Calpain-dependent disruption of nucleo-cytoplasmic transport in ALS motor neurons

Takenari Yamashita,Megumi Akamatsu,Shin Kwak,Hitoshi Aizawa,Sayaka Teramoto

doi:10.1038/srep39994

Abstract

Nuclear dysfunction in motor neurons has been hypothesized to be a principal cause of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we investigated the mechanism by which the nuclear pore complex (NPC) is disrupted in dying motor neurons in a mechanistic ALS mouse model (adenosine deaminase acting on RNA 2 (ADAR2) conditional knockout (AR2) mice) and in ALS patients. We showed that nucleoporins (Nups) that constituted the NPC were cleaved by activated calpain via a Ca2+-permeable AMPA receptor-mediated mechanism in dying motor neurons lacking ADAR2 expression in AR2 mice. In these neurons, nucleo-cytoplasmic transport was disrupted, and the level of the transcript elongation enzyme RNA polymerase II phosphorylated at Ser2 was significantly decreased. Analogous changes were observed in motor neurons lacking ADAR2 immunoreactivity in sporadic ALS patients. Therefore, calpain-dependent NPC disruption may participate in ALS pathogenesis, and inhibiting Ca2+-mediated cell death signals may be a therapeutic strategy for ALS.

Highlights

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease of unknown etiology
An in vitro calpain cleavage assay demonstrated that Flag-Nups were effectively cleaved by calpain (Fig. 1c), which suggested that the lack of Nup immunoreactivity in ADAR2-deficient anterior horn cells (AHCs) likely resulted from cleavage of Nups by activated calpain
To assess whether the lack of Nup[62] in ADAR2 knockout (AR2) mouse AHCs was induced by the activation of calpain in response to an amplified Ca2+ influx through the AMPA receptors, we examined the changes in Nup[62] and ADAR2 levels in AR2res (AR2/GluR-BR/R) mice, in which the endogenous GluA2 alleles were replaced with the GluR-BR allele, which encodes Q/R site-edited GluA220,25,26

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease of unknown etiology. Conditional ADAR2 knockout mice (AR2 mice) displayed the ALS phenotype resulting from progressive degeneration of motor neurons, and exhibited TDP-43 mislocalization that resembled TDP-43 pathology[20,21], the most reliable pathological hallmark of ALS This behaviorally and pathologically ALS-like phenotype of AR2 mice results from excess influx of Ca2+ through AMPA receptor complexes containing Q/R site-unedited GluA2 subunits[20,21]; continuous Ca2+ influx through these abnormal AMPA receptors activates the Ca2+-dependent cysteine protease calpain, which cleaves TDP-43 into aggregation-prone fragments that serve as seeds for TDP-43 pathology[20,21,22]. The motor neurons of AR2 mice possess abnormal vacuoles during the course of death, and these nuclear vacuoles disappear when Ca2+ influx through AMPA receptors is normalized[23] These pieces of evidence suggest the involvement of Ca2+-dependent dysregulation of nucleo-cytoplasmic transport in the slow death of motor neurons in AR2 mice and ALS patients. We investigated how expression of Ca2+-permeable AMPA receptors disrupts the nucleo-cytoplasmic transport machinery in AR2 mice and whether similar alterations are present in motor neurons of ALS patients

Methods

Results

Conclusion