Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

O'Shea, Ross D; Lau, Chew L.; Farso, Mark; Diwakarla, Shanti; Zagami, Chrissandra J.; Svendsen, Brian B; Feeney, Sandra J.; Callaway, Jennifer K.; Jones, Nicole M.; Pow, David V.; Danbolt, Niels Christian; Jarrott, Bevyn; Beart, Philip M

doi:10.1016/j.neuint.2005.12.028

Cited by 54 publications

(59 citation statements)

References 28 publications

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“…Transport of Glu into astrocytes is another of their functions with EAAT2 (~1% of proteome) considered an astrocyte-specific protein like the cytoskeletal marker glial fibrillary acidic protein (GFAP) (Cahoy et al, 2008) -additionally both proteins co-localize and GFAP modulates EAAT function (Hughes et al, 2004;Zhou and Sutherland, 2004;Shobha et al, 2007). Indeed, GFAP expression increases in astrocytes when they become reactive (termed 'astrogliosis') (Ridet et al, 1997;Maragakis and Rothstein, 2006) with probable alterations in EAAT biology, as we have shown an interdependence between astrocytic phenotype and EAAT function that involves altered transporter activity and/or localization (O'Shea et al, 2006;Zagami et al, 2009;Lau et al, 2010). Astrogliosis is now documented to be a graded event with both pro-survival and destructive components (Sofroniew and Vinters, 2010), and it is apparent that maintenance of EAAT function during brain perturbation (i.e.…”

Section: Introductionmentioning

confidence: 81%

The Rho kinase inhibitor Fasudil up‐regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

Lau

O'Shea

Broberg

et al. 2011

British J Pharmacology

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BACKGROUND AND PURPOSE Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology.EXPERIMENTAL APPROACH The functional activity of glutamate transport was determined in murine cultured astrocytes after exposure to the ROCK inhibitors Fasudil (HA‐1077) and Y27632 using biochemical, molecular and morphological approaches. Cytochemical analyses assessed changes in astrocytic morphology, F‐/G‐actin, and localizations of EAAT1/2.RESULTS Fasudil and Y27632 increased [3H]‐d‐aspartate (d‐Asp) uptake into astrocytes, and the action of Fasudil was time‐dependent and concentration‐related. The rapid stellation of astrocytes (glial fibrillary acidic protein immunocytochemistry) induced by Fasudil was accompanied by reduced phalloidin staining of F‐actin and increased Vmax for [3H]‐d‐Asp uptake. Immunoblotting after biotinylation demonstrated that Fasudil increased the expression of EAAT1 and EAAT2 on the cell surface. Immunocytochemistry indicated that Fasudil induced prominent labelling of astrocytic processes by EAAT1/2.CONCLUSION AND IMPLICATIONS These data show for the first time that ROCK plays a major role in determining the cell surface expression of EAAT1/2, providing new evidence for an association between transporter function and astrocytic phenotype. ROCK inhibitors, via the actin cytoskeleton, effect a consequent elevation of glutamate transporter function – this activity profile may contribute to their beneficial actions in neuropathologies.

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Section: Introductionmentioning

confidence: 81%

The Rho kinase inhibitor Fasudil up‐regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

Lau

O'Shea

Broberg

et al. 2011

British J Pharmacology

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“…they displayed expression of the glial fibrillary acidic protein (GFAP). Furthermore, LPS contributed to enhanced activity and expression of the glial membrane excitatory amino acid transporter GLT-1 (EAAT2) [13], which indicates that increased expression of the astrocytic GLT-transporter protects neurons against the toxicity of excess glutamate in the extracellular space. LPS alone administered to animals may activate astrocytes to produce and release growth factors, e.g.…”

Section: Discussionmentioning

confidence: 99%

Investigations of hippocampal astrocytes in lipopolysaccharide-preconditioned rats in the pilocarpine model of epilepsy

Jaworska-Adamu¹,

Dmowska²,

Cybulska³

et al. 2011

Folia Histochem Cytobiol.

