Septin-3 autoimmunity in patients with paraneoplastic cerebellar ataxia

Abstract

Background: Septins are cytoskeletal proteins with filament forming capabilities, which have multiple roles during cell division, cellular polarization, morphogenesis, and membrane trafficking. Autoantibodies against septin-5 are associated with non-paraneoplastic cerebellar ataxia, and autoantibodies against septin-7 with encephalopathy with prominent neuropsychiatric features. Here, we report on newly identified autoantibodies against septin-3 in patients with paraneoplastic cerebellar ataxia. We also propose a strategy for anti-septin autoantibody determination.

Methods: Sera from three patients producing similar immunofluorescence staining patterns on cerebellar and hippocampal sections were subjected to immunoprecipitation followed by mass spectrometry. The identified candidate antigens, all of which were septins, were expressed recombinantly in HEK293 cells either individually, as complexes, or combinations missing individual septins, for use in recombinant cell-based indirect immunofluorescence assays (RC-IIFA). Specificity for septin-3 was further confirmed by tissue IIFA neutralization experiments. Finally, tumor tissue sections were analyzed immunohistochemically for septin-3 expression.

Results: Immunoprecipitation with rat cerebellum lysate revealed septin-3, -5, -6, -7, and -11 as candidate target antigens. Sera of all three patients reacted with recombinant cells co-expressing septin-3/5/6/7/11, while none of 149 healthy control sera was similarly reactive. In RC-IIFAs the patient sera recognized only cells expressing septin-3, individually and in complexes. Incubation of patient sera with five different septin combinations, each missing one of the five septins, confirmed the autoantibodies' specificity for septin-3. The tissue IIFA reactivity of patient serum was abolished by pre-incubation with HEK293 cell lysates overexpressing the septin-3/5/6/7/11 complex or septin-3 alone, but not with HEK293 cell lysates overexpressing septin-5 as control. All three patients had cancers (2 × melanoma, 1 × small cell lung cancer), presented with progressive cerebellar syndromes, and responded poorly to immunotherapy. Expression of septin-3 was demonstrated in resected tumor tissue available from one patient.

Conclusions: Septin-3 is a novel autoantibody target in patients with paraneoplastic cerebellar syndromes. Based on our findings, RC-IIFA with HEK293 cells expressing the septin-3/5/6/7/11 complex may serve as a screening tool to investigate anti-septin autoantibodies in serological samples with a characteristic staining pattern on neuronal tissue sections. Autoantibodies against individual septins can then be confirmed by RC-IIFA expressing single septins.

Keywords: Autoantibodies; Autoimmune cerebellar ataxia; Autoimmune encephalitis; Autoimmunity; Cerebellitis; Cerebellum; Immunoglobulin G; Immunoprecipitation; Melanoma; Paraneoplastic cerebellar degeneration; Paraneoplastic neurological syndrome; Septin-11; Septin-3; Septin-5; Septin-6; Septin-7; Septins; Small-cell lung cancer.

Fig. 1

Immunofluorescence staining of central nervous tissues with patient sera. Cryosections of rat hippocampus, primate cerebellum and murine encephalon were incubated with patient sera (A, B Patients 1–3, 1:100; C, D Patient 1, 1:100) in the first step, and with Alexa Fluor 488 labeled goat anti-human IgG in the second step (green). A granular staining of the molecular layer (ml) was obtained on hippocampus and cerebellum (A, B). Additionally, a blotchy fluorescence of the granular layer was observed on cerebellum (B). On hippocampus, staining of the outer molecular layer (oml) is more intense compared to the inner molecular layer (iml) (A). On murine encephalon (C), the strongest reactivity was detected in the molecular layer of the cerebellum (D). Nuclei were counterstained by incubation with TO-PRO-3 iodide or DAPI (blue). (Scale bar: A, B 100 µm, C 1000 µm, D 250 µm.)

Fig. 2

Immunoprecipitation and antigen identification with patient serum. A SDS-PAGE of the immunoprecipitates of patient serum 1 (PS1) or control serum (CS) with cerebellar lysates stained with colloidal Coomassie revealed four specific bands (1–4) between 40 and 50 kDa in the eluate fraction of PS1 but not CS. Mass spectrometry analysis identified the following proteins: band 1 septin-6,-7,-11; band 2 septin-6,-7,-11; band 3 septin-3,-5; band 4 septin-3,-5 in the eluate fraction of PS1

Fig. 3

Indirect immunofluorescence analysis of single septin or septin-3/5/6/7/11 complex transfected HEK293 cells with patient serum. Acetone-fixed recombinant HEK293 cells expressing septin-3, septin-5, septin-6, septin-7 or septin-11 individually or co-expressing septin-3/5/6/7/11 or an empty vector-transfected control were incubated with the patient serum (PS1) or an anti-septin-5 or anti-septin-7 positive serum (1:100). All anti-septin positive sera showed a positive reaction with the recombinant septin-3/5/6/7/11 expressing HEK293 cells. Additionally, PS1 recognized cells expressing recombinant septin-3 but none of the other single septin expressing HEK293 cells. Recombinant septin-5 or septin-7 expressing HEK293 cells were recognized by the anti-septin-5 or anti-septin-7 positive control serum, respectively. (Scale bar 100 µm.)

Fig. 4

Neutralization of indirect immunofluorescence reaction on neuronal tissues with patient serum. Patient serum 1 (PS1, 1:100) was pre-incubated with extracts of HEK293 cells transfected with empty control vector (A) or with septin-3/5/6/7/11 (B), septin-3 (C) or septin-5 (D) before an indirect immunofluorescence assay with neuronal cryosections and Alexa Fluor 488 labeled goat anti-human IgG as secondary antibody was performed. The extract containing the septin-3/5/6/7/11 complex or septin-3 alone (B, C) abolished the immune reaction of PS1 on rat hippocampus, rat and primate cerebellum. The HEK293 control and the HEK293-septin-5 extracts (A, D) had no effect. Nuclei were counterstained with TO-PRO-3 iodide (blue). (Scale bar: 100 µm.)

Fig. 5

Expression of septin-3 in tumor specimens obtained from patient 1. Immunohistochemical staining of mouse cerebellum (A1, B1) and patient’s cancer specimens (A2-3, B2-3) incubated with anti-septin-3 rabbit commercial antibody diluted 1:1000 (A1) or 1:500 (A2-3) and control rabbit serum (B1-3). (A2, B2) Melanoma, patient 1; (A3, B3) lymph node metastasis, patient 1. (Magnification: 200-fold.)