P.C.G. Onofre-Oliveira

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies

The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmdmdx, SJL/J, Largemyd and Lama2dy2J/J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-β1 and Pro-collagen 1α2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-β1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.

Dmdmdx/Largemyd: a new mouse model of neuromuscular diseases useful for studying physiopathological mechanisms and testing therapies

SUMMARY Although muscular dystrophies are among the most common human genetic disorders, there are few treatment options available. Animal models have become increasingly important for testing new therapies prior to entering human clinical trials. The Dmdmdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD), presenting the same molecular and protein defect as seen in humans with the disease. However, this mouse is not useful for clinical trials because of its very mild phenotype. The mouse model for congenital myodystrophy type 1D, Largemyd, harbors a mutation in the glycosyltransferase Large gene and displays a severe phenotype. To help elucidate the role of the proteins dystrophin and LARGE in the organization of the dystrophin-glycoprotein complex in muscle sarcolemma, we generated double-mutant mice for the dystrophin and LARGE proteins. The new Dmdmdx/Largemyd mouse model is viable and shows a severe phenotype that is associated with the lack of dystrophin in muscle. We tested the usefulness of our new mouse model for cell therapy by systemically injecting them with normal murine mesenchymal adipose stem cells (mASCs). We verified that the mASCs were hosted in the dystrophic muscle. The new mouse model has proven to be very useful for the study of several other therapies, because injected cells can be screened both through DNA and protein analysis. Study of its substantial muscle weakness will also be very informative in the evaluation of functional benefits of these therapies.

The mdx Mutation in the 129/Sv Background Results in a Milder Phenotype: Transcriptome Comparative Analysis Searching for the Protective Factors.

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process.

G.P.40 LGMD2G with clinical presentation of congenital muscular dystrophy: A rare phenotype

Abstract LGMD2G is a relatively rare and mild autosomal recessive form of progressive neuromuscular disorder with a wide spectrum of inter and intrafamilial clinical variability. The age at onset ranges from 9 to 15 years old and loss of ambulation is uncommon, or in the third or fourth decade of life. LGMD2G is caused by mutations in the TCAP gene, causing the deficiency of the sarcomeric protein telethonin in muscle of affected patients. Up to now, only a few families have been described, mainly in Brazil. In a recent screening for mutation in the TCAP gene, including 200 additional LGMD adult patients, we identified six new cases, confirming the rarity of the disease also in Brasil. All these patients carried the same c.157C>T (Q53X) homozygous missense mutation, identified in the first families, suggesting a common origin of this mutation. In addition,we studied a five years-old patient with clinical diagnosis of congenital muscular dystrophy. Immunohistochemical analysis for muscle protein excluded the deficiency of dystrophin, the four sarcoglycans, α 2-laminin, dysferlin and calpain3 proteins. Surprisingly, telethonin analysis showed a total deficiency, both through immune fluorescence and western blot analyses. Sequencing of the TCAP gene identified the common c.157C>T mutation in a homozygous state in the patient and in heterozygosity in both non-consanguineous parents. This called our attention to the fact that the rarity of the disease could be due to the screening of the improper patients. Telethonin deficiency could be, in fact, the cause of more severe forms of muscular dystrophies. To test this hypothesis, we analyzed telethonin deficiency in the muscle of 118 patients with clinical diagnosis of congenital MD, from which we selected sixteen patients for gene sequencing. However, no alteration was found in any of them. Therefore, mutation in the TCAP gene is not a common cause of severe forms of MD. FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.

A.P.14: A new in/del in the critical splicing region of the VMA21 gene causing X-linked myopathy with excessive autophagy (XMEA)

