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

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 与死亡相关的横隔性呼吸功能障碍，呼吸道等一系列疾病 感染，并大大降低了生活质量。横隔膜由膈运动神经元直接控制 (PMN)位于C3-5层。中性粒细胞由棘上脑干神经元单突触激活 位于头端腹侧呼吸群(RVRG)。颈椎脊髓损伤导致下行rVRG轴突切断 纤维、备用PMN的失神经和沉默，以及部分至完全的半横肌麻痹。 在这项竞争性的续展应用中，我们的目标是促进rVRG-PMN的重新连接- 颈椎脊髓损伤大鼠模型中的横隔膜回路，对脊髓损伤患者来说是一个至关重要的治疗目标。 我们开发了针对PTEN(磷酸酶和张力蛋白同源：一种中枢抑制因子)的抑制肽 神经元内源性轴突生长潜能)和蛋白酪氨酸磷酸酶σ：轴突表达的 介导硫酸软骨素蛋白多糖抑制神经元外源性轴突生长的受体)。 我们令人兴奋的初步发现表明，这些多肽的系统性递送都会产生强健的-但是 脊髓损伤C2半横断模型部分恢复横隔膜功能。这些初步研究还提供了 令人信服的数据表明，抑制PTEN和PTPRG可能通过不同的σ模式促进恢复 轴突生长：(1)PTEN抑制的损伤同侧rVRG轴突的旺盛再生；(2)广泛的再生 PtPσ抑制后，备用对侧rVRG轴突发芽进入中性粒细胞池(与损伤同侧)。 重要的是，我们不知道哪种轴突生长方式可以促进横隔膜的恢复。 脊髓损伤后的功能，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操作来选择性地沉默所涉及的特定神经元群体 在呼吸控制中，为了确定导致呼吸恢复的回路重新连接模式(S) 对pTen和ptpσ操作的反应。我们将以PTEN和PtPσ为靶点，全身性递送抑制物 AAV-shRNA的多肽和rVRG神经元特异性转导。我们将使用一个 一系列用于评估rVRG-PMN电路可塑性的尖端功能和轴突/突触追踪方法。 我们假设刺激(1)损伤的rVRG轴突的再生，(2)备用纤维的萌发 (3)这些生长中的rVRG轴突与PMN的突触连接 (位于病变的尾部)将因果地促进下列患者的隔膜呼吸功能的恢复 颈椎损伤。我们还假设rVRG轴突再生和备用 在临床相关的颈椎挫伤模型中，rVRG纤维将促进健壮的横隔膜恢复。 我们将深入了解调节轴突生长抑制如何诱导rVRG- PMN电路的可塑性，以及重要的是，哪种连接模式促进了脊髓损伤后横隔膜的恢复。

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.