DS0407 - Exploration du système nerveux dans son fonctionnement normal et pathologique

Analysis of the GABAergic synapse using structural and genetic strategies in the C. elegans model organism. – GABAREL

Submission summary

GABA is the primary inhibitory neurotransmitter in the nervous system. Changes in GABAergic neurotransmission contribute to the etiology of prominent neurological and mental disorders including epilepsy, anxiety and schizophrenia. Despite extensive molecular and pharmacological characterization of GABAA receptors (GABAR), the mechanisms controlling their subcellular localization remain partially unknown. We recently identified a novel synaptic organizer of GABA synapses in C. elegans. Punctin is a member of the poorly characterized though evolutionarily conserved ADAMTS-like proteins associated with the extracellular matrix (Nature 2014) and controls clustering of GABARs at C. elegans neuromuscular junctions. We showed that Punctin is a novel ligand of the sole neuroligin ortholog in C. elegans, NLG-1, and of the DCC netrin receptor ortholog, UNC-40, which are both required to promote GABAR recruitment at synapses.

We propose to use the C. elegans neuromuscular junction as a genetically tractable system to identify additional mechanisms supporting the formation of GABAergic post-synaptic domains in vivo, and to explore the Punctin-NLG-1 partnership at the molecular level. This project relies on the complementary expertise of two teams: the team of J.-L. Bessereau, a group of cellular and molecular neurogeneticists specialists of the C. elegans model, and the team of P. Marchot and Y. Bourne, a group of biochemists and structural biologists with expertise into the structure-function relationships of synaptic macromolecules, including the neuroligins. Specifically, we propose to:

1- Decipher the Punctin/Neuroligin/UNC-40 pathway mediating GABAR clustering
Using candidate gene strategy along with a novel forward genetic screen based on in vivo visualization of GABARs in live C. elegans, we identified two components, NLG-1 and UNC-40, which act downstream of Punctin to localize GABARs in inhibitory post-synaptic domains. We now wish to understand how this is achieved and identify what molecules are involved in this pathway. Anticipated results will provide new insights on an entirely new pathway for GABAR clustering.

2- Identify the molecular determinants and mechanisms involved in the Punctin-Neuroligin partnership
The synaptic localization of neuroligins is essential for their function, and their trans-synaptic anchoring by neurexins has been extensively documented at the molecular level. Punctin is a new ligand of NLG-1 while it can also engage homomeric interactions. By combining structural and biochemical analyses with the powerful genetic tools available in C. elegans, we expect to characterize the determinants and mechanisms involved in the Punctin-NLG-1 complex formation and validate the structural interpretation functionally in vivo. Identifying the NLG-1 capability for either alternative or concomitant association with Punctin and neurexin would open new avenues into the regulation of synapse formation and functioning.

3- Conduct a visual screen for mutants with abnormal GABAR distribution
To identify novel genes required for GABAR clustering along with genes necessary for proper GABAergic synaptogenesis, we will screen a knock-in strain that permits to visualize GABAR in living animals and look for abnormal GABAR distribution after random mutagenesis, independently from behavioral phenotypes. Identification of the mutated genes will then benefit from whole genome sequencing strategies recently developed in the C. elegans field.

The human orthologs of Punctin, NLG-1 and UNC-40 have been involved in neuropsychiatric diseases such as epilepsy, autism spectrum disorders and schizophrenia, but the knowledge on their molecular and cellular functions remains fragmentary. Our project will contribute insights into the GABA synapse organization and identify novel genes and mechanisms required for synapse formation and function, and possibly involved in the onset of neuropsychiatric diseases.

Project coordination

Jean-Louis BESSEREAU (Centre de Génétique et de Physiologie Moléculaire et Cellulaire)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CGphiMC Centre de Génétique et de Physiologie Moléculaire et Cellulaire
AFMB Architecture et Fonction des Macromolécules Biologiques

Help of the ANR 458,000 euros
Beginning and duration of the scientific project: December 2015 - 36 Months

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