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 immunofuorescence assay (TBA) on rat cerebellum sections and negative for comprehensive anti-neural autoantibodies panel, were investigated for novel autoantibody identifcation. Among them, one patient with comorbid ACA and SS was qualifed for further exploration. His- immunoprecipitation (HIP) combined with mass spectrometric (MS) analysis was used to identify the target antigen, which was confrmed by recombinant cell based assay (CBA) and antibody neutralization experiments.

Results

TBA of the patient’s serum and cerebrospinal fuid (CSF) for autoantibody testing revealed binding of IgG antibody, mainly IgG1, to Purkinje cell and granular layer of rat cerebellum. Rab6A was identifed as the autoantigen by MS and Western blot, subsequently verifed by CBA with HEK293 cells expressing human Rab6A/Rab6B. Furthermore, recombinant human Rab6A/Rab6B to neutralize the autoantibodies’ tissue reaction was performed by a parallel confrmed 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 identifcation 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 infammatory 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 identifcation 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 frstly tested by tissue-based indirect immunofuorescence 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 identifcation. 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 identifcation. Additionally, 10 age- and sex-matched SS patients without neurological manifestation were enrolled to identify. Tissue based indirect immunofuorescence 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, fushed with PBS, and immersed in PBS for 5 min. Subsequently, polyclonal goat anti-human IgG (Cat. ZF-0308, ZSBio, China) labelled with fuorescein 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 modifcations applied: unconjugated sheep anti-human IgG antibodies specifc 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 specifc antibodies (Fig. 3). His-immunoprecipitation (HIP) and identifcation 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 Scientifc, 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 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 Scientifc,USA), coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientifc,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 fow 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 Scientifc, 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. The dynamic exclusion was set at 18 s to suppress the repeated detection of the same fragment ion peaks. The relative collision energy for MS2 was set at 27% for HCD. Raw MS data was analysed using the Proteome Discoverer 2.0 software (Thermo Fisher Scientifc, USA) to identify the proteins and searched against Rattus norvegicus proteins from the UniProt sequence database. The search parameters were as follows: precursor ion mass tolerance ± 15 ppm; MS/MS tolerance ± 0.1 Da; 2 missed cleavages were allowed with the enzyme of trypsin; cysteine was set as fxed modifcation of carbamidomethylation and methionine was set as variable modifcations of oxidation. The protein identifcation was supported by at least one unique peptide. The results were fltered based on a false discovery rate (FDR) of no more than 1%. Western blot

20 µl eluted proteins were electrophoresed on 12% SDS-PAGE gels and then were transferred to PVDF membrane, blocked for 1 h in 5% non-fat milk, incubated with patient’s serum (1:100) and anti-Rab6A antibody (1:1000, Cat. PA5-22127, Thermo Fisher Scientifc, USA) for overnight at 4 °C respectively. Detection of the primary antibodies were carried out with a 1:5000 diluted HRP-conjugated anti-human and anti-rabbit IgG (Cat. A0201 and A0208, Beyotime, China) for 60 min respectively, and detected with enhanced chemiluminescence (ECL) kit (Cat. CW0049, CWBio, China). Recombinant Expression of Antigens in HEK293 Cells

For the preparation of substrates for the cell-based assay, HEK293 cells (CRL-1573, ATCC, VA, USA) were cultured in high-glucose DMEM (Cat. No. 11965092, Life Technologies, USA) supplemented with 10% FBS at 37 °C and 5% CO2. For cell based assay, cells were seeded on sterile cover glasses 24 hours before transfection. The full-length human RAB6A and RAB6B cDNA expression plasmid pCMV6 was purchased from Origene (Cat. RC200432 and Cat. RC216303, Origene, USA). Transient transfection was performed by using FuGene HD transfection reagent (Cat. E2311, Promega, USA). Forty-eight hours later cover glasses were washed with PBS, fxed with 4% PFA in PBS for 20 min, and permeated with 0.3% Triton X- 100 in PBS for 10 min, after three washes in chilled PBS, blocked with 5% BSA for 1 h. For neutralization experiments, transient transfection was performed in the same way and total protein of transfected cells were obtained by lysis buffer (Cat. P0013D, Beyotime, China) 48 hours later. Cell based assay (CBA) and neutralization experiments

