Severe genetic neuromuscular diseases are debilitating diseases with a strong impact on patients, families and the society. Although rare, they represent disease models for the more common diseases and for physiological changes associated to aging.
We study rare neuromuscular disorders caused by mutations in proteins affecting organelles and membrane trafficking. Our main focus is on centronuclear myopathies (CNM), severe congenital myopathies characterized by muscle weakness, fibre atrophy and organelles mis-positioning (centralized nuclei).
Muscle biopsy showing peripheral nuclei (in violet) in control (left) vs central nuclei in CNM patient (right)
Schematic representation of myofibrils inside a muscle fiber (Ali Poursaeed)
Several CNM forms exist: a neonatal X-linked form (or myotubular myopathy), autosomal recessive forms with childhood onset, and autosomal dominant forms with adult onset. We have shown that the X-linked form is due to mutations in the phosphoinositides phosphatase myotubularin (MTM1) while some autosomal forms are due to mutations in the membrane bending protein amphiphysin 2 (BIN1); we also participated to the identification of mutations in some dominant forms in the large GTPase dynamin 2. These 3 genes mutated in the same pathology are defining a hypothetical " MAD " pathway.
The MAD pathway: Myotubularin, Amphiphysin 2 and Dynamin 2 are mutated in neuromuscular diseases
Dynamin 2 and myotubularin homologues are also mutated in Charcot-Marie-Tooth (CMT) peripheral neuropathies, while amphiphysin 2 is mis-spliced in congenital Myotonic Dystrophy. Thus, we suggest that there is a common molecular pathway implicated both in centronuclear myopathies and peripheral neuropathies, and this pathway might impact on membrane tubulation like T-tubules in skeletal muscle, or endocytosis/exocytosis in peripheral nerves.
While myotubularin has a catalytic site resembling protein tyrosine phosphatases, we and others showed that it is a 3-phosphoinositides phosphatase that dephosphorylates specifically PtdIns3P and PtdIns(3,5P)2, and produces PtdIns5P. Phosphoinositides are membrane lipids implicated in a variety of cell functions like compartimentalization and signalling. PtdIns3P and PtdIns(3,5P)2 interconversion is mediated by kinases and phosphatases and is crucial for the regulation of membrane trafficking and protein transport and degradation.
A- Endosomal phosphoinositides metabolism and localization. Interconversion of PtdIns3P and PtdIns(3,5)P2 by kinases in green ajd mymtebularin phosphatases in red (top), and associated human diseases. Localization of phosphoinositides in the endocytic pathway: early endosome, sorting endosome for receptor recycling, multi-vesicular bodies (MVB)/late endosome, and lysosome for degradation (bottom).
B- Labeling of phosphoinositides and membrane compartments. On the top, human fibroblasts labelled with an antibody against EEA1 for the early endosomes (in green) and with a PtdIns3P biosensor (in red). On the bottom, coelomocytes from a ppk-3/PIKfyve C. elegans mutant labelled with LAMP1-GFP for lysosomes (in green) and BSA-texasred for fluid-phase uptake (in red).
Phosphoinositides and membrane remodelling proteins like amphiphysin and dynamin regulate endocytosis. Amphiphysin acts by creating and sensing membrane curvature, then rectuiting dynamin. Dynamin then forms an helical collar around the neck of vesicles and tubulates membrane. Their sequential action allows the formation of vesicles budding from the plasma membrane and possibly other compartments.
DNM2 recruitment to BIN1-induced tubules in Cos-1 cells
The generation of tools to study phosphoinositides and membrane trafficking pathways and a better characterization of the roles of the implicated proteins will reveal new key players and important mechanisms for skeletal muscle and peripheral nerve functions, and allow a better comprehension of the related diseases.
The identification of genes mutated in patients with neuromuscular diseases is an important first step to provide an accurate genetic counselling, to improve health care and disease management, and to identify novel drug targets that may be more accessible for therapeutic development.
