Plasticity in the aging olfactory system

老化嗅觉系统的可塑性

基本信息

批准号：
8136361
负责人：
Harriet D. Baker
金额：
$ 12.8万
依托单位：
WINIFRED MASTERSON BURKE MED RES INST
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-14 至 2011-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8136361
关键词：
Address Adult Affect Aging Animals Autoradiography Binding Biological Assay Biological Neural Networks Cell Transplantation Cells Characteristics Clinical Trials Corpus striatum structure Data Development Dopamine EMSA ETV1 gene Embryo Engineering Future Gene Expression Genes Genetic Transcription Goals Human Immunohistochemistry In Situ Hybridization Knock-out Knockout Mice Knowledge Laboratories Life Mass Spectrum Analysis Measures Mediating Methodology Methods Molecular Molecular Genetics Mus Neuronal Differentiation Neurons Neurotransmitter Receptor Nucleic Acid Regulatory Sequences Odors Olfactory Receptor Cells PEA3 Parkinson Disease Pathway interactions Patients Phenotype Phosphotransferases Plasmids Post-Translational Protein Processing Predisposition Primary Cell Cultures Protocols documentation Publishing Receptor Signaling Regulation Research Personnel Reverse Transcriptase Polymerase Chain Reaction Signal Pathway Slice Source Specificity Stem cells Substantia nigra structure Synapses System Testing Transcriptional Regulation Transplantation Tyrosine 3-Monooxygenase base cell type cellular engineering design dopaminergic neuron embryonic stem cell gamma-Aminobutyric Acid gene function insight interest mutant nerve stem cell novel olfactory bulb programs protein expression research study response small hairpin RNA transcription factor treatment strategy

项目摘要

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.

描述（由申请人提供）：该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合移植疗法的功能性 DA 神经元，以替代帕金森病 (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丢失的 SN 细胞。干细胞（成体和胚胎）是替代细胞的有前途的来源，但产生适合移植的神经元的有效操作尚未阐明。为了设计具有适当特征的干细胞，必须了解调节 DA 神经元发育和分化的机制。 OB DA 神经元引起了人们极大的兴趣，因为它们在 PD 患者中不会退化，它们是在动物（包括人类）的整个成年过程中由神经干细胞产生的，并且新分化的 DA 细胞可以整合到预先存在的神经网络中。然而，我们知识中的一个重大差距是直接调节 DA 表型（特别是在 OB 中）的因子的身份。初步数据提供了令人信服的证据，表明 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) 表达来检验以下假设：ER81 是 DA 表型分化的 OB 特异性决定因素。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。初步数据揭示了去极化 DA 细胞中 GABA 对 TH 表达的新增强。具体目标 2 将检验以下假设：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 敲除小鼠中的免疫组织化学和原位杂交将确定 ER81 作为 OB DA 表型的特定决定因素。 OB 切片培养物中的基因功能丢失和拯救实验将证明 ER81 对于 TH 表达是必要且充分的。使用 ChIP 和 EMSA 证明的 TH 转录的直接调节将通过转录测定得到证实。药理学研究将建立影响 ER81 表达和翻译后修饰的特定受体、信号通路和激酶，以响应 OB 切片培养物中的去极化和 GABA。免疫沉淀 ER81 的翻译后修饰也将通过质谱和放射自显影来分析 32P 掺入。来自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 表达。局灶刺激和 OB 原代细胞培养研究将阐明其他非 DA 细胞类型是否有助于 ER81 响应突触活动和 GABA 调节 TH。这些研究将共同增强 PD 细胞替代策略的设计。项目叙事本提案中的研究重点关注 ER81 作为 OB DA 表型的关键决定因素。阐明这些 DA 神经元固有可塑性背后的分子遗传机制对于设计未来从干细胞中改造 DA 神经元的方案至关重要。结合 OB DA 细胞的特性（例如降低变性敏感性和易于整合到预先存在的神经网络中的能力）的前景将增强用于治疗帕金森病的细胞替代策略的设计。