Recombinant HEK293 cells over-expressing human RAB6A/ RAB6B and mock-transfected cells control were incubated with patient serum (1:100 diluted) and CSF (undiluted) or control serum (1:100 diluted) for 2 h at room temperature. Binding of human IgG was detected by incubating with FITC labelled goat anti-human IgG antibodies (Cat. ZF-0308, ZSBio, China) for 30 min. The slides were then washed again, embedded in glycerol and observed by DMi8 microscope (Leica, Germany). In neutralization experiments, serum were pre-incubated with extracts of HEK293 cells transfected with empty control vector or with the plasmid harboring RAB6A or RAB6B cDNA at 4 °C overnight and then the TBA was performed.

Page 5/18 Results Clinical data

During July 2018 to November 2019, we collected sera from patients with unexplained cerebellar ataxia for TBA on rat cerebellum sections and by extensive anti-neural autoantibodies screening. Five of them were positive in TBA but negative in all established autoantibody screenings, and in the serum of one patient we identifed the novel autoantibody (Fig. 1).

The patient is a 43-year-old woman with a medical history of elevated fasting glucose levels presented with progressive dizziness, unsteady gait, nausea and vomiting for two months. Several days before the onset of symptoms, she got injection of the yearly infuenza virus vaccine. She had feelings of dry eyes and dry mouth recently. Neurological examinations revealed intention tremor, dysdiadochokinesia, and abnormal heel-to-shin test bilaterally, with failure of heel-to-toe walking, and a wide-based, staggering gait. No nystagmus, weakness, or Babinski sign was noticed. Brain magnetic resonance imaging (MRI) showed abnormalities and atrophy in the bilateral cerebellum (Fig. 2). CSF analysis displayed infammatory features (elevated cell count, protein levels and immunoglobulin index, and positive oligoclonal bands). Other clinical examination showed that unstimulated whole saliva fow rates and Schirmer’s test were decreased, and ocular staining score was increased. Whole body (without head) PET/CT revealed no notable abnormalities. The serum was positive for anti-SSA and anti-Ro antibodies (1:8, respectively). Serum anti-aquaporin 4 antibody (CBA, Euroimmun AG, Lüebeck, Germany), anti- thyroid peroxidase and anti-thyroglobulin antibodies were negative. The tests of anti-NMDA-R, anti- CASPR2, anti-AMPA-R, anti-LGI1, anti-GABAb-R, anti-GAD-65, anti-ITPR1, anti-ZIC4, anti-PKCγ, anti-AP3B2, anti-PCA-2, anti-CARP VII, anti-Homer-3, anti-NCDN (CBA, Euroimmun AG, Lüebeck, Germany), anti- CV2/CRMP5, anti-PNMA2, anti-Ri, anti-Yo, anti-Hu and anti-amphiphysin antibodies (Euroline, Euroimmun AG, Lüebeck, Germany) were also negative in either serum or CSF. Therefore, this patient was investigated for novel antibody. In the TBA, The patient’s serum and CSF displayed a fne-granular cytoplasmic IgG staining in the Purkinje cell layer and granular cell layer of the rat cerebella (Fig. 3). The IgG subclass repertoire of the antibody was analysed in the patient and revealed mainly IgG1 antibody (Fig. 4).

