Effect of neurotensin analogs on protein phosphorylation in the mouse brain

描述(申请人提供)：建议研究的目标是：1)比较特定的神经降压素(NT)类似物NT69L和NT79与典型和非典型抗精神病药物对信号转导的影响，以及2)确定介导这些影响的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(S)，以进一步将其开发为潜在的新型抗精神病药物。有了这一知识，NT类似物可以被修改为直接针对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)是一种内源性13个氨基酸的神经肽，在大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可以产生类似于使用抗精神病药物的效果，即使在急性给药时也是如此。然而，在动物模型中，NT必须直接进入大脑观察其效果。为了绕过这个问题，Richelson实验室开发了几个NT类似物，作为已知的三个NT受体NTS1、NTS2和NTS3中的一个或多个的激动剂。NT类似物具有与NT相似的作用，但其作用机制目前尚不完全清楚。这些可以外周给药的类似物包括被认为同时结合NTS1和NTS2的NT69L，以及被认为优先结合NTS2的NT79。将这些化合物对精神分裂症相关脑区蛋白质磷酸化的影响与现有抗精神病药物的影响进行比较，将有助于深入了解抗精神病药样作用的作用机制(S)。这将通过确定可能的分子靶点来调节类似物的抗精神病药物特性以及确定潜在的不良副作用，从而进一步开发NT类似物作为潜在的抗精神病药物。现建议进行三项重点研究以阐明这些类似物的作用机制(S)：(1)鉴定赋形剂、NT69L和NT79急性处理过的野生型C57BL小鼠中差异磷酸化的蛋白质，并将它们与氟哌啶醇和氯氮平急性处理过的那些蛋白质进行比较；(2)确定赋形剂、NT69L和NT79急性处理过的NTs1基因敲除(KO)小鼠之间的差异磷酸化蛋白质；以及(3)鉴定赋形剂、NT69L和NT79急性处理过的NTS2 KO小鼠之间的差异磷酸化蛋白质。方法包括动物注射、脑解剖、提取富含突触神经体的制剂、双向凝胶电泳法分离蛋白、磷蛋白检测和质谱法鉴定差异磷酸化蛋白。 公共卫生相关性：NT69L和NT79是可能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，并将它们与现有抗精神病药物(用于治疗精神分裂症)造成的影响进行比较，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，这些化合物可能会被设计成更具针对性地针对那些与疾病有关的蛋白质，增加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.