Mechanisms of muscle membrane repair: The dysferlin model

Transcription

1 Mechanisms of muscle membrane repair: The dysferlin model Eduard Gallardo Laboratori de Neurologia Experimental Institut de Recerca Hospital Sant Pau 2nd MuscleTech Network Workshop September 27 29, 2010 Barcelona

2 Group objectives Our main focus of interest is a muscular dystrophy caused by mutations in the gene DYSF (dysferlin). Specific aims: To study possible functions of the protein (membrane repair, cell fusion, muscle differentiation, regeneration, inflammation...) To elucidate which proteins interact with dysferlin Therapeutic approaches ( minidysferlins, cell therapies). To develop new diagnostic tools and search for biomarkers of the disease.

3 Fukutin Collagen IV Sarcoglycans complex Laminin a-2 Sarcolemma Integrins complex α α-dystroglican β-dystroglican Caveolin-3 δ β α α γ γ β Sarcoplasm Sarcospan TRIM32 SEPN1 Dystrophin Filamin C β1 nnos α Distrobrevin NO sintase Sintrophins Calpain-3 Desmin Dysferlin Sarcomere Z-disk I A M A I Myotilin Telethonin Z-disk α-actinin Actin Tropomyosin Myosin Nebulin Tropomodulin Titin Desmin Nucleus Emerin Lamin A/C Por I. de Andrés

4 IMMUNOHISTOCHEMISTRY DYSF + DYSF -

5 Dysf +/+ Dysf -/- Poor differentiation, fusion defect De Luna et al J Biol Chem 2006

6 MEMBRANE REPAIR MODEL Glover et al Traffic 2007

7 In vitro studies

8 Dysferlin is also expressed in peripheral blood monocytes kb Peripheral blood Skeletal muscle CD14- Dysferlin CD14+ CD14+ CD kda Diagnostic tool minimally invasive Ho et al Ann Neurol 2002 β-actin 42 kda

9 In vivo studies

10 Skeletal muscle Phenotypic description of mouse adult mesoangioblasts Smooth muscle Myosin Low serum medium + TGF β SMA Fat Adipogenic medium + BMP Alkaline Phosphatase Oil red Bone Diaz et al Cell Death and Disease (2010)

11 IM+CTX IM IA+CTX IA Results at 1 month

12 Recovery of dysferlin expression after mesoangioblasts trasnplantation of A/J SCID mice

13 Laser induced lesions in isolated single fibers Fluorescent dye FM 4-64 Diaz et al Cell Death and Disease (2010)

14 Inflammation in dysferlin myopathy

15 MHC-I Macrophag. CD8 MHC-I H&E Macrophag. CD8 Neurology 2001

16 CHEMOTAXIS ASSAY MONOCYTES CONDITIONED MEDIA Skeletal muscle cultures CONDITIONED MEDIA Myoblasts Myotubes Contains released factors by skeletal muscle cells

17 Chemotaxis assay Results De Luna et al J. Neuropathol Exp Neurol 2010

18 Results Microarray analysis comparing dysf+/+ vs dysf -/- human myotubes (PROGENIKA) Thrombospondin-1 (TSP-1) (3.2 fold increase) Human genome U133 Plus 2.0 array De Luna et al J. Neuropathol Exp Neurol 2010

19 TSP-1 EXPRESSION in vitro ELISA De Luna et al J. Neuropathol Exp Neurol 2010

20 TSP-1 EXPRESSION IN MOUSE MODELS B.10 (Dysf +/+) A/J (Dysf-/-) De Luna et al J. Neuropathol Exp Neurol 2010

21 TSP-1 EXPRESSION in vivo Macrophages TSP-1 Merged DYSF-/- FSHD IBM

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23 HOT TOPICS IN THE AREA Phenotypic variability. How come some patients start developing the disease much later than others (from congenital forms to very late onset forms) although all of them present null or very low levels of dysferlin expression?

24 2009

25 HOT TOPICS IN THE AREA Patients with a single mutation (this is a recessive disease???)

26 HOT TOPICS Increased exercise accelerates the onset of the disease (clinical observation)?. However, recent works using mouse models, suggest that mild exercise may be benefitial for the patients. Concentric (good)versus eccentric (bad)

27 KEY QUESTIONS What are the functions of dysferlin? Membrane repair Muscle differentiation What is the origin of muscle inflammation in dysferlin myopathy? Abnormal upregulation of TSP-1 in muscle Abnormal function of monocytes due to the lack of dysferlin

28 From translational research to translational medicine Question 1:Insights into how could it be done to translate the day to day scientific discoveries into practical applications, clinical level, or patient's bedside to improve human health. Development of diagnostic tools (dysferlin expression in monocytes) Development of biomarkers of the disease : 1. Use of dysferlin expression in peripheral blood to asses the efficacy of drugs instead of repeated muscle biopsies 2. Levels of TSP-1 in serum as a specific marker of the disease (instead of CKs) or as a marker of disease progression.. Question 2: Impact the research on muscle and tendon could have on health and society, both socially and economically. Use of cell therapy to treat muscle diseases Discovery of drugs that may improve muscle membrane resealing both in patients with muscular dystrophies and in injured muscle in healthy people.

29 Unitat Neuromuscular HSCSP, Barcelona Dra. ILLA Dr. Rojas Dr. Diaz Dra. Iturriaga Dra. Hankiewicz Dra Martinez Lab Neurologia Experimental Dra. De Luna B. Flix J. Araque E. Ortiz X. Suárez Hospital St Rafaele, Milan Dr. Cossu Lab. Morfología Celular. Unidad Mixta. CIPF-UVEG Dr García-Verdugo Mario Soriano University of Massachusetts Dr. Brown