Voltage-gated calcium channel autoimmune cerebellar degeneration: Case and study of cytotoxicity

Abstract

Objectives: To describe response to treatment in a patient with autoantibodies against voltage-gated calcium channels (VGCCs) who presented with autoimmune cerebellar degeneration and subsequently developed Lambert-Eaton myasthenic syndrome (LEMS), and to study the effect of the patient's autoantibodies on Purkinje cells in rat cerebellar slice cultures.

Methods: Case report and study of rat cerebellar slice cultures incubated with patient VGCC autoantibodies.

Results: A 53-year-old man developed progressive incoordination with ataxic speech. Laboratory evaluation revealed VGCC autoantibodies without other antineuronal autoantibodies. Whole-body PET scans 6 and 12 months after presentation detected no malignancy. The patient improved significantly with IV immunoglobulin G (IgG), prednisone, and mycophenolate mofetil, but worsened after IV IgG was halted secondary to aseptic meningitis. He subsequently developed weakness with electrodiagnostic evidence of LEMS. The patient's IgG bound to Purkinje cells in rat cerebellar slice cultures, followed by neuronal death. Reactivity of the patient's autoantibodies with VGCCs was confirmed by blocking studies with defined VGCC antibodies.

Conclusions: Autoimmune cerebellar degeneration associated with VGCC autoantibodies may precede onset of LEMS and may improve with immunosuppressive treatment. Binding of anti-VGCC antibodies to Purkinje cells in cerebellar slice cultures may be followed by cell death. Patients with anti-VGCC autoantibodies may be at risk of irreversible neurologic injury over time, and treatment should be initiated early.

Figure 1. VGCC antibody uptake in cerebellar slice culture

Slice culture of rat cerebellum incubated with patient serum containing VGCC autoantibodies and examined at different points in time. (A) Slice culture incubated with normal serum for 144 hours, treated with SYTOX dyes to detect cell death, and labeled with Cy5-conjugated anti-human IgG, showing absence both of neuronal antibody binding and of SYTOX staining indicative of cell death. (B, C) Slice cultures incubated for 48 and 72 hours with serum containing VGCC autoantibodies. In these images, binding of the patient's IgG is labeled red; rare dead cells outside the Purkinje cell layer are labeled green. At 48 hours, IgG can be detected throughout the neuropil with labeling of occasional Purkinje cells (arrows) (B). By 72 hours, there is extensive binding of IgG to Purkinje cell membranes (arrows) (C). (D–F) A slice culture incubated with patient serum for 144 hours and labeled with Cy5-conjugated anti-human IgG (D), stained with SYTOX dyes to detect cell death (E), and as a merged image (F). In cultures at this time point, some Purkinje cells continued to show surface binding of antibody panels (G–I). In other areas, however, Purkinje cells showed uptake of IgG with cytoplasmic and nuclear labeling (D); some IgG-containing Purkinje cells demonstrated staining with SYTOX dyes, indicating cell membrane disruption and death (E, F), whereas others excluded SYTOX dyes, indicating antibody uptake by viable Purkinje cells. Purkinje cell antibody binding and cell death at 144 hours are shown at higher power in panels G through I. Arrows indicate the same cells in panels D–F and in G–I. Scale bars = 20 μm. IgG = immunoglobulin G; VGCC = voltage-gated calcium channel.

Figure 2. Binding of the patient's IgG to Purkinje cells is inhibited by pretreatment of rat tissue with anti-VGCC antibodies, confirming specificity of the patient's antibody response

Adult rat tissue was fixed, sectioned, and treated with an antigen retrieval system before blocking for 1 hour with serial concentrations of commercially obtained rabbit anti-VGCC antibodies directed against the α_1A subunit of the VGCC receptor before being exposed to a 1:200 dilution of patient serum for 24 hours. Tissue was then labeled with Cy5-conjugated donkey anti-human IgG to detect the patient's antibodies and fluorescein isothiocyanate–conjugated anti-rabbit IgG to detect the commercial anti-VGCC antibody. Pretreatment with commercial anti-VGCC antibody inhibited binding of the patient's IgG to Purkinje cells in a dose-dependent manner, confirming the specificity of the patient's antibody for the VGCC. Scale bars = 20 μm. IgG = immunoglobulin G; VGCC = voltage-gated calcium channel.