Cellular Model of TAR DNA-binding Protein 43 (TDP-43) Aggregation Based on Its C-terminal Gln/Asn-rich Region

Laura De Conti,Valentina Romano,Francisco E. Baralle,Cristiana Stuani,Corrado Guarnaccia,Mauricio Budini,Emanuele Buratti

doi:10.1074/jbc.m111.288720

Abstract

TDP-43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These characteristic aggregates are a "landmark" of the disease, but their role in the pathogenesis is still obscure. In previous works, we have shown that the C-terminal Gln/Asn-rich region (residues 321-366) of TDP-43 is involved in the interaction of this protein with other members of the heterogeneous nuclear ribonucleoprotein protein family. Furthermore, we have shown that the interaction through this region is important for TDP-43 splicing inhibition of cystic fibrosis transmembrane regulator exon 9, and there were indications that it was involved in the aggregation process. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331-369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients. These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. In addition, it will be a powerful tool to test novel therapeutic strategies/effectors aimed at preventing/reducing this phenomenon.

Highlights

TDP-43 is the principal protein component of cellular inclusion in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)
We have previously identified several members of the heterogeneous nuclear ribonucleoproteins (hnRNPs)-A/B protein family as specific interactors of region 321–366 of TDP-43 and have shown that these contacts are very important for maintaining the splicing functionality of TDP-43 [34, 40]
We have focused on the TDP-43-hnRNP-A2 interaction being one of the most prominent and verified by several proteomic/ interaction studies (38 – 40, 43)

Summary

Background

TDP-43 is the principal protein component of cellular inclusion in ALS and FTLD. Results: Tandem repetitions of TDP-43 residues 331–369 induce cellular aggregates that recruit endogenous TDP-43. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331–369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. Mislocalization and structural changes of two hnRNPs, TDP-43 and FUS (8 –11), have been shown to occur in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) In these two and other related diseases, the identification of TDP-43 protein as a major component of the characteristic ubiquitinated neuronal inclusions [12, 13] has opened up several new avenues of research This has lead to the generation of a TDP-43 aggregation model that recapitulates many but not all features of the aggregates observed in patients

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION