Investigating Eukaryotic Elongation Factor 2 Kinase/Eukaryotic Translation Elongation Factor 2 Pathway Regulation and Its Role in Protein Synthesis Impairment during Disuse-Induced Skeletal Muscle Atrophy.

The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. Eukaryotic elongation factor 2 kinase (eEF2k) impairs the ability of eukaryotic translation elongation factor 2 (eEF2) to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hind limb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P < 0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated/total eEF2 was robustly elevated after 3-day HS, which was completely reversed by 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and decreased by 1.7-fold (P < 0.05) by nifedipine. Transfection of C2C12 with cytomegalovirus promoter (pCMV)-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. More importantly, pharmacologic enhancement of eEF2 phosphorylation induced phosphorylated ribosomal protein S6 kinase (T389) up-regulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway was up-regulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on ribosomal protein S6 kinase activity as well as protein expression of key atrophy biomarkers, muscle atrophy F-box/atrogin-1 and muscle RING finger-1.

AR cooperates with SMAD4 to maintain skeletal muscle homeostasis.

Androgens and androgen-related molecules exert a plethora of functions across different tissues, mainly through binding to the transcription factor androgen receptor (AR). Despite widespread therapeutic use and misuse of androgens as potent anabolic agents, the molecular mechanisms of this effect on skeletal muscle are currently unknown. Muscle mass in adulthood is mainly regulated by the bone morphogenetic protein (BMP) axis of the transforming growth factor (TGF)-ß pathway via recruitment of mothers against decapentaplegic homolog 4 (SMAD4) protein. Here we show that, upon activation, AR forms a transcriptional complex with SMAD4 to orchestrate a muscle hypertrophy programme by modulating SMAD4 chromatin binding dynamics and enhancing its transactivation activity. We challenged this mechanism of action using spinal and bulbar muscular atrophy (SBMA) as a model of study. This adult-onset neuromuscular disease is caused by a polyglutamine expansion (polyQ) in AR and is characterized by progressive muscle weakness and atrophy secondary to a combination of lower motor neuron degeneration and primary muscle atrophy. Here we found that the presence of an elongated polyQ tract impairs AR cooperativity with SMAD4, leading to an inability to mount an effective anti-atrophy gene expression programme in skeletal muscle in response to denervation. Furthermore, adeno-associated virus, serotype 9 (AAV9)-mediated muscle-restricted delivery of BMP7 is able to rescue the muscle atrophy in SBMA mice, supporting the development of treatments able to fine-tune AR-SMAD4 transcriptional cooperativity as a promising target for SBMA and other conditions associated with muscle loss.

RNA Transcript Diversity in Neuromuscular Research.

Three decades since the Human Genome Project began, scientists have now identified more then 25,000 protein coding genes in the human genome. The vast majority of the protein coding genes (> 90%) are multi-exonic, with the coding DNA being interrupted by intronic sequences, which are removed from the pre-mRNA transcripts before being translated into proteins, a process called splicing maturation. Variations in this process, i.e. by exon skipping, intron retention, alternative 5' splice site (5'ss), 3' splice site (3'ss), or polyadenylation usage, lead to remarkable transcriptome and proteome diversity in human tissues. Given its critical biological importance, alternative splicing is tightly regulated in a tissue- and developmental stage-specific manner. The central nervous system and skeletal muscle are amongst the tissues with the highest number of differentially expressed alternative exons, revealing a remarkable degree of transcriptome complexity. It is therefore not surprising that splicing mis-regulation is causally associated with a myriad of neuromuscular diseases, including but not limited to amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), and myotonic dystrophy type 1 and 2 (DM1, DM2). A gene's transcript diversity has since become an integral and an important consideration for drug design, development and therapy. In this review, we will discuss transcript diversity in the context of neuromuscular diseases and current approaches to address splicing mis-regulation.

PRMT inhibitor promotes SMN2 exon 7 inclusion and synergizes with nusinersen to rescue SMA mice.

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients' life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches for improved and long-lasting benefits for patients. Protein arginine methyltransferases (PRMTs) are emerging as druggable epigenetic targets, with several small-molecule PRMT inhibitors already in clinical trials. From a screen of epigenetic molecules, we have identified MS023, a potent and selective type I PRMT inhibitor able to promote SMN2 exon 7 inclusion in preclinical SMA models. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has minimal off-target effects, and the added benefit is mainly due to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA.

LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice.

Spinobulbar muscular atrophy (SBMA) is caused by CAG expansions in the androgen receptor gene. Androgen binding to polyQ-expanded androgen receptor triggers SBMA through a combination of toxic gain-of-function and loss-of-function mechanisms. Leveraging cell lines, mice, and patient-derived specimens, we show that androgen receptor co-regulators lysine-specific demethylase 1 (LSD1) and protein arginine methyltransferase 6 (PRMT6) are overexpressed in an androgen-dependent manner specifically in the skeletal muscle of SBMA patients and mice. LSD1 and PRMT6 cooperatively and synergistically transactivate androgen receptor, and their effect is enhanced by expanded polyQ. Pharmacological and genetic silencing of LSD1 and PRMT6 attenuates polyQ-expanded androgen receptor transactivation in SBMA cells and suppresses toxicity in SBMA flies, and a preclinical approach based on miRNA-mediated silencing of LSD1 and PRMT6 attenuates disease manifestations in SBMA mice. These observations suggest that targeting overexpressed co-regulators can attenuate androgen receptor toxic gain-of-function without exacerbating loss-of-function, highlighting a potential therapeutic strategy for patients with SBMA.

Dystrophin involvement in peripheral circadian SRF signalling.

Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy.

The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.