Synaptopathies and autoantibodies (SYNATAC)

RHU BETPSY

The BETPSY project, coordinated by Prof. Honnorat, has received a 7.3 million euro “Future Investment Program (PIA)” funding. It is based on the collaboration of 5 academic research teams (UCBL, HCL, ICM, CLB and INSERM), and an industrial partner (Euroimmun), specialized in the development and marketing of autoantibody detection kits.

The aim of the BETPSY project is to better characterize EA and SNPs in order to improve the care and treatment of patients suffering from these diseases, through the identification of new biomarkers, the understanding of the mechanisms involved in these autoimmune diseases and the development of animal models: https://www.rhu-betpsy.fr/

• Jerome HONNORAT — Professor, UCBL, HCL – jerome.honnorat@chu-lyon.fr – 04 26 67 28 01
• Lucie ALIOUANE — Researcher, UCBL – lucie.aliouane@univ-lyon1.fr – 04 72 35 78 13
• Jean-Christophe ANTOINE — Professor, CNRS – j.christophe.antoine@chu-st-etienne.fr – 04 77 12 78 05
• Sarah BENKEDER — PhD student, INSERM – sarah.benkeder@univ-lyon1.fr – 04 26 67 28 01
• Roger BESANCON — Associate Professor, UCBL – roger.besancon@univ-lyon1.fr – 04 26 67 28 01
• Nadia BOUTAHAR — Associate Professor, CNRS – nadia.boutahar@chu-st-etienne.fr – 04 77 82 83 10
• Pauline BOUVET — PhD student, INSERM – pauline.bouvet@univ-lyon1.fr – 04 26 67 28 01
• Jean-Philippe CAMDESSANCHE — Professor, CNRS – j.philippe.camdessanche@chu-st-etienne.fr – 04 77 12 05 59
• Naura CHOUNLAMOUNTRI — Research Assistant, CNRS – naura.chounlamountri@univ-lyon1.fr – 04 26 67 28 01
• Sterenn CLOSS — Researcher, CNRS – sterenn.closss@chu-lyon.fr –
• Kassandre COMBET — PhD student, CNRS – Kassandre.combet@univ-lyon1.fr – 04 26 67 28 01
• Priscille DE GEA — PhD student, CNRS – priscille.de-gea@univ-lyon1.fr – 04 26 67 28 01
• Virginie DESESTRET — Professor, CNRS – virginie.desestret@univ-lyon1.fr –
• Le Duy DO — Researcher, CNRS – le-duy.do@univ-lyon1.fr – 04 26 67 28 01
• Fabrice FAURE — Research Assistant, CNRS – fabrice.faure@univ-lyon1.fr – 04 26 67 28 01
• Bastien JOUBERT — Associate Professor, CNRS – bastien.joubert@chu-lyon.fr –
• Mathilde MILLOT — Researcher, CNRS – –
• Claire MEISSEREL — CV — Researcher, INSERM – Claire.meissirel@inserm.fr – 04 26 67 28 01
• Christian MORITZ — Researcher, CNRS – christian.moritz@univ-st-etienne.fr –
• Olivier PASCUAL CV — Researcher, INSERM – olivier.pascual@inserm.fr – 04 26 68 82 74
• Veronique PELLIER-MONIN — Associate Professor, UCBL – veronique.pellier-monnin@univ-lyon1.fr – 04 26 67 28 01
• Géraldine PICARD — Researcher, HCL – geraldine.picard@chu-lyon.fr –
• Elise PETER — Researcher, UCBL – –
• Antonio FARINA — Researcher, HCL – antoniofarina100791@gmail.com –
• Macarena GARCIA — Researcher, INSERM – ext-macarena.villagran@chu-lyon.fr –
• Anne-Laurie PINTO — Researcher, HCL – anne-laure.pinto@chu-lyon.fr –
• Mélisse ROBERT — PhD student, HCL – melisse.robert@univ-lyon1.fr – 04 26 67 28 01
• Veronique ROGEMOND — Researcher, HCL – veronique.rogemond@chu-lyon.fr – 04 72 35 58 40
• Yannick THOLANCE — Associate Professor, UCBL – Yannick.Tholance@chu-st-etienne.fr –
• MArine VILLARD — Researcher, HCL – marine.villard@chu-lyon.fr –
• Clementine VINCENT — Post-doc, INSERM – clementine.vincent@univ-lyon1.fr – 04 26 67 28 01
• Valentin WUCHER — Post-doc, UCBL – valentin.wucher@univ-lyon1.fr – 04 26 73 94 04

1. Different genetic signatures of small-cell lung cancer characterize anti-GABAB R and anti-Hu paraneoplastic neurological syndromes.
   Vogrig A, Pegat A, Villagrán-García M, Wucher V, Attignon V, Sohier E, Brevet M, Rogemond V, Pinto AL, Muñiz-Castrillo S, Peter E, Robert M, Picard G, Hopes L, Psimaras D, Terra A, Perrin C, Cogne D, Tabone-Eglinger S, Martinez S, Jury D, Valantin J, Gadot N, Auclair-Perrossier J, Viari A, Dubois B, Desestret V, Honnorat J.
   Ann Neurology (2023) — Show abstract

