophic Lateral Sclerosis-Linked Mutant VAPB Inclusions Do Not Interfere with Protein Degradation Pathways or Intracellular Transport in a Cultured Cell Model.
PLoS ONE
PUBLISHED: 01-01-2014
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VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3?, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3? or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency.
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Glutamate and glutathione interplay in a motor neuronal model of amyotrophic lateral sclerosis reveals altered energy metabolism.
Neurobiol. Dis.
PUBLISHED: 02-16-2011
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Impairment of mitochondrial function might contribute to oxidative stress associated with neurodegeneration in amyotrophic lateral sclerosis (ALS). Glutamate levels in tissues of ALS patients are sometimes altered. In neurons, mitochondrial metabolism of exogenous glutamine is mainly responsible for the net synthesis of glutamate, which is a neurotransmitter, but it is also necessary for the synthesis of glutathione, the main endogenous antioxidant. We investigated glutathione synthesis and glutamine/glutamate metabolism in a motor neuronal model of familial ALS. In standard culture conditions (with glutamine) or restricting glutamine or cystine, the level of glutathione was always lower in the cell line expressing the mutant (G93A) human Cu, Zn superoxide dismutase (G93ASOD1) than in the line expressing wild-type SOD1. With glutamine the difference in glutathione was associated with a lower glutamate and impairment of the glutamine/glutamate metabolism as evidenced by lower glutaminase and cytosolic malate dehydrogenase activity. d-?-hydroxybutyrate, as an alternative to glutamine as energy substrate in addition to glucose, reversed the decreases of cytosolic malate dehydrogenase activity and glutamate and glutathione. However, in the G93ASOD1 cell line, in all culture conditions the expression of pyruvate dehydrogenase kinase l protein, which down-regulates pyruvate dehydrogenase activity, was induced, together with an increase in lactate release in the medium. These findings suggest that the glutathione decrease associated with mutant SOD1 expression is due to mitochondrial dysfunction caused by the reduction of the flow of glucose-derived pyruvate through the TCA cycle; it implies altered glutamate metabolism and depends on the different mitochondrial energy substrates.
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Characterization of detergent-insoluble proteins in ALS indicates a causal link between nitrative stress and aggregation in pathogenesis.
PLoS ONE
PUBLISHED: 09-11-2009
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Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited.
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Adaptation to G93Asuperoxide dismutase 1 in a motor neuron cell line model of amyotrophic lateral sclerosis: the role of glutathione.
FEBS J.
PUBLISHED: 05-23-2009
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Motor neuron degeneration in amyotrophic lateral sclerosis involves oxidative damage. Glutathione (GSH) is critical as an antioxidant and a redox modulator. We used a motor neuronal cell line (NSC-34) to investigate whether wild-type and familial amyotrophic lateral sclerosis-linked G93A mutant Cu,Zn superoxide dismutase (wt/G93ASOD1) modified the GSH pool and glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis. We studied the effect of various G93ASOD1 levels and exposure times. Mutant Cu,Zn superoxide dismutase induced an adaptive process involving the upregulation of GSH synthesis, even at very low expression levels. However, cells with a high level of G93ASOD1 cultured for 10 weeks showed GSH depletion and a decrease in expression of the modulatory subunit of GCL. These cells also had lower levels of GSH and GCL activity was not induced after treatment with the pro-oxidant tert-butylhydroquinone. Cells with a low level of G93ASOD1 maintained higher GSH levels and GCL activity, showing that the exposure time and the level of the mutant protein modulate GSH synthesis. We conclude that failure of the regulation of the GSH pathway caused by G93ASOD1 may contribute to motor neuron vulnerability and we identify this pathway as a target for therapeutic intervention.
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Hypoxia causes autophagic stress and derangement of metabolic adaptation in a cell model of amyotrophic lateral sclerosis.
J. Neurochem.
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons. The recruitment of autophagy (macroautophagy) and mitochondrial dysfunction are documented in ALS patients and experimental models expressing mutant forms of Cu, Zn superoxide dismutase (SOD1) protein, but their impact in the disease remains unclear. Hypoxia is a stress closely related to the disease in patients and mutant SOD1 mice; in individual cells, hypoxia activates autophagy and regulates mitochondrial metabolism as fundamental adaptive mechanisms. Our aim was to examine whether mutant SOD1 changed this response. Hypoxia (1% O2 for 22 h) caused greater loss of viability and more marked activation of caspase 3/7 in the motor neuronal NSC-34 cell line stably transfected with the G93A mutant human SOD1 (G93A-NSC) than in the one with the wild-type SOD1 (WT-NSC) or in untransfected NSC-34. In the G93A-NSC cells there was a more marked accumulation of the LC3-II autophagy protein, attributable to autophagic stress; 3-methyladenine, which acts on initiation of autophagy, fully rescued G93A-NSC viability and reduced the activation of caspase 3/7 indicating this was a secondary event; the metabolic handling of hypoxia was inappropriate possibly contributing to the autophagic stress. Our findings evidentiate that the G93A mutation of SOD1 profoundly altered the adaptive metabolic response to hypoxia and this could increase the cell susceptibility to this stress. This article is protected by copyright. All rights reserved.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.