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

突触的形成对CNS的发育、成熟和重塑至关重要。虽然三羧酸循环（KC）的中间体在许多生物学过程中起着关键作用，但关于它们在发育中的CNS中对其受体的影响知之甚少。然而，由于它们存在于胎儿和出生后早期，KC的中间体如琥珀酸和α-酮戊二酸（aKG）及其各自的受体GPR 91和GPR 99被认为参与轴突生长和突触发生。在协议中，我们最近证明了它们在轴突生长中的作用。此外，我们已经获得了初步的证据表明，他们增加突触数量。因此，本提案的主要目的是确定琥珀酸和aKG通过GPR 91/GPR 99在突触形成/重塑中的贡献以及它们发挥作用的机制。我们提出以下具体目标：1.研究糖代谢的中间产物通过GPR 91/99在体外是否有助于突触形成/重塑。我们的实验并没有区分它们是否会增加新突触的形成或干扰现有突触的维持。为了验证它，将进行突触数量/形态、突触前功能和电生理测量。为了确定它们是否增加了新突触的形成和/或现有突触的稳定性，我们将使用延时显微镜实验。2.琥珀酸盐和aKG通过其受体增加突触前体的数量。我们已经发现GPR 91和GPR 99装饰轴突和树突丝状伪足，轴突和树突棘的前体。我们将确定琥珀酸和aKG是否通过GPR 91/GPR 99增加轴突和树突丝状伪足的形成、延伸和稳定性。3.琥珀酸和aKG通过GPR 91/GPR 99增加突触前体和突触数量的机制。我们最近的研究结果表明，它们增加了突触的数量，但其发生的确切机制仍然未知。为了验证琥珀酸、aKG、GPR 91/GPR 99和ERK 1/2之间的联系，我们建议在存在或不存在ERK 1/2抑制剂的情况下进行上述相同的实验。4.琥珀酸和aKG通过GPR 91/GPR 99增加体内突触数量。我们的体外研究结果提出了一个合理的问题，即它们是否增加了体内的突触接触。为了表征GPR 91/GPR 99在体内突触形成/重塑中所起的作用，将进行形态学（EM）和功能（mEPSC）实验。该研究计划旨在确定GPR 91和GPR 99在突触形成和重塑中的作用。我们的初步研究结果表明，我们已经确定了一种新的机制，在调节神经元之间的通信中发挥着重要作用。这里提出的各种方法的结合将提供一个创新的多学科培训环境。

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.