Two distinct skeletal muscle microRNA signatures revealing the complex mechanism of sporadic ALS.

Skeletal muscle pathology is thought to have an important role in the onset and/or progression of amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disorder characterized by progressive muscle weakness. Since miRNAs are recognized as important regulatory factors of essential biological processes, we aimed to identify differentially expressed miRNAs in the skeletal muscle of sporadic ALS patients through the combination of molecular-omic technologies and bioinformatic tools. We analyzed the miRnome profiles of skeletal muscle biopsies acquired from ten sALS patients and five controls with Affymetrix GeneChip miRNA 4.0 Array. To find out differentially expressed miRNAs in patients, data were analyzed by The Institute for Genomic Research-Multi Experiment Viewer (MeV) and miRNAs whose expression difference were statistically significant were identified as candidates. The potential target genes of these miRNAs were predicted by miRWalk 2.0 and were functionally enriched by gene ontology (GO) analysis. The expression level of priority candidates was validated by quantitative real-time PCR (qRT-PCR) analysis. We identified ten differentially expressed miRNAs in patients with a fold change threshold ≥ 2.0, FDR = 0. We identified ten differentially expressed miRNAs in patients with a fold change threshold ≥ 2.0, FDR = 0. Nine out of the ten miRNAs were found to be related to top three enriched ALS-related terms. Based on the qRT-PCR validation of candidate miRNAs, patients were separated into two groups: those with upregulated miR-4429 and miR-1825 expression and those with downregulated miR-638 expression. The different muscle-specific miRNA profiles in sALS patients may indicate the involvement of etiologic heterogeneity, which may allow the development of novel therapeutic strategies.

Reduced mitochondrial fission and impaired energy metabolism in human primary skeletal muscle cells of Megaconial Congenital Muscular Dystrophy.

Megaconial Congenital Muscular Dystrophy (CMD) is a rare autosomal recessive disorder characterized by enlarged mitochondria located mainly at the periphery of muscle fibers and caused by mutations in the Choline Kinase Beta (CHKB) gene. Although the pathogenesis of this disease is not well understood, there is accumulating evidence for the presence of mitochondrial dysfunction. In this study, we aimed to investigate whether imbalanced mitochondrial dynamics affects mitochondrial function and bioenergetic efficiency in skeletal muscle cells of Megaconial CMD. Immunofluorescence, confocal and transmission electron microscopy studies revealed impaired mitochondrial network, morphology, and localization in primary skeletal muscle cells of Megaconial CMD. The organelle disruption was specific only to skeletal muscle cells grown in culture. The expression levels of mitochondrial fission proteins (DRP1, MFF, FIS1) were found to be decreased significantly in both primary skeletal muscle cells and tissue sections of Megaconial CMD by Western blotting and/or immunofluorescence analysis. The metabolomic and fluxomic analysis, which were performed in Megaconial CMD for the first time, revealed decreased levels of phosphonucleotides, Krebs cycle intermediates, ATP, and altered energy metabolism pathways. Our results indicate that reduced mitochondrial fission and altered mitochondrial energy metabolism contribute to mitochondrial dysmorphology and dysfunction in the pathogenesis of Megaconial CMD.

Microtubule-associated protein 1B dysregulates microtubule dynamics and neuronal mitochondrial transport in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating childhood disease primarily affecting lower motoneurons in the spinal cord. SMA is caused by the loss of functional survival of motoneuron (SMN) protein, leading to structural and functional alterations of the cytoskeleton in motoneurons and other cells. Loss of SMN results in impairments of microtubule architecture, but the underlying mechanisms are not completely understood. In this study, we mechanistically analyzed the effects of SMN deficiency on microtubules, demonstrating a reduced stability together with a reduction in alpha tubulin detyrosination. This was caused by increased levels of microtubule-associated protein 1B and tubulin tyrosine ligase, resulting in mitochondrial mislocalization in SMA. Our findings suggest that altered tubulin post-translational modifications and microtubule-associated proteins are involved in the pathomechanisms of SMA, such as an impaired axonal transport of mitochondria.

The Common miRNA Signatures Associated with Mitochondrial Dysfunction in Different Muscular Dystrophies.

Secondary mitochondrial damage in skeletal muscles is a common feature of different neuromuscular disorders, which fall outside the mitochondrial cytopathies. The common cause of mitochondrial dysfunction and structural changes in skeletal muscle tissue remains to be discovered. Although they are associated with different clinical, genetic, and pathologic backgrounds, the pathomechanisms underlying neuromuscular disorders might be attributed to the complex interaction and cross talk between mitochondria and the associated miRNAs. This study aimed to identify the common miRNA signatures that are associated with mitochondrial damage in different muscular dystrophies (MDs; Duchenne muscular dystrophy, megaconial congenital muscular dystrophy, Ullrich congenital muscular dystrophy, and α-dystroglycanopathy). The miRNome profiles of skeletal muscle biopsies acquired from four different MD groups and control individuals were analyzed by miRNA microarray. We identified 17 common up-regulated miRNAs in all of the tested MD groups. A specific bioinformatics approach identified 10 of these miRNAs to be specifically related to the mitochondrial pathways. Six miRNAs, miR-134-5p, miR-199a-5p, miR-382-5p, miR-409-3p, miR-497-5p, and miR-708-5p, were associated with the top four mitochondrial pathways and were thus selected as priority candidates for further validation by quantitative real-time PCR analysis. We demonstrate, for the first time, common up-regulated miRNAs that are associated with mitochondrial damage in different MD groups, therefore contributing to the pathophysiology. Our findings may open a new gate toward therapeutics.