After spinal cord injury (SCI) patients develop spasticity, a motor disorder characterized by hyperreflexia and stiffness of muscles. Spasticity results from alterations in motoneurons with an upregulation of their persistent sodium current (INaP), simultaneously with a disinhibition due to a reduced expression of chloride (Cl-) co-transporters KCC2. Up to now the origin of alterations is unknown. Meanwhile, it was established that the proteases calpains are activated in the spinal cord after injury. The typical response of Na+ channels to proteases is an increase in INaP, whereas modulation of KCC2 expression results from a dysregulation of Cl- homeostasis. In muscles, spasticity induces stiffness by calpain activation and remodeling of structural protein such as titin. The proposal will test the exciting possibility that proteolysis of the triad (Na+ channels, KCC2 and titin) by calpains represents the main mechanism responsible for spasticity. To this end we will: 1) characterize the time-course expression/activation of calpains after SCI 2) gain a better understanding of the mechanisms by which SCI-induced activation of calpains leads to spasticity 3) evaluate whether training, known to reduce spasticity, down-regulates the activation of calpains 4) demonstrate that preventing the action of calpains at the time of injury in the chronic phase of SCI, reduces the excitatory/inhibitory imbalance and stiffness of the muscles. Ultimately, an effective and minimally invasive treatment of spasticity will be designed by pharmacological or genetic tools (e.g. intramuscular injection of recombinant adenovirus associated vectors). The project will bring together two groups, located on the same campus, with complementary expertise. First, the two groups work on distinctly part of the neuromuscular system - the Brocard team on the motoneuron and the Bartoli team on the muscle. Second, the two groups have developed models and master expertise in different, yet complementary, experimental methodologies (electrophysiology, immunostaining, molecular biology, imaging, genomic, behaviour). This new collaboration is well suited for the fourth societal challenge “life, health and well-being”. It should provide both basic knowledge on SCI ethiopathology and open new avenues towards innovative therapeutics in a so far unexplored field.

Integral trans-membrane proteins are involved in different major cellular functions including e.g. cell homeostasis, cell bioenergetics and cell detoxification. Therefore their dysfunction is associated with serious human pathologies such as neurodegenerative diseases, cancer and virus infections. International consortia pursue approaches based on structural biology, cell biology and biophysics to understand the function of membrane proteins at the molecular level. Goals generally include the determination of the structure-function relationship for the proteins and also the effect of protein-lipid interactions, but often ignore the effects related to physical properties of membranes. Membrane physicists, including Partners 1 and 3, have brought evidence of the intrinsic interplay between protein shape and the properties of its membrane environment. These experiments have been limited to proteins with fixed shape and have not yet considered the role of the dynamic structural changes associated to their function. The challenge is now to investigate the feedback between the functional conformational dynamics of the protein and the physical properties of its surrounding membrane, at short and long distances. We will tackle the following, and still largely open, questions: (i) How does the conformational dynamics depend on membrane lipid composition? ii) How is it modified by the tension and curvature of the membrane? (iii) How does the conformational dynamics of the protein influence its membrane properties, such as its mobility and curvature-dependent distribution. We address these questions with proteins exhibiting large conformational changes during their ATP-ase cycle reconstituted in model membrane systems (small and giant vesicles) with controlled physico-chemical properties. This is the case of MultiDrug Resistance ABC transporters for which conformational changes associated with ATP hydrolysis are the key events of the drugs' transport. These proteins are involved in drug detoxification and protection of tissues from xenobiotics, including administered therapeutic drugs. Their structure and function are especially sensitive to the properties and composition of the surrounding membrane. We have chosen 2 ABC transporters: BmrA, a bacterial ABC from B. subtilis, fairly homologous to P-gp, and human P-gp. Tools and concepts of membrane physics can tackle from a different angle current challenges on drug transport, which is required to design new strategies of regulation and inhibition: i) dynamics and stochastic transitions between catalytic conformations of ABCs, ii) the interdependence between membrane properties and conformational dynamics iii) the role of the non-equilibrium conformational changes due to ATP hydrolysis in the lateral mobility of the proteins and in their lateral distribution. The general knowledge obtained for BmrA will be extended to investigate for the human P-gp the challenging issues of its flexibility and spatial organization during its catalytic cycle. We will combine the efforts of four groups with a unique set of expertise in structural biology, coarse-grained simulations, biomimetic systems, membrane biophysics, single-molecule biophysics and statistical physics. Thanks to a comprehensive multi-scale approach combining cryo-EM and biophysical techniques with coarse-grained simulations and theoretical models, we will be able to investigate: the shape of the ABC//membrane systems at high (with cryo-EM) or low (with CG simulations) protein density, the dynamics of the conformational changes (with single-molecule FRET measurements) and its coupling to the properties of the membrane (using micromanipulation techniques, single particle tracking and physical modeling). Overall, it will allow a novel understanding of membrane protein catalytic cycles that goes beyond the structure-function description and includes the reciprocal action between the protein and its surrounding membrane environment.

The recent expansion of the unconventional gas industry in North America and its potential advent in Europe has generated public concern regarding the protection of groundwater and surface water resources from contamination by stray gas, saline formation water and fracturing chemicals. A major scientific challenge and an indispensible prerequisite for environmental impact assessment in the context of unconventional gas development is the determination of the non-impacted baseline conditions against which potential environmental impacts on shallow water resources can be accurately and quantitatively tested. The objective of this Franco-Canadian NSERC-ANR project is to develop an innovative and comprehensive methodology of geochemical and isotopic characterization of the environmental baseline for water and gas samples from all three essential zones: (1) the production zone, including flowback waters, (2) the intermediate zone comprised of overlying formations, and (3) shallow aquifers and surface water systems where contamination may result from diverse natural or other human impacts. The outcome will be the establishment of a methodology based on innovative tracer and monitoring techniques for detecting and quantifying and modeling stray gas and leakage of saline formation water mixed onto flowback fluids into fresh groundwater resources and surface waters taking into account the mechanisms of fluid and gas migration. The new knowledge derived from this project will be of critical importance for ensuring the environmentally acceptable development of unconventional energy resources in Canada and Europe. Decision support and recommendations for stakeholders on meaningful baseline and monitoring programs in the context of unconventional energy resources will be provided as final deliverable.