The patient was fnally diagnosed as ACA associated with SS and received intravenous immunoglobulin, steroids, and cyclophosphamide (CTX) treatment. Symptoms of dizziness, nausea and vomiting were resolved, while ataxia persisted. Then the patient received tocilizumab 400 mg every month. As a result, the symptoms of ataxia improved gradually and fuorescence pattern on rat cerebella was reduced obviously (Fig. 3). Identifcation of target autoantigen

HIP of rat cerebellum and the index patient’s serum revealed a protein with apparent molecular mass of 21 kDa by SDS-PAGE. The protein was absent in HIP of therapy treatment serum or control serum (Fig. 5A). The precipitated protein was identifed as Rattus norvegicus (Rat) Rab6A (UNIPROT acc. # Q9WVB1 ) by MALDI-TOF analysis (Fig. 5B). Western blot analysis of the HIP exhibited a specifc reaction

Page 6/18 at 21 kDa using patient’s serum and anti-Rab6A antibody, respectively (Fig. 5C). Rab6A protein belongs to the Rab6 family, in human this family consists of 4 different isoforms: Rab6A, Rab6A’, Rab6B and Rab6C. Rab6A’ is generated by alternative splicing of the RAB6A and differs from Rab6A by only three amino acids. Both proteins are ubiquitously expressed and are together the most abundant Golgi- associated Rab proteins. Rab6B is encoded by a separate gene RAB6B and is preferentially expressed in brain, especially in microglial cells, pericytes and Purkinje cells [7]. RAB6C is a primate-specifc retrogene transcribed in a limited number of human tissues. Since Rab6B protein expressed in a brain specifc manner and show 91% identity with Rab6A protein[7], we doubted whether the patient’s serum also reactive with Rab6B. Indeed, both Rab6A and Rab6B antibody produced a similar fuorescence pattern on rat cerebella comparable to that generated by the patient’s serum (Fig. 6).

As a proof of correct antigen identifcation, the patient’s sample was then tested using recombinant HEK293 cells which expressed either human Rab6A or Rab6B protein (Fig. 7). Serum and CSF of patient reacted with the cells expressing Rab6A or Rab6B but not with mock transfection protein, control serum reacted with none of them.

The congruence of the autoantibody target Rab6A /Rab6B was further demonstrated by the proof of neutralization of antibody binding to brain tissue: the reaction of the patient’s autoantibody on tissue, especially in the Purkinje cell layer, could be abolished by pre-incubation with HEK293 lysate containing Rab6A or Rab6B (Fig. 8). Antibody binding was unaffected when a comparable fraction from mock- transfected HEK293 cells was used.

Specifcity of anti-Rab6a/ Rab6b autoantibody

Sera from 10 SS patients without neurological manifestation were analysed by CBA in parallel to the sample of the index patient. None of these control sera produced a similar immunofuorescence pattern to that of the index sera on the recombinant Rab6a/ Rab6b substrate (Supplementary material). It suggested that anti-Rab6a/ Rab6b autoantibody was specifc to disease of ACA, but not SS.

Discussion

We identifed an novel autoantibody against Rab6A/Rab6B in a patient with comorbid ACA and SS who met the diagnostic criteria for primary ACA recently recommended by Hadjivassiliou M. et al [1]. The patient’s serum reacted with Purkinje cell and granular layer of rat cerebellum but not with hippocampal tissue and established neural autoantigens. The following set of experiments demonstrated Rab6A/Rab6B to be the relevant autoantigen: (1) HIP of Rab6A from rat cerebellum by the patient’s IgG antibody, (2) specifc immunostaining of HEK293 cells over-expressing human RAB6A/ RAB6B gene by the patient’s IgG antibody, and (3) specifc competitive inhibition of the patient’s IgG antibody by preincubation with extracts of recombinant HEK293-RAB6A/ RAB6B cells.