   Objective: Small-cell lung cancer (SCLC) is the malignancy most frequently associated with paraneoplastic neurological syndromes (PNS) and can trigger different antibody responses against intracellular (Hu) or neuronal surface (GABAB R) antigens. Our aim was to clarify if the genomic and transcriptomic features of SCLC are different in patients with anti-GABAB R or anti-Hu PNS compared with SCLC without PNS.
   Methods: A total of 76 SCLC tumor samples were collected: 34 anti-Hu, 14 anti-GABAB R, and 28 SCLC without PNS. The study consisted of four steps: (i) pathological confirmation; (ii) Next Generation Sequencing using a panel of 98 genes, including those encoding the autoantibodies targets ELAVL1-4, GABBR1-2, KCTD16; (iii) genome-wide Copy Number Variation (CNV); (iv) whole-transcriptome RNA sequencing.
   Results: CNV analysis revealed that patients with anti-GABAB R PNS have commonly a gain in chromosome 5q, which contains KCTD16, while anti-Hu and control patients often harbor a loss. No significantly different number of mutations regarding any onconeural genes was observed. Conversely, transcriptomic profile of SCLC was different and the differentially expressed genes permitted to effectively cluster the samples into three groups, reflecting the antibody-based classification, with an overexpression of KCTD16 specific to anti-GABAB R PNS. Pathway analysis revealed that tumors of patients with anti-GABAB R encephalitis were enriched in B cell signatures, as opposed to those of patients with anti-Hu in which T cell and IFN-γ-related signatures were overexpressed.
   Interpretation: SCLC genetic and transcriptomic features differentiate anti-GABAB R, anti-Hu, and non-PNS tumors. The role of KCTD16 appears to be pivotal in the tumor immune tolerance breakdown of anti-GABAB R PNS. This article is protected by copyright. All rights reserved.

2. Detection of High-Risk Paraneoplastic Antibodies Against TRIM9 and TRIM67 proteins.
   Bartley CM, Ngo TT, Do LD, Zekeridou A, Dandekar R, Muñiz-Castrillo S, Alvarenga BD, Zorn KC, Tubati A, Pinto AL, Browne WD, Hullett PW, Terrelonge M, Schubert RD, Piquet AL, Binxia Y, Montalvo Perero MJ, Kung AF, Mann SA, Shah MP, Geschwind MD, Gelfand JM, DeRisi JL, Pittock SJ, Honnorat J, Pleasure SJ, Wilson MR.
   Ann Neurology (2023) — Show abstract

   Co-occurring anti-tripartite motif-containing protein 9 and 67 autoantibodies (TRIM9/67-IgG) have been reported in only a very few cases of paraneoplastic cerebellar syndrome. The value of these biomarkers and the most sensitive methods of TRIM9/67-IgG detection are not known. We performed a retrospective, multi-center study to evaluate the cerebrospinal fluid (CSF) and serum of candidate TRIM9/67-IgG cases by tissue-based immunofluorescence (TBIF), peptide phage display immunoprecipitation sequencing (PhIP-Seq), overexpression cell-based assay (CBA), and immunoblot. Cases in whom TRIM9/67-IgG was detected by at least two assays were considered TRIM9/67-IgG positive. Among these cases (N=13), CBA was the most sensitive (100%) and revealed that all cases had TRIM9 and TRIM67 autoantibodies. Of TRIM9/67-IgG cases with available clinical history, a subacute cerebellar syndrome was the most common presentation (N = 7/10), followed by encephalitis (N = 3/10). Of these ten, 70% had comorbid cancer (7/10), 85% of whom (N = 6/7) had confirmed metastatic disease. All evaluable cancer biopsies expressed TRIM9 protein (N = 5/5), whose expression was elevated in the cancerous regions of the tissue in 4 of 5 cases. TRIM9/67-IgG are rare but likely high-risk paraneoplastic biomarkers for which CBA appears to be the most sensitive diagnostic assay. This article is protected by copyright. All rights reserved.

3. Immune and Genetic Signatures of Breast Carcinomas Triggering Anti-Yo-Associated Paraneoplastic Cerebellar Degeneration
   Peter E, Treilleux I, Wucher V, Jougla E, Vogrig A, Pissaloux D, Paindavoine S, Berthet J, Picard G, Rogemond V, Villard M, Vincent C, Tonon L, Viari A, Honnorat J, Dubois B, Desestret V.
   Neurol Neuroimmunol Neuroinflamm (2022) — Show abstract

   Background and objectives: Paraneoplastic cerebellar degeneration (PCD) with anti-Yo antibodies is a cancer-related autoimmune disease directed against neural antigens expressed by tumor cells. A putative trigger of the immune tolerance breakdown is genetic alteration of Yo antigens. We aimed to identify the tumors' genetic and immune specificities involved in Yo-PCD pathogenesis.
   Methods: Using clinicopathologic data, immunofluorescence (IF) imaging, and whole-transcriptome analysis, 22 breast cancers (BCs) associated with Yo-PCD were characterized in terms of oncologic characteristics, genetic alteration of Yo antigens, differential gene expression profiles, and morphofunctional specificities of their in situ antitumor immunity by comparing them with matched control BCs.
   Results: Yo-PCD BCs were invasive carcinoma of no special type, which early metastasized to lymph nodes. They overexpressed human epidermal growth factor receptor 2 (HER2) but were hormone receptor negative. All Yo-PCD BCs carried at least 1 genetic alteration (variation or gain in copy number) on CDR2L, encoding the main Yo antigen that was found aberrantly overexpressed in Yo-PCD BCs. Analysis of the differentially expressed genes found 615 upregulated and 54 downregulated genes in Yo-PCD BCs compared with HER2-driven control BCs without PCD. Ontology enrichment analysis found significantly upregulated adaptive immune response pathways in Yo-PCD BCs. IF imaging confirmed an intense immune infiltration with an overwhelming predominance of immunoglobulin G-plasma cells.
   Discussion: These data confirm the role of genetic alterations of Yo antigens in triggering the immune tolerance breakdown but also outline a specific biomolecular profile in Yo-PCD BCs, suggesting a cancer-specific pathogenesis.

