IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel

Abstract

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease that selectively affects optic nerves and spinal cord. It is considered a severe variant of multiple sclerosis (MS), and frequently is misdiagnosed as MS, but prognosis and optimal treatments differ. A serum immunoglobulin G autoantibody (NMO-IgG) serves as a specific marker for NMO. Here we show that NMO-IgG binds selectively to the aquaporin-4 water channel, a component of the dystroglycan protein complex located in astrocytic foot processes at the blood-brain barrier. NMO may represent the first example of a novel class of autoimmune channelopathy.

Figure 1.

Immunofluorescence reveals NMO-IgG colocalization with AQP4 in mouse tissues. (A) Brain: Virchow-Robin space (pial–astrocyte interface) at the junction of two folia in mouse cerebellar cortex and midbrain. NMO-antigen (green, fluorescein-conjugated anti-human IgG), AQP4 water channel protein (red, rhodamine-conjugated anti–rabbit IgG); merged images yellow. Kidney: colocalization of NMO and AQP4 antigens in distal collecting tubules of medulla. Stomach: basolateral membranes of epithelial cells in deep gastric mucosa. (B) WT mouse brain binds NMO-IgG (panel 1) in a pattern that is indistinguishable from its binding of AQP4-specific IgG (panel 4); pia, subpia, and microvessels are stained. However, AQP4 knockout mouse brain (null) did not bind NMO-IgG (panel 2), and the serum of a control patient who had neuropsychiatric disease did not bind to WT brain (panel 3).

Figure 2.

NMO-IgG binds selectively to AQP4-transfected cells. NMO-IgG (red, Alexa Fluor 594–conjugated anti–human IgG) binds to the plasma membrane of cultured human embryonic kidney (HEK-293) cells transfected with AQP4 (green, [GFP]-AQP4), but not to the plasma membrane of control HEK-293 cells transfected with plasmid encoding GFP only. Nucleus is blue (stained with Hoescht). Overlap staining is yellow in the merged images. Note: GFP produced in control transfected cells is distributed throughout the nucleus and cytoplasm; GFP fused to AQP4 is membrane-bound. Cytoplasmic GFP-AQP4 (presumably in endoplasmic reticulum and transport packages) is not accessible to NMO-IgG in nonpermeabilized cells. Bars, 10 μm.

Figure 3.

NMO-IgG immunoprecipitates GFP-AQP4 but not related dystroglycan complex proteins. (A) Lysates of HEK-293 cells transfected with GFP-AQP4 or GFP vector, and nontransfected cells were separated by SDS PAGE and probed with IgG's specific for GFP, α-syntrophin, β-dystroglycan, or dystrophin (Dp71). Expression of α-syntrophin, β-dystroglycan, or dystrophin (Dp71) did not differ appreciably in any cell lysate. (B) Immune complexes were captured on protein G-agarose after incubating lysates of HEK-293 cells (stably transfected with GFP-vector or GFP-AQP4) with pooled serum from patients who had NMO, pooled serum from control patients who had neuropsychiatric disease, or a rabbit AQP4-specific IgG. The complexes were analyzed by Western blot for GFP-AQP4 and for dystroglycan complex proteins (present in both lysates). NMO-IgG and AQP4-IgG captured GFP-AQP4. Control patients' IgG did not capture GFP-AQP4. No IgG captured α-syntrophin, β-dystroglycan, or Dp71 (dystrophin). The cell lysate lane represents 1.5% of the total input used for immunoprecipitations. IgG from individual patients who had NMO yielded similar results.