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

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.

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Formation of autolysosomes is inhibited in cells expressing mFUS. (a) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and mCherry-GFP-LC3 for 18 h before being examined using confocal microscopy. White arrows indicate mCherry-LC3 (indicating lysosomes). Scale bar=10 μm. (b) Quantification of the percentage of vesicles with mCherry-LC3 from at least 10 cells of each sample, n=3. (c) SHSY5Y cells were co-transfected with HA-FUS (WT or mutant) and pcDNA-LAMP2C vectors for 18 h. Cell lysates were collected and subjected to immunoblotting using anti-LAMP2 antibodies. Blots were stripped and re-probed with β-actin as a loading control. (d) Quantification of the relative intensities of LAMP2 in (c), normalised to untransfected cells, n=2. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, **P<0.0001 versus Untr, ##P<0.0001, ###P<0.00001 versus WT.
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fig3: Formation of autolysosomes is inhibited in cells expressing mFUS. (a) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and mCherry-GFP-LC3 for 18 h before being examined using confocal microscopy. White arrows indicate mCherry-LC3 (indicating lysosomes). Scale bar=10 μm. (b) Quantification of the percentage of vesicles with mCherry-LC3 from at least 10 cells of each sample, n=3. (c) SHSY5Y cells were co-transfected with HA-FUS (WT or mutant) and pcDNA-LAMP2C vectors for 18 h. Cell lysates were collected and subjected to immunoblotting using anti-LAMP2 antibodies. Blots were stripped and re-probed with β-actin as a loading control. (d) Quantification of the relative intensities of LAMP2 in (c), normalised to untransfected cells, n=2. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, **P<0.0001 versus Untr, ##P<0.0001, ###P<0.00001 versus WT.

Mentions: We next examined whether the fusion of autophagosomes to lysosomes was impaired in mFUS-expressing cells. FUS proteins were co-expressed with double-tagged mCherry-GFP-LC3, which appears yellow (green merged with red) in non-acidic structures (autophagosomes and amiphisomes), and red in acidic autolysosomes, owing to quenching of GFP.30 In untransfected and WTFUS-expressing cells, 30–50% of LC3 vesicles were fluorescent red, indicating that lysosomes were formed. In contrast, significantly less red fluorescent LC3 vesicles were present in mFUS (P525L or R522G)-transfected cells (15–28%, Figures 3a and b, P<0.05), indicating that maturation of the autophagosome, and hence the formation of acidic autolysosomes, is inhibited in cells expressing mFUS. Similarly, the levels of lysosomal marker LAMP2 were significantly decreased in cells expressing mFUS compared with WTFUS or untransfected cells (Figures 3c and d, P<0.05). These findings indicate that fewer lysosomes were present in cells expressing mFUS.


ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1
Formation of autolysosomes is inhibited in cells expressing mFUS. (a) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and mCherry-GFP-LC3 for 18 h before being examined using confocal microscopy. White arrows indicate mCherry-LC3 (indicating lysosomes). Scale bar=10 μm. (b) Quantification of the percentage of vesicles with mCherry-LC3 from at least 10 cells of each sample, n=3. (c) SHSY5Y cells were co-transfected with HA-FUS (WT or mutant) and pcDNA-LAMP2C vectors for 18 h. Cell lysates were collected and subjected to immunoblotting using anti-LAMP2 antibodies. Blots were stripped and re-probed with β-actin as a loading control. (d) Quantification of the relative intensities of LAMP2 in (c), normalised to untransfected cells, n=2. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, **P<0.0001 versus Untr, ##P<0.0001, ###P<0.00001 versus WT.
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fig3: Formation of autolysosomes is inhibited in cells expressing mFUS. (a) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and mCherry-GFP-LC3 for 18 h before being examined using confocal microscopy. White arrows indicate mCherry-LC3 (indicating lysosomes). Scale bar=10 μm. (b) Quantification of the percentage of vesicles with mCherry-LC3 from at least 10 cells of each sample, n=3. (c) SHSY5Y cells were co-transfected with HA-FUS (WT or mutant) and pcDNA-LAMP2C vectors for 18 h. Cell lysates were collected and subjected to immunoblotting using anti-LAMP2 antibodies. Blots were stripped and re-probed with β-actin as a loading control. (d) Quantification of the relative intensities of LAMP2 in (c), normalised to untransfected cells, n=2. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, **P<0.0001 versus Untr, ##P<0.0001, ###P<0.00001 versus WT.
Mentions: We next examined whether the fusion of autophagosomes to lysosomes was impaired in mFUS-expressing cells. FUS proteins were co-expressed with double-tagged mCherry-GFP-LC3, which appears yellow (green merged with red) in non-acidic structures (autophagosomes and amiphisomes), and red in acidic autolysosomes, owing to quenching of GFP.30 In untransfected and WTFUS-expressing cells, 30–50% of LC3 vesicles were fluorescent red, indicating that lysosomes were formed. In contrast, significantly less red fluorescent LC3 vesicles were present in mFUS (P525L or R522G)-transfected cells (15–28%, Figures 3a and b, P<0.05), indicating that maturation of the autophagosome, and hence the formation of acidic autolysosomes, is inhibited in cells expressing mFUS. Similarly, the levels of lysosomal marker LAMP2 were significantly decreased in cells expressing mFUS compared with WTFUS or untransfected cells (Figures 3c and d, P<0.05). These findings indicate that fewer lysosomes were present in cells expressing 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