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Interview with C. Trollet, F. Pinto-Mariz and A. Vignaud

Capucine Trollet, Fernanda Pinto-Mariz and Alban Vignaud
Focus on UMRS 974 -Team 2: Remodelling, Regeneration and Cell Therapy of Striated Muscle
Dr. Gillian Butler-Browne leads the Remodelling, Regeneration and Cell Therapy of Striated Muscle team at the Institute of Myology. In this focus, three postdoctoral research associates, Capucine Trollet, Fernanda Pinto-Mariz and Alban Vignaud describe their latest results that have recently been published or accepted for publication in peer-reviewed journals.
Capucine Trollet
  • Therapeutic characterization in mouse models
Capucine Trollet is a post-doctoral research associate in Gillian Butler-Browne’s team. She is working on oculopharyngeal muscular dystrophy (OPMD) after 4 years of post-doc in George Dickson’s lab in Royal Holloway, London.
What is the importance of this study?
I would like first to mention that I have worked on this study both in George Dickson’s lab in the UK and then in Gillian’s team here in Paris. We have carried out a molecular and phenotypic characterisation of the skeletal muscle of a mouse model for OPMD (previously described by David Rubinsztein, Cambridge). I think the main importance of this study is that we have shown that the expression of triplet-repeat expanded PABPN1 leads to a massive gene deregulation with muscle atrophy as a major consequence. Functional and histological analysis of skeletal muscle confirmed a severe and progressive muscular atrophy and reduction in muscle strength. What was also exciting was that at the fibre-type level, we observed differences between different types of fibres, some were affected and others not, even though they all expressed the mutant PAPBN1. This is similar in OPMD patients where some muscles are not affected at all and others are. So it is quite interesting. In conclusion, this mouse recapitulates several pathological observations seen in OPMD patients: progressive muscle weakness, muscle atrophy, fibrosis, mitochondrial defects, ubiquitin-proteasome pathway dysregulation, and affected and unaffected muscles all expressing mutant PABPN1.
What do you think is the significance of this fibre type specificity?
We can’t really translate this so easily into humans because the muscles that are affected in patients are not only fast muscles, as we found in the mouse. So it’s not as simple as ‘ fast muscles are affected, slow muscles are not affected.’ But it’s interesting that for some reason, whether it be resistance or some other parameters that we have not yet deciphered, there is a muscle that has all the features necessary for it be affected but yet it is not affected whereas other muscles are greatly affected. So for the moment I can’t explain this observation but at least we possess a mouse model that contains aggregates in this muscle but no phenotype and others have aggregates and are atrophied.

Do you intend to study this in more detail?
Yes, that’s the plan now. It was not the first thing that we initially found but after looking in detail at the fibre types, we found that there really was a difference between the oxidative and glycolytic fibres. We performed a transcriptomic analysis but this was done in the whole quadriceps muscle, which is a mixture of fibre types, so now it would be really interesting to compare glycolytic fibres and oxidative fibres but that’s not a simple task considering the differences in these two types of muscle. So the muscle type differences need to be distinguished as well, according to what is specific for OPMD. But yes, it is important to investigate these two fibre types now. More generally, the molecular and pathological changes found in this study provide targets for future therapeutic strategies that may reverse some or all of these modified pathways, which are essential for muscle homeostasis and normal function.