4. Glial Fibrillary Acidic Protein Autoimmunity: A French Cohort Study
   Gravier-Dumonceau A, Ameli R, Rogemond V, Ruiz A, Joubert B, Muñiz-Castrillo S, Vogrig A, Picard G, Ambati A, Benaiteau M, Rulquin F, Ciron J, Deiva K, de Broucker T, Kremer L, Kerschen P, Sellal F, Bouldoires B, Genet R, Biberon J, Bigot A, Duval F, Issa N, Rusu EC, Goudot M, Dutray A, Devoize JL, Hopes L, Kaminsky AL, Philbert M, Chanson E, Leblanc A, Morvan E, Andriuta D, Diraison P, Mirebeau G, Derollez C, Bourg V, Bodard Q, Fort C, Grigorashvili-Coin I, Rieul G, Molinier-Tiganas D, Bonnan M, Tchoumi T, Honnorat J, Marignier R
   Neurology (2022) — Show abstract

   Background and objectives: To report the clinical, biological, and imaging features and clinical course of a French cohort of patients with glial fibrillary acidic protein (GFAP) autoantibodies. Methods: We retrospectively included all patients who tested positive for GFAP antibodies in the CSF by immunohistochemistry and confirmed by cell-based assay using cells expressing human GFAPα since 2017 from 2 French referral centers. Results: We identified 46 patients with GFAP antibodies. Median age at onset was 43 years, and 65% were men. Infectious prodromal symptoms were found in 82%. Other autoimmune diseases were found in 22% of patients, and coexisting neural autoantibodies in 11%. Tumors were present in 24%, and T-cell dysfunction in 23%. The most frequent presentation was subacute meningoencephalitis (85%), with cerebellar dysfunction in 57% of cases. Other clinical presentations included myelitis (30%) and visual (35%) and peripheral nervous system involvement (24%). MRI showed perivascular radial enhancement in 32%, periventricular T2 hyperintensity in 41%, brainstem involvement in 31%, leptomeningeal enhancement in 26%, and reversible splenial lesions in 4 cases. A total of 33 of 40 patients had a monophasic course, associated with a good outcome at last follow-up (Rankin Score ≤2: 89%), despite a severe clinical presentation. Adult and pediatric features are similar. Thirty-two patients were treated with immunotherapy. A total of 11/22 patients showed negative conversion of GFAP antibodies. Discussion: GFAP autoimmunity is mainly associated with acute/subacute meningoencephalomyelitis with prodromal symptoms, for which tumors and T-cell dysfunction are frequent triggers. The majority of patients followed a monophasic course with a good outcome.

5. Familial autoimmunity in neurological patients with GAD65 antibodies: an interview-based study
   Muñiz-Castrillo S, Vogrig A, Montagnac C, Joubert B, Benaiteau M, Casez O, Chaumont H, Hopes L, Lanoiselée HM, Navarro V, Thomas B, Ursu R, Gonçalves D, Fabien N, Ducray F, Julier C, Honnorat J
   Journal of neurology (2021) — Show abstract

   The common co-occurrence of autoimmune systemic diseases in patients with neurological disorders and antibodies against glutamic acid decarboxylase 65 (GAD65) suggests a shared genetic predisposition to these disorders. However, the nature and frequency of familial aggregation of autoimmune diseases, which might also support this hypothesis, have been poorly investigated. Herein, an exploratory, interview-based study was conducted with the aim of describing the autoimmune diseases displayed by the relatives of GAD65 neurological patients, their frequency, kinship, and potential patterns of inheritance. Patients were enrolled only if they had GAD65 antibodies in the cerebrospinal fluid and typical clinical phenotypes associated with such antibodies (stiff-person syndrome, cerebellar ataxia, limbic encephalitis, or temporal lobe epilepsy). A total of 65 patients were included in the study, and 44/65 (67.7%) reported family history of autoimmunity, including first-degree relatives in 36/65 (55.4%); the sibling recurrence risk (λS) was 5.5, reinforcing the hypothesis of an underlying strong genetic predisposition. Most pedigrees with familial autoimmunity (38/44, 86.4%) showed multiple autoimmune diseases, all but 2 of them with diabetes mellitus or autoimmune thyroid disease, therefore resembling autoimmune polyendocrine syndromes. Inheritance patterns were diverse, possibly autosomal dominant in 17/44 (38.6%) pedigrees or autosomal recessive in 5/44 (11.4%), and un-defined or complex in 24/44 (54.5%). However, a total of 21/65 (32.3%) patients had no identified family history of autoimmunity. In conclusion, these results suggest a variable and heterogeneous genetic predisposition to GAD65 neurological disorders, possibly involving multiple loci and modes of inheritance with different contribution in each family.

