Activation of GPR37 and GPR37L1 by prosaptide, a neuroprotective peptide

描述（由申请人提供）：GPR37和GPR37L1是孤儿G蛋白偶联受体，在中枢神经系统中表达最丰富。我们评估了少量孤儿神经肽对GPR37和GPR37L1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是一种源自前体蛋白prosaposin的14氨基酸肽。已知prosap肽通过未识别的G蛋白偶联受体在大脑的某些区域（如黑质）发挥神经保护作用，并且在体内也已知在神经损伤动物模型中对缓解神经性疼痛和促进髓鞘再生发挥有益作用。本探索性R21应用中所描述的实验的总体目标是测试GPR37和/或GPR37L1是否介导prosaptide刺激原代神经元中存活信号并促进体内多巴胺能神经元存活的能力。信号传导研究将集中在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上，它们分别优先表达GPR37和GPR37L1。将从野生型小鼠以及GPR37（“GPR37- ko”）或GPR37L1（“GPR37L1- ko”）表达为零的小鼠制备培养物。然后在WT和KO小鼠的培养物中评估prosap肽刺激这些神经元中磷酸化- erk和磷酸化- akt的能力。除了这些研究prosaptide对存活信号传导影响的实验外，我们还将在不同剂量prosaptide缺失和存在的情况下，用损伤（如营养因子停用或毒素MPP+治疗）挑战神经元，以评估肽对神经元存活的影响。与

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.