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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.


Expression of selected proteins in nuclear (a) or cytoplasmic (b) fractions of HEK Flp-in Flag-TDP-43-12xQ/N F4L before (−DOX) and after (+DOX) induction of TDP-43 aggregation. Relative expression of proteins was quantified with western blot (c). Fibrillarin (FBL) and GAPDH were used as loading controls for nuclear and cytoplasmic fractions. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: *for p < 0.05, ***for p < 0.001). Error bars represent s.e.m.
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f2: Expression of selected proteins in nuclear (a) or cytoplasmic (b) fractions of HEK Flp-in Flag-TDP-43-12xQ/N F4L before (−DOX) and after (+DOX) induction of TDP-43 aggregation. Relative expression of proteins was quantified with western blot (c). Fibrillarin (FBL) and GAPDH were used as loading controls for nuclear and cytoplasmic fractions. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: *for p < 0.05, ***for p < 0.001). Error bars represent s.e.m.

Mentions: In HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L cells (referred to as HEK TDP-12xQ/N-F4L hereafter) TDP-43 aggregation was induced by addition of doxycycline (DOX) to the growth medium (Fig. 1). The aggregates were observed in the nucleus and the cytoplasm and could be detected both by anti-TDP-43 and anti-Flag antibodies. After 72 hours, the nuclear levels of soluble TDP-43 significantly dropped to 37.4 ± 4.7% (mean ± s.e.m., n = 3) as determined by western blot (Fig. 2a,c).


TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins
Expression of selected proteins in nuclear (a) or cytoplasmic (b) fractions of HEK Flp-in Flag-TDP-43-12xQ/N F4L before (−DOX) and after (+DOX) induction of TDP-43 aggregation. Relative expression of proteins was quantified with western blot (c). Fibrillarin (FBL) and GAPDH were used as loading controls for nuclear and cytoplasmic fractions. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: *for p < 0.05, ***for p < 0.001). Error bars represent s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036055&req=5

f2: Expression of selected proteins in nuclear (a) or cytoplasmic (b) fractions of HEK Flp-in Flag-TDP-43-12xQ/N F4L before (−DOX) and after (+DOX) induction of TDP-43 aggregation. Relative expression of proteins was quantified with western blot (c). Fibrillarin (FBL) and GAPDH were used as loading controls for nuclear and cytoplasmic fractions. Unpaired Student’s t-test was used to determine significant differences between samples (significance levels: *for p < 0.05, ***for p < 0.001). Error bars represent s.e.m.
Mentions: In HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L cells (referred to as HEK TDP-12xQ/N-F4L hereafter) TDP-43 aggregation was induced by addition of doxycycline (DOX) to the growth medium (Fig. 1). The aggregates were observed in the nucleus and the cytoplasm and could be detected both by anti-TDP-43 and anti-Flag antibodies. After 72 hours, the nuclear levels of soluble TDP-43 significantly dropped to 37.4 ± 4.7% (mean ± s.e.m., n = 3) as determined by western blot (Fig. 2a,c).

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.