Investigations in GABAA receptor antibody-associated encephalitis

GABA-A receptor; Encephalitis; AntibodiesReceptor GABA-A; Encefalitis; AnticossosReceptor GABA-A; Encefalitis; AnticuerposOBJECTIVE: To report the clinical features, comorbidities, receptor subunit targets, and outcome in patients with anti-GABAA receptor (GABAAR) encephalitis. METHODS: Clinical study of 26 patients, including 17 new (April 2013-January 2016) and 9 previously reported patients. Antibodies to α1, β3, and γ2 subunits of the GABAAR were determined using reported techniques. RESULTS: Patients' median age was 40.5 years (interquartile range 48.5 [13.75-62.35] years; the youngest 2.5 months old; 13 female). Symptoms included seizures (88%), alteration of cognition (67%), behavior (46%), consciousness (42%), or abnormal movements (35%). Comorbidities were identified in 11 (42%) patients, including 7 tumors (mostly thymomas), 2 herpesvirus encephalitis (herpes simplex virus 1, human herpesvirus 6; coexisting with NMDAR antibodies), and 2 myasthenia without thymoma. Brain MRI was abnormal in 23 (88%) patients, showing in 20 (77%) multifocal, asynchronous, cortical-subcortical T2/fluid-attenuated inversion recovery abnormalities predominantly involving temporal (95%) and frontal (65%) lobes, but also basal ganglia and other regions. Immunologic or tumor therapy resulted in substantial improvement in 18/21 (86%) assessable patients; the other 3 (14%) died (2 status epilepticus, 1 sepsis). Compared with adults, children were more likely to have generalized seizures (p = 0.007) and movement disorders (p = 0.01) and less likely to have a tumor (p = 0.01). The main epitope targets were in the α1/β3 subunits of the GABAAR. CONCLUSIONS: Anti-GABAAR encephalitis is characterized by frequent seizures and distinctive multifocal cortical-subcortical MRI abnormalities that provide an important clue to the diagnosis. The frequency of symptoms and comorbidities differ between children (more viral-related) and adults (more tumor-related). The disorder is severe but most patients respond to treatment

Paraneoplastic cerebellar ataxia and antibodies to metabotropic glutamate receptor 2

To report the presence of a new neuronal surface antibody against the metabotropic glutamate receptor 2 antibody (mGluR2-Ab) in 2 patients with paraneoplastic cerebellar ataxia.mGluR2-Abs were initially characterized by immunohistochemistry on the rat brain and confirmed by immunofluorescence on HEK293 cells transfected with mGluR2. Additional studies included analysis of potential cross-reactivity with other mGluRs, expression of mGluR2 in patients' tumors, and the effects of mGluR2-Abs on cultures of rat hippocampal neurons.Patient 1 was a 78-year-old woman with progressive cerebellar ataxia with an initial relapsing-remitting course who developed a small-cell tumor of unknown origin. Patient 2 was a 3-year-old girl who presented a steroid-responsive acute cerebellitis preceding the diagnosis of an alveolar rhabdomyosarcoma. Patients' serum and CSF showed a characteristic immunostaining of the hippocampus and cerebellum in rat brain sections and immunolabeled the cell surface of live rat hippocampal neurons. HEK293 cells transfected with mGluR1, 2, 3, and 5 confirmed that patients' antibodies only recognized mGluR2. mGluR2-Abs were not detected in 160 controls, 120 with paraneoplastic, autoimmune, or degenerative ataxias, and 40 with autoimmune encephalitis and antibodies against mGluR5 or unknown antigens. Expression of mGluR2 in tumors was confirmed by immunohistochemistry using a commercial mGluR2-Ab. Incubation of live rat hippocampal neurons with CSF of patient 2 did not modify the density of surface mGluR2 clusters.mGluR2-Abs are a novel biomarker of paraneoplastic cerebellar ataxia. The potential pathogenic effect of the antibodies is not mediated by downregulation or internalization of neuronal surface mGluR2.Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology

