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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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Calpain-2 activity is increased by CDR-Ab internalization, but not MAP kinase. a Representative Western blot: calpain-1 and calpain-2 expression after rCDR internalization and MDL28170 co-treatment (h). bBar plots show that calpain-1 expression is not affected, but c calpain-2 expression is significantly increased after rCDR internalization (125 ng/mL; D6; nE = 21) and can be i blocked by co-treatment with calpain antagonist MDL28170 (125 ng/mL + 10 μM MDL28170; nE = 5). Calpain antagonist MDL28170 reduced the CDR-induced CB+ and L7/Pcp-2+ PC loss. d z-Stack multiphoton micrographs: co-treatment with calpain antagonist MDL28170 (10 μM) beneficially affects PC anti-CB (magenta) staining after hCDR2/2L+(PS3) internalization (4 μL/mL, 6 days); scale bars 40 μm. Stereological counting of CB+ (e) and L7/Pcp-2+ (f) cells/mm3 in these micrographs showed that hCDR2/2L+(PS3)/MDL28170 treatment (5, 10, 20 μM) reduced the CDR-induced loss of CB [nE = 6 (PS3) and nE = 4 (PS4)] and L7/Pcp-2 [nE = 3 (PS4)]. g Similar observation was found for rCDR/MDL28170 co-treatment (125 ng/mL, 10 μM MDL28170, CB: nE = 5). Calpain antagonist reduced the CDR-induced loss depending on the CDR target to up to 60 % (Table 1). This was most pronounced for CDR2. j MAP kinase antagonist U0126 (5 μM) does not influence the loss of CB+ or L7/Pcp-2+ PCs after hCDR2/2L+(PS3) internalization (1 μL/mL; 6 days; nE = 4). Data are mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.008. Table 1: CDR antibody effects in percentage
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Fig8: Calpain-2 activity is increased by CDR-Ab internalization, but not MAP kinase. a Representative Western blot: calpain-1 and calpain-2 expression after rCDR internalization and MDL28170 co-treatment (h). bBar plots show that calpain-1 expression is not affected, but c calpain-2 expression is significantly increased after rCDR internalization (125 ng/mL; D6; nE = 21) and can be i blocked by co-treatment with calpain antagonist MDL28170 (125 ng/mL + 10 μM MDL28170; nE = 5). Calpain antagonist MDL28170 reduced the CDR-induced CB+ and L7/Pcp-2+ PC loss. d z-Stack multiphoton micrographs: co-treatment with calpain antagonist MDL28170 (10 μM) beneficially affects PC anti-CB (magenta) staining after hCDR2/2L+(PS3) internalization (4 μL/mL, 6 days); scale bars 40 μm. Stereological counting of CB+ (e) and L7/Pcp-2+ (f) cells/mm3 in these micrographs showed that hCDR2/2L+(PS3)/MDL28170 treatment (5, 10, 20 μM) reduced the CDR-induced loss of CB [nE = 6 (PS3) and nE = 4 (PS4)] and L7/Pcp-2 [nE = 3 (PS4)]. g Similar observation was found for rCDR/MDL28170 co-treatment (125 ng/mL, 10 μM MDL28170, CB: nE = 5). Calpain antagonist reduced the CDR-induced loss depending on the CDR target to up to 60 % (Table 1). This was most pronounced for CDR2. j MAP kinase antagonist U0126 (5 μM) does not influence the loss of CB+ or L7/Pcp-2+ PCs after hCDR2/2L+(PS3) internalization (1 μL/mL; 6 days; nE = 4). Data are mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.008. Table 1: CDR antibody effects in percentage

