TAR-DNA-binding 43kDa protein (TDP-43), is an RNA binding protein that has been linked to the pathology of neurodegenerative diseases such as ALS and FTLD. However, pathological mechanisms involving TDP-43, remain elusive. Current hypotheses have converged on altered RNA processing, due to a loss of function of TDP-43. To gain better insight into the pathogenic mechanisms linked to TDP-43, we hypothesised that a loss of function may lead to alterations in splicing and/or changes in protein expression in transcripts potentially linked to neurodegeneration. Thus, 2-dimensional electrophoresis (2-DE) gels and splice-junction microarrays were utilised to identify targets of TDP-43 whose expression exhibited association with altered TDP-43 levels.
The 2-DE analyses depicted changes in several proteins (spots), which, upon subsequent validation, did not reveal any correlations with differential spot intensities. In contrast, the splice-junction arrays identified TDP-43 dependent changes in 2371 genes from which candidate genes for validation were selected upon fulfilment of criteria for being altered when TDP-43 was depleted and reversible by overexpression of wild type TDP-43, but not with mutant (F4L) TDP-43. 162 genes were selected and further narrowed down based on a two-fold or higher splicing score. RT-PCR validation confirmed 6/19 transcripts, POLDIP3, BCL2L11, MADD, STAG2, BRD8 and FNIP1, as undergoing TDP-43 dependent splicing. Protein level alterations, were also observed in MADD and STAG2, following which, TDP-43 binding sites were mapped. Lastly, using an inducible TDP-43 aggregation model, a loss of function effect for TDP-43 was recapitulated i. e. similar changes in splicing profiles as when TDP-43 was knocked down, for all six of the above genes, thus, supporting a loss of function hypothesis in TDP-43 aggregates.
Our results further contribute to the number of genes known to be affected by TDP-43, with the distinct observation of involvement in apoptotic and mitotic pathways, which may have implications for TDP-43 proteinopathies
