Elucidating the Contribution of Skeletal Muscle Ion Channels to Amyotrophic Lateral Sclerosis in search of new therapeutic options

Giulia Maria Camerino,Annamaria De Luca,Gabriella Dobrowolny,Michela De Bellis,Domenico Tricarico,Sabata Pierno,Antonio Musarò,Adriano Fonzino,Antonietta Mele,Antonella Liantonio,Elena Conte,Nancy Tarantino,Jean-Francois Desaphy

doi:10.1038/s41598-019-39676-3

Abstract

The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in Amyotrophic Lateral Sclerosis (ALS). Here we investigated the role of the most important ion channels in skeletal muscle of an ALS animal model (MLC/SOD1G93A) carrying a mutated SOD1 exclusively in this tissue, avoiding motor-neuron involvement. Ion channels are fundamental proteins for muscle function, and also to sustain neuromuscular junction and nerve integrity. By a multivariate statistical analysis, using machine learning algorithms, we identified the discriminant genes in MLC/SOD1G93A mice. Surprisingly, the expression of ClC-1 chloride channel, present only in skeletal muscle, was reduced. Also, the expression of Protein Kinase-C, known to control ClC-1 activity, was increased, causing its inhibition. The functional characterization confirmed the reduction of ClC-1 activity, leading to hyperexcitability and impaired relaxation. The increased expression of ion channel coupled AMPA-receptor may contribute to sustained depolarization and functional impairment. Also, the decreased expression of irisin, a muscle-secreted peptide protecting brain function, may disturb muscle-nerve connection. Interestingly, the in-vitro application of chelerythrine or acetazolamide, restored ClC-1 activity and sarcolemma hyperexcitability in these mice. These findings show that ion channel function impairment in skeletal muscle may lead to motor-neuron increased vulnerability, and opens the possibility to investigate on new compounds as promising therapy.

Highlights

Amyotrophic Lateral Sclerosis (ALS) is a progressive degenerative disease affecting motor neurons
We evaluated the expression of a series of genes involved in skeletal muscle structure and function, encoding for ion channels, their regulatory proteins and subunits, structural proteins, markers of denervation (Supplementary Fig. S1) and phenotype shift (Supplementary Fig. S2), hormones and neurotrophic factors
The involvement of these genes in ALS etiology was evaluated by an accurate multivariate statistical analysis PCA-LDA (Principal Component Analysis and Linear Discriminant Analysis), using both unsupervised and supervised machine learning algorithms, which allowed to identify new potential therapeutic targets

Summary

Introduction

Amyotrophic Lateral Sclerosis (ALS) is a progressive degenerative disease affecting motor neurons. Trophic factors secreted by myofibers, such as Insulin Like Growth Factor-1 (IGF-1) or Glial-Cell-Line-Derived Neurotrophic Factor (GDNF), can promote motor neuron survival in ALS model through stabilization of NMJ8 These observations support the view that this pathology is not solely a neurological disorder and include a “dying-back” phenomenon, by which motor unit loss and altered muscle function precede the death of motor neurons[9]. In the light of these considerations, we focused our study on ion channel expression and function in skeletal muscles of transgenic SOD1G93A mice and in muscle specific MLC/SOD1G93A mice (carrying the SOD1G93A mutant gene under the transcriptional control of Myosin Light Chain, MLC, a muscle-specific promoter) to elucidate the mechanisms and to find potential therapeutic targets in ALS

Methods

Results

Conclusion