Respiratory Motor Neuron Protection Following Cervical Spinal Cord Injury

描述(由申请人提供)：大约一半的创伤性脊髓损伤(SCI)病例影响到颈髓区域，导致虚弱，通常是慢性呼吸损害。这些损伤中的大多数影响到颈中脊髓水平，也就是支配主要吸气肌--横隔膜的重要的膈运动神经元(PMN)池的位置。在最初的颈髓损伤后，有一个宝贵的机会来防止继发性PMN变性，从而保护呼吸功能。脊髓损伤后继发性损伤的主要原因之一是兴奋性毒性细胞。 由于细胞外谷氨酸平衡失调而死亡。在中枢神经系统(CNS)中，谷氨酸被谷氨酸转运体有效地从突触和其他部位清除。星形胶质细胞是支持性神经胶质细胞，在中枢神经系统功能中发挥着重要作用。特别是，星形胶质细胞表达主要的中枢谷氨酸转运体GLT1，它负责大多数中枢神经系统区域，特别是脊髓的谷氨酸摄取。我们实验室的初步研究结果显示：1)在脊髓挫伤后，脊髓内GLT1的表达水平和GLT1介导的谷氨酸摄取减少；2)GLT1杂合小鼠脊髓损伤后的组织和功能结局恶化；3)通过注射AAV1-GLT1病毒载体增加脊髓内的GLT1水平可减少PMN丢失和横隔膜功能障碍；4)脊髓内星形胶质细胞移植可减少胸部挫伤后的继发性退行性变，而经基因工程设计为高表达GLT1的星形胶质细胞移植可进一步提高疗效。拟议的研究将检验星形胶质细胞GLT1缺失在继发性呼吸道PMN变性中起关键作用的中心假设。为了开发一种可行的治疗脊髓损伤患者的方法，研究将在颈椎挫伤模型中测试一种临床相关的细胞来源--人诱导多能干细胞(IPS)来源的星形胶质细胞(HIPSA)的脊髓内移植。通过靶向GLT1，这种基于干细胞的星形胶质细胞替代策略旨在保护PMN在继发性变性过程中免受谷氨酸兴奋毒性。由于治疗效果是疾病中枢神经系统移植整合的功能，AIM#1中的研究将表征hIPSA在体内的存活、分化和椎管内注射后的长期安全性。 小鼠颈挫伤脊髓损伤模型的移植。由于GLT1是脊髓损伤后基于移植的星形胶质细胞替代的一个有前景的靶点，AIM#2的研究将在体内检测移植的hIPSA表达GLT1的能力，以及增加颈椎挫伤后脊髓内GLT1蛋白和谷氨酸摄取水平的能力。结果表明，与脊髓挫伤后的内源性星形胶质细胞类似，移植的hIPSA是否降低了GLT1的表达和功能，这与其治疗脊髓损伤的潜力有重要关系。HIPSA还将被改造为结构性过表达GLT1，以增强治疗潜力。在目标3中，研究将评估hIPSA对PMN的保护和随后的横隔膜功能的保护的体内效果。

DESCRIPTION (provided by applicant): Approximately half of traumatic spinal cord injury (SCI) cases affect cervical spinal cord regions, resulting in debilitating and often chronic respiratory compromise. The majority of these injuries affect mid-cervical spinal cord levels, the location of the important pool of phrenic motor neurons (PMNs) that innervates the diaphragm, the primary muscle of inspiration. Following initial trauma to cervical spinal cord, a valuable opportunity exists for preventing secondary PMN degeneration and consequently preserving respiratory function. One of the major causes of secondary injury following SCI is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. In the central nervous system (CNS), glutamate is efficiently cleared from the synapse and other sites by glutamate transporters. Astrocytes are supportive glial cells that play a host of crucial roles in CNS function. In particular, astrocytes express the major CNS glutamate transporter, GLT1, which is responsible for the vast majority of functional glutamate uptake in most CNS regions, particularly spinal cord. Preliminary findings from our lab show that: 1) levels of intraspinal GLT1 expression and GLT1-mediated glutamate uptake are reduced in an animal model of cervical contusion SCI; 2) histological and functional outcomes following SCI are worsened in GLT1 heterozygous mice; 3) increasing intraspinal GLT1 levels via injection of AAV1-GLT1 viral vector decreases PMN loss and diaphragm dysfunction after cervical contusion; 4) intraspinal astrocyte transplantation decreases secondary degeneration after thoracic contusion, and transplantation of astrocytes engineered to constitutively overexpress GLT1 further enhances efficacy. Proposed studies will test the central hypothesis that astrocyte GLT1 loss plays a key role in secondary respiratory PMN degeneration. With the goal of developing a viable therapy for SCI patients, studies will test intraspinal transplantation of a clinically-relevant source of cells, human induced Pluripotent Stem (iPS) cell- derived astrocytes (hIPSAs), in a cervical contusion model. By targeting GLT1, this stem cell-based astrocyte replacement strategy aims to protect PMNs from glutamate excitotoxicity during secondary degeneration. As therapeutic efficacy is a function of transplant integration in diseased CNS, studies in Aim #1 will characterize in vivo survival, differentiation and long-term safety of hIPSAs following intraspinal transplantation in a mouse model of cervical contusion SCI. As GLT1 is a promising target for transplant-based astrocyte replacement in SCI, studies in Aim #2 will examine in vivo ability of transplanted hIPSAs to express GLT1 and to increase intraspinal GLT1 protein and glutamate uptake levels after cervical contusion. Results will show whether, similar to endogenous astrocytes after contusion SCI, transplanted hIPSAs have reduced propensity for GLT1 expression and function, which has important relevance for their therapeutic potential in SCI. hIPSAs will also be engineered to constitutively overexpress GLT1 to enhance therapeutic potential. In Aim #3, studies will evaluate in vivo efficacy of hIPSAs for PMN protection and consequent preservation of diaphragm function.