Traumatic axonal injury in the visual system: role of dual leucine zipper kinase

项目总结 创伤性脑损伤(TBI)是一个非常常见的问题，超过200万的新病例需要医疗 在美国每年都受到关注。一种在各种类型的脑外伤中广泛共享的神经病理学 严重程度包括弥漫性或创伤性轴索损伤(TAI)，这是一种超快速变形和破坏的形式 由旋转加速和脑组织剪切引起的轴突。在这个项目中，我们使用了 TAI和探索DLK-JNK信号级联的假说，已知在简单的轴突切断中被激活 像神经挤压这样的损伤，会触发TAI在视神经投射神经元中的下游退变效应 神经，即视网膜神经节细胞(RGC)。我们按逻辑顺序提出三个问题：DLK-JNK通路 是否在可视TAI中激活？如果是这样的话，DLK-JNK的激活是否会触发视网膜节细胞的退变效应 简单的轴突切断术？阻断DLK-JNK通路是否可以预防或治疗RGC变性和 相关的系统损伤，如与中枢神经系统靶点的连接中断和视力丧失？为了实施TBI，我们使用 连续产生初级视神经的弥漫性TAI撞击加速度模型 在我们的实验室里受到伤害。我们的实验对象是Dual的关键成员的条件基因敲除小鼠 亮氨酸拉链激酶(DLK-JNK)级联。我们还使用CRISPR策略来破坏启动的激酶 该药可用于治疗DLK-JNK级联反应、DLK和LZK，还可用DLK抑制剂Sunitinib治疗野生型损伤小鼠。 我们的实验读数包括激酶激活、细胞死亡、轴突和终末变性 常规和高分辨率(清晰度)神经解剖学方法，以及视觉行为测量 敏锐度。探讨DLK-JNK信号通路关键成员在核周和轴突中的作用 在视觉退行性变中，拟议的实验为保护或治疗提供了概念证明 DLK/LZK阻断对TAI的影响该项目利用了我们实验室和 设计和实验方法得到了广泛的初步研究的支持。在具体目标1中， 作为建议的基础，我们将确定TAI在视觉系统中是否与 DLK-JNK信号通路在视网膜节细胞中的激活，我们将确定关键的信号分子。具体而言 目的2，我们将探索是否通过基因敲除/基因组编辑策略来阻断JNK和DLK/LZK信号 或药物抑制可防止或中止视TAI中的RGC外膜和轴突变性。在……里面 具体目标3，我们将确定是否通过基因敲除/基因组编辑来阻止DLK/LZK激活 预防或中止神经系统损伤的策略或药物方法(视觉脱节 和功能障碍)。综上所述，这些实验检验了DLK-JNK通路在神经元中的作用。 模型中枢神经系统中TAI后的退变、断连和功能障碍及新探索 弥漫性脑损伤的保护和治疗策略。

PROJECT SUMMARY Traumatic brain injury (TBI) is a very common problem with over 2 million new cases requiring medical attention annually in the US. A neuropathology that is widely shared among various types of TBI and across spectrum of severity is diffuse or traumatic axonal injury (TAI), a form of ultra-rapid deformation and disruption of axons caused by rotational acceleration and shearing of brain tissue. In this project we use a visual model of TAI and explore the hypothesis that the DLK-JNK signaling cascade, known to be activated in simple axotomy lesions like nerve crush, triggers downstream degenerative effects of TAI in neurons projecting in the optic nerve, i.e. retinal ganglion cells (RGCs). We ask three questions, in logical order: is DLK-JNK pathway activated in visual TAI? If so, does DLK-JNK activation trigger retrograde degenerative effects in RGCs as in simple axotomy models? And does blocking of the DLK-JNK pathway prevent or treat RGC degeneration and related system impairments such as disconnection with CNS targets and loss of vision? To inflict TBI, we use the impact acceleration model of diffuse TAI with which we have consistently produced primary optic nerve injury in our laboratories. Our experimental subjects are conditional knockout mice for key members of the dual leucine zipper kinase (DLK-JNK) cascade. We also employ CRISPR strategies to disrupt the initiating kinases of the DLK-JNK cascade, DLK and LZK, and also treat wild-type injured mice with the DLK inhibitor sunitinib. Our experimental readouts include kinase activation, cell death, axonal and terminal degeneration using conventional and high-resolution (CLARITY) neuroanatomical methods, and behavioral measures of visual acuity. Besides exploring the role of key members of the DLK-JNK signaling pathway in perikaryal and axonal degeneration in visual TAI, the proposed experiments provide proof of concept for the protective or therapeutic effect of DLK/LZK blockade in TAI. The project leverages the complementary strengths of our laboratories and the design and experimental methods are supported by extensive preliminary studies. In Specific Aim 1 that serves as the foundation of the proposal, we will determine whether TAI in the visual system is associated with activation of the DLK-JNK signaling cascade in RGCs and we will identify key signaling molecules. In Specific Aim 2, we will explore whether blocking JNK and DLK/LZK signaling with knockout/genome editing strategies or pharmacological inhibition prevents or aborts RGC perikaryal and axonal degeneration in visual TAI. In Specific Aim 3, we will determine whether blocking DLK/LZK activation with knockout/genome editing strategies or pharmacological approaches prevents or aborts neural system impairments (visual disconnection and dysfunction). Taken together, these experiments examine the role of the DLK-JNK pathway in neuronal degeneration, disconnection, and dysfunction following TAI in a model CNS system and explore novel protective and therapeutic strategies for diffuse TBI.