Identification of DAGLA as an autoantibody target in cerebellar ataxia.

BACKGROUND: We aimed to investigate the clinical, imaging and fluid biomarker characteristics in patients with antidiacylglycerol lipase alpha (DAGLA)-autoantibody-associated cerebellitis. METHODS: Serum and cerebrospinal fliud (CSF) samples from four index patients were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IIFA). Immunoprecipitation, mass spectrometry and recombinant protein assays were used to identify the autoantigen. Sera from 101 patients with various neurological symptoms and a similar tissue staining pattern as the index patient samples, and 102 healthy donors were analysed in recombinant cell-based IIFA (RC-IIFA) with the identified protein. Epitope characterisation of all positive samples was performed via ELISA, immunoblot, immunoprecipitation and RC-IIFA using different DAGLA fragments. RESULTS: All index patients were relatively young (age: 18-34) and suffered from pronounced gait ataxia, dysarthria and visual impairments. Paraclinical hallmarks in early-stage disease were inflammatory CSF changes and cerebellar cortex hyperintensity in MRI. Severe cerebellar atrophy developed in three of four patients within 6 months. All patient samples showed the same unclassified IgG reactivity with the cerebellar molecular layer. DAGLA was identified as the target antigen and confirmed by competitive inhibition experiments and DAGLA-specific RC-IIFA. In RC-IIFA, serum reactivity against DAGLA was also found in 17/101 disease controls, including patients with different clinical phenotypes than the one of the index patients, and in 1/102 healthy donors. Epitope characterisation revealed that 17/18 anti-DAGLA-positive control sera reacted with a C-terminal intracellular DAGLA 583-1042 fragment, while the CSF samples of the index patients targeted a conformational epitope between amino acid 1 and 157. CONCLUSIONS: We propose that anti-DAGLA autoantibodies detected in CSF, with a characteristic tissue IIFA pattern, represent novel biomarkers for rapidly progressive cerebellitis.

KCNA2 IgG autoimmunity in neuropsychiatric diseases.

BACKGROUND: Autoantibodies against the potassium voltage-gated channel subfamily A member 2 (KCNA2) have been described in a few cases of neuropsychiatric disorders, but their diagnostic and pathophysiological role is currently unknown, imposing challenges to medical practice. DESIGN / METHODS: We retrospectively collected comprehensive clinical and paraclinical data of 35 patients with KCNA2 IgG autoantibodies detected in cell-based and tissue-based assays. Patients' sera and cerebrospinal fluid (CSF) were used for characterization of the antigen, clinical-serological correlations, and determination of IgG subclasses. RESULTS: KCNA2 autoantibody-positive patients (n = 35, median age at disease onset of 65 years, range of 16-83 years, 74 % male) mostly presented with cognitive impairment and/or epileptic seizures but also ataxia, gait disorder and personality changes. Serum autoantibodies belonged to IgG3 and IgG1 subclasses and titers ranged from 1:32 to 1:10,000. KCNA2 IgG was found in the CSF of 8/21 (38 %) patients and in the serum of 4/96 (4.2 %) healthy blood donors. KCNA2 autoantibodies bound to characteristic anatomical areas in the cerebellum and hippocampus of mammalian brain and juxtaparanodal regions of peripheral nerves but reacted exclusively with intracellular epitopes. A subset of four KCNA2 autoantibody-positive patients responded markedly to immunotherapy alongside with conversion to seronegativity, in particular those presenting an autoimmune encephalitis phenotype and receiving early immunotherapy. An available brain biopsy showed strong immune cell invasion. KCNA2 autoantibodies occurred in less than 10 % in association with an underlying tumor. CONCLUSION: Our data suggest that KCNA2 autoimmunity is clinically heterogeneous. Future studies should determine whether KCNA2 autoantibodies are directly pathogenic or develop secondarily. Early immunotherapy should be considered, in particular if autoantibodies occur in CSF or if clinical or diagnostic findings suggest ongoing inflammation. Suspicious clinical phenotypes include autoimmune encephalitis, atypical dementia, new-onset epilepsy and unexplained epileptic seizures.