LGI1 antibodies alter Kv1.1 and AMPA receptors changing synaptic excitability, plasticity and memory

Leucine-rich glioma-inactivated 1 (LGI1) is a secreted neuronal protein that forms a trans-synaptic complex that includes the presynaptic disintegrin and metalloproteinase domain-containing protein 23 (ADAM23), which interacts with voltage-gated potassium channels Kv1.1, and the postsynaptic ADAM22, which interacts with AMPA receptors. Human autoantibodies against LGI1 associate with a form of autoimmune limbic encephalitis characterized by severe but treatable memory impairment and frequent faciobrachial dystonic seizures. Although there is evidence that this disease is immune-mediated, the underlying LGI1 antibody-mediated mechanisms are unknown. Here, we used patient-derived immunoglobulin G (IgG) antibodies to determine the main epitope regions of LGI1 and whether the antibodies disrupt the interaction of LGI1 with ADAM23 and ADAM22. In addition, we assessed the effects of patient-derived antibodies on Kv1.1, AMPA receptors, and memory in a mouse model based on cerebroventricular transfer of patient-derived IgG. We found that IgG from all patients (n = 25), but not from healthy participants (n = 20), prevented the binding of LGI1 to ADAM23 and ADAM22. Using full-length LGI1, LGI3, and LGI1 constructs containing the LRR1 domain (EPTP1-deleted) or EPTP1 domain (LRR3-EPTP1), IgG from all patients reacted with epitope regions contained in the LRR1 and EPTP1 domains. Confocal analysis of hippocampal slices of mice infused with pooled IgG from eight patients, but not pooled IgG from controls, showed a decrease of total and synaptic levels of Kv1.1 and AMPA receptors. The effects on Kv1.1 preceded those involving the AMPA receptors. In acute slice preparations of hippocampus, patch-clamp analysis from dentate gyrus granule cells and CA1 pyramidal neurons showed neuronal hyperexcitability with increased glutamatergic transmission, higher presynaptic release probability, and reduced synaptic failure rate upon minimal stimulation, all likely caused by the decreased expression of Kv1.1. Analysis of synaptic plasticity by recording field potentials in the CA1 region of the hippocampus showed a severe impairment of long-term potentiation. This defect in synaptic plasticity was independent from Kv1 blockade and was possibly mediated by ineffective recruitment of postsynaptic AMPA receptors. In parallel with these findings, mice infused with patient-derived IgG showed severe memory deficits in the novel object recognition test that progressively improved after stopping the infusion of patient-derived IgG. Different from genetic models of LGI1 deficiency, we did not observe aberrant dendritic sprouting or defective synaptic pruning as potential cause of the symptoms. Overall, these findings demonstrate that patient-derived IgG disrupt presynaptic and postsynaptic LGI1 signalling, causing neuronal hyperexcitability, decreased plasticity, and reversible memory deficits

Seizure-related 6 homolog like 2 autoimmunity: Neurologic syndrome and antibody effects

