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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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NMO-IgG modulation of AQP4 from transfected HEK-293 membranes down-regulates EAAT2 expression. GFP-AQP4– or AQP5-GFP–transfected cells (green) were probed with anti-EAAT2 or –EAAT1-IgG (red) after exposure to control or NMO patient serum (37°C; 4 h). Nuclear DNA is blue. (A) NMO serum caused membrane loss of both AQP4 and EAAT2; control serum did not affect the distribution of either. Enlarged images show both EAAT2 and AQP4 internalized in cytoplasmic vesicles after exposure to NMO serum. (B) Staining with early endosome marker (EEA1, purple) after 10-min exposure to NMO serum shows colocalization of both GFP-AQP4 and EAAT2 (arrows) in endocytotic vesicles (white spots in merged image). (A and B) Boxed areas are enlarged in the bottom image. (C) NMO serum does not selectively affect EAAT2 in cells transfected with AQP5. EAAT1 is not affected by NMO or control serum in cells transfected with AQP4 (D) or AQP5 (E). (F) Western blot shows NMO-IgG or IgG specific for GFP, AQP4, or EAAT2, capture GFP-AQP4 protein; GFP-AQP4 does not coprecipitate with control patient IgG or EAAT1-specific IgG. Markers indicate kilodalton reference standards. All experiments were performed a minimum of two times. Bars: (A, C, D, and E) 10 μm; or (B) 20 μm.
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fig4: NMO-IgG modulation of AQP4 from transfected HEK-293 membranes down-regulates EAAT2 expression. GFP-AQP4– or AQP5-GFP–transfected cells (green) were probed with anti-EAAT2 or –EAAT1-IgG (red) after exposure to control or NMO patient serum (37°C; 4 h). Nuclear DNA is blue. (A) NMO serum caused membrane loss of both AQP4 and EAAT2; control serum did not affect the distribution of either. Enlarged images show both EAAT2 and AQP4 internalized in cytoplasmic vesicles after exposure to NMO serum. (B) Staining with early endosome marker (EEA1, purple) after 10-min exposure to NMO serum shows colocalization of both GFP-AQP4 and EAAT2 (arrows) in endocytotic vesicles (white spots in merged image). (A and B) Boxed areas are enlarged in the bottom image. (C) NMO serum does not selectively affect EAAT2 in cells transfected with AQP5. EAAT1 is not affected by NMO or control serum in cells transfected with AQP4 (D) or AQP5 (E). (F) Western blot shows NMO-IgG or IgG specific for GFP, AQP4, or EAAT2, capture GFP-AQP4 protein; GFP-AQP4 does not coprecipitate with control patient IgG or EAAT1-specific IgG. Markers indicate kilodalton reference standards. All experiments were performed a minimum of two times. Bars: (A, C, D, and E) 10 μm; or (B) 20 μm.

Mentions: Our data indicate that the concentration of AQP4 protein in the plasma membrane and Na+-dependent glutamate transport are both reduced by exposure of primary astrocytes to NMO-IgG (Fig. 1). Our observations in differentiated CG-4 type 2 astrocytes suggest that the effect on glutamate transport in primary astrocytes is caused by plasma membrane loss of EAAT2 (Fig. 2). To further investigate the association between AQP4 and EAAT2, we evaluated the effect of NMO and control sera on both EAAT1 and EAAT2. Control serum did not appreciably affect localization or expression of the EAAT2 transporter. However, serum containing NMO-IgG induced rapid surface down-regulation of both GFP-AQP4 and EAAT2 (Fig. 4 A). Higher magnification (Fig. 4 A) revealed apparent colocalization of EAAT2 and AQP4 in early endosomal vesicles to which we previously demonstrated AQP4 translocation after exposure to NMO serum (6). We evaluated the localization of AQP4 and the early endosome antigen-1 marker after exposing the cells to NMO serum for 10 min. The white color that resulted when the images were merged suggested partial colocalization of AQP4 and EAAT2 in early endosomes (Fig. 4 B). However, separate vesicles in close proximity or overlapping in the z axis would yield the same result.


