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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.

No MeSH data available.


Less autophagosomes and omegasomes are formed in primary cortical neurons expressing mFUS. (a) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrows indicate autophagosome formation. Scale bar=10 μm. (b) Quantification of the number of autophagosomes formed per primary neuron in (a), n=3. (c) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and EGFP-DFCP1 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrow heads indicate omegasome formation. Scale bar=10 μm. (d) Quantification of the number of omegasomes formed per primary neuron in (c), n=3. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, #P<0.05, ###P<0.00001 versus WT.
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fig5: Less autophagosomes and omegasomes are formed in primary cortical neurons expressing mFUS. (a) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrows indicate autophagosome formation. Scale bar=10 μm. (b) Quantification of the number of autophagosomes formed per primary neuron in (a), n=3. (c) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and EGFP-DFCP1 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrow heads indicate omegasome formation. Scale bar=10 μm. (d) Quantification of the number of omegasomes formed per primary neuron in (c), n=3. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, #P<0.05, ###P<0.00001 versus WT.

Mentions: We next examined the inhibition of autophagy by mFUS in intact neurons to confirm these findings. Primary mouse cortical neurons were co-transfected with FUS and either Dsred-LC3 or GFP-DFCP1, to examine autophagosome and omegasome formation, respectively. In mFUS-expressing primary neurons, there was a significant decrease in the number of autophagosomes and omegasomes detected per cell, compared with cells expressing WTFUS or untransfected cells (Figure 5, P<0.05). In WTFUS-expressing cells, there was also a significant decrease in the number of autophagosomes, but not omegasomes, present per cell compared with untransfected cells (Figure 5d, P<0.0001). These data are consistent with the findings obtained in cell lines, and thus provide further evidence that mFUS inhibits the early stages of autophagy. Similarly, WTFUS inhibits autophagy, but to a lesser extent than mFUS.


ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1
Less autophagosomes and omegasomes are formed in primary cortical neurons expressing mFUS. (a) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrows indicate autophagosome formation. Scale bar=10 μm. (b) Quantification of the number of autophagosomes formed per primary neuron in (a), n=3. (c) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and EGFP-DFCP1 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrow heads indicate omegasome formation. Scale bar=10 μm. (d) Quantification of the number of omegasomes formed per primary neuron in (c), n=3. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, #P<0.05, ###P<0.00001 versus WT.
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fig5: Less autophagosomes and omegasomes are formed in primary cortical neurons expressing mFUS. (a) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and Dsred-LC3 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrows indicate autophagosome formation. Scale bar=10 μm. (b) Quantification of the number of autophagosomes formed per primary neuron in (a), n=3. (c) Primary cortical neurons were co-transfected with HA-FUS (WT or mutant) and EGFP-DFCP1 for 18 h. Immunocytochemistry using anti-HA antibodies was then performed. White arrow heads indicate omegasome formation. Scale bar=10 μm. (d) Quantification of the number of omegasomes formed per primary neuron in (c), n=3. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. *P<0.05, ***P<0.00001 versus Untr, #P<0.05, ###P<0.00001 versus WT.
Mentions: We next examined the inhibition of autophagy by mFUS in intact neurons to confirm these findings. Primary mouse cortical neurons were co-transfected with FUS and either Dsred-LC3 or GFP-DFCP1, to examine autophagosome and omegasome formation, respectively. In mFUS-expressing primary neurons, there was a significant decrease in the number of autophagosomes and omegasomes detected per cell, compared with cells expressing WTFUS or untransfected cells (Figure 5, P<0.05). In WTFUS-expressing cells, there was also a significant decrease in the number of autophagosomes, but not omegasomes, present per cell compared with untransfected cells (Figure 5d, P<0.0001). These data are consistent with the findings obtained in cell lines, and thus provide further evidence that mFUS inhibits the early stages of autophagy. Similarly, WTFUS inhibits autophagy, but to a lesser extent than 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.