Effect of neurotensin analogs on protein phosphorylation in the mouse brain

描述（由申请人提供）：拟议研究的目标是1）将特定神经降压素（NT）类似物NT 69 L和NT 79对信号转导的影响与典型和非典型抗精神病药的影响进行比较，以及2）鉴定介导这些影响的NT受体（NTR）。拟议研究的长期目标是更好地了解NT类似物的作用机制，以进一步开发其作为潜在的新型抗精神病药物。有了这些知识，NT类似物可以被修饰为直接靶向被认为直接参与神经精神疾病治疗的蛋白质，同时避免那些被认为介导不良副作用的蛋白质。 神经降压素（NT）是一种由13个氨基酸组成的内源性神经肽，在脑中起神经递质的作用。它与包括精神分裂症在内的神经精神疾病的病理生理学以及抗精神病药物的作用机制有关。NT可以引起类似于使用抗精神病药物的效果，即使是急性给药。然而，在动物模型中，NT必须直接施用到脑中以观察其效果。为了解决这个问题，Richelson实验室已经开发了几种NT类似物，它们作为三种已知NT受体NTS1、NTS2和NTS3中的一种或多种的激动剂。NT类似物具有与NT类似的作用，但其作用机制目前尚未完全了解。这些类似物可以外周给药，包括被认为与NTS1和NTS2结合的NT69L和被认为优先与NTS2结合的NT79。将这些化合物对精神分裂症相关脑区蛋白磷酸化的影响与已确定的抗精神病药物的影响进行比较，将有助于深入了解介导抗精神病样作用的作用机制。这将通过鉴定介导类似物的抗精神病样性质的可能分子靶标以及鉴定潜在的不良副作用来进一步开发NT类似物作为潜在的抗精神病药物。 提出了三个重点研究目标，以阐明这些类似物的作用机制：（1）鉴定在用媒介物、NT 69L和NT 79急性处理的野生型C57 BL小鼠中差异磷酸化的蛋白质，并将它们与用氟哌啶醇和氯氮平急性处理的那些进行比较，（2）鉴定用载体、NT69L和NT79急性处理的NTS1敲除（KO）小鼠之间差异磷酸化的蛋白质，和（3）鉴定用载体、NT69L和NT79急性处理的NTS2 KO小鼠之间差异磷酸化的蛋白质。 方法包括动物注射、脑解剖、提取富含突触神经体的制剂、通过二维凝胶电泳进行蛋白质分离、磷蛋白检测和通过质谱鉴定差异磷酸化蛋白质。 公共卫生相关性：NT69L和NT79是可以治疗精神分裂症的化合物。确定这些化合物对大脑中蛋白质的影响，并将其与已确定的抗精神病药物（用于治疗精神分裂症）引起的影响进行比较，可以深入了解每种化合物在治疗疾病中可能发挥的作用。有了这些知识，这些化合物可以被设计成更特异性地靶向那些与疾病有关的蛋白质，提高它们作为抗精神病药物的有效性并减少潜在的副作用。

DESCRIPTION (provided by applicant): The goals of the proposed research are to 1) compare the effects on signal transduction of specific neurotensin (NT) analogs, NT69L and NT79, to those of typical and atypical antipsychotics, and 2) to identify the NT receptors (NTRs) by which these effects are mediated. The long term objective of the proposed research is to better understand the mechanism(s) of action of NT analogs to further their development as potential novel antipsychotic drugs. With this knowledge, NT analogs can be modified to directly target proteins thought to be directly involved in the treatment of neuropsychiatric disease while avoiding those thought to mediate adverse side effects. Neurotensin (NT) is an endogenous thirteen amino acid neuropeptide that acts as a neurotransmitter in the brain. It has been implicated in the pathophysiology of neuropsychiatric diseases including schizophrenia, as well as in the mechanism of action of antipsychotic drugs. NT can elicit effects similar to those seen using antipsychotic drugs, even when administered acutely. However, in animal models NT must be administered directly into the brain to observe its effects. To circumvent this problem, the Richelson laboratory has developed several NT analogs that act as agonists to one or more of the three known NT receptors, NTS1, NTS2, and NTS3. The NT analogs have similar effects as NT, but their mechanisms of action are not fully understood at this time. These analogs, which can be administered peripherally, include NT69L, thought to bind both NTS1 and NTS2, and NT79, thought to preferentially bind NTS2. Comparing the effects of these compounds on protein phosphorylation in regions of the brain implicated in schizophrenia to the effects of established antipsychotic drugs will offer insight into the mechanism(s) of action mediating antipsychotic-like effects. This will further the development of NT analogs as potential antipsychotic drugs by identifying possible molecular targets which mediate the antipsychotic-like properties of the analogs as well as identifying potential adverse side-effects. Three focused research aims are proposed to elucidate the mechanism(s) of action of these analogs: (1) Identify proteins that are differentially phosphorylated in wild-type C57BL mice treated acutely with vehicle, NT69L, and NT79, and compare them to those treated acutely with haloperidol and clozapine, (2) Identify proteins that are differentially phosphorylated between NTS1 knock-out (KO) mice treated acutely with vehicle, NT69L and NT79, and (3) Identify proteins that are differentially phosphorylated between NTS2 KO mice treated acutely with vehicle, NT69L and NT79. Methods include animal injection, brain dissection, extraction of synaptoneurosomal-enriched preparations, and protein separation via two-dimensional gel electrophoresis, phosphoprotein detection and identification of differentially phosphorylated proteins via mass spectrometry. PUBLIC HEALTH RELEVANCE: NT69L and NT79 are compounds that may be therapeutic in the treatment of schizophrenia. Determining the effects of these compounds on proteins in the brain, and comparing them to those caused by established antipsychotic drugs (used to treat schizophrenia), can offer insight to what role each compound may play in treating disease. With this knowledge, these compounds may be designed to more specifically target those proteins involved in disease, increasing their effectiveness as antipsychotics and decreasing potential side effects.