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Autoantibody to the Rab6a/Rab6b in Autoimmune Cerebellar Ataxia Associated with Sjogren’S Syndrome

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Autoantibody to the Rab6a/Rab6b in Autoimmune Cerebellar Ataxia Associated with Sjogren’S Syndrome Autoantibody to the Rab6A/Rab6B in Autoimmune Cerebellar Ataxia Associated with Sjogren’s Syndrome Liyuan Guo Institute of Psychology Chinese Academy of Sciences Haitao Ren Peking Union Medical College Hospital Department of Neurology Siyuan Fan Peking Union Medical College Hospital Department of Neurology Hongzhi Guan Peking Union Medical College Hospital Department of Neurology Jing Wang ( [email protected] ) Institute of Psychology Chinese Academy of Sciences https://orcid.org/0000-0002-2512-0223 Research Keywords: autoimmune, cerebellar ataxia, Sjogren’s syndrome, anti-Rab6A/Rab6B antibody Posted Date: December 31st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-138256/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/18 Abstract Background To report a novel autoantibody against Purkinje cell in a patient with autoimmune cerebellar ataxia (ACA) associated to Sjogren’s syndrome (SS). Methods The Patients on one centre with cerebellar ataxia of unknown cause, who were tested positive with tissue- based indirect immunouorescence assay (TBA) on rat cerebellum sections and negative for comprehensive anti-neural autoantibodies panel, were investigated for novel autoantibody identication. Among them, one patient with comorbid ACA and SS was qualied for further exploration. His- immunoprecipitation (HIP) combined with mass spectrometric (MS) analysis was used to identify the target antigen, which was conrmed by recombinant cell based assay (CBA) and antibody neutralization experiments. Results TBA of the patient’s serum and cerebrospinal uid (CSF) for autoantibody testing revealed binding of IgG antibody, mainly IgG1, to Purkinje cell and granular layer of rat cerebellum. Rab6A was identied as the autoantigen by MS and Western blot, subsequently veried by CBA with HEK293 cells expressing human Rab6A/Rab6B. Furthermore, recombinant human Rab6A/Rab6B protein to neutralize the autoantibodies’ tissue reaction was performed by a parallel conrmed approach. Conclusion Autoantibody against Rab6A/Rab6B may be a novel biomarker in diagnosis of ACA, especially in patients with comorbid ACA and SS. The role of the antibody in mechanism of ACA warrants further study. Background The disorder of suspected immune-mediated cerebellar ataxia without the identication of a well-known trigger or pathogenic neuronal antibody is refered to as autoimmune cerebellar ataxia (ACA) [1]. Some ACA patients have coexisting non-neurological autoimmune diseases, such as Sjogren’s syndrome (SS), thyroid autoimmune diseases, and vitiligo, which suggest autoimmune tendency and served as an indication in diagnosis of ACA [1, 2]. SS is an autoimmune chronic lymphocytic inammatory disease involving the exocrine glands (ocular or salivary gland) in the setting of antinuclear antibodies, particularly to Ro/SSA and La/SSB [3]. It can occur alone as primary SS or in conjunction with other connective tissue diseases (secondary SS). Primary SS primarily affects exocrine glands, but may have extra-glandular manifestations, including the neurologic system, with the prevalence of 8–49% [4]. Cerebellar ataxia is one of the described neurological manifestations [5]. Page 2/18 Antibody-mediated dysfunction is one of possible aetiologies of cerebellar ataxia related to primary SS. In 2001, Owada et al detected an antibody that reacted with a protein of 34 kDa from the extract of spinal cord, dorsal root ganglion or cerebellar cortex in a primary SS patient with motor weakness and cerebellar ataxia [6]. Here, we report the identication of a novel neural autoantibody against Rab6A/ Rab6B protein in a patient with comorbid ACA and SS. Samples And Methods Samples The samples (sera) are from the patients of cerebellar ataxia of unknown cause who were registered to the program of encephalitis and paraneoplastic syndrome (PNS) of Peking Union Medical College Hospital (PUMCH) from July 2018 to November 2019. This study was approved by the Ethics Committee of PUMCH (JS-891 and JS-2184), and informed consent was obtained from each patient. As shown in Fig. 1, sera of enrolled patients were rstly tested by tissue-based indirect immunouorescence assay (TBA) on rat cerebellum sections, positive sera were further screened for well-established anti-neural autoantibodies using recombinant protein, either immunoblot for intracellular or cell-based assay (CBA) for extracellular autoantibodies (including antibodies target to aquaporin 4, NMDA-R, CASPR2, AMPA-R, LGI1, GABAb-R, GAD-65, ITPR1, ZIC4, PKCγ, AP3B2, PCA-2, CARP VII, Homer-3, NCDN, CV2/CRMP5, PNMA2, Ri, Yo, Hu and amphiphysin). When a TBA-positive serum was negative for the above auto- antibody screenings, the sample was investigated for new antibody identication. Among them, a 43- year-old female was involved with clinically suspected primary SS combined with unexplained cerebellar