Mitochondrial Transplantation Strategies to Promote Recovery after Spinal Cord Injury

 描述（由申请人提供）：目前的提案旨在开发一个新的研究领域，旨在将线粒体移植到受损的啮齿动物脊髓中，作为创伤性脊髓损伤（SCI）后的治疗策略。我们和其他人已经发现，在挫伤SCI后维持正常的线粒体生物能学促进神经保护和功能恢复。然而，还没有研究检查在损伤后将外源性线粒体移植到脊髓组织中以试图使整体细胞生物能量正常化并使邻近宿主组织空闲的效果。因此，这项新的研究将最终测试将外源性健康线粒体移植到受损的脊髓中是否会促进功能性神经保护。使用大鼠模型的挫伤SCI，我们有初步的数据显示，同种异体转基因标记（turboGFP）的线粒体来自周围的损伤部位显微注射的PC 12细胞保持整体细胞生物能量评估24小时后。此外，使用共聚焦显微镜来评估线粒体内膜和外膜的特异性抗体，我们有初步证据表明，移植的turboGFP线粒体整合在幼稚和损伤的脊髓组织中。基于这些开创性的发现，我们建议进行一系列的移植策略，以提供损伤部位周围的线粒体。最终目标是鉴定线粒体的潜在自体细胞来源（即，腿部肌肉）以防止继发性组织损伤，所述继发性组织损伤在很大程度上由线粒体损失和功能障碍介导。总之，Aim 1a将比较性地采用剂量反应方法来评估从肌肉中分离的移植MitoTracker Red标记的线粒体是否与同种异体turboGFP线粒体在细胞生物能量学和急性挫伤SCI后的炎症程度上同样有效。目标1b将比较评估损伤后第一周内标记线粒体并入宿主细胞的频率和分布。最后，目标2将确定移植肌源性线粒体是否能增加SCI后的长期组织保留和后肢运动恢复。该项目具有翻译潜力，其中线粒体，特别是自体线粒体，可以移植到损伤部位周围以节省半影组织。基于PI在细胞移植到受损脊髓组织中的集体专业知识以及Co-I在生物能量评估和神经创伤的神经靶向治疗方面的集体专业知识，沿着我们的初步可行性数据，成功实施该项目没有预见到的技术障碍。值得注意的是，未来的实验源于当前提案的结果，将被设计为测试延迟移植范例，以延长线粒体移植的临床相关治疗时间窗，以促进更慢性SCI模型的功能恢复。

 DESCRIPTION (provided by applicant): The current proposal is designed to develop of a new research area that is aimed at transplanting mitochondria into the injured rodent spinal cord as a therapeutic strategy following traumatic spinal cord injury (SCI). We and others have found that maintaining normal mitochondrial bioenergetics after contusion SCI fosters both neuroprotection and functional recovery. However, no studies have examined the effects of transplanting exogenous mitochondria into spinal cord tissue after injury in an attempt to normalize overall cellular bioenergetics and spare adjacent host tissues. Accordingly, this new line of investigations will test, ultimately, whether transplanting exogenous healthy mitochondria into the injured spinal cord promotes functional neuroprotection. Using a rat model of contusion SCI, we have preliminary data showing that allogeneic transgenically-labeled (turboGFP) mitochondria derived from PC12 cells microinjected around the injury site maintain overall cellular bioenergetics assessed 24 hrs later. Moreover, using confocal microscopy to evaluate specific antibodies to both inner and outer mitochondrial membranes, we have preliminary evidence that grafted turboGFP mitochondria integrate within naïve and injured spinal cord tissues. Based on these seminal findings, we propose to conduct a series of transplantation strategies to deliver mitochondria around the injury sites. The ultimate goal is to identify a potential autologous cell source of mitochondria (i.e., leg muscle) to prevent secondary tissue injury that is mediated, in large part, by mitochondrial loss and dysfunction. In summary, Aim 1a will comparatively employ a dose-response approach to assess whether transplanted MitoTracker Red-labeled mitochondria isolated from muscles are equally effective as allogeneic turboGFP mitochondria on cellular bioenergetics and the extent of inflammation following acute contusion SCI. Aim 1b will then assess comparatively the frequency and distribution of labeled mitochondria incorporated into host cells over the first week post-injury. Finally, Aim 2 will establish whether transplantation of muscle-derived mitochondria increases long-term tissue sparing and recovery of hindlimb locomotion following SCI. This project has translational potential wherein mitochondria, notably autologous, can be transplanted around the injury site to spare penumbral tissues. Based on the collective expertise of the PI in cellular transplantation into injured spinal cord tissue and both Co-Is in bioenergetic assessments and mitochondrial-targeted therapeutics for neurotrauma, along with our preliminary feasibility data, there are no foreseen technical obstacles to successfully carry out this project. Notably, future experiments stemming from the results of the current proposal will be designed to test delayed grafting paradigms to extend a more clinically relevant therapeutic time window for transplantation of mitochondria to promote functional recovery in more chronic SCI models.