6. Distinctive clinical presentation and pathogenic specificities of anti-AK5 encephalitis
   Muñiz-Castrillo S, Hedou JJ, Ambati A, Jones D, Vogrig A, Pinto AL, Benaiteau M, Broucker T, Fechtenbaum L, Labauge P, Murnane M, Nocon C, Taifas I, Vialatte de Pémille C, Psimaras D, Joubert B, Dubois V, Wucher V, Desestret V, Mignot E, Honnorat J
   Brain (2021) — Show abstract

   Limbic encephalitis with antibodies against adenylate kinase 5 (AK5) has been difficult to characterize because of its rarity. In this study, we identified 10 new cases and reviewed 16 previously reported patients, investigating clinical features, IgG subclasses, human leucocyte antigen and CSF proteomic profiles. Patients with anti-AK5 limbic encephalitis were mostly male (20/26, 76.9%) with a median age of 66 years (range 48–94). The predominant symptom was severe episodic amnesia in all patients, and this was frequently associated with depression (17/25, 68.0%). Weight loss, asthenia and anorexia were also highly characteristic, being present in 11/25 (44.0%) patients. Although epilepsy was always lacking at disease onset, seizures developed later in a subset of patients (4/25, 16.0%). All patients presented CSF abnormalities, such as pleocytosis (18/25, 72.0%), oligoclonal bands (18/25, 72.0%) and increased Tau (11/14, 78.6%). Temporal lobe hyperintensities were almost always present at disease onset (23/26, 88.5%), evolving nearly invariably towards severe atrophy in subsequent MRIs (17/19, 89.5%). This finding was in line with a poor response to immunotherapy, with only 5/25 (20.0%) patients responding. IgG1 was the predominant subclass, being the most frequently detected and the one with the highest titres in nine CSF-serum paired samples. A temporal biopsy from one of our new cases showed massive lymphocytic infiltrates dominated by both CD4+ and CT8+ T cells, intense granzyme B expression and abundant macrophages/microglia. Human leucocyte antigen (HLA) analysis in 11 patients showed a striking association with HLA-B*08:01 [7/11, 63.6%; odds ratio (OR) = 13.4, 95% confidence interval (CI): 3.8–47.4], C*07:01 (8/11, 72.7%; OR = 11.0, 95% CI: 2.9–42.5), DRB1*03:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7–55.7), DQB1*02:01 (8/11, 72.7%; OR = 13.5, 95% CI: 3.5–52.0) and DQA1*05:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7–55.7) alleles, which formed the extended haplotype B8-C7-DR3-DQ2 in 6/11 (54.5%) patients (OR = 16.5, 95% CI: 4.8–57.1). Finally, we compared the CSF proteomic profile of five anti-AK5 patients with that of 40 control subjects and 10 cases with other more common non-paraneoplastic limbic encephalitis (five with antibodies against leucine-rich glioma inactivated 1 and five against contactin-associated protein-like 2), as well as 10 cases with paraneoplastic neurological syndromes (five with antibodies against Yo and five against Ma2). These comparisons revealed 31 and seven significantly upregulated proteins in anti-AK5 limbic encephalitis, respectively mapping to apoptosis pathways and innate/adaptive immune responses. These findings suggest that the clinical manifestations of anti-AK5 limbic encephalitis result from a distinct T cell-mediated pathogenesis, with major cytotoxicity-induced apoptosis leading to a prompt and aggressive neuronal loss, likely explaining the poor prognosis and response to immunotherapy.

7. Multimodal Imaging with NanoGd Reveals Spatiotemporal Features of Neuroinflammation after Experimental Stroke
   Hubert V, Hristovska I, Karpati S, Benkeder S, Dey A, Dumot C, Amaz C, Chounlamountri N, Watrin C, Comte JC, Chauveau F, Brun E, Marche P, Lerouge F, Parola S, Berthezène Y, Vorup-Jensen T, Pascual O, Wiart M.
   Adv Sci (Weinh) (2021) — Show abstract

   The purpose of this study is to propose and validate a preclinical in vivo magnetic resonance imaging (MRI) tool to monitor neuroinflammation following ischemic stroke, based on injection of a novel multimodal nanoprobe, NanoGd, specifically designed for internalization by phagocytic cells. First, it is verified that NanoGd is efficiently internalized by microglia in vitro. In vivo MRI coupled with intravenous injection of NanoGd in a permanent middle cerebral artery occlusion mouse model results in hypointense signals in the ischemic lesion. In these mice, longitudinal two-photon intravital microscopy shows NanoGd internalization by activated CX3CR1-GFP/+ cells. Ex vivo analysis, including phase contrast imaging with synchrotron X-ray, histochemistry, and transmission electron microscopy corroborate NanoGd accumulation within the ischemic lesion and uptake by immune phagocytic cells. Taken together, these results confirm the potential of NanoGd-enhanced MRI as an imaging biomarker of neuroinflammation at the subacute stage of ischemic stroke. As far as it is known, this work is the first to decipher the working mechanism of MR signals induced by a nanoparticle passively targeted at phagocytic cells by performing intravital microscopy back-to-back with MRI. Furthermore, using a gadolinium-based rather than an iron-based contrast agent raises future perspectives for the development of molecular imaging with emerging computed tomography technologies.

