Plasticity in the aging olfactory system

描述（由申请人提供）：本提案的重点是阐明多巴胺（DA）神经元在嗅球（OB）中分化的分子机制。该实验室的长期目标是合理开发适合移植治疗的功能性DA神经元，以取代帕金森病（PD）患者丢失的黑质（SN）神经元。一种新兴的治疗策略是通过将神经元移植到患者的纹状体中来功能性替代失去的SN细胞。干细胞（包括成体和胚胎）是替代细胞的一个有希望的来源，但是产生适合移植的神经元的有效操作尚未阐明。为了使干细胞具有适当的特性，了解DA神经元发育和分化的调控机制是必要的。OB - DA神经元引起了人们的极大兴趣，因为它们在PD患者中不会退化，它们是在动物（包括人类）的整个成年期由神经干细胞产生的，新分化的DA细胞可以整合到已有的神经网络中。然而，在我们的知识中，一个重要的空白是直接调节OB特异性DA表型的因子的身份。初步数据提供了令人信服的证据，证明ETS转录因子ER81是OB DA表型的关键决定因素。本提案的特异性目标1将通过直接调节酪氨酸羟化酶（TH）的表达来验证ER81是ob特异性的DA表型分化决定因素的假设。通过TH表达测量OB - DA神经元的终末分化需要嗅觉受体细胞的传入突触活动。初步数据显示，GABA在去极化DA细胞中增强TH的表达。特异性目的2将验证传入突触活性和/或GABA对TH表达的调节是由ER81的表达或翻译后修饰介导的。ER81敲除小鼠的免疫组织化学和原位杂交将证实ER81是OB - DA表型的特异性决定因素。OB切片培养的基因功能缺失和恢复实验将证明ER81是TH表达的必要和充分条件。通过ChIP和EMSA证实的TH转录的直接调控将通过转录分析得到证实。在OB切片培养中，药理学研究将建立影响ER81表达和翻译后修饰的特异性受体、信号通路和激酶，以响应去极化和GABA。免疫沉淀ER81的翻译后修饰也将通过质谱和放射自显影分析32P掺入。气味剥夺小鼠的切片培养研究和ChIP实验将确定ER81的表达或翻译后修饰是否调节TH的表达。局灶性刺激和OB原代细胞培养研究将阐明其他非da细胞类型是否参与ER81对TH的调节，以响应突触活性和GABA。总之，这些研究将增强PD细胞替代策略的设计。项目的叙述

DESCRIPTION (provided by applicant): The focus of this proposal is to elucidate molecular mechanisms of dopamine (DA) neuron differentiation in the olfactory bulb (OB). The long term goal of the laboratory is the rational development of functional DA neurons suitable for transplant therapies to replace substantia nigra (SN) neurons lost in Parkinson's Disease (PD) patients. An emerging treatment strategy is functional replacement of lost SN cells by transplantion of neurons into the patient's striatum. Stem cells (both adult and embryonic) are a promising source for replacement cells, but efficient manipulations to generate neurons suitable for transplantation have not been elucidated. To engineer stem cells with appropriate characteristics, it is imperative to understand the mechanisms that regulate development and differentiation of DA neurons. The OB DA neurons are of considerable interest because they do not degenerate in PD patients, they are generated from neural stem cells through out the adult life of animals (including humans) and newly differentiated DA cells can integrate in pre-existing neural networks. However, a significant gap in our knowledge is the identity of factors that directly regulate the DA phenotype specifically in the OB. Preliminary data provides compelling evidence that the ETS transcription factor, ER81, is a key determinant of the OB DA phenotype. Specific Aim 1 of this proposal will test the hypothesis that ER81 is an OB-specific determinant of DA phenotypic differentiation by directly regulating tyrosine hydroxylase (TH) expression. Terminal differentiation of OB DA neurons, as measured by TH expression, requires afferent synaptic activity from olfactory receptors cells. Preliminary data reveals a novel enhancement of TH expression by GABA in depolarized DA cells. Specific Aim 2 will test the hypothesis that regulation of TH expression by afferent synaptic activity and/or GABA is mediated by the expression or post- translational modification of ER81. Immunohistochemistry and in situ hybridization in ER81 knock-out mice will establish ER81 as a specific determinant of OB DA phenotype. Loss-of and rescue-of gene function experiments in OB slice cultures will demonstrate that ER81 is necessary and sufficient for TH expression. Direct regulation of TH transcription, demonstrated using both ChIP and EMSA, will be confirmed with transcription assays. Pharmacological studies will establish specific receptors, signaling pathways and kinases that effect ER81 expression and post-translational modification in response to depolarization and GABA in OB slice cultures. Post-translational modifications with immunoprecipitated ER81 will also be analyzed by mass spectrometry and by autoradiography for 32P incorporation. Slice culture studies and ChIP experiments from odor deprived mice will determine whether expression or post-translational modification of ER81 regulates TH expression. Focal stimulation and OB primary cell culture studies will elucidate whether other non-DA cell types contribute to the regulation of TH by ER81 in response to synaptic activity and GABA. Together, these studies will augment the design of cell replacement strategies for PD.Project Narrative The studies in this proposal focus on ER81 as a key determinant of the OB DA phenotype. Elucidation of the molecular genetic mechanisms that underlie the inherent plasticity of these DA neurons will be pivotal for the design of future protocols to engineer replacement DA neurons from stem cells. The prospect of incorporating characteristics of OB DA cells, such as the reduced susceptibility to degeneration and an ability to readily integrate into pre-existing neural networks, will augment the design of cell replacement strategies for the treatment of Parkinson's Disease.