We report on a serum autoantibody associated with cerebellar ataxia. Immunohistochemical studies of sera from four patients referred for autoantibody testing revealed binding of high-titer (up to 1:5,000) IgG antibodies, mainly IgG1, to the molecular layer, Purkinje cell layer, and white matter on mouse, rat, porcine, and monkey cerebellum sections. The antibody bound to PC somata, dendrites, and axons, resulting in a binding pattern similar to that reported for anti-Ca/anti-ARHGAP26, but did not react with recombinant ARHGAP26. Extensive control studies were performed to rule out a broad panel of previously described paraneoplastic and non-paraneoplastic anti-neural autoantibodies. The characteristic binding pattern as well as double staining experiments suggested inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) as the target antigen. Verification of the antigen included specific neutralization of the tissue reaction following preadsorption with ITPR1 (but not ARHGAP26) and a dot-blot assay with purified ITPR1 protein. By contrast, anti-ARHGAP26-positive sera did not bind to ITPR1. In a parallel approach, a combination of histoimmunoprecipitation and mass spectrometry also identified ITPR1 as the target antigen. Finally, a recombinant cell-based immunofluorescence assay using HEK293 cells expressing ITPR1 and ARHGAP26, respectively, confirmed the identification of ITPR1. Mutations of ITPR1 have previously been implicated in spinocerebellar ataxia with and without cognitive decline. Our findings suggest a role of autoimmunity against ITPR1 in the pathogenesis of autoimmune cerebellitis and extend the panel of diagnostic markers for this disease.

) mice, we analyzed the role of MK2/3 in cross-striated muscle by transcriptome and proteome analyses and by histology. We demonstrated enhanced expression of the slow oxidative skeletal muscle myofiber gene program, including the peroxisome proliferator-activated receptor gamma (PPAR␥) coactivator 1␣ (PGC-1␣). Using reporter gene and electrophoretic gel mobility shift assays, we demonstrated that MK2 catalytic activity directly regulated the promoters of the fast fiber-specific myosin heavy-chain IId/x and the slow fiber-specific sarco/endoplasmic reticulum Ca 2؉ -ATPase 2 (SERCA2) gene. Elevated SERCA2a gene expression caused by a decreased ratio of transcription factor Egr-1 to Sp1 was associated with accelerated relaxation and enhanced contractility in MK2/3 ؊/؊ cardiomyocytes, concomitant with improved force parameters in MK2/3 ؊/؊ soleus muscle. These results link MK2/3 to the regulation of calcium dynamics and identify enzymatic activity of MK2/3 as a critical factor for modulating cross-striated muscle function by generating a unique muscle phenotype exhibiting both reduced fatigability and enhanced force in MK2/3 ؊/؊ mice. Hence, the p38-MK2/3 axis may represent a novel target for the design of therapeutic strategies for diseases related to fiber type changes or impaired SERCA2 function.

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Objective: To report on a novel neuronal target antigen in 3 patients with autoimmune cerebellar degeneration.Methods: Three patients with subacute to chronic cerebellar ataxia and controls underwent detailed clinical and neuropsychological assessment together with quantitative high-resolution structural MRI. Sera and CSF were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IFA) and immunoblot. Immunoprecipitation with lysates of hippocampus and cerebellum combined with mass spectrometric analysis was used to identify the autoantigen, which was verified by recombinant expression in HEK293 cells and use in several immunoassays. Multiparameter flow cytometry was performed on peripheral blood and CSF, and peripheral blood was subjected to T-cell receptor spectratyping.Results: Patients presented with a subacute to chronic cerebellar and brainstem syndrome. MRI was consistent with cortical and cerebellar gray matter atrophy associated with subsequent neuroaxonal degeneration. IFA screening revealed strong immunoglobulin G1 reactivity in sera and CSF with hippocampal and cerebellar molecular and granular layers, but not with a panel of 30 recombinantly expressed established neural autoantigens. Neurochondrin was subsequently identified as the target antigen, verified by IFA and immunoblot with HEK293 cells expressing human neurochondrin as well as the ability of recombinant neurochondrin to neutralize the autoantibodies' tissue reaction. Immune phenotyping revealed intrathecal accumulation and activation of B and T cells during the acute but not chronic phase of the disease. T-cell receptor spectratyping suggested an antigen-specific T-cell response accompanying the formation of antineurochondrin autoantibodies. No such neurochondrin reactivity was found in control cohorts of various neural autoantibody-associated neurologic syndromes, relapsing-remitting multiple sclerosis, cerebellar type of multiple system atrophy, hereditary cerebellar ataxias, other neurologic disorders, or healthy donors. Conclusion:Neurochondrin is a neuronal target antigen in autoimmune cerebellar degeneration.Neurol Neuroimmunol Neuroinflamm 2017;4:e307; doi: 10.1212/NXI.0000000000000307 GLOSSARY AD 5 axial diffusivity; AI 5 antibody indices; DTI 5 diffusion tensor imaging; FA 5 fractional anisotropy; IFA 5 immunofluorescent assay; IgG 5 immunoglobulin G; IVIg 5 IV immunoglobulin; mGluR 5 metabotropic glutamate receptor; MSA-c 5 cerebellar type of multiple system atrophy; PB 5 peripheral blood; RD 5 radial diffusivity; RRMS 5 relapsing-remitting multiple sclerosis; SARA 5 Scale for the Assessment and Rating of Ataxia; TCR 5 T-cell receptor.Autoantibodies against neuronal constituents are associated with several severe immune-mediated CNS disorders. These disorders may predominantly affect gray matter structures of different brain regions such as the archicortex of the limbic system, neocortex, and basal ganglia, as well as cerebellar cortex and brainstem.

BackgroundAutoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS).MethodsThe target antigen was identified by histo-immunoprecipitation using the patients’ sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells.ResultsSerum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors.ConclusionsAutoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1–2% of patients with bona fide MS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0900-z) contains supplementary material, which is available to authorized users.

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ObjectiveTo describe phenotypes, treatment response, and outcomes of autoimmunity targeting a synaptic vesicle coat protein, the neuronal (B2) form of adaptor protein–3 (AP3).MethodsArchived serum and CSF specimens (from 616,025 screened) harboring unclassified synaptic antibodies mimicking amphiphysin–immunoglobulin G (IgG) on tissue-based indirect immunofluorescence assay (IFA) were re-evaluated for novel IgG staining patterns. Autoantigens were identified by western blot and mass spectrometry. Recombinant western blot and cell-binding assay (CBA) were used to confirm antigen specificity. Clinical data were obtained retrospectively.ResultsSerum (10) and CSF (6) specimens of 10 patients produced identical IFA staining patterns throughout mouse nervous system tissues, most prominently in cerebellum (Purkinje neuronal perikarya, granular layer synapses, and dentate regions), spinal cord gray matter, dorsal root ganglia, and sympathetic ganglia. The antigen revealed by mass spectrometry analysis and confirmed by recombinant assays (western blot and CBA) was AP3B2 in all. Of 10 seropositive patients, 6 were women; median symptom onset age was 42 years (range 24–58). Clinical information was available for 9 patients, all with subacute onset and rapidly progressive gait ataxia. Neurologic manifestations were myeloneuropathy (3), peripheral sensory neuropathy (2), cerebellar ataxia (2), and spinocerebellar ataxia (2). Five patients received immunotherapy; none improved, but they did not worsen over the follow-up period (median 36 months; range 3–94). Two patients (both with cancer) died. One of 50 control sera was positive by western blot only (but not by IFA or CBA).ConclusionAP3B2 (previously named β-neuronal adaptin-like protein) autoimmunity appears rare, is accompanied by ataxia (sensory or cerebellar), and is potentially treatable.

Background and Objectives: We sought to determine clinical significance of neuronal septin autoimmunity and evaluate for potential IgG effects. Methods: Septin-IgGs were detected by indirect immunofluorescence assays (IFAs; mouse tissue and cell based) or Western blot. IgG binding to (and internalization of) extracellular septin epitopes were evaluated for by live rat hippocampal neuron assay. The impact of purified patient IgGs on murine cortical neuron function was determined by recording extracellular field potentials in a multielectrode array platform. Results: Septin-IgGs were identified in 23 patients. All 8 patients with septin-5-IgG detected had cerebellar ataxia, and 7 had prominent eye movement disorders. One of 2 patients with co-existing septin-7-IgG had additional psychiatric phenotype (apathy, emotional blunting, and poor insight). Fifteen patients had septin-7 autoimmunity, without septin-5-IgG detected. Disorders included encephalopathy (11; 2 patients with accompanying myelopathy, and 2 were relapsing), myelopathy (3), and episodic ataxia (1). Psychiatric symptoms (≥1 of agitation, apathy, catatonia, disorganized thinking, and paranoia) were prominent in 6 of 11 patients with encephalopathic symptoms. Eight of 10 patients with data available (from 23 total) improved after immunotherapy, and a further 2 patients improved spontaneously. Staining of plasma membranes of live hippocampal neurons produced by patient IgGs (subclasses 1 and 2) colocalized with pre-and post-synaptic markers. Decreased spiking and bursting behavior in mixed cultures of murine glutamatergic and GABAergic cortical neurons produced by patient IgGs were attributable to neither antigenic crosslinking and internalization nor complement activation. Interpretation: Septin-IgGs are predictive of distinct treatment-responsive autoimmune central nervous system (CNS) disorders. Live neuron binding and induced electrophysiologic effects by patient IgGs may support septin-specific pathophysiology.