Laminin control of CNS dendrite and dendritic spine development

描述（申请人提供）：在自闭症、智力低下、中风和精神疾病中，树突和树突棘的发育、可塑性和稳定性存在缺陷。异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，小鼠中层粘连蛋白α 5亚基的神经元特异性消融会增加脊柱密度，破坏树突分支的稳定，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推定层粘连蛋白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、生化和遗传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。我们的数据强烈表明层粘连蛋白α 5控制着树突分支和树突棘的动力学。我们将使用经颅双光子显微镜观察体感觉皮层的树突，单独或结合感觉输入操作，以揭示层粘连蛋白α 5的缺失如何影响发育和活动驱动可塑性过程中的分支和脊柱动力学。我们还将使用电子显微镜和全细胞记录来验证层粘连蛋白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2。阐明功能的组成、起源和时间

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.