Page 7/18 Whether anti-Rab6A/Rab6B antibody is pathogenic is unknown. There is some indirect evidence for its potential pathogenic role. First, the antibody mainly response to Purkinje cell, which is known to be a cell type expressed exclusively in the cerebellum, and the patient presented cerebellar ataxia manifestation. Second, the antibody belonged to the IgG1 subclass, a strong complement activator, suggesting that it may act on Purkinje cell via complement-dependent mechanism, which is well-established feature in other autoantibody-associated disorders [8, 9]. Third, CSF analysis displayed infammatory features and immunosuppressive therapy was followed by clinical stabilization and ataxia improvement. Testing of serum sample taken after treatment with intravenous immunoglobulin, steroids, CTX and tocilizumab showed a decline of serum titer. On the other hand, Rab6A and Rab6B are intracellular antigens located in membranes encompassing the Golgi or (ER). Rab6A is restricted to the Golgi apparatus, whereas Rab6B is distributed over Golgi and ER membranes. It’s believed that nuclear or cytoplasmic antigens are not accessible to immune attack in situ. As antibodies targeting intracellular antigens (e.g. Hu, Yo Ri, Ta) are not pathogenic, it is therefore possible that Rab6A/ Rab6B antibody has no such impact [9].

Rab6A and Rab6B belong to GTPases of the Rab family, which are important regulators of intracellular transport and membrane trafcking in eukaryotic cells. There are about 70 members of Rab family in human, Rab6 proteins are the most abundant Golgi associated protein and they play an important role in retrograde Golgi–endoplasmic reticulum and intra-Golgi transport [10]. Although the pathogenicity of Rab6A/B hasn’t been well understood, some other Rab proteins have been reported to associated with a multitude of inherited genetic disorders and acquired diseases ranging from peripheral neuropathy [11, 12], neurodegeneration [13], to immunodefciency [14] and cancer [15]. A spastic ataxia related mutation has been identifed in the gene of VPS13 protein, a Rab6 effector [16], mutation in RAB3GAP2, gene of Rab3 GTPase-activating protein, was reported in hereditary spastic paraplegias (HSPs) [17]. It’s interesting that mutations in gene of ITPR1, another Purkinje cell antigen, are also reported in HSPs [18]. Notably, the activity of some Rabs are regulated by calcium efux [19], as most of identifed antigens that targeted by Purkinje cell antibodies are contributed in maintaining intracellular calcium homeostasis [20], there may be potential functional relationship between them.

Comprehensive evaluation of anti-Rab6A/Rab6B antibodies in disease remains challenging. By far, few publications have been reported about Rab6 in neurological disease or multi-system autoimmune diseases. Case report here may bring some hint for understanding of Rab6A/Rab6B role in disease, however we need to collect more cases and to make further investigations to explore the clinical signifcance of Rab6A/Rab6B, as well as to differentiate Rab6A from Rab6B in their contribution to the disease.

Conclusions

In summary, we identifed anti-Rab6A/B antibody as a new autoantibody against Purkinje cell in a patient with ACA and responsive to immunotherapy. Our fnding expands the spectrum of diagnostic anti-

Page 8/18 neuronal antibodies of autoimmune cerebellar ataxia. The role of this novel antibody in mechanism of ACA warrants further study.

Abbreviations

ACA: autoimmune cerebellar ataxia; AGC: automatic gain control; CBA: cell based assay; CSF: cerebrospinal fuid; CTX: cyclophosphamide; ECL: enhanced chemiluminescence; ER: endoplasmic reticulum; FDR: false discovery rate; FITC: fuorescein isothiocyanate; HIP: His-immunoprecipitation; HSPs: hereditary spastic paraplegias; MIT: maximum injection time; MRI: magnetic resonance imaging; MS: mass spectrometric; PNS: paraneoplastic syndrome; PUMCH: Peking Union Medical College Hospital; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SS: Sjogren’s syndrome; TBA: tissue-based indirect immunofuorescence assay

Declarations Ethics approval and consent to participate

This study was approved by the Institutional Review Board of Peking Union Medical College Hospital (PUMCH) (JS-891 and JS-2184). Written informed consents were obtained from all patients.

Consent for publication

Written informed consents were obtained from all patients.

Availability of data and materials

The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Key Research and Development Program of China (Grant No. 2016YFC0901500, to HG). This work was also supported by CAS Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences.