Objective: To describe the clinical syndrome of 4 new patients with seizure-related 6 homolog like 2 antibodies (SEZ6L2-abs), study the antibody characteristics, and evaluate their effects on neuronal cultures. Methods: SEZ6L2-abs were initially identified in serum and CSF of a patient with cerebellar ataxia by immunohistochemistry on rat brain sections and immunoprecipitation from rat cerebellar neurons. We used a cell-based assay (CBA) of HEK293 cells transfected with SEZ6L2 to test the serum of 95 patients with unclassified neuropil antibodies, 331 with different neurologic disorders, and 10 healthy subjects. Additional studies included characterization of immunoglobulin G (IgG) subclasses and the effects of SEZ6L2-abs on cultures of rat hippocampal neurons. Results: In addition to the index patient, SEZ6L2-abs were identified by CBA in 3/95 patients with unclassified neuropil antibodies but in none of the 341 controls. The median age of the 4 patients was 62 years (range: 54-69 years), and 2 were female. Patients presented with subacute gait ataxia, dysarthria, and mild extrapyramidal symptoms. Initial brain MRI was normal, and CSF pleocytosis was found in only 1 patient. None improved with immunotherapy. SEZ6L2-abs recognized conformational epitopes. IgG4 SEZ6L2-abs were found in all 4 patients, and it was the predominant subclass in 2. SEZ6L2-abs did not alter the number of total or synaptic SEZ6L2 or the AMPA glutamate receptor 1 (GluA1) clusters on the surface of hippocampal neurons. Conclusions: SEZ6L2-abs associate with a subacute cerebellar syndrome with frequent extrapyramidal symptoms. The potential pathogenic effect of the antibodies is not mediated by internalization of the antigen

