Identification of Genetic Modifiers of TDP-43: Inflammatory Activation of Astrocytes for Neuroinflammation.

Jae-Hong Kim,Kyoungho Suk,Donghwi Park,Myungjin Jo,Md Habibur Rahman,Hyung-Jun Kim

doi:10.3390/cells10030676

Abstract

Transactive response DNA-binding protein 43 (TDP-43) is a ubiquitously expressed DNA/RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 has been implicated in numerous aspects of the mRNA life cycle, as well as in cell toxicity and neuroinflammation. In this study, we used the toxicity of the TDP-43 expression in Saccharomyces cerevisiae as an assay to identify TDP-43 genetic interactions. Specifically, we transformed human TDP-43 cDNAs of wild-type or disease-associated mutants (M337V and Q331K) en masse into 4653 homozygous diploid yeast deletion mutants and then used next-generation sequencing readouts of growth to identify yeast toxicity modifiers. Genetic interaction analysis provided a global view of TDP-43 pathways, some of which are known to be involved in cellular metabolic processes. Selected putative loci with the potential of genetic interactions with TDP-43 were assessed for associations with neurotoxicity and inflammatory activation of astrocytes. The pharmacological inhibition of succinate dehydrogenase flavoprotein subunit A (SDHA) and voltage-dependent anion-selective channel 3 (VDAC3) suppressed TDP-43-induced expression of proinflammatory cytokines in astrocytes, indicating the critical roles played by SDHA and VDAC3 in TDP-43 pathways during inflammatory activation of astrocytes and neuroinflammation. Thus, the findings of our TDP-43 genetic interaction screen provide a global landscape of TDP-43 pathways and may help improve our understanding of the roles of glia and neuroinflammation in ALS and FTD pathogenesis.

Highlights

Transactive response DNA-binding protein 43 (TDP-43) genetic interactions, we found that overexpression of human TDP-43 cDNAs, WT, or disease-associated mutants (M337V and Q331K), caused toxicity in S. cerevisiae, as indicated by a lower number of visible spots when compared with the control vector transformation (Figure 1a–c)
In a genome-wide pooled screen to identify genetic interactions, the TDP-43 gene or its two mutant variants were first introduced into a pool of 4653 yeast homozygous deletion strains containing a 20 bp DNA barcode sequence, such that each deletion strain harbored unique barcode sequences next to the deletion locus [33,44]
The amplified barcodes were subjected to nextgeneration sequencing (Bar-seq) to quantify yeast barcode abundances and identify fitness values of TDP-43 genetic interactions [31,32,33,45] (Supplementary Table S1)

Summary

Introduction

Transactive response DNA-binding protein 43 kDa (TDP-43), a DNA/RNA-binding protein encoded by the TARDBP gene in humans, is involved in transcriptional repression and splicing and stability of RNA [1]. It is a heterogeneous nuclear ribonucleo-type protein ubiquitously expressed in eukaryotic cells, in the nucleus [2]. Inclusions of wild-type (WT) and missense mutations of TDP-43 are major causes of amyotrophic lateral sclerosis (ALS) pathology [3]. More than 50 missense mutations in the TDP-43 gene have been associated with familial and sporadic cases of ALS [4]. We built upon these findings to compare the characteristics of WT TDP-43 and these ALS-associated mutants in terms of genetic interactions and relevant signaling pathways

Objectives

Methods

Results

Conclusion