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 TDP-43 inclusions are distinct from iPOD.Cells were transfected with Httex146Q-mcherry and either mutant TDP-43, FUS or SOD1-GFP. The cells were imaged 48 h after transfection (A) and then the number of inclusions colocalized or surrounding the Htt iPOD quantified (B). Green arrows indicate distinct and separate inclusions, red arrows indicate GFP tagged inclusions surrounding Htt inclusions, yellow arrows indicate colocalization. A minimum of 100 cells with inclusions were counted in each case and the experiment was repeated n = 2. A z-test of proportions was conducted on combined dataset and ***denotes p < 0.001. (C) FRAP measurements were performed on Htt-FUS positive inclusions. 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 Htt colocalized at 80 s compared to that of either FUS-GFP or Htt-RFP alone. ***indicates p < 0.001.
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f4: SOD1 and TDP-43 inclusions are distinct from iPOD.Cells were transfected with Httex146Q-mcherry and either mutant TDP-43, FUS or SOD1-GFP. The cells were imaged 48 h after transfection (A) and then the number of inclusions colocalized or surrounding the Htt iPOD quantified (B). Green arrows indicate distinct and separate inclusions, red arrows indicate GFP tagged inclusions surrounding Htt inclusions, yellow arrows indicate colocalization. A minimum of 100 cells with inclusions were counted in each case and the experiment was repeated n = 2. A z-test of proportions was conducted on combined dataset and ***denotes p < 0.001. (C) FRAP measurements were performed on Htt-FUS positive inclusions. 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 Htt colocalized at 80 s compared to that of either FUS-GFP or Htt-RFP alone. ***indicates p < 0.001.

Mentions: Proteins that aggregate in to insoluble deposits can be recruited in to the same IPOD structure in cells regardless of their amino acid sequence3133. To test whether ALS mutants would form IPOD-like structures we co-transfected NSC-34 cells with IPOD substrate Huntingtinex146Q fused to cherry (Httex146Q-cherry) and either TDP-43M337V, FUSR495X, or SOD1A4V GFP fusions. Expression of Httex146Q consistently generated a round IPOD-like inclusion in the cytoplasm or nuclei in NSC-34 cells (Fig. 4A). Consistent with previous work, co-expression of Httex146Q-cherry with mutant SOD1 produced inclusions of SOD1 distinct from Httex146Q-cherry inclusions in 100% of cells expressing both proteins (Fig. 4A,B)303133. When TDP-43M337V was co-expressed with Httex146Q-cherry, inclusions were predominantly separate with no co-localization (Fig. 4A green arrow). However, in ~ 10% of cells containing aggregates of both TDP-43 and Htt the TDP-43M337V aggregates were tightly packed around the dense Htt core (Fig. 4A red arrow). In contrast, a statistically significant increase in FUS aggregates, compared to TDP-43 and SOD1, (n = 46; compared to SOD1 (n = 42) and TDP-43 (n = 45); z-test, p < 0.001) co-localized to Httex146Q-cherry IPOD-like structures (Fig. 4A yellow arrow). The remaining FUS aggregates were found to be similar to those observed for TDP-43 with separate inclusions, with a small percentage of those tightly packed around the Httex146Q-cherry core (Fig. 4B).


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 TDP-43 inclusions are distinct from iPOD.Cells were transfected with Httex146Q-mcherry and either mutant TDP-43, FUS or SOD1-GFP. The cells were imaged 48 h after transfection (A) and then the number of inclusions colocalized or surrounding the Htt iPOD quantified (B). Green arrows indicate distinct and separate inclusions, red arrows indicate GFP tagged inclusions surrounding Htt inclusions, yellow arrows indicate colocalization. A minimum of 100 cells with inclusions were counted in each case and the experiment was repeated n = 2. A z-test of proportions was conducted on combined dataset and ***denotes p < 0.001. (C) FRAP measurements were performed on Htt-FUS positive inclusions. 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 Htt colocalized at 80 s compared to that of either FUS-GFP or Htt-RFP alone. ***indicates p < 0.001.
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Related In: Results  -  Collection

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f4: SOD1 and TDP-43 inclusions are distinct from iPOD.Cells were transfected with Httex146Q-mcherry and either mutant TDP-43, FUS or SOD1-GFP. The cells were imaged 48 h after transfection (A) and then the number of inclusions colocalized or surrounding the Htt iPOD quantified (B). Green arrows indicate distinct and separate inclusions, red arrows indicate GFP tagged inclusions surrounding Htt inclusions, yellow arrows indicate colocalization. A minimum of 100 cells with inclusions were counted in each case and the experiment was repeated n = 2. A z-test of proportions was conducted on combined dataset and ***denotes p < 0.001. (C) FRAP measurements were performed on Htt-FUS positive inclusions. 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 Htt colocalized at 80 s compared to that of either FUS-GFP or Htt-RFP alone. ***indicates p < 0.001.
Mentions: Proteins that aggregate in to insoluble deposits can be recruited in to the same IPOD structure in cells regardless of their amino acid sequence3133. To test whether ALS mutants would form IPOD-like structures we co-transfected NSC-34 cells with IPOD substrate Huntingtinex146Q fused to cherry (Httex146Q-cherry) and either TDP-43M337V, FUSR495X, or SOD1A4V GFP fusions. Expression of Httex146Q consistently generated a round IPOD-like inclusion in the cytoplasm or nuclei in NSC-34 cells (Fig. 4A). Consistent with previous work, co-expression of Httex146Q-cherry with mutant SOD1 produced inclusions of SOD1 distinct from Httex146Q-cherry inclusions in 100% of cells expressing both proteins (Fig. 4A,B)303133. When TDP-43M337V was co-expressed with Httex146Q-cherry, inclusions were predominantly separate with no co-localization (Fig. 4A green arrow). However, in ~ 10% of cells containing aggregates of both TDP-43 and Htt the TDP-43M337V aggregates were tightly packed around the dense Htt core (Fig. 4A red arrow). In contrast, a statistically significant increase in FUS aggregates, compared to TDP-43 and SOD1, (n = 46; compared to SOD1 (n = 42) and TDP-43 (n = 45); z-test, p < 0.001) co-localized to Httex146Q-cherry IPOD-like structures (Fig. 4A yellow arrow). The remaining FUS aggregates were found to be similar to those observed for TDP-43 with separate inclusions, with a small percentage of those tightly packed around the Httex146Q-cherry core (Fig. 4B).

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