title: 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 ≥6 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(+) astrocytes that persisted for ≥6 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.
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Ke
authors.initials: K
authors.lastname: Li
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107, Neurodegeneration and Regeneration Unit, L'Unité de Recherché en Physiologie Moléculaire-Namur Research Institute for Life Sciences, University of Namur, B-5000 Namur, Belgium.
authors.forename: Charles
authors.initials: C
authors.lastname: Nicaise
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sannie
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Tamara J
authors.identifier: http://orcid.org/0000-0001-5851-271X
authors.initials: TJ
authors.lastname: Hala
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Elham
authors.initials: E
authors.lastname: Javed
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Parker
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Putatunda
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Regan
authors.affiliation: Laboratory of Histology, Neuroanatomy and Neuropathology, Université Libre de Bruxelles, B-1070 Brussels, Belgium.
authors.forename: Valérie
authors.initials: V
authors.lastname: Suain
authors.affiliation: Laboratory of Histology, Neuroanatomy and Neuropathology, Université Libre de Bruxelles, B-1070 Brussels, Belgium.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Brion
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana 59812, and.
authors.forename: Fred
authors.initials: F
authors.lastname: Rhoderick
authors.affiliation: Department of Biology, Arcadia University, Glenside, Pennsylvania 19038.
authors.forename: Megan C
authors.initials: MC
authors.lastname: Wright
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana 59812, and.
authors.forename: David J
authors.initials: DJ
authors.lastname: Poulsen
authors.affiliation: Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Philadelphia, Pennsylvania 19107, Angelo.Lepore@jefferson.edu.
authors.forename: Angelo C
authors.initials: AC
authors.lastname: Lepore
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000473:pathology*; D002574:Cervical Vertebrae; D003964:Diaphragm / Q000378:metabolism* / Q000503:physiopathology; D027342:Excitatory Amino Acid Transporter 2 / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D005552:Forelimb / Q000378:metabolism / Q000503:physiopathology*; D005786:Gene Expression Regulation / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000378:metabolism* / Q000473:pathology; D009410:Nerve Degeneration / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010791:Phrenic Nerve / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: D027342:Excitatory Amino Acid Transporter 2; C493396:Slc1a2 protein, mouse
keywords: adeno-associated virus; gene therapy; glutamate transporter; phrenic motor neuron; respiratory; spinal cord injury