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

Aggregated mutant TDP-43 and FUS recruit TDP-43wt.NSC-34 cells were transiently transfected with TDP-43wt-tomato and examined by confocal microscopy after 48 hours. (A) Mutant TDP-43M337V-GFP coexpressed with TDP-43wt-tomato. (B) Mutant FUSR495X co-expressed with TDP-43wt-tomato. (C) SOD1A4V co-transfected with TDP-43wt tomato. White arrow heads indicate areas of colocalization of GFP and tomato tagged protein. White arrows indicate distinct inclusion structures containing one tagged protein only. (D) Proportion of cells with aggregates where both mutant TDP-43, FUS and SOD1 colocalize with TDP-43wt. Data is combined from 3 independent experiments, Z-tests were used to compare differences in proportions **p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4544019&req=5

f1: Aggregated mutant TDP-43 and FUS recruit TDP-43wt.NSC-34 cells were transiently transfected with TDP-43wt-tomato and examined by confocal microscopy after 48 hours. (A) Mutant TDP-43M337V-GFP coexpressed with TDP-43wt-tomato. (B) Mutant FUSR495X co-expressed with TDP-43wt-tomato. (C) SOD1A4V co-transfected with TDP-43wt tomato. White arrow heads indicate areas of colocalization of GFP and tomato tagged protein. White arrows indicate distinct inclusion structures containing one tagged protein only. (D) Proportion of cells with aggregates where both mutant TDP-43, FUS and SOD1 colocalize with TDP-43wt. Data is combined from 3 independent experiments, Z-tests were used to compare differences in proportions **p < 0.01, ***p < 0.001.

Mentions: Given that many ALS inclusions contain TDP-43wt, we sought to examine whether overexpression of mutant TDP-43, FUS or SOD1 would induce TDP-43 aggregation and if so whether the aggregates accumulate in distinct cellular compartments. We used TDP-43wt-tomato34, which under basal conditions remains predominantly in the nucleus, as a reporter for TDP-43 recruitment into aggregates. Aggregated TDP-43 has previously been shown to recruit TDP-43wt into cytoplasmic inclusions35. Consistent with this, we observe that co-expression of TDP-43M337V-GFP and TDP-43wt-tomato produces cytosolic inclusions (defined as fluorescent foci > 2 μm) positive for both mutant and TDP-43wt in approximately 88% of cells expressing both constructs that contain inclusions (Fig. 1A,D; white arrow heads). However, while the bulk of TDP-43M337V is retained in the cytosol in inclusions, co-localization of TDP-43wt with these inclusions did not always deplete the nucleus of TDP-43wt. Indeed, in some instances inclusion formation of TDP-43M337V did not recruit TDP-43wt (Fig.1A, white arrows).


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)

Aggregated mutant TDP-43 and FUS recruit TDP-43wt.NSC-34 cells were transiently transfected with TDP-43wt-tomato and examined by confocal microscopy after 48 hours. (A) Mutant TDP-43M337V-GFP coexpressed with TDP-43wt-tomato. (B) Mutant FUSR495X co-expressed with TDP-43wt-tomato. (C) SOD1A4V co-transfected with TDP-43wt tomato. White arrow heads indicate areas of colocalization of GFP and tomato tagged protein. White arrows indicate distinct inclusion structures containing one tagged protein only. (D) Proportion of cells with aggregates where both mutant TDP-43, FUS and SOD1 colocalize with TDP-43wt. Data is combined from 3 independent experiments, Z-tests were used to compare differences in proportions **p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Aggregated mutant TDP-43 and FUS recruit TDP-43wt.NSC-34 cells were transiently transfected with TDP-43wt-tomato and examined by confocal microscopy after 48 hours. (A) Mutant TDP-43M337V-GFP coexpressed with TDP-43wt-tomato. (B) Mutant FUSR495X co-expressed with TDP-43wt-tomato. (C) SOD1A4V co-transfected with TDP-43wt tomato. White arrow heads indicate areas of colocalization of GFP and tomato tagged protein. White arrows indicate distinct inclusion structures containing one tagged protein only. (D) Proportion of cells with aggregates where both mutant TDP-43, FUS and SOD1 colocalize with TDP-43wt. Data is combined from 3 independent experiments, Z-tests were used to compare differences in proportions **p < 0.01, ***p < 0.001.
Mentions: Given that many ALS inclusions contain TDP-43wt, we sought to examine whether overexpression of mutant TDP-43, FUS or SOD1 would induce TDP-43 aggregation and if so whether the aggregates accumulate in distinct cellular compartments. We used TDP-43wt-tomato34, which under basal conditions remains predominantly in the nucleus, as a reporter for TDP-43 recruitment into aggregates. Aggregated TDP-43 has previously been shown to recruit TDP-43wt into cytoplasmic inclusions35. Consistent with this, we observe that co-expression of TDP-43M337V-GFP and TDP-43wt-tomato produces cytosolic inclusions (defined as fluorescent foci > 2 μm) positive for both mutant and TDP-43wt in approximately 88% of cells expressing both constructs that contain inclusions (Fig. 1A,D; white arrow heads). However, while the bulk of TDP-43M337V is retained in the cytosol in inclusions, co-localization of TDP-43wt with these inclusions did not always deplete the nucleus of TDP-43wt. Indeed, in some instances inclusion formation of TDP-43M337V did not recruit TDP-43wt (Fig.1A, white arrows).

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