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）病例影响颈髓区域，导致衰弱和慢性呼吸功能障碍。这些损伤中的大多数影响中颈脊髓水平，膈运动神经元（PMN）的重要池的位置，支配膈肌，吸气的主要肌肉。在颈髓初始创伤后，存在一个宝贵的机会，以防止继发性中性粒细胞变性，从而保留呼吸功能。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 由于细胞外谷氨酸稳态失调而死亡。在中枢神经系统（CNS）中，谷氨酸通过谷氨酸转运体从突触和其他部位有效地清除。星形胶质细胞是中枢神经系统中起重要作用的支持性神经胶质细胞。特别是，星形胶质细胞表达主要的CNS谷氨酸转运蛋白GLT 1，GLT 1负责大多数CNS区域（特别是脊髓）中的绝大多数功能性谷氨酸摄取。 我们实验室的初步发现表明：1）在颈挫伤SCI的动物模型中，脊髓内GLT 1表达水平和GLT 1介导的谷氨酸摄取水平降低; 2）在GLT 1杂合小鼠中，SCI后的组织学和功能结果恶化; 3）通过注射AAV 1-GLT 1病毒载体增加脊髓内GLT 1水平减少颈挫伤后的PMN损失和膈肌功能障碍; 4）脊髓内星形胶质细胞移植减少了胸部挫伤后的继发性变性，并且被工程化以组成型过表达GLT 1的星形胶质细胞的移植进一步增强了功效。 拟议的研究将测试中心的假设，星形胶质细胞GLT 1损失在继发性呼吸道PMN变性中起着关键作用。为了开发用于SCI患者的可行疗法，研究将在颈部挫伤模型中测试临床相关的细胞来源（人诱导多能干（iPS）细胞衍生的星形胶质细胞（hIPSA））的脊柱内移植。通过靶向GLT 1，这种基于干细胞的星形胶质细胞替代策略旨在保护中性粒细胞在继发性变性期间免受谷氨酸兴奋性毒性。 由于治疗功效是患病CNS中移植整合的函数，因此目的#1中的研究将表征脊柱内植入后hIPSA的体内存活、分化和长期安全性。 在颈部挫伤SCI的小鼠模型中移植。由于GLT 1是SCI中基于移植的星形胶质细胞替代的有希望的靶点，目的#2中的研究将检查移植的hIPSA在体内表达GLT 1和增加颈椎挫伤后椎管内GLT 1蛋白和谷氨酸摄取水平的能力。结果将显示，类似于挫伤SCI后的内源性星形胶质细胞，移植的hIPSA是否具有降低的GLT 1表达和功能的倾向，这与它们在SCI中的治疗潜力具有重要的相关性。hIPSA还将被工程化以组成型过表达GLT 1以增强治疗潜力。在目标#3中，研究将评价hIPSA对PMN保护和随后的膈肌功能保护的体内功效。

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.