Mentions: Changes in VGCC, AMPA receptor, and PKC signaling can lead to elevated intracellular Ca2+ levels and induce Ca2+-dependent proteases activity. To examine whether CDR internalization affects Ca2+-dependent proteases activity of calpain-1 and calpain-2, we performed Western blot analysis of rCDR-treated cOTSC lysates. Calpain-1 expression was not affected, but calpain-2 expression was significantly increased (rCDR2: 2.05 ± 0.14, ***p < 0.0001; rCDR2L: 1.72 ± 0.14, ***p = 0.0002; rCDR2/2L: 1.84 ± 0.19, **p = 0.0012; 125 ng/mL; 6 days; Fig. 8a–c). We then investigated whether calpain-2 over-activation/-expression could be antagonized by calpain-specific inhibitor MDL28170, a known neuroprotective substrate in ischemia and traumatic injury models [9, 36]. MDL28170 co-application with hCDR2/2L+(PS3/PS4) (6 days) reduced the CB+-PC loss to ~45 %, as well as the CDR-induced pathology on the dendritic arborizations in the remaining PCs (nE = 6 for PS3 and nE = 4 for PS4; Fig. 8d, e; Table 1). We found no difference between the 10 (42 ± 9 %) and 20 μM (44 ± 7 %) MDL28170/hCDR2/2L+(PS3) treatment (p = 0.3841; Fig. 8e; Table 1). The MDL28170-induced rescue of CB+-PCs differed between hCDR2/2L+(PS4) (18 %) and hCDR2/2L+(PS3) (48 %) (*p = 0.0207; 10 μM; Fig. 8e; Table 1). Furthermore, hCDR2/2L+(PS4)-induced L7/Pcp-2+-PC loss was partially rescued by 10 μM MDL28170 (***p < 0.0001; nE = 3; Fig. 8f; Table 1). MDL28170 (10 μM) co-treatment of rCDR2, rCDR2L, and rCDR2/2L-treated cOTSC (125 ng/mL, 6 days) showed an improvement of the dendritic morphology and significant reduction of CB+-PC loss (***p < 0.0001; nE = 5; Fig. 8g; Table 1). The increase of calpain-2 expression seen during CDR internalization was blocked by 10 μM MDL28170 (Fig. 8h, i; #p < 0.008; nE = 6).Fig. 8


Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis.

