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Antibodies to the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) in cerebellar ataxia.

Jarius S, Scharf M, Begemann N, Stöcker W, Probst C, Serysheva II, Nagel S, Graus F, Psimaras D, Wildemann B, Komorowski L - J Neuroinflammation (2014)

Bottom Line: 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.By contrast, anti-ARHGAP26-positive sera did not bind to ITPR1.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuroimmunology, Department of Neurology, University of Heidelberg, Im Neuenheimer Feld 400, 69120, Heidelberg, Germany. sven.jarius@med.uni-heidelberg.de.

ABSTRACT
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.

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Results from preadsorption experiments. Preadsorption of the patient serum with purified rat ITPR1 or an extract of HEK293 cells expressing murine ITPR1 resulted in complete loss of binding to cerebellar tissue sections in an indirect immunofluorescence assay (A, B, E). In contrast, binding of anti-Ca/ARHGAP26-positive sera was not affected by preadsorption with ITPR1 (C, D). Preadsorption of anti-ITPR1-positive patient serum with full-length human ARHGAP26 (not shown) or with an extract of mock-transfected HEK293 (F) did not affect binding to PCs.
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Fig7: Results from preadsorption experiments. Preadsorption of the patient serum with purified rat ITPR1 or an extract of HEK293 cells expressing murine ITPR1 resulted in complete loss of binding to cerebellar tissue sections in an indirect immunofluorescence assay (A, B, E). In contrast, binding of anti-Ca/ARHGAP26-positive sera was not affected by preadsorption with ITPR1 (C, D). Preadsorption of anti-ITPR1-positive patient serum with full-length human ARHGAP26 (not shown) or with an extract of mock-transfected HEK293 (F) did not affect binding to PCs.

Mentions: Preadsorption of the patient sera with rat ITPR1 protein resulted in complete loss of binding to cerebellum tissue sections (Figure 7); by contrast, preadsorption with ARHGAP26 did not. Interestingly, only preadsorption with full-length ITPR1 resulted in loss of PC staining, not preadsorption with a partial recombinant protein (2470 a.a. to 2577 a.a.; Abnova, Taiwan), indicating that the target epitope either depends on protein conformation or glycosylation or is located outside the residues 2470 a.a. to 2577 a.a.Figure 7


Antibodies to the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) in cerebellar ataxia.

Jarius S, Scharf M, Begemann N, Stöcker W, Probst C, Serysheva II, Nagel S, Graus F, Psimaras D, Wildemann B, Komorowski L - J Neuroinflammation (2014)

Results from preadsorption experiments. Preadsorption of the patient serum with purified rat ITPR1 or an extract of HEK293 cells expressing murine ITPR1 resulted in complete loss of binding to cerebellar tissue sections in an indirect immunofluorescence assay (A, B, E). In contrast, binding of anti-Ca/ARHGAP26-positive sera was not affected by preadsorption with ITPR1 (C, D). Preadsorption of anti-ITPR1-positive patient serum with full-length human ARHGAP26 (not shown) or with an extract of mock-transfected HEK293 (F) did not affect binding to PCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4300617&req=5

Fig7: Results from preadsorption experiments. Preadsorption of the patient serum with purified rat ITPR1 or an extract of HEK293 cells expressing murine ITPR1 resulted in complete loss of binding to cerebellar tissue sections in an indirect immunofluorescence assay (A, B, E). In contrast, binding of anti-Ca/ARHGAP26-positive sera was not affected by preadsorption with ITPR1 (C, D). Preadsorption of anti-ITPR1-positive patient serum with full-length human ARHGAP26 (not shown) or with an extract of mock-transfected HEK293 (F) did not affect binding to PCs.
Mentions: Preadsorption of the patient sera with rat ITPR1 protein resulted in complete loss of binding to cerebellum tissue sections (Figure 7); by contrast, preadsorption with ARHGAP26 did not. Interestingly, only preadsorption with full-length ITPR1 resulted in loss of PC staining, not preadsorption with a partial recombinant protein (2470 a.a. to 2577 a.a.; Abnova, Taiwan), indicating that the target epitope either depends on protein conformation or glycosylation or is located outside the residues 2470 a.a. to 2577 a.a.Figure 7

Bottom Line: 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.By contrast, anti-ARHGAP26-positive sera did not bind to ITPR1.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuroimmunology, Department of Neurology, University of Heidelberg, Im Neuenheimer Feld 400, 69120, Heidelberg, Germany. sven.jarius@med.uni-heidelberg.de.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus