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TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins

View Article: PubMed Central - PubMed

ABSTRACT

TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3β, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3α dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown.

No MeSH data available.


Cytoplasmic expression of RANBP1, STRA6, and DPCD dropped, while expression of, predominantly nuclear protein, ALYREF, increased in HEK Flp-in Flag-TDP-43-12xQ/N F4L after induction of TDP-43 aggregation.(a) Non-induced cells (−DOX) and cells after induction of TDP-43 aggregation (+DOX). Scale bars are 5 μm. (b) Immunofluorescence signal intensity quantification. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: **for p < 0.01, ***for p < 0.001). Error bars represent s.e.m.
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f4: Cytoplasmic expression of RANBP1, STRA6, and DPCD dropped, while expression of, predominantly nuclear protein, ALYREF, increased in HEK Flp-in Flag-TDP-43-12xQ/N F4L after induction of TDP-43 aggregation.(a) Non-induced cells (−DOX) and cells after induction of TDP-43 aggregation (+DOX). Scale bars are 5 μm. (b) Immunofluorescence signal intensity quantification. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: **for p < 0.01, ***for p < 0.001). Error bars represent s.e.m.

Mentions: Next we quantified the expression changes of the selected proteins following immunofluorescence (IF) staining (Figs 3 and 4). Signal intensity quantifications showed that 12 out of 13 proteins had statistically significant expression level changes after induction of aggregation (Figs 3b and 4b, Table S3). Most importantly, for 10 out of 13 proteins, the change in expression levels correlated with knockdown data from Štalekar et al.27. After TDP-43 aggregation, relative levels of nuclear proteins HNRNPL, EIF4A3, POLDIP3, and YARS increased, whereas nuclear levels of DNMT3A, HNRNPA3, and PABPC1 dropped (Fig. 3b). Although ALYREF is localised mainly in the nucleus, we observed its upregulation in the cytoplasmic fraction of HEK TDP-12xQ/N-F4L after TDP-43 aggregation (Fig. 4). We have previously shown reduced levels of RANBP1 after TDP-43 silencing in SH-SY5Y27 and herein confirmed this occurrence also in HEK TDP-12xQ/N-F4L after induction of TDP-43 aggregation (Figs 2b,c and 4). Finally, also DPCD cytoplasmic levels dropped in HEK TDP-12xQ/N-F4L after TDP-43 aggregation (Fig. 4).


TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins
Cytoplasmic expression of RANBP1, STRA6, and DPCD dropped, while expression of, predominantly nuclear protein, ALYREF, increased in HEK Flp-in Flag-TDP-43-12xQ/N F4L after induction of TDP-43 aggregation.(a) Non-induced cells (−DOX) and cells after induction of TDP-43 aggregation (+DOX). Scale bars are 5 μm. (b) Immunofluorescence signal intensity quantification. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: **for p < 0.01, ***for p < 0.001). Error bars represent s.e.m.
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Related In: Results  -  Collection

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f4: Cytoplasmic expression of RANBP1, STRA6, and DPCD dropped, while expression of, predominantly nuclear protein, ALYREF, increased in HEK Flp-in Flag-TDP-43-12xQ/N F4L after induction of TDP-43 aggregation.(a) Non-induced cells (−DOX) and cells after induction of TDP-43 aggregation (+DOX). Scale bars are 5 μm. (b) Immunofluorescence signal intensity quantification. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: **for p < 0.01, ***for p < 0.001). Error bars represent s.e.m.
Mentions: Next we quantified the expression changes of the selected proteins following immunofluorescence (IF) staining (Figs 3 and 4). Signal intensity quantifications showed that 12 out of 13 proteins had statistically significant expression level changes after induction of aggregation (Figs 3b and 4b, Table S3). Most importantly, for 10 out of 13 proteins, the change in expression levels correlated with knockdown data from Štalekar et al.27. After TDP-43 aggregation, relative levels of nuclear proteins HNRNPL, EIF4A3, POLDIP3, and YARS increased, whereas nuclear levels of DNMT3A, HNRNPA3, and PABPC1 dropped (Fig. 3b). Although ALYREF is localised mainly in the nucleus, we observed its upregulation in the cytoplasmic fraction of HEK TDP-12xQ/N-F4L after TDP-43 aggregation (Fig. 4). We have previously shown reduced levels of RANBP1 after TDP-43 silencing in SH-SY5Y27 and herein confirmed this occurrence also in HEK TDP-12xQ/N-F4L after induction of TDP-43 aggregation (Figs 2b,c and 4). Finally, also DPCD cytoplasmic levels dropped in HEK TDP-12xQ/N-F4L after TDP-43 aggregation (Fig. 4).

View Article: PubMed Central - PubMed

ABSTRACT

TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3&beta;, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3&alpha; dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown.

No MeSH data available.