P3‐022: Multiple roles for TDP‐43 in tau gene expression regulation

Abstract

Abnormal tau protein aggregation and transcriptional deregulation plays an important role in many different tauopathies. A sub-group of these was shown to be associated with abnormal clumping of a seemingly unrelated protein called TDP-43. Mutations in TDP-43 gene are found in familial frontotemporal dementia and amyotrophic lateral sclerosis. TDP-43 is an hnRNP family's member directly involved in the expression process of thousands genes. In a search for transcription factors acting as putative regulators of MAPT, we identified multiple putative binding sites for the DNA/RNA binding protein TDP-43 within the human tau gene. We overexpressed/silenced TDP-43 in human neuroblastoma cells, both undifferentiated and differentiated. Total protein extracts from overexpressing, silenced or mock-treated cells were analyzed by Western blot for the total-tau level, normalized to beta-actin. To look for a potential role of TDP-43 as a cotranscriptional regulator, we used luciferase reporter vectors for the MAPT core promoter regions containing the putative TDP-43 binding motif, co-transfected together with TDP-43 or siRNA targeting TDP-43. We used the same reporter vectors to screen for the effect of a deletion of the promoter's binding region. To look for the effect on mRNA stability, we used luciferase reporter vectors containing the full-length 3'-UTR of tau and of other TDP-43 target genes. Finally we compared the effects of different mutant TDP-43 proteins in the same reporter assay and by Western blot. We observed a bimodal effect of TDP-43 on tau-gene expression. In undifferentiated cells, TDP-43 overexpression resulted in increased levels of tau protein, whereas in retinoic acid differentiated cells, we noticed the opposite effect. Using luciferase reporter vectors for the MAPT promoter regions, we show that overexpression of TDP-43 significantly represses tau transcription for all three MAPT haplotypes, whereas an opposite effect is observed knocking-down TDP-43. TDP-43 overexpression showed to destabilize the tau mRNA through 3'-UTR and an opposite effect is observed silencing TDP-43. We found also a significantly altered tau level in cells overexpressing mutant TDP-43. Our results suggest a crucial role for TDP-43 in tau gene expression regulation at both RNA and protein level, which might change with the cellular differentiation state.