Major scientific achievements:
Genetic basis of neuromuscular disorders
-identification of myotubularin (MTM1 gene), mutated in X-linked myotubular myopathy, and of most of the myotubularin family, conserved in yeast with 14 members in human (Laporte et al., Nature Genet 1996, Hum Mol Genet 1998, 2003)
-identification and characterization of mutations in amphiphysin 2 (BIN1) in patients with centronuclear myopathy, and providing the first evidence of a molecular link between several forms of centronuclear myopathies (Nicot/Toussaint et al, Nature Genet 2007)
-participation to the identification of the first mutations of a dead phosphatase in a human disease, demyelinating CMT4B2 Charcot-Marie-Tooth neuropathy (MTMR13/Sbf2, Azzedine et al. Am J Human Genet 2003), the identification of genes mutated in dominant centronuclear myopathy (dynamin 2; Bitoun et al., Nature Genet 2005), and hypospadias (Cxorf6, Fukami et al., Nature Genet 2006)
-identification of potential dominant modifier mutations in a novel human PIs phosphatase we called hJUMPY or MTMR14 (Tosch, V. et al., Hum Mol Genet 2006).
-participation to the identification of a splicing defect of BIN1 in myotonic dystrophy (Fugier et al., Nature Medicine 2011)
-set-up and transfer to the genetic diagnosis laboratory at Strasbourg Hospital diagnosis protocols to identify CNM-causing mutations
Cellular pathways for membrane remodelling and organelles positioning
-first demonstration that myotubularin is not a protein phosphatase but represents a new class of phosphoinositides phosphatases (Blondeau et al., Hum Mol Genet 2000)
-first (bioinformatic) identification of the myotubularin dead phosphatases, and evidence that they heterodimerize with active homologues (Laporte et al., Hum Mol Genet 1998; Nandurkar et al., PNAS 2003)
- myotubularin interacts with the muscular intermediate filament desmin to regulate mitochondria dynamics (Hnia et al., J Clin Invest 2011)
Pathophysiology of centronuclear myopathies
-characterization of a murine model for myotubular myopathy, suggesting that the disease is an impairment of muscle structural maintenance rather than a block during myogenesis as previously thought (Buj-Bello et al., PNAS 2002)
-identification of a pathological mutation in MTM1 in a Labrador retriever affected by a CNM-like phenotype (Beggs, Böhm et al., PNAS 2010)
-creation of autosomal dominant CNM model in mice using our AAV expertise: expression of exogenous R465W DNM2 mutant construct in skeletal muscle of adult wild-type mice lead to most signs of CNM (central nuclei, atrophy, strong decrease in specific maximal force; Cowling et al., Am J Pathol 2011)
-characterisation of structural defects of triads in skeletal muscle from the Mtm1 KO mouse (Al-Qusairi et al., PNAS 2009), the MTM1 mutant Labrador (Beggs, Böhm et al., PNAS 2010), the AAV-DNM2-R465W mice (Cowling et al., Am J Pathol 2011) and in muscle biopsies from CNM patients with mutations in MTM1, BIN1 and DNM2 (Toussaint et al. Acta Neuropathol 2011), strongly supporting that these defects may be at the basis of the muscle weakness
Pre-clinical trials in animal models
-rescue of the histological phenotype and desmin aggregation, reverted the muscle atrophy and restored the contractile force by AAV-transduced expression of MTM1 in the Mtm1 KO (Buj-Bello et al., Hum Mol Genet 2008; Hnia et al., J Clin Invest 2011)
These achievements were performed in collaboration with the groups of B Payrastre (Toulouse), M Labouesse (Illkirch), E Leguern (Paris), A Bolino (Milan), P Guicheney / A Ferry (Paris), T Ogata (Tokyo), N Charlet-Berguerand (Illkirch) and A Beggs (Boston).
Present Research Projects:
-identification of new genes mutated in neuromuscular diseases through next generation sequencing
-establishment and validation of novel molecular diagnosis strategies
-cellular roles of amphiphysin, dynamin, myotubularins and their partners: regulation of cytoskeleton, organelles positioning and endomembrane system
-muscle specific functions and links between these proteins, implicated in centronuclear myopathies
-characterization of animal models (C. elegans and mice) to study their physiological roles and establish disease models
-therapeutic approaches in mouse
Muscular Dystrophy Association (http://www.mdausa.org/)
Myotubular Trust (http://www.myotubulartrust.com/)
French Myology Society sponsored by Genzyme
Collège de France
INSERM, CNRS, University of Strasbourg
Strasbourg genetic diagnosis laboratory: http://www-ulpmed.u-strasbg.fr/chimbio/diag/
Software used for 3D correlative microcopy: http://www.bitplane.com/go/applications/show-cases/3-d-correlative-microscopy-using-thresholding
© IGBMC 23/06/2014