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Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

Greenlee JE, Clawson SA, Hill KE, Wood B, Clardy SL, Tsunoda I, Carlson NG - PLoS ONE (2015)

Bottom Line: However, intracellular accumulation of these antibodies did not affect Purkinje cell viability.The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen.Purkinje cell death was not simply due to intraneuronal antibody accumulation.

View Article: PubMed Central - PubMed

Affiliation: Neurology Service, George E. Wahlen Veterans Affairs Health Care System, Salt Lake City, Utah, United States of America; Department of Neurology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.

ABSTRACT
Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.

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Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within Purkinje cells and IgG-mediated cytotoxicity.In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also Fig 2). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.
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pone.0123446.g001: Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within Purkinje cells and IgG-mediated cytotoxicity.In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also Fig 2). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.

Mentions: To determine whether Purkinje cell death following incubation with anti-Yo sera required immunoreactivity to the 62 kDa Yo antigen, we depleted antibodies to the 62 kDa protein from patient IgG containing high titers of anti-Yo antibody [12]. Recombinant CDR62 major Yo protein with a N-terminus His tag (to facilitate binding to a nickel column) was bound to a nickel column, and patient serum containing high titers of anti-Yo antibody was passed through the column to deplete antibody to the 62 kDa protein. As a control for nonspecific binding to the column, the same serum was also passed over a nickel column treated with E. coli containing vector lacking Yo antigen (sham control). Rat cerebellar slice cultures were then incubated for 72 hours with native anti-Yo IgG, sham-adsorbed IgG, and IgG adsorbed against the 62 kDa protein. Cultures were monitored for antibody uptake and quantified for Purkinje cell death. Internalization of IgG by Purkinje cells was confirmed using serial confocal images through individual cells (S1 Movie). Cultures incubated with native anti-Yo IgG showed both extensive antibody uptake by Purkinje cells and Purkinje cell death, as did sham-adsorbed IgG in which antibodies to the 62 kDa protein were still present (Figs 1 and 2). In contrast, antibody uptake and Purkinje cell death were abolished following adsorption with the 62 kDa Yo antigen, indicating that antibody accumulation and Purkinje cell death were specifically associated with antibodies to the major Yo antigen (Figs 1 and 2).


Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

Greenlee JE, Clawson SA, Hill KE, Wood B, Clardy SL, Tsunoda I, Carlson NG - PLoS ONE (2015)

Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within Purkinje cells and IgG-mediated cytotoxicity.In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also Fig 2). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401511&req=5

pone.0123446.g001: Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within Purkinje cells and IgG-mediated cytotoxicity.In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also Fig 2). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.
Mentions: To determine whether Purkinje cell death following incubation with anti-Yo sera required immunoreactivity to the 62 kDa Yo antigen, we depleted antibodies to the 62 kDa protein from patient IgG containing high titers of anti-Yo antibody [12]. Recombinant CDR62 major Yo protein with a N-terminus His tag (to facilitate binding to a nickel column) was bound to a nickel column, and patient serum containing high titers of anti-Yo antibody was passed through the column to deplete antibody to the 62 kDa protein. As a control for nonspecific binding to the column, the same serum was also passed over a nickel column treated with E. coli containing vector lacking Yo antigen (sham control). Rat cerebellar slice cultures were then incubated for 72 hours with native anti-Yo IgG, sham-adsorbed IgG, and IgG adsorbed against the 62 kDa protein. Cultures were monitored for antibody uptake and quantified for Purkinje cell death. Internalization of IgG by Purkinje cells was confirmed using serial confocal images through individual cells (S1 Movie). Cultures incubated with native anti-Yo IgG showed both extensive antibody uptake by Purkinje cells and Purkinje cell death, as did sham-adsorbed IgG in which antibodies to the 62 kDa protein were still present (Figs 1 and 2). In contrast, antibody uptake and Purkinje cell death were abolished following adsorption with the 62 kDa Yo antigen, indicating that antibody accumulation and Purkinje cell death were specifically associated with antibodies to the major Yo antigen (Figs 1 and 2).

Bottom Line: However, intracellular accumulation of these antibodies did not affect Purkinje cell viability.The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen.Purkinje cell death was not simply due to intraneuronal antibody accumulation.

View Article: PubMed Central - PubMed

Affiliation: Neurology Service, George E. Wahlen Veterans Affairs Health Care System, Salt Lake City, Utah, United States of America; Department of Neurology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.

ABSTRACT
Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.

Show MeSH
Related in: MedlinePlus