CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS.

Tsukasa Uchida,Naomi Sakashita,Yoshitaka Tamaki,Yoshinori Banno,Takakuni Maki,Hidefumi Ito,Kazuchika Nishitsuji,Toshifumi Morimura,Makoto Urushitani,Seiji Kaji,Ryosuke Takahashi,Takashi Ayaki,Akemi Shodai,Hirofumi Yamashita

doi:10.1038/srep19118

Abstract

The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS.

Highlights

NLS, and FLAG-CUL2, immunoprecipitated with anti-FLAG or anti-GFP affinity beads, and analyzed by Western blotting using anti-GFP or anti-FLAG antibodies, respectively. (c) Interaction of TAR DNA-binding protein 43 kDa (TDP-43) with von Hippel Lindau (VHL)
We documented that glial cytoplasmic inclusions (GCI) in Amyotrophic lateral sclerosis (ALS) patients were shown to comprise misfolded TDP-43 and VHL, which might underlie the oligodendrocyte dysfunction in ALS pathogenesis[18,19,20]
Because of the potential failure to seize labile protein complexes in specific conditions such as ubiquitination, we integrated in vitro ubiquitination and a reversible covalent immunoprecipitation assay[21], in which recombinant human TDP-43-FLAG proteins were incubated in a ubiquitination reaction solution containing detergent-soluble S100 lysates from HEK293A cells, and the reaction mixtures were incubated with disulfide cross-linker, and the immunoprecipitates with FLAG affinity beads were eluted under mild reducing condition (Fig. 1a)

Summary

Introduction

NLS (mNLS), and FLAG-CUL2, immunoprecipitated with anti-FLAG (left) or anti-GFP (right) affinity beads, and analyzed by Western blotting using anti-GFP or anti-FLAG antibodies, respectively. (c) Interaction of TDP-43 with VHL. HEK293A cell lysates transfected with HA-VHL were immunoprecipitated with anti-HA affinity beads and immunoblotted for endogenous CUL2, elongin B, elongin C, and TDP-43. Studies[12], TDP-43 in ALS tissues or with cysteine substitution mutants in RRM1 is more heavily ubiquitinated than normally folded species[4,5,10]. These lines of evidence imply the presence of misfolded protein-specific handling machinery, which may be involved in ALS pathology. We found that the von Hippel Lindau (VHL)/cullin-2 (CUL2) E3 complex recognized and ubiquitinated misfolded TDP-43 and promoted clearance of fragmented forms of TDP-43. We documented that glial cytoplasmic inclusions (GCI) in ALS patients were shown to comprise misfolded TDP-43 and VHL, which might underlie the oligodendrocyte dysfunction in ALS pathogenesis[18,19,20]

Methods

Results

Conclusion