Laminin control of synaptic function and dendritic stability

层粘连蛋白控制突触功能和树突稳定性

• 批准号: 8997015
• 负责人: Mitchell Hamed Omar
• 金额: $ 4.36万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-16 至 2017-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8997015
• 关键词: Acute; Address; Adolescence; Adult; Alzheimer' s Disease; Animal Behavior; Attenuated; Brain; Calcium; Cells; Data; Defect; Dendrites; Dendritic Spines; Development; Disease; Embryo; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Functional disorder; Genes; Genetic; Head; Health; Hippocampus (Brain); Image; Knock-out; Knockout Mice; Knowledge; Laminin; Late-Onset Disorder; Lead; Major Depressive Disorder; Measures; Mediating; Mental disorders; Morphology; Neurodegenerative Disorders; Neurons; Phenotype; Process; Property; Prosencephalon; Proteins; Research; Schizophrenia; Shapes; Slice; Source; Structural defect; Structure; Synapses; Synaptic Transmission; Testing; Time; Tissues; Vertebral column; Width; Work; cell type; cognitive function; combat; density; excitatory neuron; laminin alpha5; nervous system disorder; novel therapeutic intervention; postsynaptic; postsynaptic neurons; presynaptic; repaired; secretion process; synaptic function

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 α5 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 α5, determine when it functions to stabilize dendritic structure and synaptic transmission, and test whether activity changes cause spine stability disruption found in laminin α5 knockout neurons. Aim 1. To elucidate where the α5-containing laminin is produced and when it is necessary. My preliminary data show that loss of laminin α5 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 α5 KO mice lack laminin α5 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 α5 functions to control these phenotypes. To determine this, I will use inducible genetic inactivation of laminin α5 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 α5 knockout neurons. My preliminary studies indicate that acute hippocampal slices from excitatory- specific laminin α5 knockout mice exhibit increased currents at CA3:CA1 synapses beginning after P21. I also find cultured laminin α5 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 α5-containing laminin. A fundamental question that arises from these studies is whether the increased currents at laminin α5 KO synapses drive the loss of dendritic spine stability. To test this possibility, I will use calcium imaging to test whether rescue with exogenous v5-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 α5 KO neurons.

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。我在这个提议中提供的证据表明，细胞外基质蛋白层粘连蛋白α5的损失，特别是从兴奋性神经元破坏脊柱的稳定性，导致树突退化在青春期，并损害正常的突触传递和动物行为。在我的研究计划中，我建议确定哪种突触伴侣产生必要的层粘连蛋白α5，确定它何时起稳定树突结构和突触传递的作用，并测试活动变化是否会导致层粘连蛋白α5敲除神经元中发现的棘稳定性破坏。目标1.阐明含α5的层粘连蛋白在何处产生以及何时产生是必要的。我的初步数据表明，层粘连蛋白α5的损失，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。我还发现成年兴奋性神经元特异性层粘连蛋白α5基因敲除小鼠缺乏突触附近特异性层粘连蛋白α5蛋白。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。另一个关键问题是层粘连蛋白α5何时起作用来控制这些表型。为了确定这一点，我将在青春期之前、期间和之后的时间点使用层粘连蛋白α5的可诱导遗传失活，然后测量树突乔木、树突棘和突触电流。目的2：研究层粘连蛋白α5基因敲除神经元中突触传递缺陷是否导致树突棘不稳定。我的初步研究表明，兴奋性特异性层粘连蛋白α5基因敲除小鼠的急性海马切片在P21后开始在CA3：CA1突触处显示出增加的电流。我还发现培养的层粘连蛋白α5 KO神经元表现出相对于WT神经元的棘密度降低，棘头宽度增加和棘大小波动增加。这些表型都可以通过应用外源性含α5层粘连蛋白来挽救。从这些研究中产生的一个基本问题是，层粘连蛋白α5 KO突触处增加的电流是否导致树突棘稳定性的丧失。为了验证这种可能性，我将使用钙成像来测试在拯救脊髓波动之前，用外源性含v5的层粘连蛋白进行拯救是否会减弱钙瞬变，以及恢复WT活性水平是否会恢复层粘连蛋白α5 KO神经元的正常脊髓波动、密度和形态。