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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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In primary astrocytes, NMO-IgG induces internalization of AQP4, impairment of glutamate uptake, or complement activation. (A) Plasma membrane AQP4 (green) and cytoplasmic GFAP (red) after exposure to no human serum, control patient serum, or NMO patient serum. Serum containing NMO-IgG causes rapid translocation of surface AQP4 into cytoplasmic vesicles. Boxed area is enlarged in bottom images. Bars, 10 μm. (B) Quantitation of membrane permeability after exposure to control or NMO serum. Increase in permeability to propidium iodide (PI) greater than twofold by NMO serum required active complement (Δ C′ = inactivated complement). (C) Uptake of l-[3H]glutamate (±Na+-containing buffer) without human serum (white column) or in control (gray column) or NMO serum (crosshatched column). Excess unlabeled glutamate (black column) prevented l-[3H]glutamate uptake. NMO serum reduced l-[3H]glutamate uptake by 50%. The experiment shown in C was performed twice. All other experiments were done at least three times. The error bars represent the standard error of six and four replicates, respectively.
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fig1: In primary astrocytes, NMO-IgG induces internalization of AQP4, impairment of glutamate uptake, or complement activation. (A) Plasma membrane AQP4 (green) and cytoplasmic GFAP (red) after exposure to no human serum, control patient serum, or NMO patient serum. Serum containing NMO-IgG causes rapid translocation of surface AQP4 into cytoplasmic vesicles. Boxed area is enlarged in bottom images. Bars, 10 μm. (B) Quantitation of membrane permeability after exposure to control or NMO serum. Increase in permeability to propidium iodide (PI) greater than twofold by NMO serum required active complement (Δ C′ = inactivated complement). (C) Uptake of l-[3H]glutamate (±Na+-containing buffer) without human serum (white column) or in control (gray column) or NMO serum (crosshatched column). Excess unlabeled glutamate (black column) prevented l-[3H]glutamate uptake. NMO serum reduced l-[3H]glutamate uptake by 50%. The experiment shown in C was performed twice. All other experiments were done at least three times. The error bars represent the standard error of six and four replicates, respectively.

Mentions: We monitored AQP4 distribution after applying NMO or control serum to cerebral astrocytes. Serum containing NMO-IgG, but not control serum, induced rapid down-regulation of surface AQP4 (Fig. 1 A). AQP4 coalesced in cytoplasmic vesicles reminiscent of those observed in GFP-AQP4–transfected nonneural cells exposed to NMO-IgG (6).


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)

In primary astrocytes, NMO-IgG induces internalization of AQP4, impairment of glutamate uptake, or complement activation. (A) Plasma membrane AQP4 (green) and cytoplasmic GFAP (red) after exposure to no human serum, control patient serum, or NMO patient serum. Serum containing NMO-IgG causes rapid translocation of surface AQP4 into cytoplasmic vesicles. Boxed area is enlarged in bottom images. Bars, 10 μm. (B) Quantitation of membrane permeability after exposure to control or NMO serum. Increase in permeability to propidium iodide (PI) greater than twofold by NMO serum required active complement (Δ C′ = inactivated complement). (C) Uptake of l-[3H]glutamate (±Na+-containing buffer) without human serum (white column) or in control (gray column) or NMO serum (crosshatched column). Excess unlabeled glutamate (black column) prevented l-[3H]glutamate uptake. NMO serum reduced l-[3H]glutamate uptake by 50%. The experiment shown in C was performed twice. All other experiments were done at least three 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

fig1: In primary astrocytes, NMO-IgG induces internalization of AQP4, impairment of glutamate uptake, or complement activation. (A) Plasma membrane AQP4 (green) and cytoplasmic GFAP (red) after exposure to no human serum, control patient serum, or NMO patient serum. Serum containing NMO-IgG causes rapid translocation of surface AQP4 into cytoplasmic vesicles. Boxed area is enlarged in bottom images. Bars, 10 μm. (B) Quantitation of membrane permeability after exposure to control or NMO serum. Increase in permeability to propidium iodide (PI) greater than twofold by NMO serum required active complement (Δ C′ = inactivated complement). (C) Uptake of l-[3H]glutamate (±Na+-containing buffer) without human serum (white column) or in control (gray column) or NMO serum (crosshatched column). Excess unlabeled glutamate (black column) prevented l-[3H]glutamate uptake. NMO serum reduced l-[3H]glutamate uptake by 50%. The experiment shown in C was performed twice. All other experiments were done at least three times. The error bars represent the standard error of six and four replicates, respectively.
Mentions: We monitored AQP4 distribution after applying NMO or control serum to cerebral astrocytes. Serum containing NMO-IgG, but not control serum, induced rapid down-regulation of surface AQP4 (Fig. 1 A). AQP4 coalesced in cytoplasmic vesicles reminiscent of those observed in GFP-AQP4–transfected nonneural cells exposed to NMO-IgG (6).

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