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Abstract: Abstract: Abstract: The present paper is the first work to determine the effect of lipopolysaccharide (LPS) in the pilocarpine model of epilepsy on the morphology of rat hippocampal astrocytes in vivo. The study involved adult male Wistar rats, which 72 hours prior to administration of pilocarpine hydrochloride (PILO) were intraperitoneally (ip) preconditioned with LPS at a dose of 0.5 mg/kg b.w. The control animals were administered (ip) saline or LPS alone. The astrocytes in the control animals displayed characteristic stellate morphology. Examinations of the astrocytes were performed on days one, three and 21 of the pilocarpine model of epilepsy (i.e. in the acute, silent and chronic periods). The astrocytes of the CA1 and CA3 pyramidal layers of the hippocampus were observed and analyzed at the structural and ultrastructural levels. It was demonstrated that on days one and three, glial cells from both the nonpreconditioned and the LPS-preconditioned animals displayed similar reactive changes, manifesting themselves as swelling of cell bodies, glial processes, and astrocytosis. Moreover, reduction in cell organelles aggregated at one pole and the presence of vacuoles were observed. The most pronounced astrogliosis and cell swelling occurred on day 21. We conclude that LPS has no effect on the morphology of astrocytes in the pilocarpine model of epilepsy, unlike the results obtained by other authors in vitro.

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“…After stimulation with PMA and inhibitors, cell surface proteins were biotinylated with 0.5 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce) in PBS (with 0.1 mM CaCl 2 and 0.1 mM MgCl 2 ), pH 8.0, for 30 min at 4°C with gentle rocking (O'Shea et al, 2006). Excess biotin was quenched by washing three times in 100 mM glycin in PBS, after which the cells were lysed in RIPA buffer with Complete Protease Inhibitor Cocktail (Roche).…”

Section: Methodsmentioning

confidence: 99%

Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking

Nissen‐Meyer¹,

Popescu²,

Hamdani³

et al. 2011

J. Neurosci.

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Molecular mechanisms involved in the replenishment of the fast neurotransmitters glutamate and GABA are poorly understood. Glutamine sustains their generation. However, glutamine formation from the recycled transmitters is confined to glial processes and requires facilitators for its translocation across the glial and neuronal membranes. Indeed, glial processes are enriched with the system N transporter SN1 (Slc38a3), which, by bidirectional transport, maintains steady extracellular glutamine levels and thereby furnishes neurons with the primary precursor for fast neurotransmitters. We now demonstrate that SN1 is phosphorylated by protein kinase C␣ (PKC␣) and PKC␥. Electrophysiological characterization shows that phosphorylation reduces V max dramatically, whereas no significant effects are seen on the K m . Phosphorylation occurs specifically at a single serine residue (S52) in the N-terminal rat (Rattus norvegicus) SN1 and results in sequestration of the protein into intracellular reservoirs. Prolonged activation of PKC results in partial degradation of SN1. These results provide the first demonstration of phosphorylation of SN1 and regulation of its activity at the plasma membrane. Interestingly, membrane trafficking of SN1 resembles that of the glutamate transporter GLT and the glutamate-aspartate transporter GLAST: it involves the same PKC isoforms and occurs in the same glial processes. This suggests that the glutamate/GABA-glutamine cycle may be modified at two key points by similar signaling events and unmasks a prominent role for PKC-dependent phosphorylation. Our data suggest that extracellular glutamine levels may be fine-tuned by dynamic regulation of glial SN1 activity, which may impact on transmitter generation, contribute to defining quantal size, and have profound effects on synaptic plasticity.

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Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

Cited by 54 publications

References 28 publications

The Rho kinase inhibitor Fasudil up‐regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

The Rho kinase inhibitor Fasudil up‐regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

Investigations of hippocampal astrocytes in lipopolysaccharide-preconditioned rats in the pilocarpine model of epilepsy

Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking

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