Fused in sarcoma (FUS) is mutated in both sporadic amyotrophic lateral sclerosis (ALS) and familial ALS patients. The mechanisms underlying neurodegeneration are not fully understood, but FUS ...redistributes from the nucleus to the cytoplasm in affected motor neurons, where it triggers endoplasmic reticulum (ER) stress. Ataxin-2 is a polyglutamine protein which normally contains 22 repeats, but expanded repeats (>34) are found in Spinocerebellar Ataxia type 2. Recently ataxin-2 with intermediate length repeats (27-33) was found to increase the risk of ALS. Here we show that ataxin-2 with an ALS-linked intermediate length repeat (Q31) is a potent modifier of FUS pathology in cellular disease models. Translocation of FUS to the cytoplasm and ER stress were significantly enhanced by co-expression of mutant FUS with ataxin-2 Q31. Ataxin-2 also co-localized with FUS in sporadic and FUS-linked familial ALS patient motor neurons, co-precipitated with FUS in ALS spinal cord lysates, and co-localized with FUS in the ER-Golgi compartments in neuronal cell lines. Fragmentation of the Golgi apparatus is linked to neurodegeneration in ALS and here we show that Golgi fragmentation is induced in cells expressing mutant FUS. Moreover, Golgi fragmentation was enhanced, and the early stages of apoptosis were triggered, when ataxin-2 Q31 was co-expressed with mutant FUS. These findings describe new cellular mechanisms linking ALS with ataxin-2 intermediate length polyQ expansions and provide further evidence linking disruption to ER-Golgi compartments and FUS pathology in ALS.
Amyotrophic lateral sclerosis is a rapidly progressing fatal neurodegenerative disease characterized by the presence of protein inclusions within affected motor neurons. Endoplasmic reticulum stress ...leading to apoptosis was recently recognized to be an important process in the pathogenesis of sporadic human amyotrophic lateral sclerosis as well as in transgenic models of mutant superoxide dismutase 1-linked familial amyotrophic lateral sclerosis. Endoplasmic reticulum stress occurs early in disease, indicating a critical role in pathogenesis, and involves upregulation of an important endoplasmic reticulum chaperone, protein disulphide isomerase. We aimed to investigate the involvement of protein disulphide isomerase in endoplasmic reticulum stress induction, protein aggregation, inclusion formation and toxicity in amyotrophic lateral sclerosis. Motor neuron-like NSC-34 cell lines were transfected with superoxide dismutase 1 and protein disulphide isomerase encoding vectors and small interfering RNA, and examined by immunocytochemistry and immunoblotting. Expression of mutant superoxide dismutase 1 induced endoplasmic reticulum stress, predominantly in cells bearing mutant superoxide dismutase 1 inclusions but also in a proportion of cells expressing mutant superoxide dismutase 1 without visible inclusions. Over-expression of protein disulphide isomerase decreased mutant superoxide dismutase 1 aggregation, inclusion formation, endoplasmic reticulum stress induction and toxicity, whereas small interfering RNA targeting protein disulphide isomerase increased mutant superoxide dismutase 1 inclusion formation, indicating a protective role for protein disulphide isomerase against superoxide dismutase 1 misfolding. Aberrant modification of protein disulphide isomerase by S-nitrosylation of active site cysteine residues has previously been shown as an important process in neurodegeneration in Parkinson's and Alzheimer's disease brain tissue, but has not been described in amyotrophic lateral sclerosis. Using a biotin switch assay, we detected increased levels of S-nitrosylated protein disulphide isomerase in transgenic mutant superoxide dismutase 1 mouse and human sporadic amyotrophic lateral sclerosis spinal cord tissues. Hence, despite upregulation, protein disulphide isomerase is also functionally inactivated in amyotrophic lateral sclerosis, which may prevent its normal protective function and contribute to disease. We also found that a small molecule mimic of the protein disulphide isomerase active site, (±)-trans-1,2-bis(mercaptoacetamido)cyclohexane, protected against mutant superoxide dismutase 1 inclusion formation. These studies reveal that endoplasmic reticulum stress is important in the formation of mutant superoxide dismutase 1 inclusions, and protein disulphide isomerase has an important function in ameliorating mutant superoxide dismutase 1 aggregation and toxicity. Functional inhibition of protein disulphide isomerase by S-nitrosylation may contribute to pathophysiology in both mutant superoxide dismutase 1-linked disease and sporadic amyotrophic lateral sclerosis. Protein disulphide isomerase is therefore a novel potential therapeutic target in amyotrophic lateral sclerosis and (±)-trans-1,2-bis(mercaptoacetamido)cyclohexane and other molecular mimics of protein disulphide isomerase could be of benefit in amyotrophic lateral sclerosis and other neurodegenerative diseases related to protein misfolding.
