Laminin control of CNS dendrite and dendritic spine development

层粘连蛋白控制中枢神经系统树突和树突棘发育

• 批准号: 8796341
• 负责人: Jaime Grutzendler
• 金额: $ 35.03万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8796341
• 关键词: Ablation; Address; Adhesions; Adult; Affect; Animal Behavior; Autistic Disorder; Binding; Biochemical Genetics; Biological Assay; Brain; Brain Diseases; Cell Adhesion; Cells; Data; Defect; Dendrites; Dendritic Spines; Development; Diagnosis; Electron Microscopy; Environment; Exhibits; Extracellular Matrix Proteins; Fostering; Gel; Genetic; Glutamates; Hippocampus (Brain); Human; Image; Immunoblotting; Immunoglobulin Domain; Individual; Integrin alpha3beta1; Integrins; Knock-out; Laminin; Laminin Receptor; Lead; Mass Spectrum Analysis; Measures; Mediating; Mental Retardation; Mental disorders; Microscopy; Molecular; Morphology; Mus; Mutation; Neurons; Physiological; Play; Process; Property; Protein Binding; Proteins; Proteomics; Role; SHPS-1 protein; Sensory; Somatosensory Cortex; Stroke; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; Testing; Time; Transgenes; Vertebral column; Vibrissae; Whole-Cell Recordings; base; brain tissue; cell type; density; excitatory neuron; extracellular; genetic analysis; in vitro Assay; in vivo; in vivo imaging; knock-down; laminin alpha5; laminin-5; mutant; novel; novel diagnostics; protein expression; public health relevance; receptor; sensory cortex; synaptic function; time use; tissue culture; transmission process; treatment strategy; two-photon

DESCRIPTION (provided by applicant): The development, plasticity, and stability of dendrites and dendritic spines are defective in autism, mental retardation, stroke, and psychiatric diseases. Mutations or reduced levels of heterotrimeric laminin extracellular matrix proteins are associated with these human brain disorders. We provide evidence that neuron-specific ablation of the laminin alpha5 subunit in mice increases spine densities, destabilizes dendrite branches, and compromises normal synaptic transmission and animal behavior. We propose to elucidate the mechanisms by which laminin alpha5 and a new putative laminin alpha5 receptor we have discovered regulate dendrite and dendritic spine development and function. We will use complementary in vivo imaging, electrophysiological, biochemical, and genetic approaches to achieve the following aims: Aim 1. Determine how laminin alpha5 regulates development, plasticity, and function of dendrites, dendritic spines, and synapses. Our data strongly suggest that laminin alpha5 controls dendrite branch and dendritic spine dynamics. We will use transcranial two-photon microscopy of dendrites in the somatosensory cortex, alone and in combination with sensory input manipulation, to reveal how the loss of laminin alpha5 impacts branch and spine dynamics during development and activity-driven plasticity. We will also use electron microscopy and whole cell recording to test the hypothesis that laminin alpha5 regulates synaptic transmission by controlling the structure, transmission properties, and plasticity of individual synapses. Aim 2. Elucidate the composition, origin, and timing of function of alpha5-containing laminins in dendrite and spine development. We do not know which laminin beta and gamma chains partner with laminin alpha5, where they are produced, or when they act. We will use biochemical and genetic knockout approaches to identify laminin beta and gamma chains that associate with laminin alpha5 in neurons to regulate dendrite and spine development. We will also inactivate laminin alpha5 in specific cell types using inducible Cre transgenes to determine where and when laminin alpha5 is required to regulate dendrite and dendritic spine development. Aim 3. Characterize SIRPalpha function in laminin alpha5-mediated dendrite and dendritic spine development. We have shown that the integrin alpha3beta1 receptor for laminin alpha5 mediates dendrite branch stability, but our genetic analysis indicates that other receptors are essential to mediate the effects of laminin alpha5 on dendritic spine development. Our data strongly suggest that the Signal Regulatory Protein alpha (SIRPalpha) transmembrane receptor serves as a novel laminin alpha5 receptor in the control of spine development. We will use cell adhesion assays and in vitro binding assays with purified proteins to identify which domains in SIRPalpha and alpha5-laminins mediate these interactions. We will test how excitatory neuron-specific ablation of SIRPalpha function alone or in combination with integrin alpha3beta1 affects dendrite and spine development and synaptic function and plasticity.

描述(申请人提供)：树突和树突的发育、可塑性和稳定性在自闭症、智力低下、中风和精神疾病中是有缺陷的。异源三聚体层粘连蛋白细胞外基质蛋白的突变或水平降低与这些人类大脑疾病有关。我们提供的证据表明，神经元特异性消融小鼠层粘连蛋白α5亚单位增加了脊椎密度，破坏了树突分支的稳定性，并损害了正常的突触传递和动物行为。我们建议阐明层粘连蛋白α5和我们发现的一种新的层粘连蛋白α5受体调控树突状细胞和树突棘发育和功能的机制。我们将使用互补的体内成像、电生理、生化和遗传学方法来实现以下目标：目的1.确定层粘连蛋白α5如何调节树突、树突棘和突触的发育、可塑性和功能。我们的数据有力地表明，层粘连蛋白α5控制树突分支和树突棘的动力学。我们将单独使用经颅双光子显微镜对躯体感觉皮质中的树突进行观察，并结合感觉输入操作，揭示层粘连蛋白α5的丢失如何在发育和活动驱动的可塑性过程中影响分支和脊柱的动力学。我们还将使用电子显微镜和全细胞记录来检验层粘连蛋白α5通过控制单个突触的结构、传递特性和可塑性来调节突触传递的假设。目标2.阐明功能的组成、起源和时机 在树突和棘突发育中含有α5的层粘连蛋白。我们不知道哪些层粘连蛋白β和伽马链与层粘连蛋白α5配对，它们在哪里产生，或者它们什么时候起作用。我们将使用生化和基因敲除方法来确定层粘连蛋白β和伽马链，这些链与神经元中的层粘连蛋白α5相关，以调节树突和棘突的发育。我们还将使用可诱导的Cre转基因使特定细胞类型的层粘连蛋白α5失活，以确定何时何地需要层粘连蛋白α5来调节树突和树突棘的发育。目的3.研究SIRPalpha在层粘连蛋白α5介导的树突和树突棘发育中的功能。我们已经证明，层粘连蛋白α5的整合素α3beta1受体介导了树突状分支的稳定性，但我们的遗传分析表明，其他受体在介导层粘连蛋白α5对树突棘发育的影响中是必不可少的。我们的数据有力地表明，信号调节蛋白α(SIRPalpha)跨膜受体是一种新的层粘连蛋白α5受体，在脊柱发育中起着控制作用。我们将使用细胞黏附试验和体外与纯化蛋白的结合试验来确定SIRPalpha和Alpha5-laminins中哪些结构域介导了这些相互作用。我们将测试兴奋性神经元特异性消融SIRPalpha功能或与整合素α3beta1联合应用如何影响树突和棘突的发育以及突触功能和可塑性。