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Supplementary Figure 2. LSN2478185 increases body weight and muscle mass in non-tumor-bearing mice in the presence of gemcitabine.

Supplementary Figure Legends

Supplementary Figure 4. LY2495655 attenuates loss of muscle weight normalized to brain weight but has no effect on brain or heart weight in C26 tumor bearing mice in the presence of gemcitabine.

Supplementary Figure 1. LSN2478185 and LY2495655 neutralize 1nM myostatin in an SBE luciferase assay in HEK-293 cells.

Supplementary Figure 5. Loss of body weight, muscle mass and strength in the PC3 tumor model.

Supplementary Figure 6. LY2495655 attenuates the loss of muscle weight normalized to brain weight in the PC3 tumor model.

Supplementary Figure 3. LSN2478185 reduces the loss of skeletal muscle mass under conditions of caloric restriction.

Sclerostin (SOST), a glycoprotein primarily derived from osteocytes, is an important regulator of bone remodeling. Osteogenesis imperfecta (OI) is a heritable disorder of bone characterized by low bone mass, bone fragility, recurrent fractures, and bone deformities. Altered SOST-mediated signaling may have a role in pathogenesis of type I collagen-related OI; however, this has not been evaluated in humans. We measured serum SOST levels in adults with OI who were enrolled in a randomized, placebo-controlled clinical trial that evaluated the effects of osteoanabolic therapy with teriparatide. Compared with age- and sex-matched control participants, mean SOST levels were lower in those with type I or types III/VI OI (p

Diabetic nephropathy (DN) is a primary cause of end-stage renal disease and is becoming more prevalent because of the global rise in type 2 diabetes. A model of DN, the db/db uninephrectomized ( db/db-uni) mouse, is characterized by obesity, as well as compromised renal function. This model also manifests defects in mineral metabolism common in DN, including hyperphosphatemia, which leads to severe endocrine disease. The FGF23 coreceptor, α-Klotho, circulates as a soluble, cleaved form (cKL) and may directly influence phosphate handling. Our study sought to test the effects of cKL on mineral metabolism in db/db-uni mice. Mice were placed into either mild or moderate disease groups on the basis of the albumin-to-creatinine ratio (ACR). Body weights of db/db-uni mice were significantly greater across the study compared with lean controls regardless of disease severity. Adeno-associated cKL administration was associated with increased serum Klotho, intact, bioactive FGF23 (iFGF23), and COOH-terminal fragments of FGF23 ( P < 0.05). Blood urea nitrogen was improved after cKL administration, and cKL corrected hyperphosphatemia in the high- and low-ACR db/db-uni groups. Interestingly, 2 wk after cKL delivery, blood glucose levels were significantly reduced in db/db-uni mice with high ACR ( P < 0.05). Interestingly, several genes associated with stabilizing active iFGF23 were also increased in the osteoblastic UMR-106 cell line with cKL treatment. In summary, delivery of cKL to a model of DN normalized blood phosphate levels regardless of disease severity, supporting the concept that targeting cKL-affected pathways could provide future therapeutic avenues in DN. NEW & NOTEWORTHY In this work, systemic and continuous delivery of the “soluble” or “cleaved” form of the FGF23 coreceptor α-Klotho (cKL) via adeno-associated virus to a rodent model of diabetic nephropathy (DN), the db/db uninephrectomized mouse, normalized blood phosphate levels regardless of disease severity. This work supports the concept that targeting cKL-affected pathways could provide future therapeutic avenues for the severe mineral metabolism defects associated with DN.

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

Purpose of review This review summarizes recent progress in the development of myostatin inhibitors for the treatment of muscle wasting disorders. It also focuses on findings in myostatin biology that may have implications for the development of antimyostatin therapies. Recent findings There has been progress in evaluating antimyostatin therapies in animal models of muscle wasting disorders. Some programs have progressed into clinical development with initial results showing positive impact on muscle volume. In normal mice myostatin deficiency results in enlarged muscles with increased total force but decreased specific force (total force/total mass). An increase in myofibrillar protein synthesis without concomitant satellite cell proliferation and fusion leads to muscle hypertrophy with unchanged myonuclear number. A specific force reduction is not observed when atrophied muscle, the predominant therapeutic target of myostatin inhibitor therapy, is made myostatindeficient. Myostatin has been shown to be expressed by a number of tumor cell lines in mice and man. Summary Myostatin inhibition remains a promising therapeutic strategy for a range of muscle wasting disorders.

The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration. Developmental Dynamics 233:356–367, 2005. © 2005 Wiley-Liss, Inc.

Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs, NPs and neurons matured in culture for either 2 weeks (termed early neurons, E) or 4 weeks (termed late neurons, L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate, enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically, 328 genes were differentially expressed between BPD and control L neurons including GAD1, glutamate decarboxylase 1 (2.5 fold) and SCN4B, the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology, GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism, protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD.

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non–tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass– and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non–tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting. Mol Cancer Ther; 14(7); 1661–70. ©2015 AACR.

