ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

Highlights

• The function of nuclear TDP-43 in neurite morphology in mature neurons.

• Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus.

• Abnormal neurite morphology caused by missense mutants of TDP-43.

• The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

Abstract

Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons.

Introduction

TAR-binding protein of 43 kDa (TDP-43) is a RNA/DNA binding protein involved in functions such as transcription, RNA splicing, mRNA stability, and nucleocytoplasmic shuttling of mRNA [1], [2], [3], [4]. TDP-43 has recently been characterized as the major component of ubiquitinated inclusion in amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD) [5]. ALS is a neurodegenerative disease that shows progressive degeneration of large motor neurons in the spinal cord and cerebral cortex, which leads to paralysis and death [6]. FTLD causes focal degeneration of the frontal and temporal cortex, which is associated with defects of executive, social, and cognitive functions [7]. Although these two diseases are clinically distinct, there is significant overlap between these two disorders in patients. Surprisingly, a significant population of patients with ALS or FTLD (∼95% ALS and ∼50% FTLD) has TDP-43 positive inclusions in CNS, even though mutations in TDP-43 are absent [2], [8]. TDP-43 has characteristic hallmarks such as abnormal hyperphosphorylation, ubiquitination, and the production of abnormal C-terminal fragments, which are toxic under disease conditions [5], [9]. TDP-43 is mainly localized to the nucleus in healthy neurons, but its abnormal mislocalization from the nucleus to cytosol and neurites and aggregation of TDP-43 in disease have also been reported, indicating that loss of TDP-43 and/or gain of function may contribute to disease pathogenesis [10], [11]. Furthermore, mutations in the gene encoding TDP-43 have been found in ALS and FTLD, indicating that it plays important roles in molecular pathogenesis of diseases [2], [12]. Interestingly, the majority of mutations have been identified within the C-terminal glycine-rich region, which is known to be important for protein–protein interactions [2].

However, the underlying pathogenic mechanism conferred by these mutations has not been characterized. Moreover, little is known about earlier pathogenesis by TDP-43 with missense mutations before cell death in differentiated neurons. In addition, the exact functions of TDP-43 in differentiated neurons have not yet been established, despite several previous studies.

Herein, we investigated the physiological function of TDP-43 and the effects of missense mutant proteins in cultured differentiated cortical neurons. Reduction of TDP-43 by siRNA showed abnormal neurite morphology and cell death. Interestingly, missense mutants were prone to partial mislocalization to the cytosol and neurites and to enhanced neurite morphological changes when compared with wild-type TDP-43. However, when wild-types such as other missense mutant were expressed in the cytosol, nuclear TDP-43 was reduced, leading to abnormal neurite morphology and cell death. Therefore, our study provides an early cellular pathogenic mechanism through which mislocalization of TDP-43 with missense mutations might contribute to abnormal neuronal morphology via dominant negative action in differentiated neurons.