Neurochondrin is a neuronal target antigen in autoimmune cerebellar degeneration

Abstract

Objective: To report on a novel neuronal target antigen in 3 patients with autoimmune cerebellar degeneration.

Methods: Three patients with subacute to chronic cerebellar ataxia and controls underwent detailed clinical and neuropsychological assessment together with quantitative high-resolution structural MRI. Sera and CSF were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IFA) and immunoblot. Immunoprecipitation with lysates of hippocampus and cerebellum combined with mass spectrometric analysis was used to identify the autoantigen, which was verified by recombinant expression in HEK293 cells and use in several immunoassays. Multiparameter flow cytometry was performed on peripheral blood and CSF, and peripheral blood was subjected to T-cell receptor spectratyping.

Results: Patients presented with a subacute to chronic cerebellar and brainstem syndrome. MRI was consistent with cortical and cerebellar gray matter atrophy associated with subsequent neuroaxonal degeneration. IFA screening revealed strong immunoglobulin G1 reactivity in sera and CSF with hippocampal and cerebellar molecular and granular layers, but not with a panel of 30 recombinantly expressed established neural autoantigens. Neurochondrin was subsequently identified as the target antigen, verified by IFA and immunoblot with HEK293 cells expressing human neurochondrin as well as the ability of recombinant neurochondrin to neutralize the autoantibodies' tissue reaction. Immune phenotyping revealed intrathecal accumulation and activation of B and T cells during the acute but not chronic phase of the disease. T-cell receptor spectratyping suggested an antigen-specific T-cell response accompanying the formation of antineurochondrin autoantibodies. No such neurochondrin reactivity was found in control cohorts of various neural autoantibody-associated neurologic syndromes, relapsing-remitting multiple sclerosis, cerebellar type of multiple system atrophy, hereditary cerebellar ataxias, other neurologic disorders, or healthy donors.

Conclusion: Neurochondrin is a neuronal target antigen in autoimmune cerebellar degeneration.

Figure 1. Immunofluorescence staining of central nervous tissues

Cryosections were incubated with patient or control sera (1:100) or CSF (undiluted) in the first step, and with Alexa488-labelled goat anti-human immunoglobulin G in the second step. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). A fine granular staining of both granular and molecular layers was obtained with the strongest reaction on the granular cell layer of rat hippocampus. ×200 magnification.

Figure 2. Immunoprecipitation and antigen identification

Lysates of rat hippocampus and cerebellum were incubated with patient or control sera (1:33). Immunocomplexes were isolated with protein-G-coated magnetic beads, eluted by sodium dodecyl sulfate, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis followed by (A) staining with colloidal Coomassie or (B) Western blot using polyclonal rabbit antineurochondrin. Frames indicate the position of the immunoprecipitated antigen at about 75 kDa.

Figure 3. Double-staining of hippocampal tissue and cells with patient serum and rabbit antineurochondrin antibody

Immunofluorescence staining of rat hippocampus tissue section (A) or formalin-fixed and TritonX-100 permeabilized rat hippocampal neurons (B) with patient sera (green) and antineurochondrin antibody (red). Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). (A, B) ×200 magnification.

Figure 4. Verification of neurochondrin as the novel autoantigen by indirect immunofluorescence

(A) Indirect immunofluorescence using acetone-fixed neurochondrin or mock-transfected HEK293 cells incubated with 1:1,000 diluted serum or 1:10 diluted CSF of patient 1, patient 2, or a healthy control (green). (B) Neutralization of immunofluorescence reaction on neuronal tissues. Patient serum (green) was preincubated with extracts of HEK293 cells transfected with neurochondrin or with empty vector as control. The extract containing neurochondrin abolished the immune reaction. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). (A) Hippocampus tissue section: ×200 magnification. (B) Rat hippocampal neurons: ×400 magnification.