8. Immunopathogenesis and proposed clinical score for identifying Kelch-like protein-11 encephalitis
   Vogrig A, Péricart S, Pinto AL, Rogemond V, Muñiz-Castrillo S, Picard G, Selton M, Mittelbronn M, Lanoiselée HM, Michenet P, Benaiteau M, Pariente J, Zéphir H, Giordana C, Montaut S, Salhi H, Bachoumas P, Montcuquet A, Letovanec I, Uro-Coste E, Honnorat J
   Brain communications (2021) — Show abstract

   In this study, we report the clinical features of Kelch-like protein 11 antibody-associated paraneoplastic neurological syndrome, design and validate a clinical score to facilitate the identification of patients that should be tested for Kelch-like protein 11 antibodies, and examine in detail the nature of the immune response in both the brain and the tumour samples for a better characterization of the immunopathogenesis of this condition. The presence of Kelch-like protein 11 antibodies was retrospectively assessed in patients referred to the French Reference Center for paraneoplastic neurological syndrome and autoimmune encephalitis with (i) antibody-negative paraneoplastic neurological syndrome [limbic encephalitis (n = 105), cerebellar degeneration (n = 33)] and (ii) antibody-positive paraneoplastic neurological syndrome [Ma2-Ab encephalitis (n = 34), antibodies targeting N-methyl-D-aspartate receptor encephalitis with teratoma (n = 49)]. Additionally, since 1 January 2020, patients were prospectively screened for Kelch-like protein 11 antibodies as new usual clinical practice. Overall, Kelch-like protein 11 antibodies were detected in 11 patients [11/11, 100% were male; their median (range) age was 44 (35–79) years], 9 of them from the antibody-negative paraneoplastic neurological syndrome cohort, 1 from the antibody-positive (Ma2-Ab) cohort and 1 additional prospectively detected patient. All patients manifested a cerebellar syndrome, either isolated (4/11, 36%) or part of a multi-system neurological disorder (7/11, 64%). Additional core syndromes were limbic encephalitis (5/11, 45%) and myelitis (2/11, 18%). Severe weight loss (7/11, 64%) and hearing loss/tinnitus (5/11, 45%) were common. Rarer neurologic manifestations included hypersomnia and seizures (2/11, 18%). Two patients presented phenotypes resembling primary neurodegenerative disorders (progressive supranuclear palsy and flail arm syndrome, respectively). An associated cancer was found in 9/11 (82%) patients; it was most commonly (7/9, 78%) a spontaneously regressed (‘burned-out’) testicular germ cell tumour. A newly designed clinical score (MATCH score: male, ataxia, testicular cancer, hearing alterations) with a cut-off ≥4 successfully identified patients with Kelch-like protein 11 antibodies (sensitivity 78%, specificity 99%). Pathological findings (three testicular tumours, three lymph node metastases of testicular tumours, one brain biopsy) showed the presence of a T-cell inflammation with resulting anti-tumour immunity in the testis and one chronic, exhausted immune response—demonstrated by immune checkpoint expression—in the metastases and the brain. In conclusion, these findings suggest that Kelch-like protein 11 antibody paraneoplastic neurological syndrome is a homogeneous clinical syndrome and its detection can be facilitated using the MATCH score. The pathogenesis is probably T-cell mediated, but the stages of inflammation are different in the testis, metastases and the brain.

9. Argonaute Autoantibodies as Biomarkers in Autoimmune Neurologic Diseases
   Do LD, Moritz C, Muñiz-Castrillo S, Pinto AL, Tholance Y, Brugiere S, Couté Y, Stoevesandt O, Taussig MJ, Rogemond V, Vogrig A, Joubert B, Ferraud K, Camdessanché JP, Antoine JC, Honnorat J
   Neurol Neuroimmunol Neuroinflamm (2021) — Show abstract

   Objective To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases. Methods Two distinct approaches (immunoprecipitation/mass spectrometry–based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples. Results Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments. Conclusions AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder. Classification of Evidence This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls.

10. Clinical and Prognostic Value of Immunogenetic Characteristics in Anti-LGI1 Encephalitis
    Muñiz-Castrillo S, Haesebaert J, Thomas L, Vogrig A,  Pinto AL, Picard G, Blanc C, Do LD, Joubert B, Berzero G, Psimaras D, Alentorn A, Rogemond V, Dubois V, Ambati A, Tamouza R, Mignot E, Honnorat J
    Neurol Neuroimmunol Neuroinflamm (2021) — Show abstract

