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

Mutant TDP-43 can co-aggregate to JUNQ like SOD1 positive structures.NSC-34 cells were cotransfected with combinations of mutant SOD1, TDP-43 and FUS and imaged after 48 h (A). Mutant TDP-43 could be found colocalized with SOD1 in some cases. (B) Mutant SOD1 and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from SOD1 and TDP-43 colocalized at 80 s compared to that of either SOD1-GFP or TDP-43-tomato alone. (C) Mutant FUS and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from FUS and TDP-43 colocalized at 80 s compared to that of either FUS-GFP or TDP-43-tomato alone. *indicates p < 0.05. (D) Mutant FUS and SOD1 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 9. Histogram is data from FUS and SOD1 colocalized at 80 s compared to that of either FUS-GFP or SOD1-tomato alone.
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f5: Mutant TDP-43 can co-aggregate to JUNQ like SOD1 positive structures.NSC-34 cells were cotransfected with combinations of mutant SOD1, TDP-43 and FUS and imaged after 48 h (A). Mutant TDP-43 could be found colocalized with SOD1 in some cases. (B) Mutant SOD1 and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from SOD1 and TDP-43 colocalized at 80 s compared to that of either SOD1-GFP or TDP-43-tomato alone. (C) Mutant FUS and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from FUS and TDP-43 colocalized at 80 s compared to that of either FUS-GFP or TDP-43-tomato alone. *indicates p < 0.05. (D) Mutant FUS and SOD1 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 9. Histogram is data from FUS and SOD1 colocalized at 80 s compared to that of either FUS-GFP or SOD1-tomato alone.

Mentions: SOD1 is a well-characterized JUNQ substrate303133. To test whether TDP-43 or FUS might be JUNQ substrates we co-transfected TDP-43M337V or FUSR495X with SOD1A4V fusion proteins. In cells that contained inclusions of both TDP-43 and SOD1 we observed that inclusions were distinct 50% of the time, the remaining cells contained inclusions that were positive for both TDP-43 and SOD1 (Fig. 5A). In the cases where colocalization occurred, we observed a pattern of fluorescence in which ~ 80% of total SOD1 fluorescence was consistently associated with the dual labelled inclusions (Fig. S4B). In contrast, the amount of TDP-43 present in these inclusions was significantly lower in comparison to SOD1 (p < 0.001; Student’s t-test) and ranged from ~ 50% to ~ 70% (Fig. S4B). This suggested to us that under certain specific conditions TDP-43 could be a JUNQ substrate. Similarly, while FUS did not form round dot like inclusions that were observed when co-expressed with Httex146Q-cherry, there was co-localization with SOD1A4V inclusions in approximately 30% of cells containing inclusions of both SOD1 and FUS (Fig. 5A). In the cases where there was inclusion colocalization ~ 80% of the total cellular SOD1 and FUS was present in the inclusion suggesting an active co-aggregation process (Fig. S4C). When TDP-43M337V and FUSR495X were aggregated in the same cell around 85% of the inclusions were positive for both TDP-43 and FUS (Fig. 5A). In a manner similar to that of SOD1 we observed that colocalized inclusions consistently contained ~ 70% of total cellular FUS, these same inclusions variably contained a significantly lower amount (p < 0.05, Student’s t-test), ~ 50% of the TDP-43 signal (Fig. S4D), consistent with TDP-43 aggregation being a secondary process.


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)

Mutant TDP-43 can co-aggregate to JUNQ like SOD1 positive structures.NSC-34 cells were cotransfected with combinations of mutant SOD1, TDP-43 and FUS and imaged after 48 h (A). Mutant TDP-43 could be found colocalized with SOD1 in some cases. (B) Mutant SOD1 and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from SOD1 and TDP-43 colocalized at 80 s compared to that of either SOD1-GFP or TDP-43-tomato alone. (C) Mutant FUS and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from FUS and TDP-43 colocalized at 80 s compared to that of either FUS-GFP or TDP-43-tomato alone. *indicates p < 0.05. (D) Mutant FUS and SOD1 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 9. Histogram is data from FUS and SOD1 colocalized at 80 s compared to that of either FUS-GFP or SOD1-tomato alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: Mutant TDP-43 can co-aggregate to JUNQ like SOD1 positive structures.NSC-34 cells were cotransfected with combinations of mutant SOD1, TDP-43 and FUS and imaged after 48 h (A). Mutant TDP-43 could be found colocalized with SOD1 in some cases. (B) Mutant SOD1 and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from SOD1 and TDP-43 colocalized at 80 s compared to that of either SOD1-GFP or TDP-43-tomato alone. (C) Mutant FUS and TDP-43 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 6. Histogram is data from FUS and TDP-43 colocalized at 80 s compared to that of either FUS-GFP or TDP-43-tomato alone. *indicates p < 0.05. (D) Mutant FUS and SOD1 positive inclusions were analyzed using FRAP. White box indicates ROI, arrow indicates bleached area. Mean fluorescence intensity was followed for 80 s and data shown are means and standard deviations n = 9. Histogram is data from FUS and SOD1 colocalized at 80 s compared to that of either FUS-GFP or SOD1-tomato alone.
Mentions: SOD1 is a well-characterized JUNQ substrate303133. To test whether TDP-43 or FUS might be JUNQ substrates we co-transfected TDP-43M337V or FUSR495X with SOD1A4V fusion proteins. In cells that contained inclusions of both TDP-43 and SOD1 we observed that inclusions were distinct 50% of the time, the remaining cells contained inclusions that were positive for both TDP-43 and SOD1 (Fig. 5A). In the cases where colocalization occurred, we observed a pattern of fluorescence in which ~ 80% of total SOD1 fluorescence was consistently associated with the dual labelled inclusions (Fig. S4B). In contrast, the amount of TDP-43 present in these inclusions was significantly lower in comparison to SOD1 (p < 0.001; Student’s t-test) and ranged from ~ 50% to ~ 70% (Fig. S4B). This suggested to us that under certain specific conditions TDP-43 could be a JUNQ substrate. Similarly, while FUS did not form round dot like inclusions that were observed when co-expressed with Httex146Q-cherry, there was co-localization with SOD1A4V inclusions in approximately 30% of cells containing inclusions of both SOD1 and FUS (Fig. 5A). In the cases where there was inclusion colocalization ~ 80% of the total cellular SOD1 and FUS was present in the inclusion suggesting an active co-aggregation process (Fig. S4C). When TDP-43M337V and FUSR495X were aggregated in the same cell around 85% of the inclusions were positive for both TDP-43 and FUS (Fig. 5A). In a manner similar to that of SOD1 we observed that colocalized inclusions consistently contained ~ 70% of total cellular FUS, these same inclusions variably contained a significantly lower amount (p < 0.05, Student’s t-test), ~ 50% of the TDP-43 signal (Fig. S4D), consistent with TDP-43 aggregation being a secondary process.

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