Exploring mechanisms of axon growth and circuit connectivity for promoting respiratory function recovery following cervical spinal cord injury

探索轴突生长和回路连接促进颈脊髓损伤后呼吸功能恢复的机制

• 批准号: 10356158
• 负责人: Angelo C Lepore
• 金额: $ 40.81万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10356158
• 关键词: Address; Adult; Animal Model; Axon; Axotomy; Brain Stem; Breathing; Cervical; Cervical spinal cord injury; Cervical spinal cord structure; Chondroitin Sulfate Proteoglycan; Clinical; Contralateral; Contusions; Data; Denervation; Fiber; Functional disorder; Generations; Goals; Human; Individual; Injury; Ipsilateral; Lesion; Mediating; Methods; Modeling; Morbidity - disease rate; Motor Neurons; Muscle; Natural regeneration; Neuraxis; Neurons; Outcome; PTEN gene; Pathway interactions; Patients; Peptides; Population; Protein Tyrosine Phosphatase; Quality of life; Rattus; Recovery; Recovery of Function; Respiration Disorders; Respiratory Diaphragm; Respiratory Paralysis; Respiratory Tract Infections; Respiratory physiology; Spinal Cord Contusions; Spinal cord injury; Synapses; Testing; Therapeutic; axon growth; axon injury; axon regeneration; axonal sprouting; designer receptors exclusively activated by designer drugs; functional restoration; inhibitor; injured; innovation; knock-down; mortality; neural circuit; novel; partial recovery; prevent; receptor; reinnervation; respiratory; response; restoration; small hairpin RNA; targeted treatment; therapeutic target

Project Summary / Abstract (30-line maximum). A majority of traumatic spinal cord injury (SCI) cases occur in the cervical spinal cord, resulting in persistent diaphragmatic respiratory dysfunction that is associated with mortality, a host of morbidities such as respiratory infections, and greatly reduced quality of life. Diaphragm is directly controlled by phrenic motor neurons (PMNs) located at levels C3-5. PMNs are mono-synaptically activated by supraspinal brainstem neurons located in the rostral Ventral Respiratory Group (rVRG). Cervical SCI results in axotomy of descending rVRG fibers, denervation and silencing of spared PMNs, and partial-to-complete hemi-diaphragm paralysis. In this Competing Continuation (“Renewal”) application, we aim to promote reconnection of rVRG-PMN- diaphragm circuitry in a rat model of cervical SCI, a critically important therapeutic goal for individuals with SCI. We developed inhibitory peptides against PTEN (phosphatase and tensin homolog: a central inhibitor of neuron-intrinsic axon growth potential) and PTPσ (protein tyrosine phosphatase-sigma: an axonally-expressed receptor that mediates the neuron-extrinsic axon growth inhibitory effects of chondroitin sulfate proteoglycans). Our exciting preliminary findings show that systemic delivery of these peptides each result in robust – but partial – recovery of diaphragm function in the C2 hemisection model of SCI. These initial studies also provide compelling data suggesting that PTEN and PTPσ inhibition may promote recovery via different modes of rVRG axon growth: (1) robust regeneration of injured ipsilateral rVRG axons with PTEN inhibition; (2) extensive sprouting of spared contralateral rVRG axons into the PMN pool (ipsilateral to the lesion) with PTPσ inhibition. Importantly, we do not understand which modes of axon growth can promote recovery of diaphragm function after SCI, which significantly limits ability to develop targeted therapies. To address this critical issue, we will use chemogenetic DREAAD manipulations to selectively-silence defined neuronal populations involved in respiratory control in order to determine the mode(s) of circuit re-connectivity that causally drive recovery in response to PTEN and PTPσ manipulation. We will target PTEN and PTPσ with systemic delivery of inhibitory peptides and rVRG neuron-specific transduction with AAV-shRNA. We will compliment this approach using an array of cutting-edge functional and axonal/synaptic tracing methods to assess rVRG-PMN circuit plasticity. We hypothesize that stimulating (1) regeneration of injured rVRG axons, (2) sprouting of spared fibers originating in contralateral rVRG, and (3) synaptic connectivity of these growing rVRG axons with PMNs (located caudal to the lesion) will causally promote recovery of diaphragmatic respiratory function following cervical SCI. We also hypothesize that the combination of rVRG axon regeneration and sprouting of spared rVRG fibers will promote robust diaphragm recovery in the clinically-associated cervical contusion SCI model. We will acquire an in-depth understanding of how modulating axon growth inhibition can induce rVRG- PMN circuit plasticity and, importantly, which modes of connectivity promote diaphragm recovery after SCI.

项目摘要/摘要（最多30行）。 大多数创伤性脊髓损伤（SCI）病例发生在颈髓，导致持续性脊髓损伤。 与死亡率相关的呼吸系统功能障碍，一系列的发病率，如呼吸道疾病， 感染，生活质量大大降低。膈运动神经元直接控制膈肌 （PMNs）位于C3-5层。中性粒细胞由脊髓上脑干神经元单突触激活 位于喙侧呼吸组（rVRG）。颈脊髓损伤导致下行rVRG的轴突切断 纤维，去神经支配和沉默的备用中性粒细胞，和部分至完全半横膈膜麻痹。 在此竞争性延续（“更新”）申请中，我们的目标是促进rVRG-PMN的重新连接- 在颈脊髓损伤大鼠模型中的横膈膜回路，这是脊髓损伤患者的一个非常重要的治疗目标。 我们开发了针对PTEN（磷酸酶和张力蛋白同源物： 神经元-内在轴突生长潜力）和PTPσ（蛋白酪氨酸磷酸酶-σ：轴突表达的 受体介导硫酸软骨素蛋白聚糖的神经元-外源性轴突生长抑制作用）。 我们令人兴奋的初步研究结果表明，这些肽的全身递送各自导致稳健的-但 脊髓损伤C2半切模型中膈肌功能的部分恢复。这些初步研究还提供了 令人信服的数据表明，PTEN和PTPσ抑制可能通过不同的rVRG模式促进恢复， 轴突生长：（1）具有PTEN抑制的损伤的同侧rVRG轴突的稳健再生;（2）广泛的再生， 备用的对侧rVRG轴突发芽进入PMN池（病变的同侧），PTPσ抑制。 重要的是，我们不知道哪些轴突生长模式可以促进膈肌的恢复 SCI后的功能，这大大限制了开发靶向治疗的能力。为了解决这一关键问题， 我们将使用化学遗传学DREAAD操作来选择性沉默涉及的特定神经元群体， 在呼吸控制中，为了确定导致恢复的回路重新连接的模式， 对PTEN和PTPσ操作的响应。我们将靶向PTEN和PTPσ， 肽和用AAV-shRNA的rVRG神经元特异性转导。我们将使用 一系列尖端功能和轴突/突触追踪方法来评估rVRG-PMN回路可塑性。 我们假设刺激（1）受损rVRG轴突再生，（2）备用纤维发芽 起源于对侧rVRG，以及（3）这些生长的rVRG轴突与PMN的突触连接 （位于病变尾侧）将促进术后呼吸功能恢复， 颈椎脊髓损伤我们还假设，rVRG轴突再生和发芽的组合备用 rVRG纤维将在临床相关的颈部挫伤SCI模型中促进稳健的膈肌恢复。 我们将深入了解如何调节轴突生长抑制可以诱导rVRG- PMN回路的可塑性，以及重要的是，哪些连接模式促进了SCI后膈肌的恢复。