Astrocytes contribute to the death of motor neurons via non-cell autonomous mechanisms of injury in amyotrophic lateral sclerosis (ALS). Since mutations in the gene encoding Cu/Zn superoxide ...dismutase (SOD1) underlie the neuropathology of some forms of familial ALS, we explored how expression of mutant SOD1 protein A4V SOD1-EGFP affected the biology of secondary murine astrocytes. A4V SOD1-EGFP expressing astrocytes (72 h after transfection) displayed decreased mitochondrial activity (~45%) and
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-glutamate transport (~25%), relative to cells expressing wild-type SOD1-EGFP. A4V SOD1-EGFP altered F-actin and Hoechst staining, indicative of cytoskeletal and nuclear changes, and altered GM130 labelling suggesting fragmentation of Golgi apparatus. SOD1 inclusion formation shifted from discrete to “punctate” over 72 h with A4V SOD1-EGFP more rapidly producing inclusions than G85R SOD1-EGFP, and forming more punctate aggregates. A4V, not wild-type SOD1-EGFP, exerted a substantial, time-dependent effect on GFAP expression, and ~60% of astrocytes became stellate and hypertrophic at 72 h. Spreading toxicity was inferred since at 72 h ~80% of bystander cells exhibited hypertrophy and stellation. This evidence favours mutant SOD1-containing astrocytes releasing destructive species that alter the biology of adjacent astrocytes. This panoply of mutant SOD1-induced destructive events favours recruitment of astrocytes to non-cell autonomous injury in ALS.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Mutations in the intracellular metalloenzyme superoxide dismutase 1 (SOD1) are linked to neurotoxicity in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism. Golgi fragmentation and ...endoplasmic reticulum stress are early hallmarks of spinal motor neuron pathology in transgenic mice overexpressing mutant SOD1, suggesting that dysfunction of the neuronal secretory pathway may contribute to ALS pathogenesis. We therefore proposed that mutant SOD1 directly engages and modulates the secretory pathway based on recent evidence of SOD1 secretion in diverse human cell lines. Here, we demonstrate that a fraction of active endogenous SOD1 is secreted by NSC-34 motor neuron-like cells via a brefeldin-A (BFA)-sensitive pathway. Expression of enhanced green fluorescent protein-tagged mutant human SOD1 (hSOD1-EGFP) in NSC-34 cells induced frequent cytoplasmic inclusions and protein insolubility that correlated with toxicity. In contrast, transfection of non-neuronal COS-7 cells resulted in mutant hSOD1-EGFP cytoplasmic inclusions, oligomerization, and fragmentation without detectable toxicity. Importantly, impaired secretion of hSOD1-EGFP was common to all 10 SOD1 mutants tested relative to wild-type protein in NSC-34 cells. Treatment with BFA inhibited hSOD1-EGFP secretion with pronounced BFA-induced toxicity in mutant cells. Extracellular targeting of mutant hSOD1-EGFP via SOD3 signal peptide fusion attenuated cytoplasmic inclusion formation and toxicity. The effect of elevated extracellular SOD1 was then evaluated in a transgenic rat model of ALS. Chronic intraspinal infusion of exogenous wild-type hSOD1 significantly delayed disease progression and endpoint in transgenic SOD1(G93A) rats. Collectively, these results suggest novel extracellular roles for SOD1 in ALS and support a causal relationship between mutant SOD1 secretion and intraneuronal toxicity.
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal).
This article has been retracted at the ...request of the authors.
The authors became aware of an issue with incorrect loading control images being presented and are now retracting the article in the interests of maintaining their publication standards and those of the journal. The authors maintain that these presentational errors do not impact the underlying scientific findings of the article, some of which have been verified in other laboratories.
Figure 4 contains incorrect loading control images of SOD1G93A mouse. The b-actin images blots presented are SOD1G93A rat b-actin bands that were accidentally used when compiling the image for publication. Similarly, in Figure 1 the samples were run in two separate experiments with the controls split across the two blots. The b-actin for one set of controls was accidentally used for the second controls. The authors state that quantifications were performed using the correct b-actin bands.
In figure 1, concerns were raised that each of the panels had 13 lanes except for the ATF panel which appeared to have only twelve lanes. The authors state that the ATF6 panel has 13 lanes, but last two control lanes of the gel have no signal, making it difficult to accurately align the end part of the gel correctly for display in the figure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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