X-linked myopathy with excessive autophagy (XMEA) is an inherited, slowly progressive myopathy, characterized by membrane-bound sarcoplasmic vacuoles in muscle fibers. Proximal muscle weakness in early childhood is observed, but with no cardiac, nor cognitive impairment. Recent findings identified mutations in the vacuolar membrane ATPase activity 21 (VMA21), as causative of XMEA. Among Six different single-nucleotide substitutions in VMA21 (in 14 XMEA families), four were intronic, and in two of them, the IVS1–27A base is involved. These mutations result in a reduction in VMA21 mRNA, and protein, and a consequent elevated lysosomal pH with partial block the final degradation stage of autophagy. Only a few XMEA families have been worldwide identified. Here we describe the first XMEA Brazilian family carrying a small in/del in the VMA21 gene. The 5-year-old propositus presented a characteristic dystrophic phenotype. He walked at the age of 2 and showed difficulties for running, climbing stairs, and raising from the floor. No calf hypertrophy nor joint contractures were observed. CK level was 1330 U/l, and ECG showed altered conduction in the right branch. Muscle biopsy showed a dystrophic pattern and autophagic vacuoles. Emerin was normal. Family history revealed a recessive X-linked inheritance, with 5 affected males linked through asymptomatic females. The affected maternal grandfather, aged 48, was wheelchair bound since the age of 30, presenting also cardiac alterations and joint contractures in the upper limbs. Exome sequencing identified a small insertion-deletion, including the IVS1-27A base previously described. This new family/mutation reinforces the importance of this splice site branchpoint for the appropriate transcription/translation of VMA21, and normal lysosome function. Additionally, it expands the clinical variability, including cardiac involvement and joint contractures to the XMEA phenotype.

G.P.210

Muscle satellite cells have been widely studied, especially to understand their mechanism of action in muscle regeneration and correspondent implications in the different dystrophic processes. Two mice models for muscular dystrophies, Large myd and Lama2 dy2j / J , have a pattern of an intense and very similar degeneration, but with differences in the expression of genes involved in the regeneration cascade, as we shown in our recent work. Therefore, they are interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objective of this work was to evaluate gene expression profile of the satellite cells from both dystrophic mouse models, as compared to normal murine muscle, to try to explain the difference observed in the respective muscles. For this evaluation, we harvested muscle derived cells after enzyme dissociation of muscle tissue. The cells were then pre-plated in culture flasks (PP1) and re-plated after 24h (PP2). The different populations were than characterized by flow cytometry markers and analyzed using a murine gene expression microarray panel of more than 26,000 genes. We observed 383 differentially expressed genes in Large myd and 110 in Lama2 dy2j / J . The glycosilation alteration, exhibited by Large myd alters the expression of many genes, especially those involved with myogenesis and activation of cell differentiation. The altered genes of Lama2 dy2j / J are more related with cell membrane or extracellular matrix. These observations are corroborating our previous gene expression results, suggesting that the mutation present in Large myd mouse leads to defects in the regeneration potential of satellite cells, what does not occur in the Lama2 dy2j / J model.

T.P.38 Effects of steroid hormones on myostatin expression and on genes of muscle regeneration pathway

Abstract Myostatin is an important negative regulator of skeletal muscle growth, while decanoato de nadrolone, an anabolic steroid, is a strong positive effector. Inhibition of myostatin has been tested as an approach for treatment neuromuscular diseases. In order to investigate the possible interaction between myostatin and anabolic steroids, as a therapeutic strategy, we studied myostatin expression in the quadriceps femoris of normal mice treated with Decadurabolin® (D), flutamide (F), an antagonist of the androgen receptor, and Decadurabolin administration, post flutamide treatment (FD), as compared to controls, treated with saline (S). We also studied the relative expression of the genes, myogenin, MyoD and Myf5, involved in the pathway of muscle regeneration. We observed significant increase in the body mass in the (D) and (FD) groups, and a decrease in the group (F), when compared to groups (S). Real-time PCR quantitative analysis for myostatin expression showed no statistically significant differences between the studied groups. On the other hand, the groups (D) and (FD) showed a significant decrease in the expression of myogenin, MyoD and Myf5, while animals of the group (F) showed a significant increase in the expression of these genes. We conclude that administration of anabolic steroid, or its inhibition did not alter the expression of the myostatin gene, despite the increase or decrease in the body mass observed in group (D), (FD) and (F). However, the blockade of androgen receptor by flutamide, clearly stimulate the regeneration cascade, by increasing the expression of genes related to proliferation (MyoD and Myf5) and cell differentiation (myogenin). Additional studies will elucidate the possible role of other pathways in this stimulus for regeneration. Financial support: FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.

Myogenic Potential of Murine Embryonic Stem Cells in the Dmdmdx Mouse Model for Duchenne Muscular Dystrophy

Danielle Ayub-Guerrieri1, Poliana C. M. Martins-Machado1, Paula C.G. Onofre-Oliveira1, Lygia V. Pereira2, Camila F. Almeida1, Vanessa F. Lopes1 and Mariz Vainzof1 1Laboratory of Muscle Proteins and Comparative Histopathology, Human Genome Research Center, Biosciences Institute, University of Sao Paulo 2Dept of Genetics and Evolutionary Biology, Biosciences Institute, University of Sao Paulo Brazil