David C. Butler

AMPK inhibits myoblast differentiation through a PGC-1α-dependent mechanism

Elevated phosphorylation of AMP-activated protein kinase (AMPK) has been shown to inhibit skeletal muscle growth in both culture and animal models, but its role in differentiation of muscle cells is less clear. p21 is known to have an important role in differentiation, but AMPKs role regulating p21 in differentiation in muscle cultures is unknown. Therefore, the purpose of this study was to determine the role of p21 in differentiation of skeletal muscle cells under conditions of elevated AMPK phosphorylation. Treating C(2)C(12) myoblast cultures with 1 mM 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR) for up to 24 h induced AMPK phosphorylation. Activation of AMPK reduced p21 protein and mRNA expression, which was associated with reduced G(1)/S cell cycle transition and p21 promoter activity. AICAR-treated myoblasts undergoing differentiation also had reduced p21 protein expression, reduced myotube formation, and myosin accumulation. When myotube cultures were treated with AICAR for 24 h, p21, myosin protein expression, and MyoD were significantly reduced. Myotube atrophy was also apparent compared with control conditions. Addition of compound C, an AMPK inhibitor, attenuated AICARs negative effects on the myotube cultures. The nuclear expression of p21 protein appeared to be more affected by AICAR-treated myotubes than the cytosolic portion of p21 protein, which was attenuated with compound C treatment. Further analysis revealed that AICAR treatment increased PGC-1alpha and decreased FOXO3A protein expression, which was reversed with compound C cotreatment. Knockdown of PGC-1alpha with shRNA corroborated the compound C data, preserving nuclear FOXO3A and p21 protein expression. These data demonstrate that AICAR-induced AMPK phosphorylation inhibits cell cycle transition, reducing differentiation of myoblasts into myotubes, through PGC-1alpha-FOXO3A-p21.

Developing intrabodies for the therapeutic suppression of neurodegenerative pathology.

Many neurodegenerative diseases have misfolded proteins as a primary occurrence in pathogenesis. A combination of antibody and genetic engineering has emerged as a powerful tool for developing reagents that specifically target the misfolding process itself, and/or abnormal interactions of the misfolded protein species. This review focuses on the selection and testing of intracellular antibody fragments (intrabodies), with a particular focus on Huntingtons disease (HD) and Parkinsons disease (PD), both of which show prominent intracellular protein aggregates in affected neurons. The most dramatic advances are in HD, where in vivo efficacy of intrabodies has been demonstrated. Targets in other neurodegenerative disorders, including Alzheimers disease and prion diseases, are noted more briefly, with an emphasis on the potential for intracellular manipulations. Given the specificity and versatility of antibody-based reagents, the wide range of options for conformational and post-translationally-modified targets, and the recent improvement in gene delivery, this should be a fertile field for 21st century pharmacology.

Phospho-ablated Id2 is growth suppressive and pro-apoptotic in proliferating myoblasts.

Inhibitor of differentiation protein-2 (Id2) is a dominant negative helix-loop-helix (HLH) protein, and a positive regulator of proliferation, in various cells. The N-terminal region of Id2 contains a consensus cdk2 phosphorylation sequence SPVR, which may be involved with the induction of apoptosis, at least in myeloid 32d.3 cells. However, the role of Id2 phosphorylation at serine 5 in skeletal muscle cells is unknown. The objective of this study was to determine if the phosphorylation of Id2 at serine 5 alters its cellular localization and its role in apoptosis in C2C12 myoblasts. Overexpression of wild type Id2 decreased MyoD protein expression, which corresponded to the increased binding of Id2 to basic HLH proteins E47 and E12. Bromodeoxyuridine incorporation was significantly decreased by the overexpression of phospho-ablated Id2 (S5A); conversely, overexpression of wild type Id2 increased cellular proliferation. The subcellular localization of Id2 and phospho-mimicking Id2 (S5D) were predominantly nuclear compared to S5A. The decreased nuclear localization of S5A corresponded to a decrease in cellular proliferation, and an increase in apoptosis. These data suggest that unphosphorylated Id2 is primarily localized in the cytosol, where it is growth suppressive and potentially pro-apoptotic. These results imply that reducing unphosphorylated Id2 may improve the pool of myoblasts available for differentiation by increasing proliferation and inhibiting apoptosis.

Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ

Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson’s disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.

Dietary resveratrol confers apoptotic resistance to oxidative stress in myoblasts

High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 μM) or very high (1 mM) levels of H2O2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H2O2. Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H2O2. Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 μM of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H2O2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts.