Inhibition of TDP-43 Aggregation by Nucleic Acid Binding

Yi-Chen Huang,Ruei-Yu He,Pang-Hsien Tu,Joseph Jen-Tse Huang,Yin-Chih Hsu,Ku-Feng Lin,Jiri Koubek,Maria Gasset

doi:10.1371/journal.pone.0064002

Abstract

The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the in vitro protein expression system to evaluate the solubility change of TDP-43 in response to factors such as nucleotide binding and temperature. Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208–414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.

Highlights

The 43 kDa TAR DNA-binding protein (TDP-43) and its Cterminal proteolytic fragments have been identified as the major component of inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with unbiquitinpositive inclusions (FTLD-U) [1,2,3,4,5]
These RRM domains are followed by a glycine-rich C-terminal tail which is known to interact with different isoforms of the heterogeneous nuclear ribonucleoprotein [9,17,20,21,22,23,24] that is involved in the cystic fibrosis transmembrane conductance regulator (CFTR) exon skipping
The generated TDP-43 or its derivatives are further incubated in these systems to evaluate the temporal dependence of their solubility at different temperatures in the presence or absence of its cognate oligonucleotides by quantifying the TDP-43 proteins in the supernatant and pellet fractions of each system with western blotting (Figure 1A)

Summary

Introduction

The 43 kDa TAR DNA-binding protein (TDP-43) and its Cterminal proteolytic fragments have been identified as the major component of inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with unbiquitinpositive inclusions (FTLD-U) [1,2,3,4,5]. It has been recognized as a histopathological marker of Alzheimer’s [6], Parkinson’s [7], as well as Huntington’s diseases [8]. It has been demonstrated that certain mutations in the TDP-43 C-terminus accelerate its aggregation and increase toxicity in in vivo and in vitro studies [11,29]

Methods

Results

Conclusion