Plasticity in the aging olfactory system

老化嗅觉系统的可塑性

• 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
• 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

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）：该建议的重点是阐明嗅球中多巴胺（DA）神经元分化的分子机制（OB）。实验室的长期目标是功能性DA神经元的合理发展，适合于替代帕金森氏病（PD）患者中失去的蛋白质Nigra（SN）神经元的植物疗法。新兴的治疗策略是通过将神经元移植到患者纹状体中的功能替代丢失的SN细胞。干细胞（成年和胚胎）是替代细胞的有前途的来源，但是尚未阐明适合移植的神经元的有效操纵。对于具有适当特征的工程师干细胞，必须了解调节DA神经元发展和分化的机制。 OB DA神经元引起了极大的兴趣，因为它们在PD患者中不会退化，它们是由神经干细胞通过动物（包括人类）的成年生物产生的，而新分化的DA细胞可以集成到预先存在的神经网络中。但是，我们知识上的一个显着差距是因素的身份，这些因素是直接调节DA表型的因素。初步数据提供了令人信服的证据，即ETS转录因子ER81是OB DA表型的关键决定因素。该提案的具体目的1将检验以下假设：ER81通过直接调节酪氨酸羟化酶（Th）表达是DA表型分化的一种决定因素。通过TH表达测量，OB DA神经元的末端分化需要嗅觉受体细胞传入的突触活性。初步数据揭示了GABA在去极化DA细胞中对Th表达的新颖增强。具体目标2将检验以下假设：传入突触活动和/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建立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实验的丧失和拯救将证明ER81是必要的，足以表达TH。使用芯片和EMSA证明的TH转录的直接调节将通过转录测定确认。药理学研究将建立特定的受体，信号通路和激酶，从而影响ER81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。通过质谱法和32p掺入的自显影术分析了具有免疫沉淀的ER81的翻译后修饰。切片培养研究和来自气味的小鼠的芯片实验将决定ER81的表达或翻译后修饰是否调节表达。局灶性刺激和OB原发性细胞培养研究将阐明其他非DA细胞类型是否有助于ER81对突触活动和GABA的调节。这些研究将共同​​增强PD的细胞更换策略的设计。 该提案中的研究集中在ER81上是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设计未来方案的关键，以设计从干细胞中替换DA神经元的未来方案。纳入OB DA细胞特征的前景，例如对退化的敏感性降低以及容易融入预先存在的神经网络的能力，将增强细胞替代策略的设计，以治疗帕金森氏病的治疗。