Case report: Anti septin-5-encephalitis as a treatable cause of cerebellar ataxia and psychiatric symptoms.

Objectives: Anti-septin-5 encephalitis is a rare disease with only few published cases, mainly based on retrospective CSF and serum analyses. Predominant symptoms are cerebellar ataxia and oculomotor abnormalities. Due to the rareness of the disease, treatment recommendations are scarce. Herein, we prospectively describe the clinical course of a female patient with anti-septin-5 encephalitis. Methods: We describe diagnostic workup, treatment and follow-up of a 54-year-old patient presenting with vertigo, unsteady gait, lack of drive and behavioral changes. Results: Clinical examination revealed severe cerebellar ataxia, saccadic smooth pursuit, upbeat-nystagmus, and dysarthria. Additionally, the patient presented with a depressive syndrome. MRI of the brain and spinal cord were normal. CSF analysis showed lymphocytic pleocytosis (11 cells/µl). Extensive antibody testing revealed anti septin-5 IgG in both CSF and serum without coexisting anti-neuronal antibodies. PET/CT detected no signs of malignancy. Corticosteroids, plasma exchange, and rituximab led to transient clinical improvement followed by relapse. Re-applied treatment with plasma exchange followed by bortezomib resulted in moderate but sustained clinical improvement. Discussion: Anti septin-5 encephalitis represents a rare but treatable and therefore relevant differential diagnosis in patients with cerebellar ataxia. Psychiatric symptoms can be observed in anti septin-5 encephalitis. Immunosuppressive treatment including bortezomib is moderately effective.

Septin-3 autoimmunity in patients with paraneoplastic cerebellar ataxia.

BACKGROUND: Septins are cytoskeletal proteins with filament forming capabilities, which have multiple roles during cell division, cellular polarization, morphogenesis, and membrane trafficking. Autoantibodies against septin-5 are associated with non-paraneoplastic cerebellar ataxia, and autoantibodies against septin-7 with encephalopathy with prominent neuropsychiatric features. Here, we report on newly identified autoantibodies against septin-3 in patients with paraneoplastic cerebellar ataxia. We also propose a strategy for anti-septin autoantibody determination. METHODS: Sera from three patients producing similar immunofluorescence staining patterns on cerebellar and hippocampal sections were subjected to immunoprecipitation followed by mass spectrometry. The identified candidate antigens, all of which were septins, were expressed recombinantly in HEK293 cells either individually, as complexes, or combinations missing individual septins, for use in recombinant cell-based indirect immunofluorescence assays (RC-IIFA). Specificity for septin-3 was further confirmed by tissue IIFA neutralization experiments. Finally, tumor tissue sections were analyzed immunohistochemically for septin-3 expression. RESULTS: Immunoprecipitation with rat cerebellum lysate revealed septin-3, -5, -6, -7, and -11 as candidate target antigens. Sera of all three patients reacted with recombinant cells co-expressing septin-3/5/6/7/11, while none of 149 healthy control sera was similarly reactive. In RC-IIFAs the patient sera recognized only cells expressing septin-3, individually and in complexes. Incubation of patient sera with five different septin combinations, each missing one of the five septins, confirmed the autoantibodies' specificity for septin-3. The tissue IIFA reactivity of patient serum was abolished by pre-incubation with HEK293 cell lysates overexpressing the septin-3/5/6/7/11 complex or septin-3 alone, but not with HEK293 cell lysates overexpressing septin-5 as control. All three patients had cancers (2 × melanoma, 1 × small cell lung cancer), presented with progressive cerebellar syndromes, and responded poorly to immunotherapy. Expression of septin-3 was demonstrated in resected tumor tissue available from one patient. CONCLUSIONS: Septin-3 is a novel autoantibody target in patients with paraneoplastic cerebellar syndromes. Based on our findings, RC-IIFA with HEK293 cells expressing the septin-3/5/6/7/11 complex may serve as a screening tool to investigate anti-septin autoantibodies in serological samples with a characteristic staining pattern on neuronal tissue sections. Autoantibodies against individual septins can then be confirmed by RC-IIFA expressing single septins.

