Mechanisms of synaptic loss by the classical complement pathway in motor circuit development and disease

运动回路发育和疾病中经典补体途径突触损失的机制

• 批准号: 10517958
• 负责人: George Z Mentis
• 金额: $ 3万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10517958
• 关键词: Address; Affect; Age; Antibodies; Award; Behavior; Behavioral Assay; Biological Assay; Birth; CAV2 gene; Cause of Death; Classical Complement Pathway; Complement; Complement 1q; Confocal Microscopy; Development; Disease; Doctor of Philosophy; Excision; Fiber; Gastrocnemius Muscle; Genetic; Glutamates; Grant; Health; Hindlimb; Human; Immunohistochemistry; Intramuscular; Knockout Mice; Label; MERTK gene; Mediating; Molecular; Motor; Motor Neuron Disease; Motor Neurons; Motor output; Movement; Mus; Muscle; Neurodegenerative Disorders; Neurons; Parvalbumins; Pathologic; Phenotype; Physiological; Preparation; Proprioceptor; Proteins; Reporter; Research; Role; Sensory; Spinal; Spinal Cord; Spinal Muscular Atrophy; Synapses; Synaptic Potentials; System; Time; Viral; Virus; doctoral student; experimental study; human disease; infancy; motor function improvement; multidisciplinary; neonatal mice; neuronal circuitry; novel; recombinase; tibialis anterior muscle

Project Summary/Abstract This is an application for a diversity supplement according to FOA: Research Supplements to Promote Diversity in Health-Related Research (PA-21-071) for Ms. Tatiana M. Gonzalez, a PhD student in my lab. Ms. Gonzalez will be working on experiments for her PhD studies on the awarded and currently active R01 grant (AA027079, entitled: “Mechanisms of synaptic loss by the classical complement pathway in motor circuit development and disease”, PI: G.Z. Mentis). The essence of this grant is to unravel the molecular and cellular mechanisms responsible for the disruption of neuronal networks through synaptic elimination which leads to compromised motor output, both during normal development and in the motor neuron disease, spinal muscular atrophy (SMA). These mechanisms underlie the refinement of immature sensory-motor circuits during early normal development and are the main cause for the severe phenotypic deficits in SMA. The Specific Aims of our currently active grant are as follows: In Aim 1, we will determine whether aberrant activation of the classical complement pathway induces loss of vulnerable synapses in SMA. In Aim 2, we will investigate the requirement of the classical complement pathway for normal development of sensory-motor circuits. In Aim 3, we will determine the role of MEGF10 and MERTK in mediating C1q and C3 (proteins of the classical complement) in tagging and eliminating sensory synapses. The studies that will be investigated by Ms. Tatiana Gonzalez, will involve aspects of Aim 2 and Aim 3. Specifically, she will investigate to which extent supernumerary and inappropriate proprioceptive sensory synapses are formed through C1q and C3 involvement. To address this, Ms. Gonzalez will utilize a novel retrograde viral-mediated neuronal circuit tracing assay to selectively label and quantify hindlimb muscle-specific sensory proprioceptive synapses on muscle-identified spinal motor neurons in mice at different developmental ages. This assay relies on the expression of parvalbumin (PV) by proprioceptors; to prime fluorescent reporter expression in the proprioceptors, a parvalbumin-directed FlpO recombinase (PV::FlpO) mouse line is crossed with Cre- and Flp-dependent tdTomato reporter mice (Ai65). PV::FlpO;Ai65 neonatal mice will be injected intramuscularly at birth with CAV2-CRE virus into the tibialis anterior (TA) to activate tdTomato expression in proprioceptive sensory neurons innervating the TA. To label motor neurons innervating the antagonistic Gastrocnemius (Gs) muscle, these mice will concomitantly be injected with scAAV6-GFP virus to express GFP in Gs motor neurons. In this manner, proprioceptive TA fibers will be labelled with TdTomato (in red) and Gs motor neurons with GFP (in green). Using immunohistochemistry and confocal microscopy, excitatory glutamatergic proprioceptive sensory synapses will be labeled using an anti-VGluT1 antibody and the presence of inappropriate synapses will be detected through the co-localization of VGluT1 with tdTomato on GFP+ Gs motor neurons. With respect to Aim 3, Ms Gonzalez will investigate whether MEGF10 and MERTK are involved in the excessive pruning of spinal synapses in SMA. To do so, she will use the appropriate knock out (KO) mouse crosses as follows: she will generate MEGF10KO::SMA and MERTKKO::SMA mice and perform the righting time behavioral assay to assess whether the SMA mice lacking MEGF10 or MERTK have any improved motor function in comparison to regular SMA mice. She will perform physiological experiments on MEGF10KO::SMA and MERTKKO::SMA mice using the ex vivo spinal cord preparation, to establish the amplitude of motor neuron synaptic potentials and compared them to those from regular SMA mice and determine whether abolition of MEGF10 or MERTK confers any functional benefit in SMA mice. Lastly, she will assess whether abolition of MEGF10 and MERTK disrupts C1q and C3 tagging by utilizing immunohistochemistry and confocal microscopy.

