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Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis.

Schubert M, Panja D, Haugen M, Bramham CR, Vedeler CA - Acta Neuropathol. (2014)

Bottom Line: Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin D28K (CB) and L7/Pcp-2 as well as reduced dendritic arborizations in the remaining PCs.These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis.Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Haukeland University Hospital, 5021, Bergen, Norway, schubert_manja@hotmail.com.

ABSTRACT
Paraneoplastic cerebellar degeneration (PCD) is characterized by loss of Purkinje cells (PCs) associated with progressive pancerebellar dysfunction in the presence of onconeural Yo antibodies. These antibodies recognize the cerebellar degeneration-related antigens CDR2 and CDR2L. Response to PCD therapy is disappointing due to limited understanding of the neuropathological mechanisms. Here, we report the pathological role of CDR antibodies on the calcium homeostasis in PCs. We developed an antibody-mediated PCD model based on co-incubation of cerebellar organotypic slice culture with human patient serum or rabbit CDR2 and CDR2L antibodies. The CDR antibody-induced pathology was investigated by high-resolution multiphoton imaging and biochemical analysis. Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin D28K (CB) and L7/Pcp-2 as well as reduced dendritic arborizations in the remaining PCs. Washout of the CDR antibodies partially recovered CB immunoreactivity, suggesting a transient structural change in CB calcium-binding site. We discovered that CDR2 and CB co-immunoprecipitate. Furthermore, the expression levels of voltage-gated calcium channel Cav2.1, protein kinase C gamma and calcium-dependent protease, calpain-2, were increased after CDR antibody internalization. Inhibition of these signaling pathways prevented or attenuated CDR antibody-induced CB and L7/Pcp-2 immunoreactivity loss, morphological changes and increased protein expression. These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis. Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.

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Affinity-purified rabbit CDR-Abs (rCDR) reduce calbindin D28K immunoreactivity in a time- and concentration-dependent manner with partial recovery after CDR-Ab washout. arCDR2 and rCDR2L (400 ng/mL, 6D) are independently internalized into PCs (green). Only few CDR-marked cells (arrow) are positively correlated with calbindin D28K (CB, magenta); scale bars 20 μm. We found different expression patterns of CB immunoreactivity and PC morphology modifications for rCDR2 b, rCDR2L, and rCDR2/2L compared to rIgG control (c); scale bars 40 μm. The CB immunoreactivity loss, studied 2, 4, and 6 days of rCDR internalization by stereological counting of CB+ cells/mm3, was time- (d) and concentration- (e) dependent (time: nE = 4; concentration: nE = 3). f, g Western blot analysis of rCDR-treated cOTSC lysate (125 ng/mL, D6) showed no difference in CB protein levels to rIgG controls (nE = 10). h CB+-PC immunoreactivity loss at day 6 of rCDR internalization was partially rescued 7 days (D7wo) post-rCDR washout (scale bars 40 μm), but did not reach control level and plateaued between washout days 4 and 7 (nE = 3) (i). Data in mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001. Table 1: CDR-Ab effects in percentage
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Fig3: Affinity-purified rabbit CDR-Abs (rCDR) reduce calbindin D28K immunoreactivity in a time- and concentration-dependent manner with partial recovery after CDR-Ab washout. arCDR2 and rCDR2L (400 ng/mL, 6D) are independently internalized into PCs (green). Only few CDR-marked cells (arrow) are positively correlated with calbindin D28K (CB, magenta); scale bars 20 μm. We found different expression patterns of CB immunoreactivity and PC morphology modifications for rCDR2 b, rCDR2L, and rCDR2/2L compared to rIgG control (c); scale bars 40 μm. The CB immunoreactivity loss, studied 2, 4, and 6 days of rCDR internalization by stereological counting of CB+ cells/mm3, was time- (d) and concentration- (e) dependent (time: nE = 4; concentration: nE = 3). f, g Western blot analysis of rCDR-treated cOTSC lysate (125 ng/mL, D6) showed no difference in CB protein levels to rIgG controls (nE = 10). h CB+-PC immunoreactivity loss at day 6 of rCDR internalization was partially rescued 7 days (D7wo) post-rCDR washout (scale bars 40 μm), but did not reach control level and plateaued between washout days 4 and 7 (nE = 3) (i). Data in mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001. Table 1: CDR-Ab effects in percentage

