Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = −0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with increased Atrogin-1 expression compared to healthy control cells (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.
Highlights
Motor neuron diseases (MND) are a collection of neurological disorders that affect upper and lower motor neurons of the central nervous system (CNS)
To assess temporal changes in Akt phosphorylation in progressively denervated and atrophying mSOD1G93A mouse skeletal muscle, gastrocnemius protein from post-natal day (PD)[35], PD63, PD90 and end-stage was collected and phosphorylated and total Akt were assessed via Western blot (Fig. 1)
Though carboxyl-terminal modulator protein (CTMP) expression remained unchanged between PD35 and PD63, its expression significantly increased by PD90 compared to both PD35 (P < 0.01) and PD63 (P < 0.05) (Fig. 1B,D)
Summary
Motor neuron diseases (MND) are a collection of neurological disorders that affect upper and lower motor neurons of the central nervous system (CNS). In the past two decades, our understanding of the pre-symptomatic aspects of disease progression have expanded considerably, and we know that some of the first major outward pre-symptomatic anatomic manifestations of disease is the dismantling of the neuromuscular junction (NMJ) and progressive disconnection of motor neurons from NMJs4–7 With this information, further insights into physiologic changes within the skeletal muscle over time in humans and ALS animal models, especially the classic mutant superoxide dismutase 1 (SOD1) mouse model (mSOD1G93A)[8], have been made possible. Recent research indicated that a reduction in insulin signaling and expression and activity the serine-threonine kinase, Akt, are associated with progressive muscular atrophy in animal models and correlated with poor survival prognosis in ALS patients[12,13,14]. We report key biochemical changes in the gastrocnemius muscle of the mSOD1G93A amyotrophic mouse model and discuss for the first time, a potential association between CTMP and Akt activation overtime in the muscle of this mouse model
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