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Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2.

Hinson SR, Roemer SF, Lucchinetti CF, Fryer JP, Kryzer TJ, Chamberlain JL, Howe CL, Pittock SJ, Lennon VA - J. Exp. Med. (2008)

Bottom Line: The effect of NMO-IgG on astrocytes has not been studied.Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data.Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
Neuromyelitis optica (NMO)-immunoglobulin G (IgG) is a clinically validated serum biomarker that distinguishes relapsing central nervous system (CNS) inflammatory demyelinating disorders related to NMO from multiple sclerosis. This autoantibody targets astrocytic aquaporin-4 (AQP4) water channels. Clinical, radiological, and immunopathological data suggest that NMO-IgG might be pathogenic. Characteristic CNS lesions exhibit selective depletion of AQP4, with and without associated myelin loss; focal vasculocentric deposits of IgG, IgM, and complement; prominent edema; and inflammation. The effect of NMO-IgG on astrocytes has not been studied. In this study, we demonstrate that exposure to NMO patient serum and active complement compromises the membrane integrity of CNS-derived astrocytes. Without complement, astrocytic membranes remain intact, but AQP4 is endocytosed with concomitant loss of Na(+)-dependent glutamate transport and loss of the excitatory amino acid transporter 2 (EAAT2) . Our data suggest that EAAT2 and AQP4 exist in astrocytic membranes as a macromolecular complex. Transport-competent EAAT2 protein is up-regulated in differentiating astrocyte progenitors and in nonneural cells expressing AQP4 transgenically. Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data. Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.

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CG-4 glial cells in astrocytic differentiation medium up-regulate both AQP4 and EAAT2 expression; NMO serum down-regulates both. (A) CG-4 cells cultured in proliferation medium (CG4-PM) do not express cytoplasmic GFAP (red). After 7 d in astrocytic differentiation medium (CG4-AM), a subset of cells contains brilliant cytoplasmic GFAP. (B) The Western blot shows that undifferentiated CG4-PM (PM) cell lysates contain minimal EAAT2 or AQP4; both proteins are up-regulated in CG4-AM (AM). Markers indicate kilodalton reference standards. (C) AQP4 (green) and EAAT2 (red) are negligible in CG4-PM cells, but are up-regulated on the surface of CG4-AM cells; NMO serum, but not control patient serum, depletes both. DNA is blue (A and C). All experiments were performed a minimum of two times. Bars, 5 μm.
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fig2: CG-4 glial cells in astrocytic differentiation medium up-regulate both AQP4 and EAAT2 expression; NMO serum down-regulates both. (A) CG-4 cells cultured in proliferation medium (CG4-PM) do not express cytoplasmic GFAP (red). After 7 d in astrocytic differentiation medium (CG4-AM), a subset of cells contains brilliant cytoplasmic GFAP. (B) The Western blot shows that undifferentiated CG4-PM (PM) cell lysates contain minimal EAAT2 or AQP4; both proteins are up-regulated in CG4-AM (AM). Markers indicate kilodalton reference standards. (C) AQP4 (green) and EAAT2 (red) are negligible in CG4-PM cells, but are up-regulated on the surface of CG4-AM cells; NMO serum, but not control patient serum, depletes both. DNA is blue (A and C). All experiments were performed a minimum of two times. Bars, 5 μm.

Mentions: Attenuation of astrocytic l-[3H]glutamate uptake after exposure to NMO-IgG parallels the loss of AQP4 protein. To investigate the possibility that EAAT2 protein may be lost secondary to loss of surface AQP4, we used EAAT2-specific IgG to follow the fate of EAAT2 after exposing astrocytes to NMO-IgG. As noted by Stanimirovic et al. (16), we confirmed that EAAT1 and EAAT2 glutamate transporter levels in primary rat astrocyte membranes are too low to detect by immunofluorescence staining. We therefore investigated EAAT2 expression in the bipotential glial cell line CG-4, which is derived from O2-A progenitor cells in the developing rat CNS. In prescribed culture conditions (17), CG-4 cells differentiate into type 2 astrocytes. In proliferation-promoting medium (containing bFGF and PDGF), the cells (CG4-PM) lack GFAP intermediate filaments (Fig. 2 A). However, in medium with growth factors replaced by a high concentration of fetal bovine serum, type 2 astrocyte differentiation is evident in the cells (CG4-AM) by morphology and GFAP immunoreactivity (Fig. 2 A). In cells grown for 7 d in astrocyte differentiation medium, plasma membrane expression of both AQP4 and EAAT2 is enhanced (Fig. 2, B and C). However, when NMO serum is added to CG4-AM cells at day 7, both AQP4 and EAAT2 are depleted from the plasma membrane (Fig. 2 C). Addition of control sera in identical conditions does not discernibly affect expression of either AQP4 or EAAT2 (Fig. 2 C). Concomitant loss of both EAAT2 and AQP4 plausibly explains the reduced glutamate transport that we observed in cultured astrocytes exposed to NMO-IgG.


Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2.