Autoantibodies Against the Purkinje Cell Protein RGS8 in Paraneoplastic Cerebellar Syndrome.

OBJECTIVE: To describe the identification of regulator of G-protein signaling 8 (RGS8) as an autoantibody target in patients with cerebellar syndrome associated with lymphoma. METHODS: Sera of 4 patients with a very similar unclassified reactivity against cerebellar Purkinje cells were used in antigen identification experiments. Immunoprecipitations with cerebellar lysates followed by mass spectrometry identified the autoantigen, which was verified by recombinant immunofluorescence assay, immunoblot, and ELISA with the recombinant protein. RESULTS: The sera and CSF of 4 patients stained the Purkinje cells and molecular layer of the cerebellum. RGS8 was identified as the target antigen in all 4 sera. In a neutralization experiment, recombinant human RGS8 was able to neutralize the autoantibodies' tissue reaction. Patient sera and CSF showed a specific reactivity against recombinant RGS8 in ELISA and immunoblot, whereas no such reactivity was detectable in the controls. Clinical data were available for 2 of the 4 patients, remarkably both presented with cerebellar syndrome accompanied by B-cell lymphoma of the stomach (patient 1, 53 years) or Hodgkin lymphoma (patient 2, 74 years). CONCLUSION: Our results indicate that autoantibodies against the intracellular Purkinje cell protein RGS8 represent new markers for paraneoplastic cerebellar syndrome associated with lymphoma. CLASSIFICATION OF EVIDENCE: This study provided Class IV evidence that autoantibodies against the intracellular Purkinje cell protein RGS8 are associated with paraneoplastic cerebellar syndrome in lymphoma.

Onconeural Antibodies in Acute Psychiatric Inpatient Care.

Paraneoplastic neurological disorders associated with onconeural antibodies often appear with neuropsychiatric symptoms. To study the prevalence of onconeural antibodies in patients admitted to acute psychiatric inpatient care, the serum of 585 such patients was tested for antibodies targeting MOG, GLRA1B, DPPX, GRM1, GRM5, DNER, Yo, ZIC4, GAD67, amphiphysin, CV2, Hu, Ri, Ma2, and recoverin. Only one sample was positive (antirecoverin IgG). The present findings suggest that serum onconeural antibody positivity is rare among patients acutely admitted for inpatient psychiatric care. The clinical implications of this finding are discussed.

Seroprevalence of autoantibodies against brain antigens in health and disease.

OBJECTIVE: We previously reported an unexpectedly high seroprevalence (~10%) of N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1) autoantibodies (AB) in healthy and neuropsychiatrically ill subjects (N = 2,817). This finding challenges an unambiguous causal relationship of serum AB with brain disease. To test whether similar results would be obtained for other brain antigen-directed AB previously connected with pathological conditions, we systematically screened serum samples of 4,236 individuals. METHODS: Serum samples of healthy (n = 1,703) versus neuropsychiatrically ill subjects (schizophrenia, affective disorders, stroke, Parkinson disease, amyotrophic lateral sclerosis, personality disorder; total n = 2,533) were tested. For analysis based on indirect immunofluorescence, we used biochip mosaics of frozen brain sections (rat, monkey) and transfected HEK293 cells expressing respective recombinant target antigens. RESULTS: Seroprevalence of all screened AB was comparable in healthy and ill individuals. None of them, however, reached the abundance of NMDAR1 AB (again ~10%; immunoglobulin [Ig] G ~1%). Appreciable frequency was noted for AB against amphiphysin (2.0%), ARHGAP26 (1.3%), CASPR2 (0.9%), MOG (0.8%), GAD65 (0.5%), Ma2 (0.5%), Yo (0.4%), and Ma1 (0.4%), with titers and Ig class distribution similar among groups. All other AB were found in ≤0.1% of individuals (anti-AMPAR-1/2, AQP4, CV2, Tr/DNER, DPPX-IF1, GABAR-B1/B2, GAD67, GLRA1b, GRM1, GRM5, Hu, LGl1, recoverin, Ri, ZIC4). The predominant Ig class depended on antigen location, with intracellular epitopes predisposing to IgG (chi-square = 218.91, p = 2.8 × 10(-48) ). INTERPRETATION: To conclude, the brain antigen-directed AB tested here are comparably detectable in healthy subjects and the disease groups studied here, thus questioning an upfront pathological role of these serum AB.

