Yazhong Tao

Skeletal muscle satellite cells are adult stem cells responsible for postnatal skeletal muscle growth and regeneration. Paired-box transcription factor Pax7 plays a central role in satellite cell survival, self-renewal, and proliferation. However, how Pax7 is regulated during the transition from proliferating satellite cells to differentiating myogenic progenitor cells is largely unknown. In this study, we find that miR-1 and miR-206 are sharply up-regulated during satellite cell differentiation and down-regulated after muscle injury. We show that miR-1 and miR-206 facilitate satellite cell differentiation by restricting their proliferative potential. We identify Pax7 as one of the direct regulatory targets of miR-1 and miR-206. Inhibition of miR-1 and miR-206 substantially enhances satellite cell proliferation and increases Pax7 protein level in vivo. Conversely, sustained Pax7 expression as a result of the loss of miR-1 and miR-206 repression elements at its 3' untranslated region significantly inhibits myoblast differentiation. Therefore, our experiments suggest that microRNAs participate in a regulatory circuit that allows rapid gene program transitions from proliferation to differentiation.

Alemtuzumab (anti-CD52 mAb) provides long-lasting disease activity suppression in relapsing-remitting multiple sclerosis (RRMS). The objective of this study was to characterize the immunological reconstitution of T cell subsets and its contribution to the prolonged RRMS suppression following alemtuzumab-induced lymphocyte depletion. The study was performed on blood samples from RRMS patients enrolled in the CARE-MS II clinical trial, which was recently completed and led to the submission of alemtuzumab for U.S. Food and Drug Administration approval as a treatment for RRMS. Alemtuzumab-treated patients exhibited a nearly complete depletion of circulating CD4(+) lymphocytes at day 7. During the immunological reconstitution, CD4(+)CD25(+)CD127(low) regulatory T cells preferentially expanded within the CD4(+) lymphocytes, reaching their peak expansion at month 1. The increase in the percentage of TGF-β1-, IL-10-, and IL-4-producing CD4(+) cells reached a maximum at month 3, whereas a significant decrease in the percentages of Th1 and Th17 cells was detected at months 12 and 24 in comparison with the baseline. A gradual increase in serum IL-7 and IL-4 and a decrease in IL-17A, IL-17F, IL-21, IL-22, and IFN-γ levels were detected following treatment. In vitro studies have demonstrated that IL-7 induced an expansion of CD4(+)CD25(+)CD127(low) regulatory T cells and a decrease in the percentages of Th17 and Th1 cells. In conclusion, our results indicate that differential reconstitution of T cell subsets and selectively delayed CD4(+) T cell repopulation following alemtuzumab-induced lymphopenia may contribute to its long-lasting suppression of disease activity.

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.

The molecular events that modulate chromatin structure during skeletal muscle differentiation are still poorly understood. We report in this paper that expression of the H3-K4 histone methyltransferase Set7 is increased when myoblasts differentiate into myotubes and is required for skeletal muscle development, expression of muscle contractile proteins, and myofibril assembly. Knockdown of Set7 or expression of a dominant-negative Set7 mutant impairs skeletal muscle differentiation, accompanied by a decrease in levels of histone monomethylation (H3-K4me1). Set7 directly interacts with MyoD to enhance expression of muscle differentiation genes. Expression of myocyte enhancer factor 2 and genes encoding contractile proteins is decreased in Set7 knockdown myocytes. Furthermore, we demonstrate that Set7 also activates muscle gene expression by precluding Suv39h1-mediated H3-K9 methylation on the promoters of myogenic differentiation genes. Together, our experiments define a biological function for Set7 in muscle differentiation and provide a molecular mechanism by which Set7 modulates myogenic transcription factors during muscle differentiation.

Subsequent to the clinical trial of simvastatin in patients with relapsing remitting multiple sclerosis (RR MS), which demonstrated the ability of simvastatin to inhibit new inflammatory CNS lesion formation, the current in vitro study has characterized the mechanisms through which simvastatin inhibits Th17 cell differentiation. The anti-inflammatory effects of statins are mediated by the inhibition of isoprenylation, which ensures proper membrane insertion and function of proteins. Small GTPases, involved in multiple signal transduction pathways, are the key targets for isoprenylation. We report that simvastatin, one of the most hydrophobic statins with good CNS penetration, inhibited Th17 cell differentiation and IL-17A, IL-17F, IL-21, and IL-22 secretion in in vitro-differentiated naive CD4(+) T cells from RR MS patients. Simvastatin exerted a less prominent effect on the cells from healthy controls, as it inhibited only IL-17F secretion. The inhibition of Th17 cell differentiation was mediated via inhibition of IFN regulatory factor 4 (IRF4) expression, which was identified as a key transcription factor for human Th17 cell differentiation using both IRF4 gene knockdown and overexpression experiments. In studies addressing which isoprenylation pathway--geranylgeranylation or farnesylation--is inhibited by simvastatin, we demonstrated that the geranylgeranyl transferase inhibitor replicated the effect of simvastatin. Selective inhibition of geranylgeranylated RhoA-associated kinase replicated the effect of simvastatin on the inhibition of IRF4 expression and IL-17A, IL-17F, IL-21, and IL-22 secretion, presenting a promising new therapeutic approach for this disabling disease.