Laminin control of CNS dendrite and dendritic spine development

描述（由申请人提供）：树突和树突棘的发育、可塑性和稳定性在自闭症、精神发育迟滞、中风和精神疾病中存在缺陷。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脑疾病相关。我们提供的证据表明，神经元特异性消融层粘连蛋白α 5亚基在小鼠中增加棘密度，不稳定树突分支，并损害正常的突触传递和动物行为。我们建议阐明层粘连蛋白α 5和我们发现的一种新的假定层粘连蛋白α 5受体调节树突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，生物化学和遗传学的方法来实现以下目标：目的1。确定层粘连蛋白α 5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α 5控制树突分支和树突棘动力学。我们将使用经颅双光子显微镜的树突在体感皮层，单独和结合感觉输入操作，揭示层粘连蛋白α 5的损失如何影响分支和脊柱动态发展和活动驱动的可塑性。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α 5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。目标二。阐明功能的组成、起源和时机 在树突和棘发育中含有α 5的层粘连蛋白。我们不知道哪些层粘连蛋白β和γ链与层粘连蛋白α 5合作，它们在哪里产生，或者它们何时起作用。我们将使用生物化学和基因敲除的方法来确定层粘连蛋白β和γ链，与层粘连蛋白α 5在神经元中调节树突和棘发育。我们还将使用诱导型Cre转基因在特定细胞类型中表达层粘连蛋白α 5，以确定何时何地需要层粘连蛋白α 5来调节树突和树突棘的发育。目标3。表征SIRP α在层粘连蛋白α 5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α 5的整合素α 3 β 1受体介导了树突分支稳定性，但我们的遗传分析表明其他受体对于介导层粘连蛋白α 5对树突棘发育的影响至关重要。我们的数据有力地表明，信号调节蛋白α（SIRP α）跨膜受体作为一种新的层粘连蛋白α 5受体在脊柱发育的控制。我们将使用细胞粘附试验和纯化蛋白的体外结合试验来鉴定SIRP α和α 5-层粘连蛋白中哪些结构域介导这些相互作用。我们将测试SIRP α功能的兴奋性神经元特异性消融单独或与整合素α 3 β 1组合如何影响树突和棘发育以及突触功能和可塑性。

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.