Identifying novel regenerative treatments for CNS injury in adult mammals

摘要 我们建议研究是否同时针对转录因子ZNF362和细胞骨架非肌肉 肌球蛋白IIA和IIB(NMIIA&B)将促进脊髓后轴突再生和功能恢复 损伤(SCI)比任何一种单独的方法都要好。脊髓损伤后，切断的轴突不能再生，主要是因为 成年中枢神经系统神经元的内在生长能力降低，轴突延伸的环境较差。治疗 无法恢复瘫痪和其他丧失的功能，脊髓损伤患者往往是永久性的 已禁用。许多基因已经被确定可以控制成熟神经元的再生失败，但没有一个基因能够做到这一点。 已转化为临床使用。人们一直需要确定更好的基因靶点和治疗方法 战略。ZNF362在中枢神经系统中高度表达，但其在哺乳动物中的主要功能仍不清楚。 Pi的团队建立了ZNF362条件性基因敲除(CKO)小鼠，并设计了新的小肽来阻断 ZNF362具有选择性作用。我们的初步研究表明，ZNF362强烈抑制小鼠的生长能力 成年啮齿动物的中枢神经系统神经元。中枢神经系统损伤后，损伤周围的各种抑制分子激活神经元 RhoA，进而激活细胞骨架NMIIA&B，凝聚肌动蛋白细丝，限制微管 突起和轴突延长。因为神经元细胞骨架是驱动轴突生长和 控制轴突生长、操纵神经元的多条信号通路的汇聚靶点 细胞骨架在促进中枢神经系统轴突再生方面也很有吸引力。我们假设这种抑制作用 ZNF362和NMIIA&B都代表了增强神经元生长能力和减少 病变周围的环境抑制。我们建议剖析ZNF362和NMIIA&B的潜在关键作用 控制成熟中枢神经系统神经元的再生并开发新的有效的促进中枢神经系统的策略 轴突再生。我们的目标是通过以下方法刺激脊髓损伤啮齿动物强大的轴突再生和功能恢复 使用CKO小鼠和我们实验室设计的新的选择性拮抗肽来抑制这些基因。在目标1中， 我们将研究转基因缺失ZNF362和NMIIA&B是否协同促进轴突 成年小鼠脊髓损伤后的再生与恢复。目标2建议确定阻止个体 ZNF362和NMIIA&B通过新型选择性拮抗剂促进轴突再生的药理信号 患有脊髓损伤的成年啮齿动物的康复。在目标3中，我们计划开发阻止两者的联合疗法 ZNF362和NMIIA&B信号，旨在产生强大的轴突再生和功能恢复的成年啮齿动物 SCI。基于我们试点研究的有希望的结果，我们预计我们的新的再生策略将 显著提高了我们治疗脊髓损伤的能力。如果我们的多肽在啮齿动物脊髓损伤模型上取得成功，我们计划 将这项工作转移到多肽安全性评估和进一步的翻译研究(例如，颈部/慢性脊髓损伤和 临床试验)。因此，该项目可能有助于寻找新的中枢神经系统修复和修复的分子靶点 治疗中枢神经系统病变的高效策略。

Abstract We propose to study whether targeting both the transcription factor ZNF362 and cytoskeletal non-muscle myosin IIA and IIB (NMIIA&B) will promote better axon regeneration and functional recovery after spinal cord injury (SCI) than either approach alone. After SCI, severed axons fail to regenerate largely because of the reduced intrinsic growth capacity of adult CNS neurons and the poor environment for axon extension. Treatments to recover paralysis and other lost functions are not available and patients with SCI are often permanently disabled. Many genes have been determined to control the regrowth failure of mature neurons, but none have been translated to clinical use. There is a persistent need to identify better gene targets and therapeutic strategies. ZNF362 is highly expressed in the CNS, but its major function in mammals remains largely unknown. The PI’s group generated ZNF362 conditional knockout (cKO) mice and designed novel small peptides to block ZNF362 function selectively. Our pilot studies suggest that ZNF362 strongly suppresses the growth capacity of CNS neurons in adult rodents. After CNS injury, various inhibitory molecules around the lesion activate neuronal RhoA, which, in turn, activates cytoskeletal NMIIA&B to condense actin filaments and restricts microtubule protrusion and axon elongation. Because neuronal cytoskeleton is the major machinery to drive axon growth and the converging targets of multiple signaling pathways that control axon growth, manipulating neuronal cytoskeleton is also very attractive to promote significant CNS axon regeneration. We hypothesize that inhibiting both ZNF362 and NMIIA&B represents a dual approach for enhancing neuronal growth capacity and reducing environmental inhibition around the lesion. We propose to dissect potential critical roles of ZNF362 and NMIIA&B for controlling regrowth of mature CNS neurons and to develop novel and effective strategies for promoting CNS axon regeneration. We aim to stimulate robust axon regrowth and functional recovery in SCI rodents by suppressing these genes using cKO mice and new selective antagonist peptides designed in our lab. In Aim 1, we will study whether transgenically deleting ZNF362 and NMIIA&B acts synergistically to promote axon regeneration and recovery in adult mice with SCI. Aim 2 proposes to determine whether blocking individual ZNF362 and NMIIA&B signals pharmacologically with novel selective antagonists promotes axon regeneration and recovery in adult rodents with SCI. In Aim 3, we plan to develop combination therapies that block both ZNF362 and NMIIA&B signals, aiming to yield robust axon regrowth and functional recovery in adult rodents with SCI. Based on the promising results of our pilot studies, we anticipate that our novel regenerative strategies will significantly advance our ability to treat SCI. If our peptides are successful with rodent SCI models, we plan to move this work to peptide safety assessments and further translational studies (e.g., cervical/chronic SCI and clinical trials). Therefore, this project may facilitate identifying novel molecular targets for CNS repair and highly effective strategies for treating CNS lesions.