Autoantibodies Against the Purkinje Cell Protein RGS8 in Paraneoplastic Cerebellar Syndrome

Abstract

Objective: To describe the identification of regulator of G-protein signaling 8 (RGS8) as an autoantibody target in patients with cerebellar syndrome associated with lymphoma.

Methods: Sera of 4 patients with a very similar unclassified reactivity against cerebellar Purkinje cells were used in antigen identification experiments. Immunoprecipitations with cerebellar lysates followed by mass spectrometry identified the autoantigen, which was verified by recombinant immunofluorescence assay, immunoblot, and ELISA with the recombinant protein.

Results: The sera and CSF of 4 patients stained the Purkinje cells and molecular layer of the cerebellum. RGS8 was identified as the target antigen in all 4 sera. In a neutralization experiment, recombinant human RGS8 was able to neutralize the autoantibodies' tissue reaction. Patient sera and CSF showed a specific reactivity against recombinant RGS8 in ELISA and immunoblot, whereas no such reactivity was detectable in the controls. Clinical data were available for 2 of the 4 patients, remarkably both presented with cerebellar syndrome accompanied by B-cell lymphoma of the stomach (patient 1, 53 years) or Hodgkin lymphoma (patient 2, 74 years).

Conclusion: Our results indicate that autoantibodies against the intracellular Purkinje cell protein RGS8 represent new markers for paraneoplastic cerebellar syndrome associated with lymphoma.

Classification of evidence: This study provided Class IV evidence that autoantibodies against the intracellular Purkinje cell protein RGS8 are associated with paraneoplastic cerebellar syndrome in lymphoma.

Figure 1. Immunofluorescence Staining of the Cerebellum

(A) Cerebellar cryosections were incubated with patient sera (1:32) or CSF (1:10) (green) in the first step and with Alexa488-labeled goat anti-human IgG in the second step. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). A fine-granular staining of cerebellar molecular layer and Purkinje cells was obtained with the strongest reaction on the Purkinje cells (scale bar 100 µm). (B) Sagittal sections of the murine whole brain were incubated with patient serum 2 (1:32) in the first step and with Alexa488-labeled goat anti-human IgG in the second step. Nuclei were counterstained by incubation with DAPI (blue). The patient serum mainly reacted with the cerebellum. No obvious reactivity against other parts of the brain was observed (scale bar 500 µm).

Figure 2. Immunoprecipitation and Antigen Identification

Lysates of the rat cerebellum were incubated with patient or control serum. Immunocomplexes were enriched with protein-G–coated magnetic beads, eluted by SDS, and subjected to SDS-PAGE analysis followed by staining with colloidal Coomassie (left) or immunoblot with patient or control serum (1:200) (right). Arrows indicate the position of the immunoprecipitated antigen at about 25 kDa, which was identified as RGS8 by MALDI-TOF MS.

Figure 3. Neutralization of Antibody Reaction on the Cerebellum With Recombinant RGS8

Patient sera (1:32, green) were preincubated with 1:10 diluted extracts of HEK293 cells transfected with RGS8 or with empty vector as control. The extract containing RGS8 abolished the immune reaction. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue) (scale bar 100 µm).

Figure 4. Anti-RGS8 Autoantibody Detection in Patient Sera and CSF by Different Immunoassays

(A) Immunoblots with lysates of RGS8-transfected HEK293 cells incubated with 1:200 diluted patient or control sera and anti-human IgG-AP or anti-His antibody and anti-mouse IgG-AP. (B) Results of RGS8 line blot assay with patient or control sera (CS) (1:100). (C) ELISA with purified recombinant RGS8-His with patient or control sera (1:100) and patient or control CSF (1:10) and anti-human IgG POD. Positive and total numbers of sera and CSF are given below the diagrams. (D) IFA with patient or control sera (1:100) and RGS8- or Mock-transfected HEK293 cells incubated with anti-human IgG-Alexa488. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue) (scale bar 50 µm).