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

Calpain-2 activity is increased by CDR-Ab internalization, but not MAP kinase. a Representative Western blot: calpain-1 and calpain-2 expression after rCDR internalization and MDL28170 co-treatment (h). bBar plots show that calpain-1 expression is not affected, but c calpain-2 expression is significantly increased after rCDR internalization (125 ng/mL; D6; nE = 21) and can be i blocked by co-treatment with calpain antagonist MDL28170 (125 ng/mL + 10 μM MDL28170; nE = 5). Calpain antagonist MDL28170 reduced the CDR-induced CB+ and L7/Pcp-2+ PC loss. d z-Stack multiphoton micrographs: co-treatment with calpain antagonist MDL28170 (10 μM) beneficially affects PC anti-CB (magenta) staining after hCDR2/2L+(PS3) internalization (4 μL/mL, 6 days); scale bars 40 μm. Stereological counting of CB+ (e) and L7/Pcp-2+ (f) cells/mm3 in these micrographs showed that hCDR2/2L+(PS3)/MDL28170 treatment (5, 10, 20 μM) reduced the CDR-induced loss of CB [nE = 6 (PS3) and nE = 4 (PS4)] and L7/Pcp-2 [nE = 3 (PS4)]. g Similar observation was found for rCDR/MDL28170 co-treatment (125 ng/mL, 10 μM MDL28170, CB: nE = 5). Calpain antagonist reduced the CDR-induced loss depending on the CDR target to up to 60 % (Table 1). This was most pronounced for CDR2. j MAP kinase antagonist U0126 (5 μM) does not influence the loss of CB+ or L7/Pcp-2+ PCs after hCDR2/2L+(PS3) internalization (1 μL/mL; 6 days; nE = 4). Data are mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.008. Table 1: CDR antibody effects in percentage
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Fig8: Calpain-2 activity is increased by CDR-Ab internalization, but not MAP kinase. a Representative Western blot: calpain-1 and calpain-2 expression after rCDR internalization and MDL28170 co-treatment (h). bBar plots show that calpain-1 expression is not affected, but c calpain-2 expression is significantly increased after rCDR internalization (125 ng/mL; D6; nE = 21) and can be i blocked by co-treatment with calpain antagonist MDL28170 (125 ng/mL + 10 μM MDL28170; nE = 5). Calpain antagonist MDL28170 reduced the CDR-induced CB+ and L7/Pcp-2+ PC loss. d z-Stack multiphoton micrographs: co-treatment with calpain antagonist MDL28170 (10 μM) beneficially affects PC anti-CB (magenta) staining after hCDR2/2L+(PS3) internalization (4 μL/mL, 6 days); scale bars 40 μm. Stereological counting of CB+ (e) and L7/Pcp-2+ (f) cells/mm3 in these micrographs showed that hCDR2/2L+(PS3)/MDL28170 treatment (5, 10, 20 μM) reduced the CDR-induced loss of CB [nE = 6 (PS3) and nE = 4 (PS4)] and L7/Pcp-2 [nE = 3 (PS4)]. g Similar observation was found for rCDR/MDL28170 co-treatment (125 ng/mL, 10 μM MDL28170, CB: nE = 5). Calpain antagonist reduced the CDR-induced loss depending on the CDR target to up to 60 % (Table 1). This was most pronounced for CDR2. j MAP kinase antagonist U0126 (5 μM) does not influence the loss of CB+ or L7/Pcp-2+ PCs after hCDR2/2L+(PS3) internalization (1 μL/mL; 6 days; nE = 4). Data are mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.008. Table 1: CDR antibody effects in percentage
Mentions: Changes in VGCC, AMPA receptor, and PKC signaling can lead to elevated intracellular Ca2+ levels and induce Ca2+-dependent proteases activity. To examine whether CDR internalization affects Ca2+-dependent proteases activity of calpain-1 and calpain-2, we performed Western blot analysis of rCDR-treated cOTSC lysates. Calpain-1 expression was not affected, but calpain-2 expression was significantly increased (rCDR2: 2.05 ± 0.14, ***p < 0.0001; rCDR2L: 1.72 ± 0.14, ***p = 0.0002; rCDR2/2L: 1.84 ± 0.19, **p = 0.0012; 125 ng/mL; 6 days; Fig. 8a–c). We then investigated whether calpain-2 over-activation/-expression could be antagonized by calpain-specific inhibitor MDL28170, a known neuroprotective substrate in ischemia and traumatic injury models [9, 36]. MDL28170 co-application with hCDR2/2L+(PS3/PS4) (6 days) reduced the CB+-PC loss to ~45 %, as well as the CDR-induced pathology on the dendritic arborizations in the remaining PCs (nE = 6 for PS3 and nE = 4 for PS4; Fig. 8d, e; Table 1). We found no difference between the 10 (42 ± 9 %) and 20 μM (44 ± 7 %) MDL28170/hCDR2/2L+(PS3) treatment (p = 0.3841; Fig. 8e; Table 1). The MDL28170-induced rescue of CB+-PCs differed between hCDR2/2L+(PS4) (18 %) and hCDR2/2L+(PS3) (48 %) (*p = 0.0207; 10 μM; Fig. 8e; Table 1). Furthermore, hCDR2/2L+(PS4)-induced L7/Pcp-2+-PC loss was partially rescued by 10 μM MDL28170 (***p < 0.0001; nE = 3; Fig. 8f; Table 1). MDL28170 (10 μM) co-treatment of rCDR2, rCDR2L, and rCDR2/2L-treated cOTSC (125 ng/mL, 6 days) showed an improvement of the dendritic morphology and significant reduction of CB+-PC loss (***p < 0.0001; nE = 5; Fig. 8g; Table 1). The increase of calpain-2 expression seen during CDR internalization was blocked by 10 μM MDL28170 (Fig. 8h, i; #p < 0.008; nE = 6).Fig. 8

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