Determining Mechanisms of DLK Regulation in Axon Injury Responses

 描述(申请人提供)：神经细胞死亡和轴突损伤是许多神经疾病和神经退行性疾病的特征，包括阿尔茨海默氏症和帕金森氏病，脊髓损伤，青光眼，多发性硬化症，创伤性和缺血性脑损伤，以及100多种不同的外周神经病。双亮氨酸拉链激酶(DLK，MAP3K12)是轴突损伤的关键感受器，参与三种不同的神经元结局：(1)细胞死亡，(2)轴突变性(AxD)，(3)轴突再生(AXR)。因此，DLK信号是一种令人兴奋的新的治疗靶点，可用于治疗广泛的神经疾病和损伤。尽管靶向DLK信号的临床重要性，但令人震惊的是，我们对DLK是如何调节的，以及对损伤后神经元结局的贡献知之甚少。这项建议将解决我们知识中的这一根本差距，并将确定DLK的调节因子，并测试它们在神经元损伤信号和结果中的作用。我进行了初步筛选，以确定限制DRG神经元中DLK信号转导的蛋白质，并确定MAP3K12结合抑制蛋白(MBIP)是DLK的负调节因子。在目标1中，我将确定MBIP调节DLK信号的机制。这将涉及DLK信号的遗传和药理学操作，以及对该途径的每个水平的评估。在目标1中，我还将测试损伤缓解MBIP的假设-DLK的抑制和操纵MBIP足以改变DLK依赖的细胞死亡、AxD和AXR模型中的损伤表型。在目标2中，我将通过评估自发的DLK通路激活来验证MBIP在体内调节基础DLK信号的假设。此外，我还将测试MBIP在周围神经损伤中的作用。这将通过坐骨神经中AxD和AXR的多次读数和后肢肌肉的重新神经支配来实现。目标1和2将(1)阐明MBIP调节DLK信号转导的机制以及损伤对其的影响，(2)测试损伤后靶向MBIP的治疗潜力，以及(3)确定MBIP介导的抑制DLK信号转导的作用 体内的DLK信号转导。由于我的试验性筛选的成功，我建议在AIM 3中建立一个更大的基于候选的筛查来识别DLK的其他正向和负向调控因子。将评估候选对象抑制或增强DLK信号通路的分子标志物的能力。HITS将通过在多种损伤模型中测试它们对神经元预后的贡献来进一步评估。这一目标将识别哺乳动物感觉神经元中DLK通路的新调节因子，并将导致可检验的假说，以揭示DLK调节的机制及其对损伤表型的影响。这些目标将填补关于DLK调节机制以及DLK调节因子与神经元损伤表型之间的关系的知识的关键空白。这些结果将显著提高我们对DLK信号的理解，并可能为广泛的神经元损伤和疾病的治疗确定新的分子靶点。

 DESCRIPTION (provided by applicant): Neuronal cell death and axonal damage are hallmarks of many neurological disorders and neurodegenerative diseases including, Alzheimer's and Parkinson's diseases, spinal cord injury, glaucoma, multiple sclerosis, traumatic and ischemic brain injuries, and over 100 different peripheral neuropathies. The dual leucine zipper kinase (DLK, MAP3K12) is a key sensor of axon injury and contributes to three distinct neuronal outcomes: (1) cell death, (2) axon degeneration (AxD), and (3) axon regeneration (AxR). Thus DLK signaling is an exciting new therapeutic target for a wide range of neurological disorders and injury. In spite of the clinical importance for targeting DLK signaling, we know shockingly little about how DLK is regulated and contributes to neuronal outcome after injury. This proposal will address this fundamental gap in our knowledge and will identify regulators of DLK and test their roles in neuronal injury signaling and outcomes. I performed a pilot screen to identify proteins that restrict DLK signaling in DRG neurons, and identified MAP3K12 binding inhibitor protein (MBIP) as a negative regulator of DLK. In Aim 1, I will determine the mechanism by which MBIP regulates DLK signaling. This will involve genetic and pharmacological manipulation of DLK signaling and assessment of each level of the pathway. In Aim 1, I will also test the hypothesis that injury relieves MBIP-inhibition of DLK and manipulating MBIP is sufficient to alter the injury phenotype in models of DLK-dependent cell death, AxD, and AxR. In Aim 2, I will test the hypothesis that MBIP regulates basal DLK-signaling in vivo by assessing spontaneous DLK pathway activation. Furthermore, I will test the role of MBIP in peripheral nerve injury. This will be accomplished using multiple readouts of AxD and AxR in the sciatic nerve and re-innervation of hindlimb muscles. Aims 1 and 2 will (1) elucidate the mechanism by which MBIP regulates DLK signaling and how this is affected by injury, (2) test the therapeutic potential of targeting MBIP after injury, and (3) determine the role of MBIP-mediated inhibition of DLK signaling in vivo. Due to the success of my pilot screen, I propose a larger candidate-based screen to identify additional positive and negative regulators of DLK in Aim 3. Candidates will be evaluated for their ability to suppress or enhance molecular markers of the DLK signaling pathway. Hits will be further evaluated by testing their contributions to neuronal outcome in multiple injury models. This aim will identify novel regulators of the DLK pathway in mammalian sensory neurons and will lead to testable hypotheses to uncover mechanisms of DLK regulation and the impact on injury phenotypes. These aims will fill a critical gap in knowledge regarding mechanisms of DLK regulation and the relationship between DLK regulators and neuronal injury phenotypes. These results will significantly enhance our understanding of DLK signaling and may identify novel molecular targets for therapeutic intervention in a wide range of neuronal injuries and diseases.