Mitochondrial Transplantation Strategies to Promote Recovery after Spinal Cord Injury

 描述(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。我们和其他人发现，在脊髓挫伤后保持正常的线粒体生物能量有助于神经保护和功能恢复。然而，还没有研究在损伤后将外源性线粒体移植到脊髓组织中，试图使整个细胞生物能量学正常化，并保留邻近宿主组织的影响。因此，这一新的研究路线最终将测试将外源健康线粒体移植到受损脊髓是否促进功能性神经保护。使用大鼠挫伤脊髓损伤模型，我们有初步数据显示，从损伤部位周围显微注射的PC12细胞衍生的同种异体转基因标记(TurboGFP)线粒体维持24小时后评估的整体细胞生物能量学。此外，使用共聚焦显微镜评估内外线粒体膜的特异性抗体，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移植策略，将线粒体运送到损伤部位周围。最终目标是确定线粒体(即腿部肌肉)的潜在自体细胞来源，以防止在很大程度上由线粒体丢失和功能障碍所介导的继发性组织损伤。总之，Aim 1a将采用剂量-反应方法来评估从肌肉中分离的移植MitoTracker Red标记线粒体在细胞生物能量学和急性挫伤后炎症程度上是否与同种异体TurboGFP线粒体一样有效。目的1b随后将比较评估损伤后第一周内被标记的线粒体并入宿主细胞的频率和分布。最后，目标2将确定肌源性线粒体移植是否增加了脊髓损伤后长期组织的保留和后肢运动的恢复。这个项目具有翻译潜力，其中线粒体，特别是自体线粒体，可以移植到损伤部位周围，以备用半影体组织。根据PI在细胞移植到受损脊髓组织方面的集体专业知识，以及Co-IS在生物能量评估和线粒体靶向治疗神经创伤方面的专业知识，以及我们的初步可行性数据，成功实施该项目没有可预见的技术障碍。值得注意的是，根据当前提议的结果，未来的实验将被设计用于测试延迟移植范例，以延长更具临床相关性的线粒体移植的治疗时间窗口，以促进更慢性脊髓损伤模型的功能恢复。

 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.