Antigens and mechanisms of immune-mediated encephalitis

[eng] BACKGROUND: The autoimmune encephalitis represents a new category of immune- mediated diseases of the brain that are mediated by antibodies against neurotransmitter receptors, ion channels or neuronal cell-surface proteins. Among the many encephalitis considered idiopathic, there are subtypes that are probably immune-mediated and are pending to be discovered. In the last 11 years, 16 subtypes of idiopathic encephalitis have been found to be immune mediated. The recognition of these disorders is important because despite being potentially lethal, they are curable if promptly recognized and treated. On a more basic level, the study of these diseases has uncovered novel antibody-mediated mechanisms of synaptic dysfunction that lead to changes in memory and behavior, epilepsy, abnormal movements, or decreased level of consciousness. OBJECTIVES: 1) Identify patients with encephalitis of unclear etiology but whose clinical features and initial investigations strongly suggest an antibody-mediated cause; 2) characterize the new autoantibodies associated with these disorders along with the neuronal target antigens, and develop unambiguous diagnostic tests, and 3) determine in animal models how autoantibodies alter the level and function of neuronal synaptic antigens (NMDAR and LGI1) potentially resulting in an impairment of memory and behavior. METHODS: Rat brain sections and primary cultures of dissociated rat hippocampal neurons served to identify patients whose serum and CSF samples contained novel neuronal antibodies. Techniques of tissue immunohistochemistry, neuronal immunocytochemistry and cell-based assays were used to identify the antibodies. Cell- surface neuronal protein immunoprecipitation with patients’ antibodies was used to isolate the target antigen, which was subsequently characterized by mass spectrometry. Cerebroventricular transfer of patients’ antibodies to mice through subcutaneously implanted mini-osmotic pumps was used to determine the pathogenic effect of patients’ antibodies on the corresponding synaptic targets and how these changes altered memory and behavior. An extensive combination of techniques was used for these studies, including quantitative confocal microscopy analysis of the levels of synaptic targets, immunoprecipitation of brain-bound antibodies, immunoblot of precipitated proteins, electrophysiology on acute sections of mice hippocampus, and a comprehensive panel of standard behavioral tests. All studies were conducted with sets of mice at different time points during the antibody infusion (disease phase) and after the infusion was stopped (recovery phase). RESULTS: (1) Two novel autoimmune encephalitis were identified: anti-GABAaR encephalitis and anti-neurexin-3α encephalitis. Anti-GABAaR encephalitis can affect children and adults, associates with prominent seizures and highly suggestive MRI abnormalities, and is treatable with immunotherapy. In some patients the immune response is triggered by the presence of a tumor. Anti-neurexin-3α encephalitis manifests with a less distinctive syndrome, often associates with seizures and is also treatable with immunotherapy. No tumor association has been identified. (2) Immunoprecipitation of the target antigen of anti-GABAaR encephalitis demonstrated that the epitopes are mainly located in the alpha1 and beta3 subunits, and less frequently in the γ2 subunit. Whereas the antibodies against alpha1 and beta3 subunits are disease relevant, the presence of additional antibodies to γ2 does not modify the disease phenotype. Patients’ GABAaR antibodies cause a decrease in the total and synaptic levels of GABAaR clusters, supporting their pathogenicity. (3) Immunoprecipitation of the target antigen of patients with anti-neurexin-3α encephalitis demonstrated that the epitopes are specifically located in neurexin-3α, but not in its postsynaptic ligand LRRTM2. Patients’ antibodies cause a reduction of the total number of synapses as well as the levels of the presynaptic protein Bassoon and the post-synaptic protein Homer1, supporting the pathogenicity of the antibodies. (4) Recombinant expression of the indicated subunits of the GABAaR and neurexin-3α in HEK293 cells can be used as a test to diagnose patients with these autoimmune encephalitis. (5) The infusion of patients’ NMDAR antibodies into the cerebroventricular system of mice, cause memory deficits, anhedonia, and depressive-like behavior. The infused antibodies specifically bind to brain NMDAR resulting in a highly specific reduction of the density of these receptors at synaptic and extrasynaptic levels. The behavioral and molecular effects caused by patients’ antibodies are reversible upon stopping the infusion of antibodies. (6) The administration of ephrin-B2 antagonizes the pathogenic effects of patients’ NMDAR antibodies in all the investigated paradigms, including memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients’ antibody effects. (7) The antibodies of patients with anti-LGI1 encephalitis abrogate the binding of the neuronal secreted LGI1 with the presynaptic ADAM23 and with the postsynaptic ADAM22. (8) The infusion of patients’ LGI1 antibodies into the cerebroventricular system of mice cause protracted memory deficits, together with a decrease of presynaptic Kv1.1 potassium channels and post- synaptic AMPAR. These structural effects associate with impairment of synaptic plasticity and increase of neuronal excitability, which are in line with the models of genetic depletion of LGI1. CONCLUSIONS: (1) Anti-GABAaR and anti-neurexin-3α encephalitis are two new forms of antibody-mediated encephalitis for which there is evidence of antibody-mediated pathogenicity. These findings support the concept that among the many types of encephalitis of unclear etiology, there are some that are immune mediated but are still pending to characterize. (2) Direct neuronal antigen precipitation using patients’ antibodies is an excellent strategy to isolate and characterize disease-relevant antigens, and subsequently develop diagnostic screening tests. (3) My studies have contributed to develop two animal models of antibody-mediated symptoms (NMDAR, LGI1) and uncover the underlying antibody-mediated changes in synaptic function and plasticity. Both models fulfill the Witebsky’s criteria for antibody-mediated disease, and provide the basis for modeling other antibody-mediated neurological disorders.