项目摘要/摘要 这是根据FOA：研究补充剂来促进多样性补充的申请 我实验室中的博士生Tatiana M. Gonzalez女士的健康相关研究多样性（PA-​​21-071）。多发性硬化症。 冈萨雷斯将研究她对奖励且目前活跃的R01 Grant的博士学位研究的实验 （AA027079，标题为：“马达电路中经典完成途径的合成损失机制 发育与疾病”，PI：G.Z。Mentis）。 这项赠款的本质是阐明负责的分子和细胞机制 神经元网络通过突触紧急紧急情况破坏，导致电动机输出受损 在正常发育和运动神经元疾病中，脊柱肌肉萎缩（SMA）。这些 在正常发育和 是SMA严重表型防御的主要原因。我们目前积极赠款的具体目标是 如下：在AIM 1中，我们将确定经典补体途径的异常激活是否会影响 SMA中脆弱突触的丧失。在AIM 2中，我们将调查经典完成的要求 正常发展感觉运动电路的途径。在AIM 3中，我们将确定MEGF10和 MERTK在介导C1Q和C3（经典补体的蛋白质）中进行标记和消除感觉 突触。 Tatiana Gonzalez女士将研究的研究将涉及AIM 2和AIM 3的各个方面。 具体而言，她将在多大程度上调查超级和不适当的感觉 突触是通过C1Q和C3参与形成的。为了解决这个问题，冈萨雷斯女士将利用一本小说 逆行病毒介导的神经元电路追踪测定法，有选择地标记并量化后肢肌肉特异性 在不同发育的小鼠肌肉识别的脊柱运动神经元上的感觉本体感受突触 年龄。该评估依赖于本体感受器的白蛋白（PV）的表达。到达荧光记者 在本体感受器中的表达，横断小蛋白酶指导的FLPO重组酶（PV :: FLPO）小鼠系 与CRE和FLP依赖性TDTOMATO报告者小鼠（AI65）。 PV :: FLPO; AI65新生儿小鼠将被注入 用Cav2-cre病毒出生时肌肉内到胫骨前（TA）激活TDTOMATO表达 支配TA的本体感受的感觉神经元。标记运动神经元的拮抗作用 甲状腺素（GS）肌肉，这些小鼠将同时注射SCAAV6-GFP病毒以表达GFP 在GS运动神经元中。以这种方式，将用TDTOMATO（红色）和GS标记本体感受性TA纤维 带有GFP的运动神经元（绿色）。使用免疫组织化学和共聚焦显微镜，兴奋性 谷氨酸能的本体感知性感觉突触将使用抗VGLUT1抗体标记 通过在GFP+ GS上与TDTOMATO的VGLUT1共定位，将检测到不适当的突触 运动神经元。关于AIM 3，Gonzalez女士将调查MEGF10和MERTK是否涉及 在SMA中脊柱突触的过度修剪中。为此，她将使用适当的敲除（KO）鼠标 十字架如下：她将生成Megf10ko :: SMA和Mertkko :: SMA老鼠并执行右时间 行为评估以评估缺乏MEGF10或MERTK的SMA小鼠是否有任何改进的电动机 与常规SMA小鼠相比，功能。她将在megf10ko :: SMA上进行物理实验 和Mertkko :: SMA小鼠使用离体脊髓制备，以建立运动神经元的放大器 突触电位，并将其与常规SMA小鼠的那些进行比较，并确定是否取消 MEGF10或MERTK在SMA小鼠中承担任何功能益处。最后，她将评估废除 MEGF10和MERTK通过使用免疫组织化学和共聚焦显微镜破坏C1Q和C3标记。