Anti-Ca/anti-ARHGAP26 antibodies associated with cerebellar atrophy and cognitive decline.

Recently, we identified a novel Purkinje cell-specific autoantibody (termed anti-Ca) targeting rhoGTPase-activating-protein-26 (ARHGAP26) in a patient with cerebellar ataxia. Here we describe a new case of anti-Ca/ARHGAP26 antibody-positive cerebellar ataxia. Cerebellar ataxia was associated with signs of possible limbic encephalitis in this case. The 24-year-old man presented with subacute pancerebellar ataxia, flattened affect, and cognitive decline. Neuropsychological testing revealed working memory deficits, compromised verbal learning and recall, attention deficits, slowed information processing, interference difficulty, and reduced spatial recognition. MRI showed severe pancerebellar atrophy. Serological examinations revealed high-titre anti-Ca/anti-ARHGAP26 antibodies. The antibodies belonged to the IgG1 subclass and were produced intrathecally. This case further corroborates the association of anti-Ca antibodies with cerebellar ataxia, expands the clinical spectrum, and highlights the necessity of antigen-specific diagnostic testing in immune-mediated cerebellar ataxia.

Two new cases of anti-Ca (anti-ARHGAP26/GRAF) autoantibody-associated cerebellar ataxia.

Recently, we discovered a novel serum and cerebrospinal fluid (CSF) autoantibody (anti-Ca) to Purkinje cells in a patient with autoimmune cerebellar ataxia (ACA) and identified the RhoGTPase-activating protein 26 (ARHGAP26; alternative designations include GTPase regulator associated with focal adhesion kinase pp125, GRAF, and oligophrenin-1-like protein, OPHN1L) as the target antigen. Here, we report on two new cases of ARHGAP26 autoantibody-positive ACA that were first diagnosed after publication of the index case study. While the index patient developed ACA following an episode of respiratory infection with still no evidence for malignancy 52 months after onset, neurological symptoms heralded ovarian cancer in one of the patients described here. Our finding of anti-Ca/anti-ARHGAP26 antibodies in two additional patients supports a role of autoimmunity against ARHGAP26 in the pathogenesis of ACA. Moreover, the finding of ovarian cancer in one of our patients suggests that anti-Ca/anti-ARHGAP26-positive ACA might be of paraneoplastic aetiology in some cases. In conclusion, testing for anti-Ca/anti-ARHGAP26 should be included in the diagnostic work-up of patients with ACA, and an underlying tumour should be considered in patients presenting with anti-Ca/ARHGAP26 antibody-positive ACA.

N-methyl-D-aspartate receptor antibodies in herpes simplex encephalitis.

OBJECTIVE: To determine the presence and kinetics of antibodies against synaptic proteins in patients with herpes simplex virus encephalitis (HSE). METHODS: Retrospective analysis of 44 patients with polymerase chain reaction-proven HSE for the presence of a large panel of onconeuronal and synaptic receptor antibodies. The effect of patients' serum was studied in cultures of primary mouse hippocampal neurons. RESULTS: N-Methyl-D-aspartate receptor (NMDAR) antibodies of the immunoglobulin (Ig) subtypes IgA, IgG, or IgM were detected in 13 of 44 patients (30%) in the course of HSE, suggesting secondary autoimmune mechanisms. NMDAR antibodies were often present at hospital admission, but in some patients developed after the first week of HSE. Antibody-positive sera resulted in downregulation of synaptic marker proteins in hippocampal neurons. INTERPRETATION: Some patients with HSE develop IgA, IgG, or IgM autoantibodies against NMDAR. Sera from these patients alter the density of neuronal synaptic markers, suggesting a potential pathogenic disease-modifying effect. These findings have implications for the understanding of autoimmunity in infectious diseases, and prospective studies should reveal whether the subgroup of patients with HSE and NMDAR antibodies may benefit from immunotherapy. .