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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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Double labeling of primate intestine sections with patient serum and with commercial antibodies to anti-ITPR1 or ARHGAP26, respectively. The anti-Sj index serum and the commercial antibody to ITPR1 (A) stained both the stratum circulare (SC) and the stratum longitudinale (SL) of the tunica muscularis as well as the muscularis mucosae (MM) and structures adjacent to the enteric villi (V), with a perfect overlay, but spared the plexus myentericus Auerbach (MP), which is located between SC and SL. Conversely, the anti-Ca index serum [30] and the commercial antibody to ARHGAP26 (B) both stained the MP (and the plexus submucosus Meissner; not shown) but spared the enteric muscle cells. Anti-ITPR1 or anti-ARHGAP26 reactivity is depicted in red (Alexa Fluor® 568), the patient antibody in green (Alexa Fluor® 488), and yellow indicates overlay of the two antibodies. Nuclei are shown in blue (DAPI).
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Fig4: Double labeling of primate intestine sections with patient serum and with commercial antibodies to anti-ITPR1 or ARHGAP26, respectively. The anti-Sj index serum and the commercial antibody to ITPR1 (A) stained both the stratum circulare (SC) and the stratum longitudinale (SL) of the tunica muscularis as well as the muscularis mucosae (MM) and structures adjacent to the enteric villi (V), with a perfect overlay, but spared the plexus myentericus Auerbach (MP), which is located between SC and SL. Conversely, the anti-Ca index serum [30] and the commercial antibody to ARHGAP26 (B) both stained the MP (and the plexus submucosus Meissner; not shown) but spared the enteric muscle cells. Anti-ITPR1 or anti-ARHGAP26 reactivity is depicted in red (Alexa Fluor® 568), the patient antibody in green (Alexa Fluor® 488), and yellow indicates overlay of the two antibodies. Nuclei are shown in blue (DAPI).

Mentions: The staining pattern observed with the patients’ serum highly resembled the pattern observed by us in a previous study with a commercial antibody to the inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1) [30]. Double staining of cerebellum sections with that commercial antibody, which is used as a well-established marker of PCs in our laboratory, showed a perfect overlay with the staining pattern found with the patient antibody in the ML, PCL and WM (Figure 3). By contrast, sera positive for anti-ARHGAP26 had shown only partial overlay using the same commercial anti-ITRP1 antibody (see Figure 13 in ref. [30]). In addition, an overlay between the patients’ IgG and the commercial anti-ITPR1 antibody was also observed on other tissue sections, including intestine (Figure 4) and bulbus oculi sections (Figure 5), corroborating ITPR1 as the target antigen. In line with these findings, IgG from the patients’ sera but not from healthy controls bound to ITPR1 purified from rat cerebellum in a dot-blot assay (Figure 6).Figure 3


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)

Double labeling of primate intestine sections with patient serum and with commercial antibodies to anti-ITPR1 or ARHGAP26, respectively. The anti-Sj index serum and the commercial antibody to ITPR1 (A) stained both the stratum circulare (SC) and the stratum longitudinale (SL) of the tunica muscularis as well as the muscularis mucosae (MM) and structures adjacent to the enteric villi (V), with a perfect overlay, but spared the plexus myentericus Auerbach (MP), which is located between SC and SL. Conversely, the anti-Ca index serum [30] and the commercial antibody to ARHGAP26 (B) both stained the MP (and the plexus submucosus Meissner; not shown) but spared the enteric muscle cells. Anti-ITPR1 or anti-ARHGAP26 reactivity is depicted in red (Alexa Fluor® 568), the patient antibody in green (Alexa Fluor® 488), and yellow indicates overlay of the two antibodies. Nuclei are shown in blue (DAPI).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Double labeling of primate intestine sections with patient serum and with commercial antibodies to anti-ITPR1 or ARHGAP26, respectively. The anti-Sj index serum and the commercial antibody to ITPR1 (A) stained both the stratum circulare (SC) and the stratum longitudinale (SL) of the tunica muscularis as well as the muscularis mucosae (MM) and structures adjacent to the enteric villi (V), with a perfect overlay, but spared the plexus myentericus Auerbach (MP), which is located between SC and SL. Conversely, the anti-Ca index serum [30] and the commercial antibody to ARHGAP26 (B) both stained the MP (and the plexus submucosus Meissner; not shown) but spared the enteric muscle cells. Anti-ITPR1 or anti-ARHGAP26 reactivity is depicted in red (Alexa Fluor® 568), the patient antibody in green (Alexa Fluor® 488), and yellow indicates overlay of the two antibodies. Nuclei are shown in blue (DAPI).
Mentions: The staining pattern observed with the patients’ serum highly resembled the pattern observed by us in a previous study with a commercial antibody to the inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1) [30]. Double staining of cerebellum sections with that commercial antibody, which is used as a well-established marker of PCs in our laboratory, showed a perfect overlay with the staining pattern found with the patient antibody in the ML, PCL and WM (Figure 3). By contrast, sera positive for anti-ARHGAP26 had shown only partial overlay using the same commercial anti-ITRP1 antibody (see Figure 13 in ref. [30]). In addition, an overlay between the patients’ IgG and the commercial anti-ITPR1 antibody was also observed on other tissue sections, including intestine (Figure 4) and bulbus oculi sections (Figure 5), corroborating ITPR1 as the target antigen. In line with these findings, IgG from the patients’ sera but not from healthy controls bound to ITPR1 purified from rat cerebellum in a dot-blot assay (Figure 6).Figure 3

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