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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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Expression of AQP proteins in HEK-293 cells induces EAAT2 protein expression. DNA is stained blue (A and B). (A) EAAT1 (red) is expressed constitutively, regardless of transfection. (B) EAAT2 (red) is localized in the plasma membrane of cells expressing AQP4 or AQP5 (green), but is undetectable in cells transfected with GFP-vector lacking an AQP cDNA. Bars, 10 μm. (C) For RT-PCR, all three cell lines express EAAT2 mRNA, regardless of AQP expression. Markers indicate basepair reference standards. (D) The Western blot shows that EAAT2 protein is up-regulated in cells transfected with AQP4 or AQP5, but not in GFP-vector–transfected cells. (E) EAAT1 protein and actin are expressed constitutively. Markers indicate kilodalton reference standards (D and E). (F) In glutamate transport experiments, GFP-AQP4 cells take up approximately threefold more l-[3H]glutamate than vector-transfected cells (note: Na+ dependence). All experiments were performed a minimum of two times. The error bars represent the standard error of six and four replicates, respectively.
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fig3: Expression of AQP proteins in HEK-293 cells induces EAAT2 protein expression. DNA is stained blue (A and B). (A) EAAT1 (red) is expressed constitutively, regardless of transfection. (B) EAAT2 (red) is localized in the plasma membrane of cells expressing AQP4 or AQP5 (green), but is undetectable in cells transfected with GFP-vector lacking an AQP cDNA. Bars, 10 μm. (C) For RT-PCR, all three cell lines express EAAT2 mRNA, regardless of AQP expression. Markers indicate basepair reference standards. (D) The Western blot shows that EAAT2 protein is up-regulated in cells transfected with AQP4 or AQP5, but not in GFP-vector–transfected cells. (E) EAAT1 protein and actin are expressed constitutively. Markers indicate kilodalton reference standards (D and E). (F) In glutamate transport experiments, GFP-AQP4 cells take up approximately threefold more l-[3H]glutamate than vector-transfected cells (note: Na+ dependence). All experiments were performed a minimum of two times. The error bars represent the standard error of six and four replicates, respectively.

Mentions: AQP4 and EAAT2 are both enriched in the astrocytic endfoot membrane (18). Our finding that both are depleted from plasma membranes of cultured astrocytes exposed to NMO-IgG is consistent with the notion that EAAT2 expression depends on AQP4 expression. To further evaluate the relationship between AQP4 and glutamate transport, we studied the HEK-293 nonneural cell line, comparing EAAT2 expression in cells stably expressing GFP, GFP-AQP4, or a nonneural AQP, AQP5-GFP (Fig. 3). We readily detected EAAT1 in the plasma membranes of cell lines transfected with either GFP vector or GFP-AQP4 (Fig. 3 A). We did not detect EAAT2 in GFP vector-transfected cells (Fig. 3 B), but EAAT2 was strikingly upregulated in the plasma membrane of cells transgenically expressing either AQP4 or AQP5 (Fig. 3 B).


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)

Expression of AQP proteins in HEK-293 cells induces EAAT2 protein expression. DNA is stained blue (A and B). (A) EAAT1 (red) is expressed constitutively, regardless of transfection. (B) EAAT2 (red) is localized in the plasma membrane of cells expressing AQP4 or AQP5 (green), but is undetectable in cells transfected with GFP-vector lacking an AQP cDNA. Bars, 10 μm. (C) For RT-PCR, all three cell lines express EAAT2 mRNA, regardless of AQP expression. Markers indicate basepair reference standards. (D) The Western blot shows that EAAT2 protein is up-regulated in cells transfected with AQP4 or AQP5, but not in GFP-vector–transfected cells. (E) EAAT1 protein and actin are expressed constitutively. Markers indicate kilodalton reference standards (D and E). (F) In glutamate transport experiments, GFP-AQP4 cells take up approximately threefold more l-[3H]glutamate than vector-transfected cells (note: Na+ dependence). All experiments were performed a minimum of two times. The error bars represent the standard error of six and four replicates, respectively.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571922&req=5

fig3: Expression of AQP proteins in HEK-293 cells induces EAAT2 protein expression. DNA is stained blue (A and B). (A) EAAT1 (red) is expressed constitutively, regardless of transfection. (B) EAAT2 (red) is localized in the plasma membrane of cells expressing AQP4 or AQP5 (green), but is undetectable in cells transfected with GFP-vector lacking an AQP cDNA. Bars, 10 μm. (C) For RT-PCR, all three cell lines express EAAT2 mRNA, regardless of AQP expression. Markers indicate basepair reference standards. (D) The Western blot shows that EAAT2 protein is up-regulated in cells transfected with AQP4 or AQP5, but not in GFP-vector–transfected cells. (E) EAAT1 protein and actin are expressed constitutively. Markers indicate kilodalton reference standards (D and E). (F) In glutamate transport experiments, GFP-AQP4 cells take up approximately threefold more l-[3H]glutamate than vector-transfected cells (note: Na+ dependence). All experiments were performed a minimum of two times. The error bars represent the standard error of six and four replicates, respectively.
Mentions: AQP4 and EAAT2 are both enriched in the astrocytic endfoot membrane (18). Our finding that both are depleted from plasma membranes of cultured astrocytes exposed to NMO-IgG is consistent with the notion that EAAT2 expression depends on AQP4 expression. To further evaluate the relationship between AQP4 and glutamate transport, we studied the HEK-293 nonneural cell line, comparing EAAT2 expression in cells stably expressing GFP, GFP-AQP4, or a nonneural AQP, AQP5-GFP (Fig. 3). We readily detected EAAT1 in the plasma membranes of cell lines transfected with either GFP vector or GFP-AQP4 (Fig. 3 A). We did not detect EAAT2 in GFP vector-transfected cells (Fig. 3 B), but EAAT2 was strikingly upregulated in the plasma membrane of cells transgenically expressing either AQP4 or AQP5 (Fig. 3 B).

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