Abstract
The astrocytic glutamate transporters, EAAT1 and EAAT2, remove released L-glutamate from the synaptic milieu thereby maintaining normal excitatory transmission. EAAT dysfunction during the excitotoxicity and oxidative stress of neurological insults may involve homoeostatic mechanisms associated with astrocytic function. We investigated aspects of EAAT function and expression in concert with astrocytic phenotype in primary cultures of cortical astrocytes and mixed cells of the spinal cord. In spinal cord mixed cultures, hydrogen peroxide (300 µM) reduced both EAAT activity and cellular viability to half of their basal values at 24 h post-treatment, but at 2 h EAAT activity was unaltered, while cellular viability was significantly decreased, suggestive of a mechanism for the maintenance of EAAT activity. Cytochemistry for MAP2, GFAP and propidium iodide revealed that neurons and astrocytes were damaged in a time-dependent manner. A change in astrocyte morphology was observed, with astrocyte cell bodies becoming larger and processes becoming more stellate and often shorter in length. EAAT1 immunoreactivity was reduced at 24 h post-treatment and a re-distribution of the protein was noted after 2 h treatment. In pure astrocytes, lipopolysaccharide (1 µg/ml, 3 d) increased [3H]D-aspartate uptake by 90%, as well as EAAT1 immunoreactivity and astrocyte stellation, as shown by immunofluorescent labelling for GFAP. In both culture systems, prominent changes were noted in EAAT function and localization in conjunction with altered astrocytic phenotype. Our findings are indicative of a relationship between astrocytic phenotype and the level of EAAT activity that may be a vital component of astrocytic homeostatic responses in brain injury.
Similar content being viewed by others
References
Aprico K, PM Beart, D Crawford and RD O’Shea (2004) Binding and transport of [3H](2S,4R)-4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes.J. Neurosci. Res. 75, 751–759.
Beal MF, BT Hyman and W Koroshetz (1993) Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases?Trends Neurosci. 16, 125–131.
Danbolt NC (2001) Glutamate uptake.Prog. Neurobiol. 65, 1–105.
Dirnagl U, C Iadecola and MA Moskowitz (1999) Pathobiology of ischaemic stroke: an integrated view.Trends Neurosci. 22, 391–397.
Duan S, CM Anderson, BA Stein and RA Swanson (1999) Glutamate induces rapid upregulation of astrocyte glutamate transport and cell-surface expression of GLAST.J. Neurosci. 19, 10193–10200.
Figiel M and J Engele (2000) Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuron-derived peptide regulating glial glutamate transport and metabolism.J. Neurosci. 20, 3596–3605.
Gegelashvili G, NC Danbolt and A Schousboe (1997) Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia.J. Neurochem. 69, 2612–2615.
Gegelashvili G, Y Dehnes, NC Danbolt and A Schousboe (2000) The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms.Neurochem. Int. 37, 163–170.
Ginsberg SD, LJ Martin and JD Rothstein (1995) Regional deafferentation down-regulates subtypes of glutamate transporter proteins.J. Neurochem. 65, 2800–2803.
Gurney ME, R Liu, JS Althaus, ED Hall and DA Becker (1998) Mutant CuZn superoxide dismutase in motor neuron disease.J. Inherited Metab. Dis. 21, 587–597.
Hughes EG, JL Maguire, MT McMinn, RE Scholz and ML Sutherland (2004) Loss of glial fibrillary acidic protein results in decreased glutamate transport and inhibition of PKA-induced EAAT2 cell surface trafficking.Brain Res. Mol. Brain Res. 124, 114–123.
Levy LM, KP Lehre, SI Walaas, J Storm-Mathisen and NC Danbolt (1995) Down-regulation of glial glutamate transporters after glutamatergic denervation in the rat brain.Eur. J. Neurosci. 7, 2036–2041.
Louvel E, J Hugon and A Doble (1997) Therapeutic advances in amyotrophic lateral sclerosis.Trends Pharmacol. Sci. 18, 196–203.
Olney JW (1989) Excitatory amino acids and neuropsychiatric disorders.Biol. Psychiatry 26, 505–525.