Mentions: Multiphoton images were collected with a Ti Sapp laser (Coherent Chameleon Ultra2) and DM6000 CFS-TCS SP5 microscope (Leica) using an HCX PL APO 20 × 1.0 water-immersion objective (with a digital zoom of ×1.7 or ×3.0 [Fig. 3b]). Excitation was performed at 740 nm (6.8–7.2 mW laser power); emission was detected for Alexa Fluor® 488/594 with the NDD1/NDD2 external detectors, respectively. The fluorescence intensity was adjusted to 75 % of the maximum in untreated controls for each experiment. Z-stack images were taken at 0.5–1 μm intervals. Pictures were superimposed using LASAF software version 2.5.1 (Leica Microsystems CMS GmbH). Additionally, images from Alexa Fluor® 488/594 Donkey Anti-Mouse IgG staining in Figs. 2b, 3b, c, h were pseudo-colored in gray and inverted for clarity using Fiji. Figure S1b was created as 3D projections with orthogonal section from z-stacks using the Fiji plug-in 3D Viewer. Calbindin D28K (CB+)- and L7/Pcp-2 (L7/Pcp-2+)-positive PCs were counted manually and automatically, but blind in three to eight images of 750 × 750 × 100 μm in each slice for each experiment (nE) and group and projected to mm3 (Fig. 1c). The automatic count was performed with Fiji: (1) image → stack → plug-in: z projection [max intensity]; (2.) image → adjustment → plug-in: auto threshold [yen]; (3) analyze → plug-in: 3D objects counter [threshold: 50; size filter: min: 200 and max: 1000]. The manual and automatic counts produced equivalent numbers.Fig. 2


Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis.

Schubert M, Panja D, Haugen M, Bramham CR, Vedeler CA - Acta Neuropathol. (2014)

