New mechanisms for the fine-tuning of inhibitory synaptic receptor localization – Synaptune

In the past few years, we have developed optical, imaging and analytical tools to monitor the behavior of neurotransmitter receptors at the single molecule level. This allowed us to access novel dimensions of neurotransmission regulation. Based on this novel understanding of synaptic biology, we have demonstrated that glycine receptor (Gly-R) and GABAA-R lateral diffusion and capture at synapses are regulated by activity. We also showed that the coupling excitation-inhibition is regulated by calcium dependant phosphorylation. Based on these tools and the new understanding of synapse, we now propose a new project to access novel dimensions of the physiological non-cell autonomous regulation of inhibitory neurotransmitter dynamic and synaptic strength. It will also decipher their pharmacological controls.
Task 1: we found that the diffusion dynamic of Gly-Rs is regulated via a PKC-dependent mechanism. We identified the S403 in the intracellular loop of the Gly-R as the target of PKC. We will now unravel the regulation of GABAAR diffusion capture. We will then establish how these regulations are used to fine-tune the strength of synaptic inhibition upon neuronal activity.
Task 2: our preliminary data establish that microglial activation down regulates Gly-R and GABAA-R trapping at synapses leading to a disinhibition. We propose to determine how microglia and astrocytes are involved in this disinhibition. We will then focus on the role of TNFa and Thrombospondin that are glial factors for which we have preliminary results. This will lead us to describe how the homeostatic balance between excitation and inhibition is (dys)-regulated upon microglial activation.
Task 3: we have evidence that the metabotropic GABAB receptors (mGABAB-Rs) modulate Gly-R and GABAA-R lateral diffusion and interactions with scaffolding proteins, ultimately controlling their accumulation at synapses. We will determine how the trafficking of Gly-R and GABAA-R are impacted by GABAB signaling, and unravel the mechanisms of this regulation. This will provide a basis for pharmacological actions targeting inhibition.
Finally (Task 4), we have developed a multicolor super-resolutive PALM-STORM microscope (resolution about 20nm) to access novel dimensions of the dynamic of membrane proteins. We will combine the super-resolution with Single Particle Tracking. Our aim is to collect quantitative molecular data together with dynamic aspects of receptor binding to allow a biochemical study in cellulo. Ultimately, we will put a strong effort to access super-resolution imaging of Gly-R and GABAAR diffusion in integrated systems such as organotypic slice cultures. The tools developed in this part of the project will be used in all the aspects of the project related to receptor dynamic.
Ultimately this research may find potential applications in situations were the homeostatic excitation/inhibition regulation is altered following inflammatory microglial activation resulting in increased glutamatergic neurotransmission or loss of tonic inhibitory control as seen in sensitization of pain or in other synaptopathies such as spasticity and epilepsy.

Selected publications of the partners as corresponding authors: Pascual et al (2011) PNAS ; Pinaud & Dahan (2011) PNAS; Specht et al (2011) EMBO J; Charrier et al. (2010). Nature Neurosci; Renner et al. (2010) Neuron; Pinaud et al. (2010) Nature Meth; Bernard et al. (2010) EMBO J; Bannai et al. (2009) Neuron; Lévi et al. (2008) Neuron; Triller & Choquet (2008) Neuron; Bouzigues et al (2007) PNAS; Dahan et al.(2003) Science 302:442.; Meier et al. (2001) Nature Neurosci 4:253