O’Shea RD (2002) Roles and regulation of glutamate transporters in the central nervous system.Clin. Exp. Pharmacol. Physiol. 29, 1018–1023.
Rembach A, BJ Turner, S Bruce, IK Cheah, RL Scott, EC Lopes, CJ Zagami, PM Beart, NS Cheung, SJ Langford and SS Cheema (2004) Antisense peptide nucleic acid targeting GluR3 delays disease onset and progression in the SOD1 G93A mouse model of familial ALS.J. Neurosci. Res. 77, 573–582.
Ridet JL, SK Malhotra, A Privat and FH Gage (1997) Reactive astrocytes: cellular and molecular cues to biological function.Trends Neurosci. 20, 570–577.
Rosen DR, T Siddique, D Patterson, DA Figlewicz, P Sapp, A Hentati, D Donaldson, J Goto, JP O’Regan and HX Deng (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.Nature 362, 59–62.
Rothstein JD, M Dykes-Hoberg, CA Pardo, LA Bristol, L Jin, RW Kuncl, Y Kanai, MA Hediger, Y Wang, JP Schielke and DF Welty (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.Neuron 16, 675–686.
Rothstein JD, G Tsai, RW Kuncl, L Clawson, DR Cornblath, DB Drachman, A Pestronk, BL Stauch and JT Coyle (1990) Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis.Ann. Neurol. 28, 18–25.
Rothstein JD,LJ Martin and RW Kuncl (1992) Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis.New Engl. J. Med. 326, 1464–1468.
Rothstein JD, M Van Kammen, AI Levey, LJ Martin and RW Kuncl (1995) Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis.Ann. Neurol. 38, 73–84.
Schiffer D and V Fiano (2004) Astrogliosis in ALS: possible interpretations according to pathogenetic hypotheses.ALS and Other Motor Neuron Disord. 5, 22–25.
Schlag BD, JR Vondrasek, M Munir, A Kalandadze, OA Zelenaia, JD Rothstein and MB Robinson (1998) Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.Mol. Pharmacol. 53, 355–369.
Scott HL, DV Pow, AE Tannenberg and PR Dodd (2002) Aberrant expression of the glutamate transporter excitatory amino acid transporter 1 (EAAT1) in Alzheimer’s disease.J.Neurosci. 22, RC206 (1–5).
Shaw PJ and CJ Eggett (2000) Molecular factors underlying selective vulnerability of motor neurons to neurodegeneration in amyotrophic lateral sclerosis.J. Neurol. 247, I17–27.
Stanimirovic DB, R Ball and JP Durkin (1997) Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults.Glia 19, 123–134.
Tanaka H, SY Grooms, MV Bennett and RS Zukin (2000) The AMPAR subunit GluR2: still front and center-stage.Brain Res. 886, 190–207.
Trotti D, NC Danbolt and A Volterra (1998) Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration?Trends Pharmacol. Sci. 19, 328–334.
Volterra A, D Trotti, C Tromba, S Floridi and G Racagni (1994) Glutamate uptake inhibition by oxygen free radicals in rat cortical astrocytes.J. Neurosci. 14, 2924–2932.
Voutsinos-Porche B, G Bonvento, K Tanaka, P Steiner, E Welker, JY Chatton, PJ Magistretti and L Pellerin (2003) Glial glutamate transporters mediate a functional metabolic crosstalk between neurons and astrocytes in the mouse developing cortex.Neuron 37, 275–286.
Watanabe T, K Morimoto, T Hirao, H Suwaki, K Watase and K Tanaka (1999) Amygdala-kindled and pentylenetetrazoleinduced seizures in glutamate transporter GLAST-deficient mice.Brain Res. 845, 92–96.
Wu VW and JP Schwartz (1998) Cell culture models for reactive gliosis: new perspectives.J. Neurosci. Res. 51, 675–681.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zagami, C.J., O’shea, R.D., Lau, C.L. et al. Regulation of glutamate transporters in astrocytes: Evidence for a relationship between transporter expression and astrocytic phenotype. neurotox res 7, 143–149 (2005). https://doi.org/10.1007/BF03033783
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF03033783