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)

NMO-IgG modulation of AQP4 from transfected HEK-293 membranes down-regulates EAAT2 expression. GFP-AQP4– or AQP5-GFP–transfected cells (green) were probed with anti-EAAT2 or –EAAT1-IgG (red) after exposure to control or NMO patient serum (37°C; 4 h). Nuclear DNA is blue. (A) NMO serum caused membrane loss of both AQP4 and EAAT2; control serum did not affect the distribution of either. Enlarged images show both EAAT2 and AQP4 internalized in cytoplasmic vesicles after exposure to NMO serum. (B) Staining with early endosome marker (EEA1, purple) after 10-min exposure to NMO serum shows colocalization of both GFP-AQP4 and EAAT2 (arrows) in endocytotic vesicles (white spots in merged image). (A and B) Boxed areas are enlarged in the bottom image. (C) NMO serum does not selectively affect EAAT2 in cells transfected with AQP5. EAAT1 is not affected by NMO or control serum in cells transfected with AQP4 (D) or AQP5 (E). (F) Western blot shows NMO-IgG or IgG specific for GFP, AQP4, or EAAT2, capture GFP-AQP4 protein; GFP-AQP4 does not coprecipitate with control patient IgG or EAAT1-specific IgG. Markers indicate kilodalton reference standards. All experiments were performed a minimum of two times. Bars: (A, C, D, and E) 10 μm; or (B) 20 μm.
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Related In: Results  -  Collection

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fig4: NMO-IgG modulation of AQP4 from transfected HEK-293 membranes down-regulates EAAT2 expression. GFP-AQP4– or AQP5-GFP–transfected cells (green) were probed with anti-EAAT2 or –EAAT1-IgG (red) after exposure to control or NMO patient serum (37°C; 4 h). Nuclear DNA is blue. (A) NMO serum caused membrane loss of both AQP4 and EAAT2; control serum did not affect the distribution of either. Enlarged images show both EAAT2 and AQP4 internalized in cytoplasmic vesicles after exposure to NMO serum. (B) Staining with early endosome marker (EEA1, purple) after 10-min exposure to NMO serum shows colocalization of both GFP-AQP4 and EAAT2 (arrows) in endocytotic vesicles (white spots in merged image). (A and B) Boxed areas are enlarged in the bottom image. (C) NMO serum does not selectively affect EAAT2 in cells transfected with AQP5. EAAT1 is not affected by NMO or control serum in cells transfected with AQP4 (D) or AQP5 (E). (F) Western blot shows NMO-IgG or IgG specific for GFP, AQP4, or EAAT2, capture GFP-AQP4 protein; GFP-AQP4 does not coprecipitate with control patient IgG or EAAT1-specific IgG. Markers indicate kilodalton reference standards. All experiments were performed a minimum of two times. Bars: (A, C, D, and E) 10 μm; or (B) 20 μm.
Mentions: Our data indicate that the concentration of AQP4 protein in the plasma membrane and Na+-dependent glutamate transport are both reduced by exposure of primary astrocytes to NMO-IgG (Fig. 1). Our observations in differentiated CG-4 type 2 astrocytes suggest that the effect on glutamate transport in primary astrocytes is caused by plasma membrane loss of EAAT2 (Fig. 2). To further investigate the association between AQP4 and EAAT2, we evaluated the effect of NMO and control sera on both EAAT1 and EAAT2. Control serum did not appreciably affect localization or expression of the EAAT2 transporter. However, serum containing NMO-IgG induced rapid surface down-regulation of both GFP-AQP4 and EAAT2 (Fig. 4 A). Higher magnification (Fig. 4 A) revealed apparent colocalization of EAAT2 and AQP4 in early endosomal vesicles to which we previously demonstrated AQP4 translocation after exposure to NMO serum (6). We evaluated the localization of AQP4 and the early endosome antigen-1 marker after exposing the cells to NMO serum for 10 min. The white color that resulted when the images were merged suggested partial colocalization of AQP4 and EAAT2 in early endosomes (Fig. 4 B). However, separate vesicles in close proximity or overlapping in the z axis would yield the same result.

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