Affinity-purified rabbit CDR-Abs (rCDR) reduce calbindin D28K immunoreactivity in a time- and concentration-dependent manner with partial recovery after CDR-Ab washout. arCDR2 and rCDR2L (400 ng/mL, 6D) are independently internalized into PCs (green). Only few CDR-marked cells (arrow) are positively correlated with calbindin D28K (CB, magenta); scale bars 20 μm. We found different expression patterns of CB immunoreactivity and PC morphology modifications for rCDR2 b, rCDR2L, and rCDR2/2L compared to rIgG control (c); scale bars 40 μm. The CB immunoreactivity loss, studied 2, 4, and 6 days of rCDR internalization by stereological counting of CB+ cells/mm3, was time- (d) and concentration- (e) dependent (time: nE = 4; concentration: nE = 3). f, g Western blot analysis of rCDR-treated cOTSC lysate (125 ng/mL, D6) showed no difference in CB protein levels to rIgG controls (nE = 10). h CB+-PC immunoreactivity loss at day 6 of rCDR internalization was partially rescued 7 days (D7wo) post-rCDR washout (scale bars 40 μm), but did not reach control level and plateaued between washout days 4 and 7 (nE = 3) (i). Data in mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001. Table 1: CDR-Ab effects in percentage
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Fig3: Affinity-purified rabbit CDR-Abs (rCDR) reduce calbindin D28K immunoreactivity in a time- and concentration-dependent manner with partial recovery after CDR-Ab washout. arCDR2 and rCDR2L (400 ng/mL, 6D) are independently internalized into PCs (green). Only few CDR-marked cells (arrow) are positively correlated with calbindin D28K (CB, magenta); scale bars 20 μm. We found different expression patterns of CB immunoreactivity and PC morphology modifications for rCDR2 b, rCDR2L, and rCDR2/2L compared to rIgG control (c); scale bars 40 μm. The CB immunoreactivity loss, studied 2, 4, and 6 days of rCDR internalization by stereological counting of CB+ cells/mm3, was time- (d) and concentration- (e) dependent (time: nE = 4; concentration: nE = 3). f, g Western blot analysis of rCDR-treated cOTSC lysate (125 ng/mL, D6) showed no difference in CB protein levels to rIgG controls (nE = 10). h CB+-PC immunoreactivity loss at day 6 of rCDR internalization was partially rescued 7 days (D7wo) post-rCDR washout (scale bars 40 μm), but did not reach control level and plateaued between washout days 4 and 7 (nE = 3) (i). Data in mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001. Table 1: CDR-Ab effects in percentage
Mentions: Multiphoton images were collected with a Ti Sapp laser (Coherent Chameleon Ultra2) and DM6000 CFS-TCS SP5 microscope (Leica) using an HCX PL APO 20 × 1.0 water-immersion objective (with a digital zoom of ×1.7 or ×3.0 [Fig. 3b]). Excitation was performed at 740 nm (6.8–7.2 mW laser power); emission was detected for Alexa Fluor® 488/594 with the NDD1/NDD2 external detectors, respectively. The fluorescence intensity was adjusted to 75 % of the maximum in untreated controls for each experiment. Z-stack images were taken at 0.5–1 μm intervals. Pictures were superimposed using LASAF software version 2.5.1 (Leica Microsystems CMS GmbH). Additionally, images from Alexa Fluor® 488/594 Donkey Anti-Mouse IgG staining in Figs. 2b, 3b, c, h were pseudo-colored in gray and inverted for clarity using Fiji. Figure S1b was created as 3D projections with orthogonal section from z-stacks using the Fiji plug-in 3D Viewer. Calbindin D28K (CB+)- and L7/Pcp-2 (L7/Pcp-2+)-positive PCs were counted manually and automatically, but blind in three to eight images of 750 × 750 × 100 μm in each slice for each experiment (nE) and group and projected to mm3 (Fig. 1c). The automatic count was performed with Fiji: (1) image → stack → plug-in: z projection [max intensity]; (2.) image → adjustment → plug-in: auto threshold [yen]; (3) analyze → plug-in: 3D objects counter [threshold: 50; size filter: min: 200 and max: 1000]. The manual and automatic counts produced equivalent numbers.Fig. 2

Bottom Line: Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin D28K (CB) and L7/Pcp-2 as well as reduced dendritic arborizations in the remaining PCs.These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis.Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Haukeland University Hospital, 5021, Bergen, Norway, schubert_manja@hotmail.com.

ABSTRACT
Paraneoplastic cerebellar degeneration (PCD) is characterized by loss of Purkinje cells (PCs) associated with progressive pancerebellar dysfunction in the presence of onconeural Yo antibodies. These antibodies recognize the cerebellar degeneration-related antigens CDR2 and CDR2L. Response to PCD therapy is disappointing due to limited understanding of the neuropathological mechanisms. Here, we report the pathological role of CDR antibodies on the calcium homeostasis in PCs. We developed an antibody-mediated PCD model based on co-incubation of cerebellar organotypic slice culture with human patient serum or rabbit CDR2 and CDR2L antibodies. The CDR antibody-induced pathology was investigated by high-resolution multiphoton imaging and biochemical analysis. Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin D28K (CB) and L7/Pcp-2 as well as reduced dendritic arborizations in the remaining PCs. Washout of the CDR antibodies partially recovered CB immunoreactivity, suggesting a transient structural change in CB calcium-binding site. We discovered that CDR2 and CB co-immunoprecipitate. Furthermore, the expression levels of voltage-gated calcium channel Cav2.1, protein kinase C gamma and calcium-dependent protease, calpain-2, were increased after CDR antibody internalization. Inhibition of these signaling pathways prevented or attenuated CDR antibody-induced CB and L7/Pcp-2 immunoreactivity loss, morphological changes and increased protein expression. These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis. Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.

Show MeSH
Related in: MedlinePlus