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ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1

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ABSTRACT

Amyotrophic lateral sclerosis (ALS) is characterised by the formation of intracellular misfolded protein inclusions that form in motor neurons. Autophagy is the major degradation pathway for aggregate-prone proteins within lysosomes. Autophagy begins by the production of the omegasome, forming the autophagosome membrane, which then fuses with the lysosome. Mutations in fused in sarcoma (FUS) cause 5% of familial ALS cases and FUS-positive inclusions are also formed in sporadic ALS tissues. In this study, we demonstrate that the expression of ALS-associated mutant FUS impairs autophagy in neuronal cells. In mutant FUS-expressing neuronal cells, accumulation of ubiquitinated proteins and autophagy substrates p62 and NBR1 was detected, and formation of both the omegasome and autophagosome was inhibited in these cells. However, overexpression of Rab1 rescued these defects, suggesting that Rab1 is protective in ALS. The number of LC3-positive vesicles was also increased in motor neurons from the spinal cord of an ALS patient carrying a FUS (R521C) mutation compared with a control patient, providing additional evidence that autophagy is dysregulated in mutant FUS-associated ALS. This study provides further understanding of the intricate autophagy system and neurodegeneration in ALS.

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Overexpression of mFUS inhibits the clearance of ubiquitinylated proteins. (a) Effect of mFUS overexpression on the levels of p62. EGFP-p62 and EGFP (1.5 : 1) were co-transfected with HA-FUS (WT or mutant) in Neuro2a cells for 18 h. EGFP was used as a control for transfection efficiency of EGFP-p62. Cell lysates were collected and subjected to immunoblotting using anti-GFP antibodies. (b) Quantification of the relative intensities of EGFP-p62 from the immunoblots in (a), normalised to untransfected cells, n=5. (c) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Cells were then fixed and immunocytochemistry using anti-NBR1 antibodies was performed. Merge images and insets demonstrating co-localisation of LC3 and NBR1 are shown. Scale bar=10 μm. (d) Quantification of the percentage of vesicles with co-localisation of LC3 and NBR1, n=3. Approximately 10 to 20 cells were scored in each experiments. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, ###P<0.00001 versus WT.
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fig2: Overexpression of mFUS inhibits the clearance of ubiquitinylated proteins. (a) Effect of mFUS overexpression on the levels of p62. EGFP-p62 and EGFP (1.5 : 1) were co-transfected with HA-FUS (WT or mutant) in Neuro2a cells for 18 h. EGFP was used as a control for transfection efficiency of EGFP-p62. Cell lysates were collected and subjected to immunoblotting using anti-GFP antibodies. (b) Quantification of the relative intensities of EGFP-p62 from the immunoblots in (a), normalised to untransfected cells, n=5. (c) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Cells were then fixed and immunocytochemistry using anti-NBR1 antibodies was performed. Merge images and insets demonstrating co-localisation of LC3 and NBR1 are shown. Scale bar=10 μm. (d) Quantification of the percentage of vesicles with co-localisation of LC3 and NBR1, n=3. Approximately 10 to 20 cells were scored in each experiments. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, ###P<0.00001 versus WT.

Mentions: We next examined autophagy substrates, p62 and NBR1, which bind ubiquitin and accumulate when autophagy is inhibited.29 In cells expressing mFUS, increased levels of p62 were detected by immunoblotting compared with WTFUS or untransfected cells (Figures 2a and b, P<0.05). Similarly, in cells expressing mFUS, LC3 and NBR1 co-localised with fewer vesicles compared with WTFUS-expressing cells or untransfected cells (Figures 2c and d, P<0.00001). These data imply that less NBR1 is recruited to the autophagosome in mFUS expressing cells, suggesting that ubiquitinated proteins are removed less efficiently, providing further evidence that autophagy is impaired by mFUS.


ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1
Overexpression of mFUS inhibits the clearance of ubiquitinylated proteins. (a) Effect of mFUS overexpression on the levels of p62. EGFP-p62 and EGFP (1.5 : 1) were co-transfected with HA-FUS (WT or mutant) in Neuro2a cells for 18 h. EGFP was used as a control for transfection efficiency of EGFP-p62. Cell lysates were collected and subjected to immunoblotting using anti-GFP antibodies. (b) Quantification of the relative intensities of EGFP-p62 from the immunoblots in (a), normalised to untransfected cells, n=5. (c) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Cells were then fixed and immunocytochemistry using anti-NBR1 antibodies was performed. Merge images and insets demonstrating co-localisation of LC3 and NBR1 are shown. Scale bar=10 μm. (d) Quantification of the percentage of vesicles with co-localisation of LC3 and NBR1, n=3. Approximately 10 to 20 cells were scored in each experiments. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, ###P<0.00001 versus WT.
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fig2: Overexpression of mFUS inhibits the clearance of ubiquitinylated proteins. (a) Effect of mFUS overexpression on the levels of p62. EGFP-p62 and EGFP (1.5 : 1) were co-transfected with HA-FUS (WT or mutant) in Neuro2a cells for 18 h. EGFP was used as a control for transfection efficiency of EGFP-p62. Cell lysates were collected and subjected to immunoblotting using anti-GFP antibodies. (b) Quantification of the relative intensities of EGFP-p62 from the immunoblots in (a), normalised to untransfected cells, n=5. (c) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Cells were then fixed and immunocytochemistry using anti-NBR1 antibodies was performed. Merge images and insets demonstrating co-localisation of LC3 and NBR1 are shown. Scale bar=10 μm. (d) Quantification of the percentage of vesicles with co-localisation of LC3 and NBR1, n=3. Approximately 10 to 20 cells were scored in each experiments. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, ###P<0.00001 versus WT.
Mentions: We next examined autophagy substrates, p62 and NBR1, which bind ubiquitin and accumulate when autophagy is inhibited.29 In cells expressing mFUS, increased levels of p62 were detected by immunoblotting compared with WTFUS or untransfected cells (Figures 2a and b, P<0.05). Similarly, in cells expressing mFUS, LC3 and NBR1 co-localised with fewer vesicles compared with WTFUS-expressing cells or untransfected cells (Figures 2c and d, P<0.00001). These data imply that less NBR1 is recruited to the autophagosome in mFUS expressing cells, suggesting that ubiquitinated proteins are removed less efficiently, providing further evidence that autophagy is impaired by mFUS.

View Article: PubMed Central - PubMed

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is characterised by the formation of intracellular misfolded protein inclusions that form in motor neurons. Autophagy is the major degradation pathway for aggregate-prone proteins within lysosomes. Autophagy begins by the production of the omegasome, forming the autophagosome membrane, which then fuses with the lysosome. Mutations in fused in sarcoma (FUS) cause 5% of familial ALS cases and FUS-positive inclusions are also formed in sporadic ALS tissues. In this study, we demonstrate that the expression of ALS-associated mutant FUS impairs autophagy in neuronal cells. In mutant FUS-expressing neuronal cells, accumulation of ubiquitinated proteins and autophagy substrates p62 and NBR1 was detected, and formation of both the omegasome and autophagosome was inhibited in these cells. However, overexpression of Rab1 rescued these defects, suggesting that Rab1 is protective in ALS. The number of LC3-positive vesicles was also increased in motor neurons from the spinal cord of an ALS patient carrying a FUS (R521C) mutation compared with a control patient, providing additional evidence that autophagy is dysregulated in mutant FUS-associated ALS. This study provides further understanding of the intricate autophagy system and neurodegeneration in ALS.

No MeSH data available.


Related in: MedlinePlus