Hinson SR, Roemer SF, Lucchinetti CF, Fryer JP, Kryzer TJ, Chamberlain JL, Howe CL, Pittock SJ, Lennon VA - J. Exp. Med. (2008)

CG-4 glial cells in astrocytic differentiation medium up-regulate both AQP4 and EAAT2 expression; NMO serum down-regulates both. (A) CG-4 cells cultured in proliferation medium (CG4-PM) do not express cytoplasmic GFAP (red). After 7 d in astrocytic differentiation medium (CG4-AM), a subset of cells contains brilliant cytoplasmic GFAP. (B) The Western blot shows that undifferentiated CG4-PM (PM) cell lysates contain minimal EAAT2 or AQP4; both proteins are up-regulated in CG4-AM (AM). Markers indicate kilodalton reference standards. (C) AQP4 (green) and EAAT2 (red) are negligible in CG4-PM cells, but are up-regulated on the surface of CG4-AM cells; NMO serum, but not control patient serum, depletes both. DNA is blue (A and C). All experiments were performed a minimum of two times. Bars, 5 μm.
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fig2: CG-4 glial cells in astrocytic differentiation medium up-regulate both AQP4 and EAAT2 expression; NMO serum down-regulates both. (A) CG-4 cells cultured in proliferation medium (CG4-PM) do not express cytoplasmic GFAP (red). After 7 d in astrocytic differentiation medium (CG4-AM), a subset of cells contains brilliant cytoplasmic GFAP. (B) The Western blot shows that undifferentiated CG4-PM (PM) cell lysates contain minimal EAAT2 or AQP4; both proteins are up-regulated in CG4-AM (AM). Markers indicate kilodalton reference standards. (C) AQP4 (green) and EAAT2 (red) are negligible in CG4-PM cells, but are up-regulated on the surface of CG4-AM cells; NMO serum, but not control patient serum, depletes both. DNA is blue (A and C). All experiments were performed a minimum of two times. Bars, 5 μm.
Mentions: Attenuation of astrocytic l-[3H]glutamate uptake after exposure to NMO-IgG parallels the loss of AQP4 protein. To investigate the possibility that EAAT2 protein may be lost secondary to loss of surface AQP4, we used EAAT2-specific IgG to follow the fate of EAAT2 after exposing astrocytes to NMO-IgG. As noted by Stanimirovic et al. (16), we confirmed that EAAT1 and EAAT2 glutamate transporter levels in primary rat astrocyte membranes are too low to detect by immunofluorescence staining. We therefore investigated EAAT2 expression in the bipotential glial cell line CG-4, which is derived from O2-A progenitor cells in the developing rat CNS. In prescribed culture conditions (17), CG-4 cells differentiate into type 2 astrocytes. In proliferation-promoting medium (containing bFGF and PDGF), the cells (CG4-PM) lack GFAP intermediate filaments (Fig. 2 A). However, in medium with growth factors replaced by a high concentration of fetal bovine serum, type 2 astrocyte differentiation is evident in the cells (CG4-AM) by morphology and GFAP immunoreactivity (Fig. 2 A). In cells grown for 7 d in astrocyte differentiation medium, plasma membrane expression of both AQP4 and EAAT2 is enhanced (Fig. 2, B and C). However, when NMO serum is added to CG4-AM cells at day 7, both AQP4 and EAAT2 are depleted from the plasma membrane (Fig. 2 C). Addition of control sera in identical conditions does not discernibly affect expression of either AQP4 or EAAT2 (Fig. 2 C). Concomitant loss of both EAAT2 and AQP4 plausibly explains the reduced glutamate transport that we observed in cultured astrocytes exposed to NMO-IgG.

Bottom Line: The effect of NMO-IgG on astrocytes has not been studied.Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data.Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
Neuromyelitis optica (NMO)-immunoglobulin G (IgG) is a clinically validated serum biomarker that distinguishes relapsing central nervous system (CNS) inflammatory demyelinating disorders related to NMO from multiple sclerosis. This autoantibody targets astrocytic aquaporin-4 (AQP4) water channels. Clinical, radiological, and immunopathological data suggest that NMO-IgG might be pathogenic. Characteristic CNS lesions exhibit selective depletion of AQP4, with and without associated myelin loss; focal vasculocentric deposits of IgG, IgM, and complement; prominent edema; and inflammation. The effect of NMO-IgG on astrocytes has not been studied. In this study, we demonstrate that exposure to NMO patient serum and active complement compromises the membrane integrity of CNS-derived astrocytes. Without complement, astrocytic membranes remain intact, but AQP4 is endocytosed with concomitant loss of Na(+)-dependent glutamate transport and loss of the excitatory amino acid transporter 2 (EAAT2) . Our data suggest that EAAT2 and AQP4 exist in astrocytic membranes as a macromolecular complex. Transport-competent EAAT2 protein is up-regulated in differentiating astrocyte progenitors and in nonneural cells expressing AQP4 transgenically. Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data. Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.

Show MeSH
Related in: MedlinePlus