    Objective Antibodies against leucine-rich glioma-inactivated 1 (LGI1-Abs) characterize a limbic encephalitis (LE) strongly associated with HLA-DRB1*07:01, although some patients lack LGI1-Abs in CSF or do not carry this allele. Whether they represent a different subtype of disease or have different prognoses is unclear. Methods Retrospective analysis of clinical features, IgG isotypes, and outcome according to LGI1-Ab CSF positivity and DRB1*07:01 in a cohort of anti-LGI1 LE patients. Results Patients with LGI1-Abs detected in both CSF and serum (105/134, 78%) were compared with those who were CSF negative (29/134, 22%). Both groups had similar clinical features and serum levels, but CSF-positive patients had shorter diagnostic delay, more frequently hyponatremia, inflammatory CSF, and abnormal MRI (p < 0.05). Human leukocyte antigen (HLA) genotyping was performed in 72/134 (54%) patients and 63/72 (88%) carried DRB1*07:01. Noncarriers (9/72, 12%) were younger, more commonly women, and had less frequently psychiatric and frontal symptoms (p < 0.05). No difference in IgG isotypes according to CSF positivity or HLA was found (p > 0.05). HLA and IgG isotypes were not associated with poor outcome (mRS >2 at last follow-up) in univariate analyses; CSF positivity was only identified as a poor outcome predictor in the multivariate analysis including the complete follow-up, whereas age and female sex also remained when just the first year was considered. Conclusions LE without CSF LGI1-Abs is clinically indistinguishable and likely reflects just a lesser LGI1-Ab production. HLA association is sex and age biased and presents clinical particularities, suggesting subtle differences in the immune response. Long-term outcome depends mostly on demographic characteristics and the intensity of the intrathecal synthesis.

11. Microglia modulate gliotransmission through the regulation of VAMP2 proteins in astrocytes
    Takata-Tsuji F, Chounlamountri N, Do LD, Philippot C, Novion Ducassou J, Couté Y, Ben Achour S, Honnorat J, Place C, Pascual O.
    Glia (2021) — Show abstract

    Vesicular release is one of the release mechanisms of various signaling molecules. In neurons, the molecular machinery involved in vesicular release has been designed through evolution to trigger fast and synchronous release of neurotransmitters. Similar machinery with a slower kinetic and a slightly different molecular assembly allows astrocytes to release various transmitters such as adenosine triphosphate (ATP), glutamate, and D-serine. Astrocytes are important modulators of neurotransmission through gliotransmitter release. We recently demonstrated that microglia, another type of glia, release ATP to modulate synaptic transmission using astrocytes as intermediate. We now report that microglia regulate astrocytic gliotransmission through the regulation of SNARE proteins in astrocytes. Indeed, we found that gliotransmission triggered by P2Y1 agonist is impaired in slices from transgenic mice devoid of microglia. Using total internal reflection fluorescence imaging, we found that the vesicular release of gliotransmitter by astrocytes was different in cultures lacking microglia compared to vesicular release in astrocytes cocultured with microglia. Quantification of the kinetic of vesicular release indicates that the overall release appears to be faster in pure astrocyte cultures with more vesicles close to the membrane when compared to astrocytes cocultured with microglia. Finally, biochemical investigation of SNARE protein expression indicates an upregulation of VAMP2 in absence of microglia. Altogether, these results indicate that microglia seems to be involved in the regulation of an astrocytic phenotype compatible with proper gliotransmission. The mechanisms described in this study could be of importance for central nervous system diseases where microglia are activated.

12. VEGF counteracts amyloid-β-induced synaptic dysfunction
    Martin L, Bouvet P, Chounlamountri N, Watrin C, Besançon R, Pinatel D, Meyronet D, Honnorat J, Buisson A, Salin PA, Meissirel C.
    Cell Rep (2021) — Show abstract

    The vascular endothelial growth factor (VEGF) pathway regulates key processes in synapse function, which are disrupted in early stages of Alzheimer's disease (AD) by toxic-soluble amyloid-beta oligomers (Aβo). Here, we show that VEGF accumulates in and around Aβ plaques in postmortem brains of patients with AD and in APP/PS1 mice, an AD mouse model. We uncover specific binding domains involved in direct interaction between Aβo and VEGF and reveal that this interaction jeopardizes VEGFR2 activation in neurons. Notably, we demonstrate that VEGF gain of function rescues basal synaptic transmission, long-term potentiation (LTP), and dendritic spine alterations, and blocks long-term depression (LTD) facilitation triggered by Aβo. We further decipher underlying mechanisms and find that VEGF inhibits the caspase-3-calcineurin pathway responsible for postsynaptic glutamate receptor loss due to Aβo. These findings provide evidence for alterations of the VEGF pathway in AD models and suggest that restoring VEGF action on neurons may rescue synaptic dysfunction in AD.

13. Anti-CASPR2 clinical phenotypes correlate with HLA and immunological features
    Muñiz-Castrillo S, Joubert B, Elsensohn ME, Pinto AL, Saint-Martin M, Vogrig A, Picard G, Rogemond V, Dubois V, Tamouza R, Maucort-Boulch D, Honnorat J
    J Neurol Neurosurg Psychiatry (2020) — Show abstract

