ONGOING RESEARCH

Aim 1 : Characterization of new presynaptic determinants of GABAergic transmission.
By studying 55 VGAT and GAT homologous genes in C. elegans, we identified eight candidate genes for GABA transport. Some of these genes are members of the SLC36, SLC7 and SLC16 families, which are not known to be involved in GABA transport in the vertebrate nervous system. If one of them turns out to be a GABA transporter, this would highlight a new function for an entire family. To test the contribution of these genes, we are developing a GABA sensor. We will take advantage of a genetically encoded fluorescent reporter whose intensity reflects the amount of GABA detected, iGABASnFR, which has recently been developed and tested in mice and zebrafish. This sensor is a combination of a periplasmic GABA-binding protein from Pseudomonas fluorescens and the circularly permuted green fluorescent protein superfolder with optimized linkers and mutations. We were able to express this sensor in all C. elegans neurons using a pan-neuronal promoter. Characterization and validation of this strain is currently underway. We will then use it to perform an EMS genetic screen to isolate mutants with a deficiency in GABA transport.

Aim 2 : Effects of caffeine on the pruning of glutamatergic synapses during development.
Caffeine is the most widely consumed psychoactive substance in the world and one of the main food chemicals. It acts as a non-selective competitive antagonist of the four subtypes of G protein-coupled adenosine receptors, the A1, A2A, A2B and A3 receptors. When ingested during pregnancy at doses ≥200 mg/day, children may show a reduction in IQ. We have shown that exposure to caffeine or a selective A2A receptor antagonist in early life disrupts brain development, with long-term adverse consequences for offspring. Daily exposure to caffeine during lactation, which coincides with the period of intense synapse remodeling (between P3 and P16), results in a loss of inhibitory GABAergic synapses and a gain in excitatory glutamatergic synapses at the time of weaning (P21). This affects the functioning of the hippocampal network, since exposure to caffeine during this period of synaptic remodeling makes animals more susceptible to epileptic seizures and spatial memory deficits. Thus, the “impaired construction” of the brain following exposure to caffeine in early life makes offspring more susceptible to brain disorders. The main aim of our project is to discover the cellular and molecular mechanisms involved in this effect of caffeine on the development of synapses, in order to target it in neurodevelopmental diseases. Our recent data show an unexpected role for the A2A receptor in stabilizing inhibitory GABAergic synapses during development (Science 2021). Our unpublished data suggest a broader involvement of the A2A receptor in the pruning of hippocampal glutamatergic synapses via neuroglial interactions. We are currently analyzing the respective role of the neuronal, astrocytic and microglial A2A receptor in the fate of glutamatergic synapses and the underlying mechanisms.

Aim 3 : Characterization of a new signaling pathway in the control of inhibition at mature inhibitory synapses.
The WNK/SPAK pathway controls the neuronal Cl- extruder KCC2 and the Cl- importer NKCC1, both of which regulate chloride homeostasis and are critical for GABAergic transmission. We have also shown that this pathway is abnormally activated in the epileptic brain. Our project aims to reveal the virtually unknown role of this signaling pathway, which is widespread in the nervous system, and to determine whether interference with this pathway has beneficial effects in epilepsy and stress.
In particular, we are attempting to answer the following questions :
• Does this pathway only control chloride transporters or does its action extend to other molecules in the inhibitory synapse ?
• Is there a neuronal type (interneurons vs. pyramidal cells) that is particularly sensitive to this signaling pathway ?
• Are WNK/SPAK kinases recruited to the neuronal membrane following their activation, and if so, by what mechanism ?
• Can we target this mechanism and thus prevent the deregulation of chloride transporters in pathology ?