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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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Comparison of uptake and cytotoxicity of anti-Yo antibody versus three other antibodies specific for the intracellular Purkinje cell proteins: calbindin, calmodulin, and PCP-2.The top row of panels demonstrates uptake and accumulation of IgG (red) within Purkinje cells after 96 hours in cultures incubated with anti-calmodulin, anti-calbindin, anti PCP-2, or anti-Yo IgGs. Entry of SYTOX green into Purkinje cells containing IgG, indicative of cell membrane injury and death (yellow), was seen in only in cultures incubated with anti-Yo IgGs (examples shown by arrows). The lower panels show only SYTOX green staining of Purkinje cells in cultures incubated with the antibodies indicated. In the culture incubated with anti-PCP-2. SYTOX staining indicative of cell death is seen in a single cell outside the Purkinje cell layer (asterisk) but not in Purkinje cells. Cultures were followed through 144 hours. There was progression of cell death in cultures incubated with anti-Yo antibodies. In contrast, cultures incubated calbindin, calmodulin, and PCP-2 did not exhibit Purkinje cell death above background levels seen in controls incubated with normal human IgG (data not shown). Scale bar = 20μ.
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pone.0123446.g004: Comparison of uptake and cytotoxicity of anti-Yo antibody versus three other antibodies specific for the intracellular Purkinje cell proteins: calbindin, calmodulin, and PCP-2.The top row of panels demonstrates uptake and accumulation of IgG (red) within Purkinje cells after 96 hours in cultures incubated with anti-calmodulin, anti-calbindin, anti PCP-2, or anti-Yo IgGs. Entry of SYTOX green into Purkinje cells containing IgG, indicative of cell membrane injury and death (yellow), was seen in only in cultures incubated with anti-Yo IgGs (examples shown by arrows). The lower panels show only SYTOX green staining of Purkinje cells in cultures incubated with the antibodies indicated. In the culture incubated with anti-PCP-2. SYTOX staining indicative of cell death is seen in a single cell outside the Purkinje cell layer (asterisk) but not in Purkinje cells. Cultures were followed through 144 hours. There was progression of cell death in cultures incubated with anti-Yo antibodies. In contrast, cultures incubated calbindin, calmodulin, and PCP-2 did not exhibit Purkinje cell death above background levels seen in controls incubated with normal human IgG (data not shown). Scale bar = 20μ.

Mentions: As previously described, anti-Yo IgG was initially detected in Purkinje cell processes within 4 to 8 hours, then in Purkinje cell cytoplasm by 24 hours, and in Purkinje cell nuclei by 48 hours [12]. Uptake of IgG specific for calbindin, calmodulin, PCP-2, and IgG from patients 1, 2, or 3 was observed over this same time interval. At early time points (48 hours or earlier), Purkinje cells and other neurons containing IgG excluded SYTOX green, confirming cell viability at the time of antibody uptake. Although normal IgG is readily cleared from Purkinje cells, none of the intracellularly bound commercial or patient IgGs were cleared from Purkinje cells after removing unbound antibody from the media as is typically seen with normal human IgG [11]. By 48 hours, cultures incubated with anti-Yo antisera began to exhibit abnormal Purkinje cell morphology and by 72–96 hours, multiple Purkinje cells showed intracellular staining with SYTOX green, indicating cell membrane disruption and death (Figs 4, 5, and 6). Increasing numbers of Purkinje cells containing anti-Yo IgG and SYTOX green incorporation were observed at subsequent time intervals (data not shown) [12]. In contrast, although intracellular binding and accumulation of antibodies to calbindin, calmodulin, PCP-2, or from patients 1, 2, or 3 in cultures could be readily detected, incubation of cultures with these antibodies for periods of up to 144 hours did not result in detectable death of Purkinje cells in excess of background levels present in controls incubated with normal IgG (Figs 4, 5, and 6; data at 144 hours not shown). In cultures incubated with sera from patients 1 and 2 (with ovarian carcinomas), antibody uptake and accumulation were also observed almost exclusively in Purkinje cells. In cultures incubated with sera from patient 3 (with an ovarian mixed mesodermal sarcoma), IgG was detected not only in Purkinje cells but also in numerous small neurons within and near the Purkinje cell layer (Fig 7).


