Plasticity in the aging olfactory system

描述(申请人提供)：本提案的重点是阐明嗅球(OB)中多巴胺(DA)神经元分化的分子机制。该实验室的长期目标是合理开发适用于移植治疗的功能性DA神经元，以取代帕金森病(PD)患者失去的黑质(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来功能替代丢失的SN细胞。干细胞(包括成体和胚胎)是一种很有前途的替代细胞来源，但产生适合移植的神经元的有效操作尚未阐明。为了设计出具有合适特性的干细胞，必须了解调控DA神经元发育和分化的机制。OB DA神经元在帕金森病患者中不会退化，它们是从动物(包括人类)的成年生活中产生的，新分化的DA细胞可以整合到预先存在的神经网络中，因此引起了人们的极大兴趣。然而，我们知识中的一个显著差距是直接调节DA表型的因素的一致性，特别是在OB中。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。该提案的具体目标1将通过直接调节酪氨酸羟化酶(TH)的表达来检验Er81是DA表型分化的OB特异性决定因素的假设。通过TH表达来衡量OB DA神经元的终末分化，需要来自嗅觉受体细胞的传入突触活动。初步数据显示，在去极化的DA细胞中，GABA对TH表达有新的促进作用。特定目的2将检验这样一种假设，即传入突触活动和/或GABA对TH表达的调节是通过ER81的表达或翻译后修饰来介导的。Er81基因敲除小鼠的免疫组织化学和原位杂交将确定Er81是OB DA表型的特异性决定因素。在OB切片培养中的基因功能丧失和修复实验将证明Er81是TH表达所必需的和充分的。使用CHIP和EMSA证实的对TH转录的直接调控，将通过转录检测得到证实。药理学研究将建立特定的受体、信号通路和激酶，以影响OB切片培养中Er81的表达和翻译后修饰，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分析和32P掺入的放射自显影进行分析。来自气味剥夺小鼠的切片培养研究和芯片实验将确定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对突触活性和GABA反应的TH调节。总之，这些研究将加强PD细胞替换策略的设计。项目叙述 这项建议中的研究集中在Er81作为OB DA表型的关键决定因素。阐明这些DA神经元固有可塑性的分子遗传学机制将对未来设计从干细胞替代DA神经元的方案至关重要。融合OB DA细胞特性的前景，如降低变性的敏感性和容易整合到现有神经网络中的能力，将增强细胞替代策略的设计，用于治疗帕金森氏病。

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.