Investigation of the role of GPR91 and GPR99 during Central Nervous System Development

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The formation of synapses is crucial to development, maturation, and remodeling of the CNS. While intermediates of Krebs cycle (KC) have key roles in many biological processes, much less is known about their effects on their receptors in the developing CNS. However, due to their presence at the fetal and early postnatal periods, intermediates of KC such as succinate and alpha-ketoglutarate (aKG) and their respective receptors GPR91 and GPR99 were proposed to participate to axon growth and synaptogenesis. In agreement, we have recently demonstrated their role in axon growth. Furthermore, we have obtained preliminary evidence indicating that they increase synapse number. Therefore, the main objective of this proposal is to identify the contribution of succinate and aKG via GPR91/GPR99 in synapse formation/remodeling as well as the mechanisms by which they exert their effects. We propose the following specific objectives: 1. To study if the intermediates of carbohydrate metabolism via GPR91/99 contribute to synapse formation/remodeling in vitro. Our experiments do not distinguish whether they may increase the formation of new synapses or interfere with the maintenance of existing ones. In order to verify it; synapse number/morphology, presynaptic functionality, and electrophysiological measurements will be performed. To determine if they increase the formation of new synapses and/or the stabilization of existing ones, we will use time-lapse microscopy experiments. 2. Succinate and aKG via their receptors increase the number synaptic precursors. We have found that GPR91 and GPR99 decorate axon and dendritic filopodia, precursors of axonal and dendritic spines. We will determine if succinate and aKG via GPR91/GPR99 increase the formation, extension, and stability of axon and dendritic filopodia. 3. The mechanisms by which succinate and aKG via GPR91/GPR99 increase synapse precursors and synapse number. Our recent results suggest that they increase the number of synapses but the exact mechanism by which it occurs is still unknown. In order to verify the link between succinate, aKG, GPR91/GPR99 and ERK1/2, we propose to perform the same experiments described above in the presence or the absence of ERK1/2 inhibitors. 4. Succinate and aKG via GPR91/GPR99 increase synapse number in vivo. Our in vitro results raise the legitimate question of whether they increase synaptic contacts in vivo. In order to characterize the role played by GPR91/GPR99 in synapse formation/remodeling in vivo, morphological (EM) and functional (mEPSCs) experiments will be undertaken. Summary: The proposed research program aims to identify the effects of GPR91 and GPR99 in synapse formation and remodeling. Our preliminary findings suggest that we have identified a novel mechanism that plays a fundamental role in regulating the communication between neurons. The combination of approaches proposed here will provide an innovative multidisciplinary training environment.