Laminin control of synaptic function and dendritic stability

描述（由申请人提供）：在发育过程中，树突和树突棘动态形成和翻转。然而，在成年人的大脑中，大多数树突分支和许多树突棘是稳定的。树突乔木和树突棘稳定性的缺陷是许多精神和神经疾病的基础，包括迟发性疾病，如精神分裂症、重度抑郁症和阿尔茨海默病。我提供的证据表明，在这一建议的细胞外基质蛋白层粘连蛋白的损失？特别是来自兴奋性神经元的5破坏脊柱稳定性，导致青春期树突退化，并损害正常的突触传递和动物行为。在我的研究计划中，我建议确定哪一个突触伴侣产生必要的层粘连蛋白？5、确定它何时起稳定树突结构和突触传递的作用，并测试活性变化是否导致层粘连蛋白中发现的棘稳定性破坏？5个敲除神经元。目标1。为了阐明在哪里？5-含有层粘连蛋白的蛋白质被生产出来，并且在必要时。我的初步数据显示层粘连蛋白的丢失？5，特别是从兴奋性前脑神经元引起树突损失和突触功能障碍的CA1神经元开始后P21。我还发现成人兴奋性神经元特异性层粘连蛋白？5只KO小鼠缺乏层粘连蛋白？5蛋白质特异性地靠近突触。哪一个突触伴侣提供这种层粘连蛋白是一个基本的和未解决的问题。了解其来源对于了解其表达、加工和分泌是如何控制的，以及最终是什么因素控制树突稳定性至关重要。为了解决这一问题，我将选择性地在突触前（CA3）或突触后（CA1）细胞中插入lama5基因，然后测量突触后神经元中的树突乔木、树突棘密度和突触电流。另一个关键的问题是层粘连蛋白何时出现？5功能来控制这些表型。为了确定这一点，我将使用诱导遗传失活层粘连蛋白？5在青春期之前，期间和之后的时间点，然后测量树突乔木，树突棘和突触电流。目的2：确定是否突触传递缺陷驱动树突棘层粘连蛋白不稳定？5个敲除神经元。我的初步研究表明兴奋性层粘连蛋白的急性海马切片？5敲除小鼠在P21后开始在CA3：CA1突触处表现出增加的电流。我还发现了培养的层粘连蛋白5 KO神经元相对于WT神经元表现出降低的棘密度、增加的棘头宽度和增加的棘大小波动。这些表型都可以挽救与应用外源性？5-含有层粘连蛋白。一个基本的问题，从这些研究中产生的是是否增加电流层粘连蛋白？5 KO突触驱动树突棘稳定性的丧失。为了检验这种可能性，我将用钙显像来检验是否用外源性抢救？5-含有层粘连蛋白的蛋白质在拯救脊波动之前减弱钙瞬变，以及恢复WT活性水平是否恢复层粘连蛋白中的正常脊波动、密度和形态？5个KO神经元。

DESCRIPTION (provided by applicant): During development, dendrites and dendritic spines form and turn over dynamically. In adult brains, however, most dendrite branches and many dendritic spines are stable. Defects in dendrite arbor and dendritic spine stability underlie numerous psychiatric and neurological diseases, including late-onset disorders such as schizophrenia, Major Depressive Disorder, and Alzheimer's disease. I provide evidence in this proposal that loss of the extracellular matrix protein laminin ?5 specifically from excitatory neurons disrupts spine stability, causes dendrite regression during adolescence, and compromises normal synaptic transmission and animal behavior. In my research plan, I propose to identify which synaptic partner produces the necessary laminin ?5, determine when it functions to stabilize dendritic structure and synaptic transmission, and test whether activity changes cause spine stability disruption found in laminin ?5 knockout neurons. Aim 1. To elucidate where the ?5-containing laminin is produced and when it is necessary. My preliminary data show that loss of laminin ?5 specifically from excitatory forebrain neurons causes dendrite loss and synaptic dysfunction in CA1 neurons starting after P21. I also show that adult excitatory neuron- specific laminin ?5 KO mice lack laminin ?5 protein specifically near synapses. Which synaptic partner provides this laminin is a fundamental and unresolved question. Knowledge of its source is critical to understanding how its expression, processing, and secretion are controlled, and ultimately what factors govern dendritic stability. To address this, I will selectively inactivate the lama5 gene in presynaptic (CA3) or postsynaptic (CA1) cells and then measure dendritic arbors, dendritic spine density, and synaptic currents in the postsynaptic neuron. Another critical question is when laminin ?5 functions to control these phenotypes. To determine this, I will use inducible genetic inactivation of laminin ?5 at time points before, during, and after adolescence and then measure dendrite arbors, dendritic spines, and synaptic currents. Aim 2: To determine whether synaptic transmission defects drive dendritic spine destabilization in laminin ?5 knockout neurons. My preliminary studies indicate that acute hippocampal slices from excitatory- specific laminin ?5 knockout mice exhibit increased currents at CA3:CA1 synapses beginning after P21. I also find cultured laminin ?5 KO neurons exhibit decreased spine density, increased spine head width, and increased spine size fluctuations relative to WT neurons. These phenotypes can all be rescued with application of exogenous ?5-containing laminin. A fundamental question that arises from these studies is whether the increased currents at laminin ?5 KO synapses drive the loss of dendritic spine stability. To test this possibility, I will use calcium imaging to test whether rescue with exogenous ?5-containing laminin attenuates calcium transients before rescuing spine fluctuation and also whether restoring WT activity levels restores normal spine fluctuation, density, and morphology in laminin ?5 KO neurons.