Department of Neuroscience, Farber Institute for Neurosciences, Sidney Kimmel Medical College at Thomas Jefferson University, 900 Walnut Street, JHN 469, Philadelphia, PA 19107, USA.
Department of Biomedical and Pharmaceutical Sciences, University of Montana, 32 Campus Dr., Missoula, MT 59812, USA.
Neurobiol Dis. 2015 Jun;78:12-23. doi: 10.1016/j.nbd.2015.03.010. Epub 2015 Mar 25.
Amyotrophic lateral sclerosis (ALS) is characterized by relatively rapid degeneration of both upper and lower motor neurons, with death normally occurring 2-5years following diagnosis primarily due to respiratory paralysis resulting from phrenic motor neuron (PhMN) loss and consequent diaphragm denervation. In ALS, cellular abnormalities are not limited to MNs. For example, decreased levels and aberrant functioning of the major central nervous system (CNS) glutamate transporter, GLT1, occur in spinal cord and motor cortex astrocytes of both humans with ALS and in SOD1(G93A) rodents, a widely studied ALS animal model. This results in dysregulation of extracellular glutamate homeostasis and consequent glutamate excitotoxicity, a primary mechanism responsible for MN loss in ALS animal models and in the human disease. Given these observations of GLT1 dysfunction in areas of MN loss, as well as the importance of testing therapeutic strategies for preserving PhMNs in ALS, we evaluated intraspinal delivery of an adeno-associated virus type 8 (AAV8)-Gfa2 vector to the cervical spinal cord ventral horn of SOD1(G93A) ALS mice for focally restoring intraspinal GLT1 expression. AAV8 was specifically injected into the ventral horn bilaterally throughout the cervical enlargement at 110days of age, a clinically-relevant time point coinciding with phenotypic/symptomatic disease onset. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in robust transduction primarily of GFAP(+) astrocytes that persisted until disease endstage, as well as a 2-3-fold increase in total intraspinal GLT1 protein expression in the ventral horn. Despite this robust level of astrocyte transduction and GLT1 elevation, GLT1 overexpression did not protect PhMNs, preserve histological PhMN innervation of the diaphragm NMJ, or prevent decline in diaphragmatic respiratory function as assessed by phrenic nerve-diaphragm compound muscle action potential (CMAP) recordings compared to control AAV8-Gfa2-eGFP injected mice. In addition, AAV-Gfa2-GLT1 did not delay forelimb disease onset, extend disease duration (i.e. time from either forelimb or hindlimb disease onsets to endstage) or prolong overall animal survival. These findings suggest that focal restoration of GLT1 expression in astrocytes of the cervical spinal cord using AAV delivery is not an effective therapy for ALS.
肌萎缩侧索硬化症（ALS）的特征是上下运动神经元相对迅速退化，通常在诊断后 2-5 年内死亡，主要原因是膈神经运动神经元（PhMN）丧失和随之而来的膈肌去神经支配导致呼吸瘫痪。在 ALS 中，细胞异常不仅限于 MNs。例如，主要中枢神经系统（CNS）谷氨酸转运体 GLT1 的水平降低和功能异常，发生在人类 ALS 的脊髓和运动皮层星形胶质细胞中，以及广泛研究的 ALS 动物模型 SOD1（G93A）鼠中。这导致细胞外谷氨酸稳态失调和随后的谷氨酸兴奋性毒性，这是 ALS 动物模型和人类疾病中 MN 丧失的主要机制。鉴于在 MN 丧失区域观察到 GLT1 功能障碍，以及测试保护 PhMNs 的治疗策略的重要性，我们评估了腺相关病毒 8 型（AAV8）-Gfa2 载体在 SOD1（G93A）ALS 小鼠颈脊髓腹角的鞘内递送来局部恢复脊髓内 GLT1 表达。AAV8 特异性地在 110 天大龄时双侧注射到颈膨大的腹角，这是一个与表型/症状发病时间一致的临床相关时间点。AAV8-Gfa2-GLT1 的鞘内递送至疾病终末期，主要转导 GFAP（+）星形胶质细胞，持续存在，同时脊髓腹角的总 GLT1 蛋白表达增加 2-3 倍。尽管这种星形胶质细胞转导和 GLT1 升高水平非常显著，但与对照 AAV8-Gfa2-eGFP 注射小鼠相比，GLT1 过表达并不能保护 PhMNs，保留膈神经肌接头 NMJ 的组织学 PhMN 神经支配，或防止膈肌呼吸功能下降，如通过膈神经-膈肌复合肌肉动作电位（CMAP）记录评估。此外，AAV-Gfa2-GLT1 并未延迟前肢疾病发病，延长疾病持续时间（即从前肢或后肢疾病发病到终末期的时间）或延长整体动物存活时间。这些发现表明，使用 AAV 递送在颈脊髓星形胶质细胞中局部恢复 GLT1 表达不是治疗 ALS 的有效方法。