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)

Comparison of uptake and cytotoxicity of anti-Yo antibody versus three other antibodies specific for the intracellular Purkinje cell proteins: calbindin, calmodulin, and PCP-2.The top row of panels demonstrates uptake and accumulation of IgG (red) within Purkinje cells after 96 hours in cultures incubated with anti-calmodulin, anti-calbindin, anti PCP-2, or anti-Yo IgGs. Entry of SYTOX green into Purkinje cells containing IgG, indicative of cell membrane injury and death (yellow), was seen in only in cultures incubated with anti-Yo IgGs (examples shown by arrows). The lower panels show only SYTOX green staining of Purkinje cells in cultures incubated with the antibodies indicated. In the culture incubated with anti-PCP-2. SYTOX staining indicative of cell death is seen in a single cell outside the Purkinje cell layer (asterisk) but not in Purkinje cells. Cultures were followed through 144 hours. There was progression of cell death in cultures incubated with anti-Yo antibodies. In contrast, cultures incubated calbindin, calmodulin, and PCP-2 did not exhibit Purkinje cell death above background levels seen in controls incubated with normal human IgG (data not shown). Scale bar = 20μ.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401511&req=5

pone.0123446.g004: Comparison of uptake and cytotoxicity of anti-Yo antibody versus three other antibodies specific for the intracellular Purkinje cell proteins: calbindin, calmodulin, and PCP-2.The top row of panels demonstrates uptake and accumulation of IgG (red) within Purkinje cells after 96 hours in cultures incubated with anti-calmodulin, anti-calbindin, anti PCP-2, or anti-Yo IgGs. Entry of SYTOX green into Purkinje cells containing IgG, indicative of cell membrane injury and death (yellow), was seen in only in cultures incubated with anti-Yo IgGs (examples shown by arrows). The lower panels show only SYTOX green staining of Purkinje cells in cultures incubated with the antibodies indicated. In the culture incubated with anti-PCP-2. SYTOX staining indicative of cell death is seen in a single cell outside the Purkinje cell layer (asterisk) but not in Purkinje cells. Cultures were followed through 144 hours. There was progression of cell death in cultures incubated with anti-Yo antibodies. In contrast, cultures incubated calbindin, calmodulin, and PCP-2 did not exhibit Purkinje cell death above background levels seen in controls incubated with normal human IgG (data not shown). Scale bar = 20μ.
Mentions: As previously described, anti-Yo IgG was initially detected in Purkinje cell processes within 4 to 8 hours, then in Purkinje cell cytoplasm by 24 hours, and in Purkinje cell nuclei by 48 hours [12]. Uptake of IgG specific for calbindin, calmodulin, PCP-2, and IgG from patients 1, 2, or 3 was observed over this same time interval. At early time points (48 hours or earlier), Purkinje cells and other neurons containing IgG excluded SYTOX green, confirming cell viability at the time of antibody uptake. Although normal IgG is readily cleared from Purkinje cells, none of the intracellularly bound commercial or patient IgGs were cleared from Purkinje cells after removing unbound antibody from the media as is typically seen with normal human IgG [11]. By 48 hours, cultures incubated with anti-Yo antisera began to exhibit abnormal Purkinje cell morphology and by 72–96 hours, multiple Purkinje cells showed intracellular staining with SYTOX green, indicating cell membrane disruption and death (Figs 4, 5, and 6). Increasing numbers of Purkinje cells containing anti-Yo IgG and SYTOX green incorporation were observed at subsequent time intervals (data not shown) [12]. In contrast, although intracellular binding and accumulation of antibodies to calbindin, calmodulin, PCP-2, or from patients 1, 2, or 3 in cultures could be readily detected, incubation of cultures with these antibodies for periods of up to 144 hours did not result in detectable death of Purkinje cells in excess of background levels present in controls incubated with normal IgG (Figs 4, 5, and 6; data at 144 hours not shown). In cultures incubated with sera from patients 1 and 2 (with ovarian carcinomas), antibody uptake and accumulation were also observed almost exclusively in Purkinje cells. In cultures incubated with sera from patient 3 (with an ovarian mixed mesodermal sarcoma), IgG was detected not only in Purkinje cells but also in numerous small neurons within and near the Purkinje cell layer (Fig 7).

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