    Objective Antibodies against contactin-associated protein-like 2 (CASPR2-Abs) have been described in acquired neuromyotonia, limbic encephalitis (LE) and Morvan syndrome (MoS). However, it is unknown whether these constitute one sole spectrum of diseases with the same immunopathogenesis or three distinct entities with different mechanisms. Methods A cluster analysis of neurological symptoms was performed in a retrospective cohort of 56 CASPR2-Abs patients. In parallel, immunological features and human leucocyte antigen (HLA) were studied. Results Cluster analysis distinguished patients with predominant limbic symptoms (n=29/56) from those with peripheral nerve hyperexcitability (PNH; n=27/56). In the limbic-prominent group, limbic features were either isolated (LE/−; 18/56, 32.1%), or combined with extralimbic symptoms (LE/+; 11/56, 19.6%). Those with PNH were separated in one group with severe PNH and extralimbic involvement (PNH/+; 16/56, 28.6%), resembling historical MoS descriptions; and one group with milder and usually isolated PNH (PNH/−; 11/56, 19.6%). LE/− and LE/+ patients shared immunogenetic characteristics demonstrating a homogeneous entity. HLA-DRB1*11:01 was carried more frequently than in healthy controls only by patients with LE (94.1% vs 18.3%; p=1.3×10−10). Patients with LE also had serum titres (median 1:40 960) and rates of cerebrospinal fluid positivity (93.1%) higher than the other groups (p<0.05). Conversely, DRB1*11:01 association was absent in PNH/+ patients, but only they had malignant thymoma (87.5%), serum antibodies against leucine-rich glioma-inactivated 1 protein (66.7%) and against netrin-1 receptor deleted in colorectal carcinoma (53.8%), and myasthenia gravis (50.0%). Interpretation Symptoms’ distribution supports specific clinical phenotypes without overlap between LE and MoS. The distinct immunogenetic characteristics shared by all patients with LE and the particular oncological and autoimmune associations of MoS suggest two very different aetiopathogenesis.

14. Ketamine/xylazine and barbiturates modulate microglial morphology and motility differently in a mouse model
    Hristovska I, Verdonk F, Comte JC, Tsai ES, Desestret V, Honnorat J, Chrétien F, Pascual O.
    PLoS One (2020) — Show abstract

    Myelination leads to the formation of myelin sheaths surrounding neuronal axons and is crucial for function, plasticity and repair of the central nervous system (CNS). It relies on the interaction of the axons and the oligodendrocytes: the glial cells producing CNS myelin. Here, we have investigated the role of a crucial component of the Sonic hedgehog (Shh) signalling pathway, the co-receptor Boc, in developmental and repairing myelination. During development, Boc mutant mice display a transient decrease in oligodendroglial cell density together with delayed myelination. Despite recovery of oligodendroglial cells at later stages, adult mutants still exhibit a lower production of myelin basic protein correlated with a significant decrease in the calibre of callosal axons and a reduced amount of the neurofilament NF-M. During myelin repair, the altered OPC differentiation observed in the mutant is reminiscent of the phenotype observed after blockade of Shh signalling. In addition, Boc mutant microglia/macrophages unexpectedly exhibit the apparent inability to transition from a highly to a faintly ramified morphology in vivo Altogether, these results identify Boc as an important component of myelin formation and repair.

15. Structural mapping of hot spots within human CASPR2 discoidin domain for autoantibody recognition
    Liang W, Zhang J, Saint-Martin M, Xu F, Noraz N, Liu J, Honnorat J, Liu H.
    J Autoimmun (2019) — Show abstract

    Accumulating evidence has showed that anti-CASPR2 autoantibodies occur in a long list of neurological immune disorders including limbic encephalitis (LE). Belonging to the well-known neurexin superfamily, CASPR2 has been suggested to be a central node in the molecular networks controlling neurodevelopment. Distinct from other subfamilies in the neurexin superfamily, the CASPR subfamily features a unique discoidin (Disc) domain. As revealed by our and others' recent studies, CASPR2 Disc domain bears a major epitope for autoantibodies. However, structural information on CASPR2 recognition by autoantibodies has been lacking. Here, we report the crystal structure of human CASPR2 Disc domain at a high resolution of 1.31 Å, which is the first atomic-resolution structure of the CASPR subfamily members. The Disc domain adopts a total β structure and folds into a distorted jellyroll-like barrel with a conserved disulfide-bond interlocking its N- and C-termini. Defined by four loops and located in one end of the barrel, the "loop-tip surface" is totally polar and easily available for protein docking. Based on structure-guided epitope prediction, we generated nine mutants and evaluated their binding to autoantibodies of cerebrospinal fluid from twelve patients with limbic encephalitis. The quadruple mutant G69N/A71S/S77N/D78R impaired CASPR2 binding to autoantibodies from eleven LE patients, which indicates that the loop L1 in the Disc domain bears hot spots for autoantibody interaction. Structural mapping of autoepitopes within human CASPR2 Disc domain sheds light on how autoantibodies could sequester CASPR2 ectodomain and antagonize its functionalities in the pathogenic processes.

16. Impact of anti-CASPR2 autoantibodies from patients with autoimmune encephalitis on CASPR2/TAG-1 interaction and Kv1 expression
    Saint-Martin M, Pieters A, Déchelotte B, Malleval C, Pinatel D, Pascual O, Karagogeos D, Honnorat J, Pellier-Monnin V, Noraz N.
    J Autoimmun (2019) — Show abstract

