Innate Immune Mechanisms of Motor Neuron Injury

描述(申请人提供)：与健康相关：神经退行性疾病的一个中心问题是特定的神经元群体如何经历细胞死亡。运动神经元的选择性退化发生在各种侮辱的背景下，包括RNA病毒，如艾滋病毒和西尼罗河病毒，以及普遍表达的基因的可遗传突变，如SOD1。目前尚不清楚为什么无处不在表达的蛋白质，或者为什么不是特定嗜神经性的病毒，会使运动神经元选择性地容易死亡。运动神经元的选择性脆弱性可能与它们独特的代谢需求有关，或者，它们可能拥有一系列蛋白质，这些蛋白质在上下文中有利于细胞死亡途径。我们已经鉴定了TLR3，一种双链RNA的天然免疫模式识别受体，在多个神经元群体中表达。虽然TLR3在所有被检测的神经元中都有表达，但对皮质神经元或感觉神经元的激活对细胞存活没有影响。相反，TLR3的激活会导致原代分离的脊髓培养中运动神经元的选择性丢失。给新生小鼠注射TLR3激动剂会导致腹角内胰岛-1阳性神经元的丢失。选择性运动神经元死亡的原因可能是观察到TLR3招募了细胞死亡激活或抑制蛋白，我们发现它的激活仅在运动神经元中诱导裂解的caspase-3。长期目标：我们的长期目标是确定TLR3在运动神经元疾病的翻译模型中作为运动神经元死亡的潜在介体的功能。我们的假设是，由于运动神经元独特的脆弱性，TLR3通路的激活会导致运动神经元选择性死亡。研究方法：在目标1中，我们将在现有初步数据的基础上，进一步研究运动神经元对TLR3介导的死亡的选择性易感性，并研究相关的细胞机制。与运动神经元相比，TLR3激活后不同的神经元群体将被评估细胞死亡。然后，我们将确定TLR3是否在运动神经元中自主发挥作用导致死亡，或者是否TLR3在另一种胶质细胞类型中发挥作用，通过神经胶质细胞-神经元共培养导致运动神经元死亡。我们还将通过比较TLR3基因敲除和菌株匹配的对照来确定TLR3是否在体内发挥作用，在新生动物和成年动物中诱导运动神经元的选择性死亡。在目标2中，我们将研究TLR3介导运动神经元选择性死亡的分子基础。我们将使用细胞培养和体内模型来确定近端接头蛋白TRIF是否对TLR3介导的运动神经元死亡是必需的。我们将研究TLR3信号的基本分支点，以确定NF-kB和caspase-8激活的平衡是否有利于运动神经元的细胞死亡和其他类型神经元的细胞存活。由于存在TLR3的病毒和天然细胞配体，这一途径代表了感染、细胞损伤和运动神经元疾病之间的一种新的和独特的联系。公共卫生相关性：选择性运动损伤发生在许多神经退行性疾病中，如原发性外侧硬化症、纯遗传性痉挛截瘫、肯尼迪病、脊髓肌萎缩、进行性肌肉萎缩和肌萎缩侧索硬化症，以及病毒性疾病，如脊髓灰质炎、艾滋病毒和西尼罗河。为什么特定的运动神经元群体选择性地容易受到与这些疾病相关的遗传和环境条件的影响，这一点尚不清楚。我们已经确定了一种称为TLR3的受体，存在于所有神经元上，当被激活时，只会导致运动神经元死亡。这项应用程序进行研究，以确定这一发现与运动神经元疾病的相关性。

DESCRIPTION (provided by applicant): Health Relatedness: A central question in neurodegenerative diseases is how specific populations of neurons undergo cell death. Selective degeneration of motor neurons occurs in the setting of diverse insults including RNA viruses such as HIV and West Nile, and in heritable mutations of ubiquitously expressed genes such as SOD1. It is unclear why proteins that are ubiquitously expressed, or why viruses that are not specifically neurotropic, should render motor neurons selectively vulnerable to death. The selective vulnerability of motor neurons may relate to their unique metabolic demands or, alternatively, they may possess a repertoire of proteins that contextually favor cell-death pathways. We have identified expression of TLR3, an innate immune pattern recognition receptor for duplex RNA, on multiple neuronal populations. While TLR3 is expressed in all neurons examined, activation on cortical neurons or sensory neurons has no effect on cell survival. By contrast, activation of TLR3 results in selective loss of motor neurons in primary dissociated spinal cord cultures. Administration of a TLR3 agonist in neonatal mice results in a loss of islet-1 positive neurons within the ventral horns. A clue into the cause of selective motor neuron death may be the observation that TLR3 recruits either cell-death activating or repressing proteins and we found its activation induces cleaved caspase-3 in motor neurons exclusively. Long-term goals: Our long-term goal is to define the function of TLR3 as a potential mediator of motor neuron death in translational models of motor neuron disease. Our hypothesis is that activation of the TLR3 pathway induces selective death in motor neurons as a consequence of their unique vulnerability. Research Approach: In aim 1, we will further investigate the selective vulnerability of motor neurons to TLR3 mediated death by expanding on our existing preliminary data and studying the cellular mechanisms involved. Different neuronal populations will be assessed for cell death following TLR3 activation in comparison to motor neurons. We will then determine if TLR3 functions autonomously in motor neurons to cause death or if TLR3 functions in an additional glial cell type to cause motor neuron death using glial- neuronal co-cultures. We will also determine if TLR3 functions in vivo, in both neonatal and adult animals, to induce selective death of motor neurons by comparing TLR3 knock-outs with strain matched controls. In aim 2 we will study the molecular basis by which TLR3 mediates selective death of motor neurons. We will determine if the proximal adaptor protein TRIF is necessary for TLR3 mediated death of motor neurons using cell culture and in vivo models. We will examine the essential branch points of TLR3 signaling to determine if the balance of NF-kB and caspase-8 activation favors cell death in motor neurons and cell survival in other neuronal types. Since both viral and native cellular ligands exist for TLR3, this pathway represents a new and unique link between infection, cell injury, and motor neuron disease. PUBLIC HEALTH RELEVANCE: Selective motor injury occurs in a number of neurodegenerative diseases such as Primary Lateral Sclerosis, pure Hereditary Spastic Paraplegia, Kennedy's disease, Spinal Muscular Atrophy, Progressive Muscular Atrophy, and amyotrophic lateral sclerosis, as well as in viral diseases such as Polio, HIV, and West Nile. Why specific populations of motor neurons are selectively vulnerable to the genetic and environmental conditions associated with these diseases is unknown. We have identified a receptor termed TLR3 present on all neurons, that when activated, induces death to only motor neurons. This application pursues research to define the relevance of this finding to diseases of the motor neuron.