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

Ubiquitin associates with TDP-43 and FUS inclusions late compared to SOD1.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP with mRFP-ubiquitin and examined by confocal microscopy at various times after transfection (A–C). Quantification of proportion of inclusions in dual transfected cells that contain both GFP fusion proteins and ubiquitin (D). 8 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiment was performed in triplicate and is representative of 2 independent experiments. (E) Human ALS post mortem tissue was stained for both TDP-43 and ubiquitin. 40 inclusions were imaged across two cases of sporadic ALS. Representative images of small foci (not colocalizing with ubiquitin) and large skeins that colocalize to ubiquitin staining are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Ubiquitin associates with TDP-43 and FUS inclusions late compared to SOD1.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP with mRFP-ubiquitin and examined by confocal microscopy at various times after transfection (A–C). Quantification of proportion of inclusions in dual transfected cells that contain both GFP fusion proteins and ubiquitin (D). 8 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiment was performed in triplicate and is representative of 2 independent experiments. (E) Human ALS post mortem tissue was stained for both TDP-43 and ubiquitin. 40 inclusions were imaged across two cases of sporadic ALS. Representative images of small foci (not colocalizing with ubiquitin) and large skeins that colocalize to ubiquitin staining are shown.

Mentions: Ubiquitin positive inclusions are a hallmark of ALS, however the mechanism and dynamics of ubiquitylation and inclusion formation cannot be determined from postmortem tissue. Recent work on Huntingtin systems shows that although ubiquitylated Htt positive inclusions are present in post mortem tissue, in cell culture systems ubiquitin co-localizes to inclusions relatively late32. To compare the association of ubiquitin with ALS inclusions we initially co-transfected NSC-34 cells with mRFP-ubiquitin and SOD1A4V, TDP-43M337V or FUSR495X GFP fusions. SOD1A4V inclusions were always observed to co-localize with ubiquitin starting at our earliest observations 24 hours after transfection (Fig. 7C,D). In contrast, TDP-43M337V and FUSR495X did not always co-localize with ubiquitin with around 15 and 30% of inclusions respectively becoming positive for mRFP-ubiquitin after 72 h (Fig. 7A,B,D). To confirm this was not an artefact of the co-expression with mRFP-ubiquitin fusion we performed ubiquitin- immunostaining on cells expressing mutant FUS, TDP-43 and SOD1-GFP alone and observed similar ubiquitin co-localization patterns at 48–72 hours (Fig. S5).


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)

Ubiquitin associates with TDP-43 and FUS inclusions late compared to SOD1.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP with mRFP-ubiquitin and examined by confocal microscopy at various times after transfection (A–C). Quantification of proportion of inclusions in dual transfected cells that contain both GFP fusion proteins and ubiquitin (D). 8 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiment was performed in triplicate and is representative of 2 independent experiments. (E) Human ALS post mortem tissue was stained for both TDP-43 and ubiquitin. 40 inclusions were imaged across two cases of sporadic ALS. Representative images of small foci (not colocalizing with ubiquitin) and large skeins that colocalize to ubiquitin staining are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Ubiquitin associates with TDP-43 and FUS inclusions late compared to SOD1.NSC-34 cells were transiently transfected with mutant TDP-43, FUS, or SOD1-GFP with mRFP-ubiquitin and examined by confocal microscopy at various times after transfection (A–C). Quantification of proportion of inclusions in dual transfected cells that contain both GFP fusion proteins and ubiquitin (D). 8 fields of view from each timepoint were counted (minimum 30 cells per field) and scored. Experiment was performed in triplicate and is representative of 2 independent experiments. (E) Human ALS post mortem tissue was stained for both TDP-43 and ubiquitin. 40 inclusions were imaged across two cases of sporadic ALS. Representative images of small foci (not colocalizing with ubiquitin) and large skeins that colocalize to ubiquitin staining are shown.
Mentions: Ubiquitin positive inclusions are a hallmark of ALS, however the mechanism and dynamics of ubiquitylation and inclusion formation cannot be determined from postmortem tissue. Recent work on Huntingtin systems shows that although ubiquitylated Htt positive inclusions are present in post mortem tissue, in cell culture systems ubiquitin co-localizes to inclusions relatively late32. To compare the association of ubiquitin with ALS inclusions we initially co-transfected NSC-34 cells with mRFP-ubiquitin and SOD1A4V, TDP-43M337V or FUSR495X GFP fusions. SOD1A4V inclusions were always observed to co-localize with ubiquitin starting at our earliest observations 24 hours after transfection (Fig. 7C,D). In contrast, TDP-43M337V and FUSR495X did not always co-localize with ubiquitin with around 15 and 30% of inclusions respectively becoming positive for mRFP-ubiquitin after 72 h (Fig. 7A,B,D). To confirm this was not an artefact of the co-expression with mRFP-ubiquitin fusion we performed ubiquitin- immunostaining on cells expressing mutant FUS, TDP-43 and SOD1-GFP alone and observed similar ubiquitin co-localization patterns at 48–72 hours (Fig. S5).

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