ataxias. The patient experienced extensive tests, yet no infectious, metabolic nor genetic cause was revealing. However, oligoclonal bands of the patient's CSF was positive and detection of anti-SSA and anti-Ro antibodies strongly indicated immune-mediated pathogenesis. TBA test revealed strong IgG1 reactivity with cerebellar granular cell layers and Purkinje cell layers, but not with a broad panel of recombinant expressed anti-neural autoantibodies. Therefore, the patient (serum) was selected for novel antibody identication. Additionally, 10 age- and sex-matched SS patients without neurological manifestation were enrolled to identify. Tissue based indirect immunouorescence assay (TBA) Slides with rat cerebellum sections were used for TBA. Each slide was incubated with 30 µl of sample diluted in phosphate-buffered saline (PBS) (1:100) at 4 °C for 3 h, ushed with PBS, and immersed in PBS for 5 min. Subsequently, polyclonal goat anti-human IgG (Cat. ZF-0308, ZSBio, China) labelled with uorescein isothiocyanate (FITC), were incubated at room temperature for 30 min. The slides were then washed again; embedded in glycerol (approximately 10 µl per cryosection), and examined by two independent observers using DMi8 microscope (Leica, Germany). Positive and negative controls were included. Samples were categorized based on tissue patterns in direct comparison with control samples. For co-localization test, slides were incubated with 30 µl of sample diluted in PBS (1:100) and anti-Rab6A (Cat. 10187-2-AP, Proteintech, USA) or anti-Rab6B (Cat. 10340-1-AP, Proteintech, USA) antibody at 4 °C for Page 3/18 3 h. After washes in PBST, slides were incubated with FITC labelled goat anti-human IgG antibody (Cat. ZF-0308, ZSBio, China) and Alexa Fluor 555 labelled goat anti-rabbit IgG antibody (Cat. Ab150078, Abcam, UK) at room temperature for 30 min. The slides were then washed again, embedded in glycerol and observed by DMi8 microscope (Leica, Germany). For evaluation of IgG subclasses, patient serum was tested on rat cerebellum sections as described above, with the following modications applied: unconjugated sheep anti-human IgG antibodies specic for IgG subclasses 1 to 4 (Nodics-Mubio, Netherlands, 1:100) were substituted for the FITC labelled goat anti-human IgG antibody, and AF568 labelled donkey anti-sheep IgG (Invitrogen; absorbed against human IgG, 1:200) was used to detect the subclass specic antibodies (Fig. 3). His-immunoprecipitation (HIP) and identication of the antigen Slides with rat cerebellum cryosections were incubated with serum (diluted 1:100) or CSF (undiluted) at 4 °C for 3 h followed by three washes with PBS containing 0.2% (w/v) Tween 20. The immunocomplexes were then extracted with lysis buffer (Cat. P0013D, Beyotime, China) containing protease inhibitors and the detached material was lysed at 4 °C for 1 h. The resulting suspension was homogenized and centrifuged at 16,000 × g at 4 °C for 15 min. Immunocomplexes were precipitated from the clear supernatant with Protein G Dynabeads (Cat. 10003D, Thermo Fisher Scientic, USA) at 4 °C overnight, washed three times with PBS, and eluted with 60 ul lysis buffer with laemmli sample buffer (Cat. S3401, SIGMA, USA). The elution products were boiled for 10 minutes and were used for sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and Mass spectrometry (MS) 20 µl eluted proteins were electrophoresed on 12% SDS–PAGE gels. After visualizing with Coomassie Brilliant Blue G-250, the divisible protein bands were cut and destained with 25 mM ammonium bicarbonate/50% Acetonitrile. Proteins in the gel particles were extracted by PAGE gel protein extraction kit (Sangon Biotech, China). Dried extracts were resuspended in lysis buffer and subjected to online reverse phase nano LC-MS/MS analysis with 50% of sample loading using an Easy nLC 1000 (Thermo Fisher Scientic,USA), coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientic,USA). Peptide samples were concentrated on a 2 cm trap column (100 µm diameter) and separated on a 12 cm capillary column (75 µm diameter), both packed in-house with 1.9 µm C18 reverse-phase fused silica (Michrom Bioresources, Inc., Auburn, CA). The samples were separated at a ow rate of 0.6 µL/min with a 71 min linear gradient from 5–30% mobile phase B (phase A: 0.1% formic acid in water, phase B: 0.1% FA in ACN), followed by a quick ramp from 30% mobile phase B to 95% mobile phase B within 1 min, where samples were held for 6 min before a quick ramp down; then, the column was re-equilibrated. Eluted peptides were analysed with a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientic, USA). The MS survey scan was analysed over a mass range of 300–1400 Da with a resolution of 70000 at m/z 200. The isolation width was 3 m/z for precursor ion selection. The automatic gain control (AGC) was set to 3e6, and the maximum injection time (MIT) was 60 ms. The MS2 was analysed using data-dependent Page 4/18 mode searching for the 20 most intense ions fragmented in the HCD. For each scan with a resolution of 17500 at m/z 200, the AGC was set at 5e4 and the MIT was 80 ms.
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