Functional Testing of KLF7 in Spinal Cord Injury: An Optogenetic Approach

KLF7 在脊髓损伤中的功能测试：光遗传学方法

• 批准号: 9067525
• 负责人: Murray G Blackmore
• 金额: $ 32.92万
• 依托单位: MARQUETTE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9067525
• 关键词: Address; Adult; Animals; Axon; Behavioral; Brain; Cells; Central Nervous System Diseases; Chondroitin Sulfate Proteoglycan; Ciliary Neurotrophic Factor; Corticospinal Tracts; Development; Disease; Electrophysiology (science); FRAP1 gene; Failure; Family; Fiber; Forelimb; Gene Delivery; Genes; Goals; Growth; Health; Injury; Lasers; Lead; Light; Mediating; Methods; Modality; Molecular; Molecular Target; Natural regeneration; Nerve Regeneration; Nervous system structure; Neuraxis; Neurons; Output; Parkinson Disease; Pathway interactions; Population; Process; Reading; Recovery; Recovery of Function; Recruitment Activity; Red nucleus structure; Regenerative response; Rodent Model; Sensory; Signal Pathway; Signal Transduction; Site; Specificity; Spinal Cord; Spinal cord injury; Stroke; Synapses; Techniques; Testing; VP 16; Viral; awake; axon growth; axon regeneration; base; behavior test; behavioral outcome; central nervous system injury; clinically relevant; combinatorial; efficacy testing; extracellular; functional restoration; gene therapy; in vivo; injured; meetings; mutant; novel; optogenetics; overexpression; particle; prevent; regenerative; research study; synaptogenesis; therapeutic target; tool; transcription factor; viral gene delivery

DESCRIPTION (provided by applicant): The failure of axon regeneration in the central nervous system (CNS) prevents treatment of a wide range of CNS afflictions, including spinal cord injury, stroke, and diseases like Parkinson's. One major reason that regeneration fails is that many CNS undergo a developmental loss in their intrinsic capacity for axon growth. To restore function after CNS injury or disease it is essential that we devise means to enhance the intrinsic growth state of CNS neurons. We recently identified the Kr¿ppel-like family of transcription factors (KLFs) as key regulators of intrinsic regenerative capacity in CNS neurons. Critically, a transcriptionally active mutant, VP16-KLF7, promotes axon regeneration by injured corticospinal tract (CST) neurons, an important therapeutic target. Robust regenerative responses were achieved using viral delivery to adult wild-type animals, raising potential clinical relevance for this novel pro-regenerative tool. To further explore the potential of KLF7 activity t promote CNS regeneration, three key questions must be addressed, corresponding to our aims. First because recovery from SCI depends on multiple functional modalities carried by diverse fiber tracts, we will use viral-mediated gene delivery to test the ability of VP16-KLF7 to promote regeneration in additional neuronal populations. Targeting additional neuronal subtypes has the potential to broaden the range of behavioral improvement after SCI. Second, we will examine the expression of pro-regenerative genes in neurons stimulated by VP16-KLF7 in order to determine the potential relationship between with known pro-regenerative (e.g. mTOR, CNTF) or anti-regenerative (e.g. chondroitin sulfate proteoglycan (CSPG)) pathways and signals. Based on this information we will rationally combine VP16/KLF7 with viral particles that target complementary pathways, potentially inducing additive or synergistic improvements in axon regeneration. Finally, we will examine the extent to which KLF7-stimulated axons succeed in forming functional synapses on appropriate target cells and ultimately contribute to functional recovery. Using viral gene delivery we will co-express VP16- KLF7 with optogenetic constructs that enable the treated neurons to be reversibly activated or silenced. This technique will allow us to determine the specific contribution of the treated neurons to electrophysiological and behavioral output as regeneration proceeds. Taken together, these experiments have a strong potential to extend the use of a promising new pro-regenerative tool and lead to the development of novel combinatorial methods to promote axon regeneration and functional recovery in the injured CNS, leading to novel treatment options for people suffering from CNS afflictions.

描述（由申请人提供）：中枢神经系统（CNS）轴突再生失败阻碍了多种中枢神经系统疾病的治疗，包括脊髓损伤、中风和帕金森病等疾病。再生失败的一个主要原因是许多中枢神经系统轴突生长的内在能力发生发育损失。为了在中枢神经系统损伤或疾病后恢复功能，我们必须设计出增强中枢神经系统神经元内在生长状态的方法。我们最近发现 Kr¿ppel 样转录因子家族 (KLF) 是中枢神经系统神经元内在再生能力的关键调节因子。至关重要的是，转录活性突变体 VP16-KLF7 可促进受损皮质脊髓束 (CST) 神经元的轴突再生，这是一个重要的治疗靶点。通过将病毒传递给成年野生型动物，实现了强大的再生反应，提高了潜在的临床效果 与这种新颖的促再生工具的相关性。为了进一步探索 KLF7 活性促进中枢神经系统再生的潜力，必须解决与我们的目标相对应的三个关键问题。首先，由于 SCI 的恢复取决于不同纤维束携带的多种功能模式，因此我们将使用病毒介导的基因传递来测试 VP16-KLF7 促进其他神经元群体再生的能力。针对其他神经元亚型有可能扩大 SCI 后行为改善的范围。其次，我们将检查 VP16-KLF7 刺激的神经元中促再生基因的表达，以确定与已知的促再生（例如 mTOR、CNTF）或抗再生（例如硫酸软骨素蛋白聚糖（CSPG））途径和信号之间的潜在关系。基于这些信息，我们将合理地将 VP16/KLF7 与针对互补途径的病毒颗粒结合起来，潜在地诱导轴突再生的附加或协同改善。最后，我们将检查 KLF7 刺激的轴突在适当的靶细胞上成功形成功能性突触并最终有助于功能恢复的程度。使用病毒基因传递，我们将与光遗传学构建体共表达 VP16-KLF7，使处理的神经元能够可逆地激活或沉默。这项技术将使我们能够确定经过治疗的神经元在再生过程中对电生理和行为输出的具体贡献。总而言之，这些实验具有巨大的潜力，可以扩展有前途的新型促再生工具的使用，并导致开发新的组合方法来促进受损中枢神经系统的轴突再生和功能恢复，从而为患有中枢神经系统疾病的人提供新的治疗选择。