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.
Highlights
HUGO symbol eukaryotic translation initiation factor 4A3 (EIF4A3) TARDBP YARS DNMT3A POLDIP3 HNRNPL ZNF326 PABPC1 HNRNPA3 TIAL1 STRA6 DPCD ALYREF RANBP1
We have investigated whether the aggregation/sequestration of TDP-43 correlated with its loss of function by comparing the expression changes of selected proteins responsive to silencing of TDP-43 in an aggregation and sequestration model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L
Our results show that differential expression of proteins in TDP-12xQ/N-F4L cells correlated with proteomic results of TDP-43 knockdown in SH-SY5Y, revealing a common set of proteins whose expression is influenced via TDP-43 aggregation or knockdown
Summary
HUGO symbol EIF4A3 TARDBP YARS DNMT3A POLDIP3 HNRNPL ZNF326 PABPC1 HNRNPA3 TIAL1 STRA6 DPCD ALYREF RANBP1. It is of prime importance to better characterize its consequences at the cellular level. In this respect, previous studies demonstrated the effect of TDP-43 knockdown on the transcriptome[2,26] and recently on proteome of SH-SY5Y cells[27] and cytotoxicity has been observed to increase following cytoplasmic internalisation of TDP-43 containing inclusions bodies[28]. Our results show that differential expression of proteins in TDP-12xQ/N-F4L cells correlated with proteomic results of TDP-43 knockdown in SH-SY5Y, revealing a common set of proteins whose expression is influenced via TDP-43 aggregation or knockdown
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