Limits...
Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions.

Farrawell NE, Lambert-Smith IA, Warraich ST, Blair IP, Saunders DN, Hatters DM, Yerbury JJ - Sci Rep (2015)

Bottom Line: Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin.TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure.We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.

View Article: PubMed Central - PubMed

Affiliation: Illawarra Health and Medical Research Institute, Wollongong, NSW 2522 Australia.

ABSTRACT
Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.

No MeSH data available.


Related in: MedlinePlus

Microtubule destabilization prevents SOD1 inclusions but not TDP-43 and FUS inclusions.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP and after 24 hours incubated with or without 33 μM nocodazole. (A) Confocal images of cells expressing GFP fusion proteins in the presence or absence of nocodazole. Dotted white line represents cell outline, solid white line represents nuclear outline obtained from transmission image (denoted N), white arrows indicate inclusions. (B) Quantification of the proportion of cells with inclusions in transfected cells. At least 6 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiments were performed 3 times and bar charts represent mean and standard deviation. ** indicates p < 0.01 (C) After treatment with nocodazole cells were lysed and lysates were used in filter trap assays. Trapped material represents aggregates. Western blotting of resulting filter trap assay and quantification using imageJ. Experiments were performed 3 times and bar charts represent mean and standard deviation. **indicates p < 0.01.
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f2: Microtubule destabilization prevents SOD1 inclusions but not TDP-43 and FUS inclusions.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP and after 24 hours incubated with or without 33 μM nocodazole. (A) Confocal images of cells expressing GFP fusion proteins in the presence or absence of nocodazole. Dotted white line represents cell outline, solid white line represents nuclear outline obtained from transmission image (denoted N), white arrows indicate inclusions. (B) Quantification of the proportion of cells with inclusions in transfected cells. At least 6 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiments were performed 3 times and bar charts represent mean and standard deviation. ** indicates p < 0.01 (C) After treatment with nocodazole cells were lysed and lysates were used in filter trap assays. Trapped material represents aggregates. Western blotting of resulting filter trap assay and quantification using imageJ. Experiments were performed 3 times and bar charts represent mean and standard deviation. **indicates p < 0.01.

Mentions: Previous work indicates that microtubule disruption promotes TDP-43 inclusions40 and halts the formation of a very specific FUS inclusion type formed by the 1–359 FUS fragment41. Similarly, aggresomes, IPOD and JUNQ structures are suppressed when microtubules are disrupted2930. To compare the effects of destabilization of microtubules on ALS inclusions we treated NSC-34 cells transfected with mutant FUS, TDP-43 or SOD1 fused to GFP with microtubule destabilizer nocodazole. We observed large cytoplasmic inclusions that are cloud or sponge-like in appearance (referred to as conglomerate in ALS pathology) in approximately 15% of SOD1A4V -GFP transfected cells (Fig. 2A,B) but after nocodazole treatment this was significantly reduced (p < 0.01; Student’s t-test) to fewer than 5% of transfected cells had large inclusions. The remaining cells did not appear to have smaller granular foci as observed in other aggregating systems such as occurs in the Htt42 model but remained in a diffuse pattern of fluorescence. This is consistent with previous observations of mutant SOD1 forming JUNQ-like aggresomes that are microtubule dependent43. In contrast, NSC-34 cells transfected with either TDP-43M337V or FUSR495X showed no significant difference in percentage of inclusions present regardless of nocodazole treatment (Fig. 2A,B). This suggests that neither FUS nor TDP-43 aggregate in a manner consistent with aggresomes, IPOD or JUNQ structures. However, it must be noted that although there is evidence that IPOD–like inclusions require intact microtubules to aggregate, evidence also exists to show that polyQ-104 protein aggregates similarly in the presence or absence of nocodazole44. To complement image analysis we also performed filter trap assays to examine any difference in total trappable aggregated protein under the same conditions. We found that although the number of SOD1 inclusions decreased upon nocodazole treatment (Fig. 2B, p < 0.05; Student’s t-test), total aggregation measured by filter trap did not (Fig. 2C). Similarly, FUS aggregation did not change after nocodazole treatment. In contrast, total trappable TDP-43 aggregates significantly increased upon nocodazole treatment (Fig. 2C; p < 0.01; Student’s t-test).


Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions.

Farrawell NE, Lambert-Smith IA, Warraich ST, Blair IP, Saunders DN, Hatters DM, Yerbury JJ - Sci Rep (2015)

Microtubule destabilization prevents SOD1 inclusions but not TDP-43 and FUS inclusions.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP and after 24 hours incubated with or without 33 μM nocodazole. (A) Confocal images of cells expressing GFP fusion proteins in the presence or absence of nocodazole. Dotted white line represents cell outline, solid white line represents nuclear outline obtained from transmission image (denoted N), white arrows indicate inclusions. (B) Quantification of the proportion of cells with inclusions in transfected cells. At least 6 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiments were performed 3 times and bar charts represent mean and standard deviation. ** indicates p < 0.01 (C) After treatment with nocodazole cells were lysed and lysates were used in filter trap assays. Trapped material represents aggregates. Western blotting of resulting filter trap assay and quantification using imageJ. Experiments were performed 3 times and bar charts represent mean and standard deviation. **indicates p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4544019&req=5

f2: Microtubule destabilization prevents SOD1 inclusions but not TDP-43 and FUS inclusions.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP and after 24 hours incubated with or without 33 μM nocodazole. (A) Confocal images of cells expressing GFP fusion proteins in the presence or absence of nocodazole. Dotted white line represents cell outline, solid white line represents nuclear outline obtained from transmission image (denoted N), white arrows indicate inclusions. (B) Quantification of the proportion of cells with inclusions in transfected cells. At least 6 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiments were performed 3 times and bar charts represent mean and standard deviation. ** indicates p < 0.01 (C) After treatment with nocodazole cells were lysed and lysates were used in filter trap assays. Trapped material represents aggregates. Western blotting of resulting filter trap assay and quantification using imageJ. Experiments were performed 3 times and bar charts represent mean and standard deviation. **indicates p < 0.01.
Mentions: Previous work indicates that microtubule disruption promotes TDP-43 inclusions40 and halts the formation of a very specific FUS inclusion type formed by the 1–359 FUS fragment41. Similarly, aggresomes, IPOD and JUNQ structures are suppressed when microtubules are disrupted2930. To compare the effects of destabilization of microtubules on ALS inclusions we treated NSC-34 cells transfected with mutant FUS, TDP-43 or SOD1 fused to GFP with microtubule destabilizer nocodazole. We observed large cytoplasmic inclusions that are cloud or sponge-like in appearance (referred to as conglomerate in ALS pathology) in approximately 15% of SOD1A4V -GFP transfected cells (Fig. 2A,B) but after nocodazole treatment this was significantly reduced (p < 0.01; Student’s t-test) to fewer than 5% of transfected cells had large inclusions. The remaining cells did not appear to have smaller granular foci as observed in other aggregating systems such as occurs in the Htt42 model but remained in a diffuse pattern of fluorescence. This is consistent with previous observations of mutant SOD1 forming JUNQ-like aggresomes that are microtubule dependent43. In contrast, NSC-34 cells transfected with either TDP-43M337V or FUSR495X showed no significant difference in percentage of inclusions present regardless of nocodazole treatment (Fig. 2A,B). This suggests that neither FUS nor TDP-43 aggregate in a manner consistent with aggresomes, IPOD or JUNQ structures. However, it must be noted that although there is evidence that IPOD–like inclusions require intact microtubules to aggregate, evidence also exists to show that polyQ-104 protein aggregates similarly in the presence or absence of nocodazole44. To complement image analysis we also performed filter trap assays to examine any difference in total trappable aggregated protein under the same conditions. We found that although the number of SOD1 inclusions decreased upon nocodazole treatment (Fig. 2B, p < 0.05; Student’s t-test), total aggregation measured by filter trap did not (Fig. 2C). Similarly, FUS aggregation did not change after nocodazole treatment. In contrast, total trappable TDP-43 aggregates significantly increased upon nocodazole treatment (Fig. 2C; p < 0.01; Student’s t-test).

Bottom Line: Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin.TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure.We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.

View Article: PubMed Central - PubMed

Affiliation: Illawarra Health and Medical Research Institute, Wollongong, NSW 2522 Australia.

ABSTRACT
Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.

No MeSH data available.


Related in: MedlinePlus