Could there be a relationship between the mitochondrial abnormalities and fibre type specificity?
We only looked at the activity of the complex in the mitochondria, where we found differences. However, we looked in the TA (tibialis anterior). So similarly it would be interesting to now study and compare, for example, the soleus muscle (a ‘slow’ muscle) versus the EDL (extensor digitorum longus, a ‘fast’ muscle) to determine whether there are differences in the mitochondria. This is exciting because thus far, all of the papers that describe OPMD patients have just highlighted abnormal mitochondrial structures, mainly using electron microscopy. In our study we really show a difference in mitochondrial activity in this mouse model. Whether it has any significance for OPMD or not, whether it is just age-related, is yet to be revealed, but at least with the transcriptomic data and this mitochondrial activity, we demonstrate that there are significant differences.
Alban Vignaud
Alban Vignaud is a research engineer associate in Yves Fromes’ team, ‘Experimental physiology platform and in vivo evaluation of biotherapies’. This paper mainly concerns muscle function evaluation. Even though contraction represents the main role of muscle, few physiologists study contractile properties.
What is the relevance of studying the variability of muscle function during disease progression in DM1?
Myotonic Dystrophy type 1 (DM1) is an autosomal dominant disorder and patients have CTG repeat expansions ranging from 50 to 1000. The size of the expansion is correlated with the age of onset of the disease. In DM1 patients, weakness and atrophy appear in skeletal muscles, inducing one of the most disabling aspects of DM1. To study DM1, we used a transgenic mouse model, which has the human genomic DM1 region with 550 CTG repeats. This model was initially characterized in very old mice (Seznec et al. 2001) showing some muscle defects. We decided to study the emergence of the weakness and atrophy using two different ages (3- and 10-month old mice). We also wanted to show if there was a possible link between both.
What results did you obtain?
The main results were progressive muscle weakness and atrophy that was observed only in 10-months old mice. We also showed that the muscle weakness was induced by atrophy, since the force normalized to muscle mass was unchanged. We also confirmed that muscle atrophy was induced by a decreased fibre cross sectional area (CSA). We then asked the question: “Is muscle wasting mediated by an atrophic process?” The ubiquitin proteasome pathway and activity were analysed and results indicated a decrease in Fbxo32 (Atrogin-1) expression and the trypsin-like proteasome activity. Our results also suggest that triggering of the Fbox32/ubiquitin-proteasome pathway is an early event (an increase was observed in 3-month old mice) leading to atrophy and finally muscle weakness.
What are the next steps?
Many studies are engaged concerning an transgenic model that carries a longer expansion. We also plan to try different therapeutic approaches based on genetic tools, since some of those strategies already gave encouraging results in other muscle diseases. It is also important to know at what age these strategies should be put in place, to see if we can prevent the appearance of symptoms or reverse the symptoms back to normal levels.

Vignaud A et coll. Progressive skeletal muscle weakness in transgenic mice expressing CTG expansions is associated with the activation of the ubiquitin-proteasome pathway. Neuromuscul Disord. 2010 May;20(5):319-25. Epub 2010 Mar 25
Fernanda Pinto-Mariz
  • Clinical application in humans
Fernanda Pinto-Mariz is a Brazilian M.D./Ph.D fellow, presently working as a post-doc in Gillian Butler-Browne’s Laboratory with a "Poste vert" provided by Inserm to work in the context of the French/Brazilian International Associated Laboratory on Cell Therapy and Immunotherapy with the Laboratory of Thymus Research, Rio de Janeiro, Brazil, directed by Wilson Savino.

What was the aim of your study?
Studies on animal models as well as in DMD patients have suggested that the immune response observed in this disease could also contribute to the pathophysiology of DMD. In our study we aimed to look at the molecules potentially involved in the migration of the T cells from the blood to the muscle of DMD patients. We analysed the expression of the integrin-type extracellular matrix (ECM) receptors (VLA-4, VLA-5 and VLA-6) on the surface of the T cells obtained from the blood, as well as the ECM and endothelial-driven migratory response of these T cells in vitro. In addition the inflammatory infiltrate in the muscle tissue of DMD patients was characterized.
What is the clinical importance of your study?
In our first paper we demonstrated that in DMD patients the relative numbers of T cells expressing a higher level of VLA-4 and VLA-5 (VLA-4Hi and VLA-5Hi population) in the blood and the fibronectin-driven migration is higher than healthy control. Now, in a second paper, which will soon be submitted for publication, we have more than 60 DMD patients subdivided in different groups according to the severity and the evolution of the disease. We have observed that the expression of VLA-4Hi, and not VLA-5, on the surface of T lymphocytes can be used as a biomarker for the prognosis of DMD and that the levels of T+/VLA-4Hi cells increase according to the severity of the disease. The number of T+/VLA-4Hi cells in the blood in the beginning of the disease is higher in patients who will lose the ability to walk before 10 years age. Our results in skeletal muscles shows a higher number of activated TCD8+ cells in the tissue of the DMD patients who become wheel chair bound before 10 years age, which also suggests a role for the immune system in this disease. Another point that I think is significant is that in vitro experiments showed that the transendothelial and fibronectin-driven migration of T cells can be blocked after the treatment with the antibody anti-VLA-4. Considering that VLA-4 antagonists, including anti-VLA-4 antibodies are already employed in clinical trials in other diseases, it’s exciting to think of its potential therapeutic application in DMD, to slow down disease progression, ameliorating the patients’ quality of life.
Pinto-Mariz F et coll. Differential integrin expression by T lymphocytes: Potential role in DMD muscle damage. J Neuroimmunol. 2010 Apr 9. [Epub ahead of print]

April 2010
Interview by Racquel N. Cooper