Using a multidisciplinary approach, this project addressed:
1) the molecular and cellular mechanisms underlying Cx30 regulation of synaptic function
To do so, we first identified interacting protein partners of Cx30, and found two particularly interesting proteins, which control the expression and localization of Cx30, and also play a role in the structural and functional membrane properties of astrocytes, notably on the structural reorganization and dynamics of astrocytes during their migration, which alters their local physical surface. In addition deficiency for one of this partner mimics the change in astroglial morphology (ramification) and excitatory synaptic transmission induced by Cx30 deficiency, suggesting its involvement in the effect of Cx30. Altogether, these data reveal the importance of Cx30 protein interactions in the inhibition of astrocyte structural and mechanical plasticity.
We then explored the role of Cx30 at the cellular level, and found that it regulates astrocyte polarization during postnatal development. We indeed discovered that Cx30, independently of its gap-junction mediated biochemical coupling function, controls the orientation of astroglial protrusion during polarized migration via modulation of a well-known polarity pathway.
Finally, at the subcellular level, we found that Cx30 regulation of synaptic function is cell specific as we discovered that Cx30 regulates both excitatory and inhibitory synapses in the hippocampus, but differentially according to their presence on principal cells and interneurons.
2) The activity-dependent dynamics of Cx30 function at synapses
Here we first investigated whether Cx30 expression, localization and function was activity-dependent. Interestingly, we found that bursting activity increases hippocampal Cx30 protein levels, in particular at membranes and perisynaptic processes, via a calcium-dependent and post-translational mechanism regulating lysosomal degradation. This regulation translated at the functional level in the activation of Cx30 hemichannels, and in Cx30-mediated remodeling of astrocyte morphology independently of gap junction biochemical coupling. Remarkably, we found in turn that astroglial Cx30 sustains neuronal population bursts and increases the severity of associated behavioral seizures, also independently of gap-junction mediated biochemical coupling. Altogether, these data point to a positive feedback loop that could favor aberrant bursting and possibly increase the severity of seizures in vivo.
Finally, we studied the local and dynamic synthesis of connexins and associated partners in perisynaptic astroglial processes (PAP). We found in hippocampal PAPs the presence of a whole translational machinery including ribosomes, mRNAs and organelles for protein maturation, which permits the translation of a specific set of proteins, including Cx30, which are involved in a variety of processes including cell signaling or cytoskeletal dynamics, and whose expression is dynamically regulated by memory related processes.
3) The role for Cx30 in wiring synaptic circuits during critical developmental periods
We here investigated the role of astroglial Cx30 in a form of more integrated plasticity occurring during the development: the critical period plasticity in the visual cortex. Postnatal development is indeed characterized by critical periods of experience-dependent enhanced brain plasticity. If these periods do not end properly, this can lead to neurodevelopmental disorders due to the absence of proper synaptic connections. We found that astrocytes close the visual cortex critical period via a novel mechanism involving a Cx30-Rho-ROCK signaling pathway, which regulates the expression of the matrix metalloproteinase 9 that controls the abundance of the extracellular matrix, and thus the ability to structurally and functionally remodel synaptic circuits in an activity-dependent manner. This work reveals that Cx30 is an astroglial molecular lock involved in the termination of the critical period, and thereby offers a new cellular and molecular target to alleviate dysfunctions associated with failure to close critical developmental periods.
In all, these data provide fundamental knowledge on the molecular and cellular mechanisms mediating astroglial regulation of synaptic networks.
The results of this research have been well disseminated in international and national meetings or institutes via oral presentations and posters, as well as via the publication of several important articles in high impact journals (Science, Nature Communications, Nature Reviews Neuroscience...).
