Activation of GPR37 and GPR37L1 by prosaptide, a neuroprotective peptide

描述（由申请人提供）：GPR 37和GPR 37 L1是孤儿G蛋白偶联受体，在中枢神经系统中表达最丰富。我们评估了少数孤儿神经肽对GPR 37和GPR 37 L1的潜在激活作用，发现这两种受体都可以在转染的非神经元细胞中被prosaptide激活，prosaptide是一种来源于前体蛋白saposin的14个氨基酸的肽。已知普罗萨肽通过未鉴定的G蛋白偶联受体在脑的某些区域（例如黑质）中发挥神经保护作用，并且还已知在体内对减轻神经性疼痛和促进神经损伤动物模型中的髓鞘再生发挥有益作用。在该探索性R21申请中描述的实验的总体目标是测试GPR 37和/或GPR 37 L1是否介导丙沙肽刺激原代神经元中的存活信号传导和促进多巴胺能神经元体内存活的能力。信号传导研究将集中于来自腹侧中脑和皮质神经元的多巴胺能神经元的原代培养物，其分别优先表达GPR 37和GPR 37 L1。将从野生型小鼠以及从不表达GPR 37（“GPR 37-KO”）或GPR 37 L1（“GPR 37 L1-KO”）的小鼠制备培养物。然后，在从WT与KO小鼠制备的培养物中评估普罗沙肽刺激这些神经元中的磷酸化ERK和磷酸化Akt的能力。除了这些研究prosaptide对存活信号传导的影响的实验之外，我们还将在不存在和存在不同剂量的prosaptide的情况下用损伤（例如营养因子撤除或用毒素MPP+处理）来挑战神经元，以评估肽对神经元存活的影响。平行于 在对培养的神经元的实验中，整体动物研究将评估在用多巴胺能神经毒素MPTP处理后，丙沙肽对黑质中多巴胺能神经元的神经保护作用。我们将采用2种不同的MPTP方案（急性和慢性）来评估GPR 37和GPR 37 L1在体内的丙沙肽的神经保护作用中的作用。这些MPTP毒性研究将与WT、GPR 37-KO和GPR 37 L1-KO小鼠平行进行。如果在该提议中描述的工作可以确定GPR 37和/或GPR 37 L1作为介导普罗沙肽在原代神经元培养物和体内的神经保护作用的受体，那么未来的研究可以集中于发现这些受体的小分子激动剂和/或正变构调节剂，其可以用作治疗帕金森病、中风、神经性疼痛和髓鞘形成障碍。 公共卫生相关性：前列腺素是一种神经肽，已知其通过未鉴定的G蛋白偶联受体在脑的某些区域（例如黑质）中发挥神经保护作用，并且还已知其在体内对减轻神经性疼痛和促进神经损伤动物模型中的髓鞘再生发挥有益作用。我们发现，prosaptide可以激活孤儿G蛋白偶联受体GPR 37和GPR 37 L1在转染的非神经元细胞，我们现在试图了解GPR 37和/或GPR 37 L1介导的神经保护作用的prosaptide在培养的神经元，以及在帕金森氏病的动物模型。考虑到普罗沙肽在体内的已知作用，介导这种神经保护肽的生理作用的受体的鉴定可以为治疗帕金森病、中风、神经性疼痛和髓鞘形成障碍的新疗法铺平道路。

DESCRIPTION (provided by applicant): GPR37 and GPR37L1 are orphan G protein-coupled receptors that are most abundantly expressed in the central nervous system. We assessed potential activation of GPR37 and GPR37L1 by a handful of orphan neuropeptides and found that both receptors can be activated in transfected non-neuronal cells by prosaptide, which is a 14-amino-acid peptide derived from the precursor protein prosaposin. Prosaptide is known to exert neuroprotective actions in certain areas of the brain, such as the substantia nigra, via unidentified G protein-coupled receptors, and also known to exert beneficial effects in vivo on relieving neuropathic pain and promoting remyelination in animal models of nerve damage. The overall goals of the experiments described in this exploratory R21 application are to test whether GPR37 and/or GPR37L1 mediate the ability of prosaptide to stimulate survival signaling in primary neurons and promote the survival of dopaminergic neurons in vivo. The signaling studies will focus on primary cultures of dopaminergic neurons from the ventral mesencephalon and cortical neurons, which preferentially express GPR37 vs. GPR37L1, respectively. Cultures will be prepared from wild-type mice as well as from mice that are null for expression of GPR37 ("GPR37-KO") or GPR37L1 ("GPR37L1-KO"). The ability of prosaptide to stimulate phospho-ERK and phospho-Akt in these neurons will then be assessed in cultures prepared from the WT vs. KO mice. In addition to these experiments studying the effects of prosaptide on survival signaling, we will also challenge the neurons with insults (such as trophic factor withdrawal or treatment with the toxin MPP+) in the absence and presence of varying doses of prosaptide to assess the effects of the peptide on neuronal survival. In parallel with the experiments on cultured neurons, the whole-animal studies will assess the neuroprotective effects of prosaptide on dopaminergic neurons in the substantia nigra following treatment with the dopaminergic neurotoxin MPTP. We will employ 2 different MPTP regimens (both acute and chronic) to assess the role of GPR37 and GPR37L1 in the neuroprotective effects of prosaptide in vivo. These MPTP toxicity studies will be performed in parallel with WT, GPR37-KO and GPR37L1-KO mice. If the work described in this proposal can establish GPR37 and/or GPR37L1 as the receptor(s) that mediate the neuroprotective actions of prosaptide in primary neuronal cultures and in vivo, then future studies can focus on the discovery of small molecule agonists and/or positive allosteric modulators of these receptors that might serve as novel therapeutics in the treatment of Parkinson's Disease, stroke, neuropathic pain and myelination disorders. PUBLIC HEALTH RELEVANCE: Prosaptide is a neuropeptide that is known to exert neuroprotective actions in certain areas of the brain, such as the substantia nigra, via unidentified G protein-coupled receptors, and also known to exert beneficial effects in vivo on relieving neuropathic pain and promoting remyelination in animal models of nerve damage. We found that prosaptide can activate the orphan G protein-coupled receptors GPR37 and GPR37L1 in transfected non-neuronal cells, and we now seek to understand if GPR37 and/or GPR37L1 mediate the neuroprotective actions of prosaptide in cultured neurons as well as in an animal model of Parkinson's Disease. Given the known actions of prosaptide in vivo, identification of the receptor(s) mediating the physiological effects of this neuroprotective peptide may pave the way for novel therapeutics in the treatment of Parkinson's Disease, stroke, neuropathic pain and myelination disorders.