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: 8835605
• 负责人: Mitchell Hamed Omar
• 金额: $ 4.27万
• 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/8835605
• 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; Hippocampus (Brain); Image; Knock-out; Knockout Mice; Knowledge; Laminin; Late-Onset Disorder; Lead; Major Depressive Disorder; Measures; Mediating; Mental disorders; Modeling; Morphology; Mus; Neurodegenerative Disorders; Neurons; Phenotype; Process; Property; Prosencephalon; Proteins; Relative (related person); Research; Schizophrenia; Shapes; Slice; Source; Structure; Synapses; Synaptic Transmission; Testing; Time; Tissues; Vertebral column; Width; Work; cell type; cognitive function; combat; density; excitatory neuron; laminin-5; late disease onset; nervous system disorder; novel therapeutics; postsynaptic; presynaptic; public health relevance; 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 ?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.

描述(申请人提供)：在发育过程中，树枝和树枝状刺形成并动态翻转。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。树突枝和树突棘的稳定性缺陷是许多精神和神经疾病的基础，包括精神分裂症、严重抑郁障碍和阿尔茨海默病等晚发性疾病。我在这项建议中提供了证据，证明兴奋性神经元特异性地丢失细胞外基质蛋白Laminin？5会破坏脊柱的稳定性，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，我建议确定哪个突触伙伴产生必要的层粘连蛋白？5，确定它何时发挥稳定树突结构和突触传递的功能，并测试在层粘连蛋白？5基因敲除神经元中发现的活动变化是否会导致脊柱稳定性的破坏。目的1.阐明含层粘连蛋白的来源和需要的时间。我的初步数据显示，兴奋性前脑神经元特异性的层粘连蛋白？5的丢失导致了从P21开始的CA1神经元的树突丢失和突触功能障碍。我还发现，成年兴奋性神经元特异性的层粘连蛋白？5KO小鼠在突触附近缺乏专门的层粘连蛋白？5蛋白。哪个突触伙伴提供这种层粘连蛋白是一个基本且尚未解决的问题。了解它的来源对于了解它的表达、加工和分泌是如何控制的，以及最终是什么因素控制树突状细胞的稳定性至关重要。为了解决这个问题，我将选择性地灭活突触前(CA3)或突触后(CA1)细胞中的lama5基因，然后测量突触后神经元中的树突、树突棘密度和突触电流。另一个关键问题是层粘连蛋白？5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？5的可诱导遗传失活，然后测量树突分枝、树突棘和突触电流。目的2：确定突触传递缺陷是否导致层粘连蛋白？5基因敲除神经元中树突棘的不稳定。我的初步研究表明，兴奋性特异性层粘连蛋白？5基因敲除小鼠的急性海马片在P21后开始在CA3：CA1突触处显示电流增加。我还发现，与WT神经元相比，培养的LN？5 KO神经元表现出脊柱密度降低，脊柱头部宽度增加，脊柱大小波动增加。这些表型都可以通过应用外源性含层粘连蛋白来挽救。这些研究提出的一个基本问题是，层粘连蛋白？5KO突触电流的增加是否导致树突棘稳定性的丧失。为了测试这种可能性，我将使用钙成像来测试在挽救脊柱波动之前，外源性含层粘连蛋白的复苏是否减弱了钙瞬变，以及恢复WT活动水平是否恢复了正常的脊柱波动、密度和层粘连蛋白5KO神经元的形态。