Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies

Travis L. Unger,Linda K. Kwong,John Q. Trojanowski,Manuela Neumann,Yan Xu,Virginia M.-Y. Lee,Matthew J. Winton,Alice Chen-Plotkin,Lionel M. Igaz

doi:10.1074/jbc.m809462200

Abstract

The disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) was identified recently as the TDP-43 (TAR DNA-binding protein 43), thereby providing a molecular link between these two disorders. In FTLD-U and ALS, TDP-43 is redistributed from its normal nuclear localization to form cytoplasmic insoluble aggregates. Moreover, pathological TDP-43 is abnormally ubiquitinated, hyperphosphorylated, and N-terminally cleaved to generate C-terminal fragments (CTFs). However, the specific cleavage site(s) and the biochemical properties as well as the functional consequences of pathological TDP-43 CTFs remained unknown. Here we have identified the specific cleavage site, Arg(208), of a pathological TDP-43 CTF purified from FTLD-U brains and show that the expression of this and other TDP-43 CTFs in cultured cells recapitulates key features of TDP-43 proteinopathy. These include the formation of cytoplasmic aggregates that are ubiquitinated and abnormally phosphorylated at sites found in FTLD-U and ALS brain and spinal cord samples. Furthermore, we observed splicing abnormalities in a cell culture system expressing TDP-43 CTFs, and this is significant because the regulation of exon splicing is a known function of TDP-43. Thus, our results show that TDP-43 CTF expression recapitulates key biochemical features of pathological TDP-43 and support the hypothesis that the generation of TDP-43 CTFs is an important step in the pathogenesis of FTLD-U and ALS.

Highlights

TDP-43 (TAR DNA-binding protein 43) is the major disease protein of sporadic and familial frontotemporal lobar degeneration (FTLD)4 with ubiquitin-positive, tau-negative inclusions (FTLD-U) with or without motor neuron disease as well as sporadic and the majority of familial amyotrophic lateral sclerosis (ALS) cases [1, 2]
Cortical urea extracts of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) brains containing high levels of C-terminal fragments (CTFs) were immunoprecipitated with anti-TDP-43 mAb, and the resultant proteins were resolved on SDS-PAGE gels and immunoblotted with a polyclonal antibody (pAb) raised to the extreme C terminus (C-t) of TDP-43
The identification of the N terminus of a CTF together with our previous liquid chromatography/tandem mass spectrometry studies on TDP-43 CTFs showing the presence of residues at the extreme C terminus [1] allow us to conclude that we have identified a pathological TDP-43 fragment spanning amino acid residues 208 – 414

Summary

Introduction

TDP-43 (TAR DNA-binding protein 43) is the major disease protein of sporadic and familial frontotemporal lobar degeneration (FTLD)4 with ubiquitin-positive, tau-negative inclusions (FTLD-U) with or without motor neuron disease as well as sporadic and the majority of familial amyotrophic lateral sclerosis (ALS) cases [1, 2]. Because pathological TDP-43 CTFs recovered from FTLD-U and ALS brains are hyperphosphorylated at multiple sites including hyperphosphorylation at Ser409 and Ser410 (p409/ 410), we asked whether the CTFs expressed in transfected cells are hyperphosphorylated [19].

Results

Conclusion