[spa] INTRODUCCIÓN: La encefalitis autoimmune representa una nueva categoria de enfermedades immunomediadas del cerebro que estan mediadas por anticuerpos dirigidos contra receptores sinápticos, canales iónicos o proteinas neuronales de superfície. De entre todas las encefalitis consideradas idiopaticas, hay un subgrupo, probablemente immunomedidos, pendientes de ser identificadas. En los ultimos 11 años, 16 subtipos de encefalitis idiopaticas han sido identificadas como immunomediadas. La identificación de estas enfermedades es importante porque, aunque son potencialmente letales, son curables si se identifican rapid de nuevas en amente y se tratan. El estudio de estas enfermedades ha permitido el descubrimiento de nuevos mecanismos de alteración de la sinapsi medidados por anticuerpos que dan lugar a alteraciones de comportamiento y de memoria, epilepsia, movimientos anormales, o bajo nivel de consciencia. OBJETIVOS: 1) Identificar pacientes con encefalitis de etiologia desconocida, pero con caracteristicas clinicas y paraclinicas que sugieren una causa immunomedidada; 2) Caracterizar nuevos autoanticuerpos asociados a estos trastornos asi como las dianas antigenicas, y desarrollar pruebas diagnosticas inequivocas, y 3) determinar en modelos animales como los autoanticuerpos alteran el nivel y funcion de de los antigenos neuronales sinápticos (NMDAR y LGI1). MÉTODOS: Se usaron secciones de cerebro de rata y cultivos primarios de neuronas hipocampales de rata para la identificación de nuevos anticuerpos contra proteinas neuronales en muestras de suero y liquido cefaloraquideo (LCR) de pacientes. Para la detección de los anticuerpos se usó immunohistoquimica, immunocitoquimica de neuronas y en células HEK293 transfectadas. El aislamiento de nuevos antigeno diana se hizo mediante la immunoprecipitacion de proteinas de superficie neuronal con anticuerpos de los pacientes, y posterior caracterización por espectometria de masas. Para determinar el efecto patogenico de los anticuerpos de los pacientes, durante 14 dias los ratones fueron expuestos de manera cerebroventricular a los anticuerpos mediante la implantación subcutanea de bombas osmoticas, y posteriormente se analizó el efecto sobre las dianas antigenicas asi como alteraciones de comportamiento y memoria. Para ello, se usaron tecnicas de microscopia confocal cuantitativa de los niveles de antigenos sinápticos, immunoprecipitación de los anticuerpos unidos al tejido cerebral, immunoblot de las proteinas precipitadas, electrofisiologia en secciones de cerebro de raton, y una bateria de pruebas de comportamiento estandarizadas. Todos los estudios se realizaron en dias determinados de exposición a los anticuerpos (fase de la enfermedad), asi como después de la infusión (fase de recuperación). RESULTADOS: (1) Se identificaron dos nuevas encefalitis autoimmunes: encefalitis anti-GABAaR y encefalitis anti-neurexin-3α. La encefalitis anti-GABAaR puede afectar niños y adultos, esta asociada con crisis epilepticas y anormalidades en la resonancia magnetica del cerebro, y es tratable con immunoterapia. En algunos pacientes la respuesta immune se desencadena por la presencia de un tumor. La encefalitis anti-neurexin-3α manifiesta con un sindrome menos distintivo, a menudo se asocia con crisis epilepticas y tambien es tratable con immunoterapia. No se ha identificado asociacion tumoral. (2) La diana antigenica de los anticuerpos de la encefalitis anti-GABAaR se encuentra principalmente en las subunidades alfa1 y β3, y menos frecuentemente en la subunidad γ2. Mientras que las subunidades alfa1 y β3 son relevantes para la enfermedad, la presencia de anticuerpos adicionales contra γ2 no modifica el fenotipo de la enfermedad. Los anticuerpos anti-GABAaR producen una disminución de los niveles de GABAaR totales y sinapticos, apoyando su patogenicidad. (3) La immunoprecipitación de la diana antigenica de pacientes con encefalitis anti-neurexin-3α demostró que los epitopos estan localizados en la subunidad 3alfa, y no en su ligando postsinaptico LRRTM2. Estos autoanticuerpos causan una reducción del numero total de sinapsis asi como de la proteina presinaptica Bassoon, y de la postsinaptica Homer1, apoyando su patogenicidad. (4) La expresion recombinante de las subunidades antigenicas en celulas HEK293, pueden ser usadas como prueba diagnostica para identificar pacientes con estas encefalitis autoimmunes. (5) La infusión de anticuerpos NMDAR en el sistema cerebroventricular de ratones causa problemas de memoria, anhedonia, y comportamientos depresivos. Los anticuerpos infusionados se unen especificamente a los receptores NMDA del cerebro y provocan la disminución de la densidad de dichos receptores a nivel sinaptico y extrasinaptico. Estos efectos son reversibles despues de la suspension de la infusion de anticuerpos. (6) La administracion de ephrin-B2 antagoniza el efecto patogenico de los anticuerpos contra NMDAR de los pacientes, incluyendo los efectos de memoria, depresion, densidad de receptores NMDA y EphB2, y plasticidad sinaptica a largo plazo, pudiendo ser usado como estrategia terapeutica. (7) Los anticuerpos de los pacientes con encefalitis anti-LGI1 impiden la unión entre la proteina neuronal secretada LGI1 y la proteina presinaptica ADAM23 y la postsinaptica ADAM22. (8) La infusión de anticuerpos de los pacientes contra LGI1 en el sistema cerebroventricular de los ratones causa problemas de memoria, asi como disminución de los niveles de canales de potasio Kv1.1 presinapticos y de receptores AMPA postsinapticos. Estos efectos se asocian a una alteración de la plasticidad sinaptica e incremento de excitabilidad neuronal, de manera similar a los modelos de depleción genetica de LGI1. CONCLUSIONES: (1) Las encefalitis Anti-GABAaR y anti-neurexin-3α encephalitis son dos nuevas formas de encefalitis mediadas por anticuerpos. Hay evidencia que sostiene que estos anticuerpos son patogenicos, apoyando el concepto de que hay encefalitis sin caracterizar dentro de las muchas formas de encefalitis de causa desconocida. (2) La precipitación del antigeno neuronal es una estrategia excelente para aislar y caracterizar antigenos relevantes para la enfermedad, y poder desarrollar pruebas de detección. (3) Estos estudios han contribuido a desarrollar dos modelos animales de infusión de anticuerpos de encefalitis autoimmunes (NMDAR, LGI1) y a descubrir los cambios subyacentes mediados por anticuerpos en la función sináptica y la plasticidad. Los dos modelos cumplen con los criterios de Witebsky para enfermedades immunomediadas, y proporcionan la base para modelar otros trastornos neurológicos mediados por anticuerpos