The ski oncogene was originally isolated as an avian retroviral gene with the ability to induce quail embryonic cells to differentiate into muscle. Mice containing a chicken c-ski transgene exhibit postnatal hypertrophy of skeletal muscle. Xenopus ski (Xski) protein is maternal and present throughout early development. We show that overexpression of Xski RNA in Xenopus embryos results in the cell-autonomous induction of secondary neural axis formation. Injection of Xski RNA into prospective endodermal cells resulted in the formation of an ectopic neural tube-like structure and cells derived from the injected blastomeres populated the spinal cord. Injected Xski RNA was able to induce neural-specific gene expression directly in ectodermal explants in the absence of the expression of mesodermal markers. The widespread distribution of ski protein in the early gastrula embryo including the dorsal animal region supports a role for ski in neural axis formation in vivo.

The FGF23 coreceptor αKlotho (αKL) is expressed as a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as an endoproteolytic cleavage product of mKL (cKL). Previously, a patient with increased plasma cKL as the result of a translocation [t(9;13)] in the αKLOTHO (KL) gene presented with rickets and a complex endocrine profile, including paradoxically elevated plasma FGF23, despite hypophosphatemia. The goal of this study was to test whether cKL regulates phosphate handling through control of FGF23 expression. To increase cKL levels, mice were treated with an adeno-associated virus producing cKL. The treated groups exhibited dose-dependent hypophosphatemia and hypocalcemia, with markedly elevated FGF23 (38 to 456 fold). The animals also manifested fractures, reduced bone mineral content, expanded growth plates, and severe osteomalacia, with highly increased bone Fgf23 mRNA (>150 fold). cKL activity in vitro was specific for interactions with FGF23 and was FGFR dependent. These results demonstrate that cKL potently stimulates FGF23 production in vivo, which phenocopies the KL translocation patient and metabolic bone syndromes associated with elevated FGF23. These findings have important implications for the regulation of αKL and FGF23 in disorders of phosphate handling and biomineralization.

This investigation examined the effects of acute resistance exercise (RE), progressive resistance training (PRT), and age on the human skeletal muscle Transcriptome. Two cohorts of young and old adults [ study A: 24 yr, 84 yr ( n = 28); study B: 25 yr, 78 yr ( n = 36)] were studied. Vastus lateralis biopsies were obtained pre- and 4 h post-RE in conjunction with the 1st and 36th (last) training session as part of a 12-wk PRT program in study A, whereas biopsies were obtained in the basal untrained state in study B. Additionally, the muscle fiber type specific (MHC I and MHC IIa) Transcriptome response to RE was examined in a subset of young and old women from study A. Transcriptome profiling was performed using HG U133 Plus 2.0 Arrays. The main findings were 1) there were 661 genes affected by RE during the 1st and 36th training bout that correlated with gains in muscle size and strength with PRT (termed the Transcriptome signature of resistance exercise adaptations); 2) the RE gene response was most pronounced in fast-twitch (MHC IIa) muscle fibers and provided additional insight into the skeletal muscle biology affected by RE; 3) skeletal muscle of young adults is more responsive to RE at the gene level compared with old adults and age also affected basal level skeletal muscle gene expression. These skeletal muscle Transcriptome findings provide further insight into the molecular basis of sarcopenia and the impact of resistance exercise at the mixed muscle and fiber type specific level.

Background Matrix metalloproteinase-28 (MMP-28) is a poorly understood member of the matrix metalloproteinase family. Metalloproteinases are important mediators in the development of the nervous system and can contribute to the maturation of the neural micro-environment. Results MMP-28 added to myelinating rat dorsal root ganglion (DRG) co-cultures reduces myelination and two antibodies targeted to MMP-28 (pAb180 and pAb183) are capable of binding MMP-28 and inhibiting its activity in a dose-dependent manner. Addition of 30 nM pAb180 or pAb183 to rat DRG cultures resulted in the 2.6 and 4.8 fold enhancement of myelination respectively while addition of MMP-28 to DRG co-cultures resulted in enhanced MAPK, ErbB2 and ErbB3 phosphorylation. MMP-28 protein expression was increased within demyelinated lesions of mouse experimental autoimmune encephalitis (EAE) and human multiple sclerosis lesions compared to surrounding normal tissue. Conclusion MMP-28 is upregulated in conditions of demyelination in vivo, induces signaling in vitro consistent with myelination inhibition and, neutralization of MMP-28 activity can enhance myelination in vitro. These results suggest inhibition of MMP-28 may be beneficial under conditions of dysmyelination.

Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.

Xenopus oocyte maturation can be induced in vitro by a number of agents including hormones, proto-oncogenes and cyclins. Xenopus oocytes matured by a variety of treatments, including progesterone, insulin, testosterone and injection of Xenopus c-mos RNA, Xenopus cyclin B2 RNA and egg extracts, synthesize a predominant 180 kDa protein (p180). p180 synthesis begins during maturation prior to germinal vesicle breakdown (GVBD) but is prominent only in oocytes that have undergone GVBD. p180 synthesis is thus a useful, reliable molecular marker of oocyte maturation. p180 continues to be synthesized in eggs and embryos up to the gastrula stage of embryogenesis, but not in the neurula or tailbud embryo.

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37 °C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37 °C for 4 weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4 + T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.