Nurit Yanay

Losartan, a therapeutic candidate in congenital muscular dystrophy: Studies in the dy2J/dy2J Mouse

Lamininα2‐deficient congenital muscular dystrophy type 1A (MDC1A) is a cureless disease associated with severe disability and shortened lifespan. Previous studies have shown reduced fibrosis and restored skeletal muscle remodeling following treatment with losartan, an angiotensin II type I receptor blocker. We therefore evaluated the effect of losartan treatment in the dy2J/dy2J mouse model of MDC1A.

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

The Ras Antagonist, Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy. The dy2J/dy2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy2J/dy2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy2J/dy2J mouse model of congenital muscular dystrophy.

Improved muscle strength and mobility in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.

Sustained expression and safety of human GNE in normal mice after gene transfer based on AAV8 systemic delivery

GNE myopathy is an autosomal recessive adult onset disorder caused by mutations in the GNE gene. GNE encodes the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase, the key enzyme in the biosynthesis pathway of sialic acid. Additional functions for GNE have been described recently, but the mechanism leading from GNE mutation to this myopathy is unclear. Therefore a gene therapy approach could address all potential defects caused by GNE mutations in muscle. We show that AAV8 viral vectors carrying wild type human GNE cDNA are able to transduce murine muscle cells and human GNE myopathy-derived muscle cells in culture and to express the transgene in these cells. Furthermore, the intravenous administration of this viral vector to healthy mice allows expression of the GNE transgene mRNA and of the coexpressed luciferase protein, for at least 6months in skeletal muscles, with no clinical or pathological signs of focal or general toxicity, neither from the virus particles nor from the wild type human GNE overexpression. Our results support the future use of an AAV8 based vector platform for a safe and efficient therapy of muscle in GNE myopathy.

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene.

T.P.6 Combination anti-inflammatory and anti-fibrotic treatment in muscular dystrophy – The more the better?

Abstract Congenital muscular dystrophy is an incurable disorder with no effective treatment. Anti-inflammatory and anti-fibrotic agents have been suggested as potential therapies. As previously demonstrated by our group, Glatiramer acetate (GA), an anti-inflammatory agent and the anti-fibrotic agent Losartan exert their effect through different pathways and have distinct beneficial effects on strength, mobility and fibrosis. Thus, the aim of the present study was to evaluate the effect of combination therapy of GA with Losartan in the dy2J/dy2J mouse model of congenital muscular dystrophy. Fore and hind limb muscle strength, fibrosis and mobility parameters were assessed. dy2J/dy2J mice receiving the combination of GA/Losartan or GA alone showed mild improvement in forelimb muscle strength (11%) in contrast to treatment with Losartan alone (74%). Hind limb muscle strength was unchanged in dy2J/dy2J mice receiving the combination therapy, while the two drugs alone showed significant improvement in strength (GA; 52.7% vs. Losartan; 74%). The combined treatment showed only mild reduction in muscle fibrosis (20%) compared with Losartan alone (42.3%. No significant change for GA). GA/Losartan combination therapy or Losartan alone showed no change in mobility parameters. However, treatment with GA alone resulted in a marked improvement in mobility parameters. In conclusion, the improvements seen with GA/Losartan combination therapy were less noticeable than the improvements of each of the two agents administered as a single medication. We conclude that combination therapy should be administered with caution. Combining two medications with theoretical synergistic effect and previous positive effect may result in a reduction of the therapeutic effect compared to the use of each of these agents separately.

G.P.211

Merosin deficient congenital muscular dystrophy type 1A (MDC1A) is a devastating incurable disorder of childhood. We previously studied the effect of Glatiramer acetate (GA), an anti-inflammatory agent and S- farnesylthiosalicilate acid (FTS), an anti-fibrotic agent, as a single agent in the dy2J/dy2J mouse model of MDC1A. Each of the two medications showed a different pattern of disease on three primary outcome measures: strength, fibrosis and animal mobility. GA monotheraphy resulted in significant improvement in hind limb muscle strength and movement parameters, but without a decrease in fibrosis measurement. FTS monotheraphy was associated with increased hind limb muscle strength, decreased fibrosis, but with no improvement in movement parameters. In the present study, the combination of GA and FTS was found to increase both fore (32%) and hind limb (75.8%) muscle strength, more than treatment with either drug alone (fore limb: no significant change for GA and for FTS; hind limb: GA 52.7% vs. FTS 42%). In addition, GA-FTS treatment resulted in significant reduction in muscle fibrosis (35.4%) compared to untreated dy2J/dy2J mice. However, no improvement in animal mobility was shown. According to the results, combined GA and FTS increased both fore and hind limb muscle strength more than either drug by itself and significantly ameliorated muscle fibrosis. Thus, GA-FTS treatment might be considered as a potential candidate for future clinical trials in children with CMD.

P.8.10 Losartan up-regulates NFκB signaling pathway and favors survival versus apoptosis in the dy2J/dy2J mouse model of Congenital Muscular Dystrophy

Congenital Muscular Dystrophy (CMD) is a group of genetic disorders characterized by progressive loss of muscle strength and integrity. Merosin deficient congenital muscular dystrophy type 1A (MDC1A) is a common form of this disorder. Children affected with MDC1A suffer from early onset severe hypotonia and weakness with significant motor milestone delay. Often they do not achieve independent ambulation and die in the second or third decade. Despite extensive advances in diagnosis, cellular and molecular understanding, MDC1A remains a disease without a cure or any proven therapeutic option to relieve or slow disease progression. Our experimental data suggest that treatment with Losartan, an Angiotensin II type I receptor antagonist, results in significant clinical improvements and amelioration of fibrosis in the dy2J/dy2J mouse model of CMD through inhibition of TGFβ and MAPK signaling. We further examined Losartan’s effect on the cellular network, focusing on NFκB signaling. Previous studies suggested a role of NFκB in promoting muscle inflammation, necrosis and degeneration in Duchenne muscular dystrophy patients and animal models. Contrary to this, here we show that Losartan’s beneficial effect in the dy2J/dy2J of CMD is associated with NFκB signaling up-regulation manifested by enhanced serum TNFα level, decreased IκB-β protein level (NFκB inhibitor) and P65 accumulation in gastrocnemius nuclei of the dy2J/dy2J mice. A more in-depth investigation revealed that Losartan induced a modification in the NFκB gene expression towards pro-survival profile as cIAP2, TRAF2 and FTH mRNA levels were markedly increased following treatment. Losartan also induced the expression of anti-apoptotic BCl2 protein and down-regulated the expression of pro-apoptotic caspase 3 protein. Our study indicates that in the dy2J/dy2J mice of CMD, Losartan treatment resulted in NFκB activation with shifting from apoptosis/damage targeting pathway to a profile favoring cell survival.

P3.48 Losartan improves muscle strength and ameliorates fibrosis in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy

Losartan improves muscle strength and ameliorates fibrosis in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy M. Elbaz , S. Aga-Mizrachi , N. Yanay , O. Dadush , K. Ettinger , Z. Brunschwig , M. Rabie , V. Barak , Y. Nevo 1 1 Hadassah, Hebrew University Hospital, Pediatric Neurology Unit, Jerusalem, Israel, 2 Hadassah, Hebrew University Hospital, Immunology Laboratory for Tumor Diagnosis, Jerusalem, Israel