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

SOD1 and FUS inclusions contain a mobile fraction.FRAP analysis of inclusions formed in NSC-34 cells expressing mutant TDP-43, FUS or SOD1-GFP. (A) Mean fluorescence intensity (from within the ROI) plotted over time. Prebleach intensity was recorded, and recovery was recorded for up to 40 s. Results are means and standard deviation from n = 10. Mean fluorescence at 40 s is quantified in histogram. ***indicates p < 0.001. (B) Representative confocal images of prebleach, post bleach and recovery endpoint. ROI is marked in white, timing of image relative to data collection is marked in panel A.
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f3: SOD1 and FUS inclusions contain a mobile fraction.FRAP analysis of inclusions formed in NSC-34 cells expressing mutant TDP-43, FUS or SOD1-GFP. (A) Mean fluorescence intensity (from within the ROI) plotted over time. Prebleach intensity was recorded, and recovery was recorded for up to 40 s. Results are means and standard deviation from n = 10. Mean fluorescence at 40 s is quantified in histogram. ***indicates p < 0.001. (B) Representative confocal images of prebleach, post bleach and recovery endpoint. ROI is marked in white, timing of image relative to data collection is marked in panel A.

Mentions: IPOD structures are thought to be predominantly immobile, while JUNQ and aggresome structures contain a mobile fraction3145. Here we used fluorescence recovery after photo bleaching (FRAP) to examine the mobility of molecules within ALS inclusions. Using a high powered laser pulse we bleached a region of GFP positive inclusions that corresponded to < 25% of any given inclusion and then recorded the subsequent fluorescence over 40 s. We found that there was minimal fluorescence recovery after bleaching TDP-43M337V inclusions (Fig. 3A,B) confirming recent observations46. In contrast, our data suggests that both SOD1 and FUS inclusions recover approximately 25% of their original fluorescence, significantly more recovery than TDP-43 (Tukey’s multiple comparison test; p < 0.001), suggesting a mobile fraction or porous structure within the inclusions (Fig. 3A,B). The recovery patterns of each of the aggregates was similar to that of the soluble protein tested in both wt and mutant expressing cells (Fig. S2). Therefore we cannot rule out the possibility that the protein species diffusing in to the inclusion are soluble species from outside of the aggregate and do not represent diffusion from within the aggregate itself.


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)

SOD1 and FUS inclusions contain a mobile fraction.FRAP analysis of inclusions formed in NSC-34 cells expressing mutant TDP-43, FUS or SOD1-GFP. (A) Mean fluorescence intensity (from within the ROI) plotted over time. Prebleach intensity was recorded, and recovery was recorded for up to 40 s. Results are means and standard deviation from n = 10. Mean fluorescence at 40 s is quantified in histogram. ***indicates p < 0.001. (B) Representative confocal images of prebleach, post bleach and recovery endpoint. ROI is marked in white, timing of image relative to data collection is marked in panel A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: SOD1 and FUS inclusions contain a mobile fraction.FRAP analysis of inclusions formed in NSC-34 cells expressing mutant TDP-43, FUS or SOD1-GFP. (A) Mean fluorescence intensity (from within the ROI) plotted over time. Prebleach intensity was recorded, and recovery was recorded for up to 40 s. Results are means and standard deviation from n = 10. Mean fluorescence at 40 s is quantified in histogram. ***indicates p < 0.001. (B) Representative confocal images of prebleach, post bleach and recovery endpoint. ROI is marked in white, timing of image relative to data collection is marked in panel A.
Mentions: IPOD structures are thought to be predominantly immobile, while JUNQ and aggresome structures contain a mobile fraction3145. Here we used fluorescence recovery after photo bleaching (FRAP) to examine the mobility of molecules within ALS inclusions. Using a high powered laser pulse we bleached a region of GFP positive inclusions that corresponded to < 25% of any given inclusion and then recorded the subsequent fluorescence over 40 s. We found that there was minimal fluorescence recovery after bleaching TDP-43M337V inclusions (Fig. 3A,B) confirming recent observations46. In contrast, our data suggests that both SOD1 and FUS inclusions recover approximately 25% of their original fluorescence, significantly more recovery than TDP-43 (Tukey’s multiple comparison test; p < 0.001), suggesting a mobile fraction or porous structure within the inclusions (Fig. 3A,B). The recovery patterns of each of the aggregates was similar to that of the soluble protein tested in both wt and mutant expressing cells (Fig. S2). Therefore we cannot rule out the possibility that the protein species diffusing in to the inclusion are soluble species from outside of the aggregate and do not represent diffusion from within the aggregate itself.

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