Page 9/18 All funding bodies didn’t play any roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Authors' contributions

JW and HG designed the study and drafted the manuscript. LG and HR performed the experiments. SF performed clinical examination. All authors have read and approved the fnal manuscript.

Acknowledgements

We acknowledge funding support from National Key Research and Development Program of China (Grant No. 2016YFC0901500, to HG).

References

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Page 10/18 8. Sven Jarius KPW, Sigrun Horn, Heike Heuer, Brigitte Wildemann: A new Purkinje cell antibody (anti- Ca) associated with CA Journal of Neuroinfammation 2010. 9. Jarius S, Scharf M, Begemann N, Stocker W, Probst C, Serysheva, II, Nagel S, Graus F, Psimaras D, Wildemann B, Komorowski L: Antibodies to the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) in cerebellar ataxia. J Neuroinfammation 2014, 11:206. 10. Zerial M, McBride H: Rab proteins as membrane organizers. Nature Reviews Molecular Cell Biology 2001, 2:107-117. 11. Verhoeven K, De Jonghe P, Coen K, Verpoorten N, Auer-Grumbach M, Kwon JM, FitzPatrick D, Schmedding E, De Vriendt E, Jacobs A, et al: Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy. American Journal of Human Genetics 2003, 72:722-727. 12. Wang X, Han CM, Liu WQ, Wang P, Zhang XQ: A novel RAB7 mutation in a Chinese family with Charcot-Marie-Tooth type 2B disease. Gene 2014, 534:431-434. 13. Wilson GR, Sim JCH, McLean C, Giannandrea M, Galea CA, Riseley JR, Stephenson SEM, Fitzpatrick E, Haas SA, Pope K, et al: Mutations in RAB39B Cause X-Linked Intellectual Disability and Early-Onset Parkinson Disease with alpha-Synuclein Pathology. American Journal of Human Genetics 2014, 95:729-735. 14. Menasche G, Pastural E, Feldmann J, Certain S, Ersoy F, Dupuis S, Wulffraat N, Bianchi D, Fischer A, Le Deist F, de Saint Basile G: Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nature Genetics 2000, 25:173-176. 15. Banworth MJ, Li G: Consequences of Rab GTPase dysfunction in genetic or acquired human diseases. Small GTPases 2018, 9:158-181. 16. Seong E, Insolera R, Dulovic M, Kamsteeg EJ, Trinh J, Bruggemann N, Sandford E, Li S, Ozel AB, Li JZ, et al: Mutations in VPS13D lead to a new recessive ataxia with spasticity and mitochondrial defects. Annals of Neurology 2018, 83:1075-1088. 17. Novarino G, Fenstermaker AG, Zaki MS, Hofree M, Silhavy JL, Heiberg AD, Abdellateef M, Rosti B, Scott E, Mansour L, et al: Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders. Science 2014, 343:506-511. 18. Elert-Dobkowska E, Stepniak I, Krysa W, Ziora-Jakutowicz K, Rakowicz M, Sobanska A, Pilch J, Antczak-Marach D, Zaremba J, Sulek A: Next-generation sequencing study reveals the broader variant spectrum of hereditary spastic paraplegia and related phenotypes. Neurogenetics 2019, 20:27-38. 19. Parkinson K, Baines AE, Keller T, Gruenheit N, Bragg L, North RA, Thompson CRL: Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel. Nature Cell Biology 2014, 16:87. 20. Jarius S, Wildemann B: 'Medusa-head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 1: Anti-mGluR1, anti-Homer-3, anti-Sj/ITPR1 and anti-CARP VIII. Journal of Neuroinfammation 2015, 12.