    Autoantibodies against CASPR2 (contactin-associated protein-like 2) have been linked to autoimmune limbic encephalitis that manifests with memory disorders and temporal lobe seizures. According to the growing number of data supporting a role for CASPR2 in neuronal excitability, CASPR2 forms a molecular complex with transient axonal glycoprotein-1 (TAG-1) and shaker-type voltage-gated potassium channels (Kv1.1 and Kv1.2) in compartments critical for neuronal activity and is required for Kv1 proper positioning. Whereas the perturbation of these functions could explain the symptoms observed in patients, the pathogenic role of anti-CASPR2 antibodies has been poorly studied. In the present study, we find that patient autoantibodies alter Caspr2 distribution at the cell membrane promoting cluster formation. We confirm in a HEK cellular model that the anti-CASPR2 antibodies impede CASPR2/TAG-1 interaction and we identify the domains of CASPR2 and TAG-1 taking part in this interaction. Moreover, introduction of CASPR2 into HEK cells induces a marked increase of the level of Kv1.2 surface expression and in cultures of hippocampal neurons Caspr2-positive inhibitory neurons appear to specifically express high levels of Kv1.2. Importantly, in both cellular models, anti-CASPR2 patient autoAb increase Kv1.2 expression. These results provide new insights into the pathogenic role of autoAb in the disease.

17. Contactin-associated protein-like 2, a protein of the neurexin family involved in several human diseases
    Saint-Martin M, Joubert B, Pellier-Monnin V, Pascual O, Noraz N, Honnorat J.
    Eur J Neurosci (2018) — Show abstract

    Contactin-associated protein-like 2 (CASPR2) is a cell adhesion protein of the neurexin family. Proteins of this family have been shown to play a role in the development of the nervous system, in synaptic functions, and in neurological diseases. Over recent years, CASPR2 function has gained an increasing interest as demonstrated by the growing number of publications. Here, we gather published data to comprehensively review CASPR2 functions within the nervous system in relation to CASPR2-related diseases in humans. On the one hand, studies on Cntnap2 (coding for CASPR2) knockout mice revealed its role during development, especially, in setting-up the inhibitory network. Consistent with this result, mutations in the CNTNAP2 gene coding for CASPR2 in human have been identified in neurodevelopmental disorders such as autism, intellectual disability, and epilepsy. On the other hand, CASPR2 was shown to play a role beyond development, in the localization of voltage-gated potassium channel (VGKC) complex that is composed of TAG-1, Kv1.1, and Kv1.2. This complex was found in several subcellular compartments essential for action potential propagation: the node of Ranvier, the axon initial segment, and the synapse. In line with a role of CASPR2 in the mature nervous system, neurological autoimmune diseases have been described in patients without neurodevelopmental disorders but with antibodies directed against CASPR2. These autoimmune diseases were of two types: central with memory disorders and temporal lobe seizures, or peripheral with muscular hyperactivity. Overall, we review the up-to-date knowledge on CASPR2 function and pinpoint confused or lacking information that will need further investigation.

18. Antifibroblast growth factor receptor 3 antibodies identify a subgroup of patients with sensory neuropathy
    Antoine JC, Boutahar N, Lassablière F, Reynaud E, Ferraud K, Rogemond V, Paul S, Honnorat J, Camdessanché JP
    Neurol Neurosurg Psychiatry (2015) — Show abstract

    Background: Immunological mechanisms are suspected in sensory neuropathy (SN) occurring with systemic autoimmune diseases and in some idiopathic cases, but so far there are no antibodies (Abs) identifying these neuropathies. Methods: In the search for such specific antibodies, serum samples were collected from 106 patients with SN of these 72 fulfilled the diagnosis criteria of sensory neuronopathy (SNN) and 211 control subjects including patients with sensorimotor neuropathies, other neurological diseases (ONDs), systemic autoimmune diseases and healthy blood donors. Results: In the first step, a protein array with 8000 human proteins allowed identification of the intracellular domain of the fibroblast growth factor receptor 3 (FGFR3) as a target of Abs in 7/16 SNN and 0/30 controls. In the second step, an ELISA method was used to test the 317 patients and controls for anti-FGFR3 Abs. Abs were detected in 16/106 patients with SN and 1/211 controls (p<0.001). Among the 106 patients with SN, anti-FGFR3 Abs were found in 11/38 patients with autoimmune context, 5/46 with idiopathic neuropathy and 0/22 with neuropathy of other aetiology (p=0.006). The only control patient with anti-FGFR3 Abs had lupus and no recorded neuropathy. Sensitivity, specificity, and positive and negative predictive values of anti-FGFR3 Abs for a diagnosis of idiopathic or dysimmune SN were 19%, 99.6%, 94.1% and 77.3%, respectively. A cell-based assay confirmed serum reactivity against the intracellular domain of FGFR3. The neuropathy in patients with anti-FGF3 Abs was non-length dependent in 87% of patients and fulfilled the criteria of probable SNN in 82%. Trigeminal nerve involvement and pain were frequent features. Conclusions: A anti-FGFR3 Abs identify a subgroup of patients with SN in whom an underlying autoimmune disorder affecting sensory neurons in the dorsal root and trigeminal nerve ganglia is suspected.

• Ministry of Health: Reference center on rare diseases. Autoimmune encephalitis and paraneoplastic neurological syndromes.
• Inca: Protéines de guidage axonal et tumeurs neuroendocrines gastro-entero-pancréatiques: rôle dans la progression tumorale
• ANR: Identification de biomarqueurs diagnostiques, physiopathologiques, pronostiques et de progression des troubles
• ANR-PRTS: MECANO : Mechanisms of autoimmune encephalitis
• FRC: Mechanisms of autoimmune abnormal behaviour