Overexpression of the Astrocyte Glutamate Transporter GLT1 Exacerbates Phrenic Motor Neuron Degeneration, Diaphragm Compromise, and Forelimb Motor Dysfunction following Cervical Contusion Spinal Cord Injury

Abstract

A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for Ն6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP ϩ astrocytes that persisted for Ն6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.

Transplantation of stem cell-derived astrocytes for the treatment of amyotrophic lateral sclerosis and spinal cord injury

“…Failure of long-term extrasynaptic glutamate clearance is suspected to be one major cause of secondary cell loss following SCI. Noteworthy, we and others demonstrated that astrocyte GLT1 was chronically lost at the injury epicenter following SCI but also downregulated in spinal cord regions distant from the lesion core [103][104][105][106] . Furthermore, experimental data showed that the newlygenerated astrocytes arising during the SCI repair phase lacked GLT1 expression, possibly compromising long-term astrocyte glutamate homeostasis [107] .…”

“…The rationale behind such an approach is based on the findings that, following SCI, astrocyte GLT1 expression and function are compromised, resulting in excitotoxicity-induced cell death during the delayed secondary injury phase [105] . Unpublished data from our group show that transplantation of astrocytes engineered to overexpress GLT1 can prevent excitotoxicity and protect respiratory phrenic motor neurons following cervical SCI.…”

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

“…Adult Sprague-Dawley rats received either laminectomy only (uninjured control) or unilateral hemi-contusion SCI at the C4 spinal cord level 10–12 . At 5 weeks post-surgery, peak CMAP amplitude recorded from the hemi-diaphragm ipsilateral to the laminectomy/injury site was significantly reduced in SCI rats (Figure 2C) compared to laminectomy-only control (Figure 2B).…”

Long-Term Anesthetic-Dependent Hypoactivity after Repetitive Mild Traumatic Brain Injuries in Adolescent Mice

“…Preliminary assessment of a subset of samples yielded a minimum threshold for detecting GFAP expression, which was used to determine the integrated intensity of GFAP expression for all sections. The percent GFAP intensity [(the integrated intensity with minimum threshold divided by the integrated intensity without minimum threshold) multiplied by 100] was calculated per section and averaged per brain [42]. …”