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

描述（由申请人提供）：中枢神经系统（CNS）中轴突再生的失败阻碍了对广泛的CNS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CNS经历轴突生长的内在能力的发育丧失。为了在CNS损伤或疾病后恢复功能，我们必须设计方法来增强CNS神经元的内在生长状态。我们最近确定了Krppel样转录因子家族（KLF）作为中枢神经系统神经元内在再生能力的关键调节因子。关键的是，转录活性突变体VP 16-KLF 7通过受损的皮质脊髓束（CST）神经元促进轴突再生，这是一个重要的治疗靶点。使用病毒递送至成年野生型动物实现了稳健的再生反应，提高了潜在的临床应用。 与这种新型再生工具的相关性。为了进一步探索KLF 7活性促进CNS再生的潜力，必须解决三个关键问题，这与我们的目标相对应。首先，由于SCI的恢复依赖于不同纤维束携带的多种功能模式，我们将使用病毒介导的基因递送来测试VP 16-KLF 7促进其他神经元群体再生的能力。靶向其他神经元亚型有可能扩大SCI后行为改善的范围。第二，我们将检查VP 16-KLF 7刺激的神经元中促再生基因的表达，以确定与已知促再生（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。基于此信息，我们将合理地将联合收割机VP 16/KLF 7与靶向互补途径的病毒颗粒组合，从而潜在地诱导轴突再生的累加或协同改善。最后，我们将检查KLF 7刺激的轴突在多大程度上成功地在适当的靶细胞上形成功能性突触并最终有助于功能恢复。使用病毒基因递送，我们将与光遗传学构建体共表达VP 16-KLF 7，所述光遗传学构建体使得经处理的神经元能够被可逆地激活或沉默。这项技术将使我们能够确定特定的贡献，治疗神经元的电生理和行为输出的再生进行。总之，这些实验具有很强的潜力来扩展有前途的新的促再生工具的使用，并导致开发新的组合方法来促进受损CNS中的轴突再生和功能恢复，从而为患有CNS疾病的人提供新的治疗选择。

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.