Plasticity in the aging olfactory system

老化嗅觉系统的可塑性

• 批准号: 7857515
• 负责人: Harriet D. Baker
• 金额: $ 25.3万
• 依托单位: WINIFRED MASTERSON BURKE MED RES INST
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7857515
• 关键词: Address; Adult; Affect; Aging; Animals; Autoradiography; Binding; Biological Assay; Biological Neural Networks; Cations; Cells; Characteristics; Clinical Trials; Corpus striatum structure; Data; Development; Disease; 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; Reverse Transcriptase Polymerase Chain Reaction; Signal Pathway; Slice; Source; Specificity; Stem cells; Substantia nigra structure; Synapses; System; Testing; Transcriptional Regulation; Transplantation; Tyrosine 3-Monooxygenase; Wild Type Mouse; cell type; cellular engineering; design; dopaminergic neuron; embryonic stem cell; gamma-Aminobutyric Acid; gene function; interest; mutant; nerve stem cell; novel; olfactory bulb; protein expression; research study; response; small hairpin RNA; transcription factor; treatment strategy

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）：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）患者中丢失的黑质（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（成人和胚胎）是替代细胞的一个有前途的来源，但有效的操作，以产生适合移植的神经元尚未阐明。为了使干细胞具有合适的特性，必须了解调节DA神经元发育和分化的机制。OB DA神经元是相当令人感兴趣的，因为它们在PD患者中不会退化，它们在动物（包括人类）的成年生活中由神经干细胞产生，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，在我们的知识的一个显着的差距是直接调节DA表型，特别是在OB的因素的身份。初步数据提供了令人信服的证据，ETS转录因子，ER 81，是一个关键的决定因素的OB DA表型。具体目标1的这一建议将测试的假设，ER 81是一个OB特异性决定因素DA表型分化，直接调节酪氨酸羟化酶（TH）的表达。通过TH表达测量的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。初步数据揭示了一种新的增强TH的表达GABA在去极化DA细胞。具体目标2将检验以下假设：通过传入突触活动和/或GABA调节TH表达是由ER 81的表达或翻译后修饰介导的。ER 81基因敲除小鼠的免疫组织化学和原位杂交将确立ER 81作为OB DA表型的特异性决定因素。OB切片培养物中基因功能的丧失和拯救实验将证明ER 81对于TH表达是必要的和足够的。使用ChIP和EMSA证明的TH转录的直接调节将通过转录测定来证实。药理学研究将建立特定的受体，信号通路和激酶，影响ER 81的表达和翻译后修饰，以响应OB切片培养物中的去极化和GABA。还将通过质谱法和放射自显影法分析免疫沉淀ER 81的翻译后修饰，以确定32 P掺入。来自气味剥夺小鼠的切片培养研究和ChIP实验将确定ER 81的表达或翻译后修饰是否调节TH表达。局灶性刺激和OB原代细胞培养研究将阐明是否其他非DA细胞类型有助于调节TH的ER 81在响应突触活动和GABA。总之，这些研究将增强PD细胞替代策略的设计。项目叙述 本提案中的研究重点是ER 81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机制将是未来设计干细胞替代DA神经元的方案的关键。结合OB DA细胞的特性，例如对变性的敏感性降低和容易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。