Page 11/18 Figures

Figure 1

Screening chart of patient samples followed in our study for new antigen identifcation. We enrolled patients with clinically suspected immune-mediated cerebellar ataxias who attended our hospital during July 2018 to December 2019. All individuals gave written informed consent for participation in the study. We reviewed their medical records and the patients didn’t have a known trigger or any pathogenic neuronal antibodies. Patients’ sera were tested by TBA on rat cerebellum sections and screened for anti- AQP4, thyroid peroxidase, thyroglobulin, NMDA-R, CASPR2, AMPA-R, LGI1, GABAb-R, GAD-65, CV2/CRMP5, PNMA2, Ri, Yo, Hu, amphiphysin, ITPR1, ZIC4, PKCγ, AP3B2, PCA-2, CARP VII, Homer-3, and NCDN antibodies. After the screening, 5 patients with a distinct pattern on positive TBA and negative detection of the above antibodies were further investigated for novel antibodies. Additionally, 10 age- and sex-matched SS patients were enrolled to test the specifcity.

Page 12/18 Figure 2

Brain magnetic resonance imaging showed abnormalities and atrophy in the bilateral cerebellum. A-C: T1 weighted image showed atrophy of cerebellum. D-F: fuid attenuated inversion recovery sequence showed hyper-intensities in the bilateral dentate nucleus.

Page 13/18 Figure 3

Immunofuorescence staining of rat cerebellar tissues. Cryosections of rat cerebellum were incubated with patient serum (before therapy and after therapy, 1:100) and CSF (before therapy, undiluted) or control serum (1:100) in the frst step and with FITC-labeled goat anti-human IgG in the second step (green). A fne IgG staining of the Purkinje cell layer and granular cell layer was obtained. Upper row: X200 magnifcation; lower row: X400 magnifcation.

Page 14/18 Figure 4

Subclass analysis revealed that the antibody belongs mainly to the IgG1 subclass. Subclass analysis revealed mainly IgG1 antibody (depicted in red) and no IgG2, IgG3 or IgG4 antibodies (not shown).

Figure 5

His-immunoprecipitation and antigen identifcation. Section of rat cerebellum was incubated with the patient’s serum (1:100), washed in PBS, and solubilized using 1% Triton X-100. The solution was incubated with protein-G-coated magnetic beads. The immunocomplexes were eluted by SDS and subjected to SDS-PAGE analysis and Western blot. A: SDS-PAGE and staining with colloidal Coomassie. His-immunoprecipitate of the patient serum (before therapy, lane 1; after therapy, lane 2) and control

Page 15/18 serum (lane 3). Arrow indicates the position of the immunoprecipitated antigen at ~21 kDa. B: MALDI- TOF sequence analysis identifed target antigen as Rab6A. C: Western blot analysis with patient’s serum (lane 1) or anti-Rab6A antibody (lane 2).

Figure 6

Double-staining of rat cerebellar tissues with patient serum and anti-Rab6A or anti-Rab6B antibody. Immunofuorescence staining of rat cerebellum tissue section with patient’s serum (green) and anti- Rab6A or anti-Rab6A (red) antibody. The merged images (right, yellow) show localization of the reactivity in the same region. Nuclei were counterstained by incubation with DAPI (blue). X200 magnifcation.

Page 16/18 Figure 7

Immunofuorescence staining of recombinant Rab6A or Rab6B. Immunofuorescence analysis of transfected HEK293 cells. Formaldehyde-fxed recombinant HEK293 cells expressing human RAB6A, RAB6B, or mock transfected control were incubated with patient serum (1:100) and CSF (undiluted) or control serum (1:100).

Page 17/18 Figure 8

Neutralization of antibody reaction on tissue. Neutralization of immunofuorescence reaction on neuronal tissues. Serum was pre-incubated with extracts of HEK293 cells transfected with empty control vector or with the plasmid harboring human RAB6A or RAB6B cDNA. The extract containing Rab6A or Rab6B greatly reduced the immune reaction of the serum on rat cerebellum tissue. X200 magnifcation.

Supplementary Files

This is a list of supplementary fles associated with this preprint. Click to download.

FigureS1.jpg

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