Antibodies to inhibitory synaptic proteins in neurological syndromes associated with glutamic acid decarboxylase autoimmunity

Antibodies to glutamic acid decarboxylase (GAD-ab) associate to different neurological syndromes. It is unknown if the diversity in syndrome association represents epitopes in different immunodominant domains or co-existence of antibodies to other proteins of the inhibitory synapsis. We examined the serum and CSF of 106 patients with anti-GAD related syndromes (39 cerebellar ataxia, 32 stiff-person syndrome [SPS], 18 epilepsy, and 17 limbic encephalitis [LE]). GAD65-ab titres were quantified by ELISA. Immunoblot was used to determine if the antibody-targeted epitopes of GAD65 and GAD67 were linear. A cell-based assay (CBA) with HEK293 cells expressing the GAD65 N-terminal, central catalytic domain, or C-terminal was used to investigate the immunodominant domains. Antibodies to GAD67, gamma-aminobutyric acid A receptor (GABAaR), glycine receptor (GlyR), GABAaR-associated protein (GABARAP), and gephyrin were determined with CBA. GAD-ab internalization was investigated using cultured rat hippocampal neurons. CSF GAD65-ab titres were higher in patients with cerebellar ataxia and LE compared to those with SPS (p = 0.02). GAD67-ab were identified in 81% of sera and 100% of CSF. GAD65-ab recognized linear epitopes in 98% of the patients and GAD67-ab in 42% (p<0.001). The GAD65 catalytic domain was recognized by 93% of sera, and the three domains by 22% of sera and 74% of CSF (p<0.001). Six patients had GABAaR-ab and another 6 had GlyR-ab without association to distinctive symptoms. None of the patients had gephyrin- or GABARAP-ab. GAD65-ab were not internalized by live neurons. Overall, these findings show that regardless of the neurological syndrome, the CSF immune response against GAD is more widespread than that of the serum and that there is no specific association between clinical phenotype and the presence of antibodies against other proteins of the inhibitory synapsis

Antibodies to inhibitory synaptic proteins in neurological syndromes associated with glutamic acid decarboxylase autoimmunity.

Antibodies to glutamic acid decarboxylase (GAD-ab) associate to different neurological syndromes. It is unknown if the diversity in syndrome association represents epitopes in different immunodominant domains or co-existence of antibodies to other proteins of the inhibitory synapsis. We examined the serum and CSF of 106 patients with anti-GAD related syndromes (39 cerebellar ataxia, 32 stiff-person syndrome [SPS], 18 epilepsy, and 17 limbic encephalitis [LE]). GAD65-ab titres were quantified by ELISA. Immunoblot was used to determine if the antibody-targeted epitopes of GAD65 and GAD67 were linear. A cell-based assay (CBA) with HEK293 cells expressing the GAD65 N-terminal, central catalytic domain, or C-terminal was used to investigate the immunodominant domains. Antibodies to GAD67, gamma-aminobutyric acid A receptor (GABAaR), glycine receptor (GlyR), GABAaR-associated protein (GABARAP), and gephyrin were determined with CBA. GAD-ab internalization was investigated using cultured rat hippocampal neurons. CSF GAD65-ab titres were higher in patients with cerebellar ataxia and LE compared to those with SPS (p = 0.02). GAD67-ab were identified in 81% of sera and 100% of CSF. GAD65-ab recognized linear epitopes in 98% of the patients and GAD67-ab in 42% (p<0.001). The GAD65 catalytic domain was recognized by 93% of sera, and the three domains by 22% of sera and 74% of CSF (p<0.001). Six patients had GABAaR-ab and another 6 had GlyR-ab without association to distinctive symptoms. None of the patients had gephyrin- or GABARAP-ab. GAD65-ab were not internalized by live neurons. Overall, these findings show that regardless of the neurological syndrome, the CSF immune response against GAD is more widespread than that of the serum and that there is no specific association between clinical phenotype and the presence of antibodies against other proteins of the inhibitory synapsis

Antibodies to inhibitory synaptic proteins in neurological syndromes associated with glutamic acid decarboxylase autoimmunity

Antibodies to glutamic acid decarboxylase (GAD-ab) associate to different neurological syndromes. It is unknown if the diversity in syndrome association represents epitopes in different immunodominant domains or co-existence of antibodies to other proteins of the inhibitory synapsis. We examined the serum and CSF of 106 patients with anti-GAD related syndromes (39 cerebellar ataxia, 32 stiff-person syndrome [SPS], 18 epilepsy, and 17 limbic encephalitis [LE]). GAD65-ab titres were quantified by ELISA. Immunoblot was used to determine if the antibody-targeted epitopes of GAD65 and GAD67 were linear. A cell-based assay (CBA) with HEK293 cells expressing the GAD65 N-terminal, central catalytic domain, or C-terminal was used to investigate the immunodominant domains. Antibodies to GAD67, gamma-aminobutyric acid A receptor (GABAaR), glycine receptor (GlyR), GABAaR-associated protein (GABARAP), and gephyrin were determined with CBA. GAD-ab internalization was investigated using cultured rat hippocampal neurons. CSF GAD65-ab titres were higher in patients with cerebellar ataxia and LE compared to those with SPS (p = 0.02). GAD67-ab were identified in 81% of sera and 100% of CSF. GAD65-ab recognized linear epitopes in 98% of the patients and GAD67-ab in 42% (p<0.001). The GAD65 catalytic domain was recognized by 93% of sera, and the three domains by 22% of sera and 74% of CSF (p<0.001). Six patients had GABAaR-ab and another 6 had GlyR-ab without association to distinctive symptoms. None of the patients had gephyrin- or GABARAP-ab. GAD65-ab were not internalized by live neurons. Overall, these findings show that regardless of the neurological syndrome, the CSF immune response against GAD is more widespread than that of the serum and that there is no specific association between clinical phenotype and the presence of antibodies against other proteins of the inhibitory synapsis