Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis

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. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1351-6) contains supplementary material, which is available to authorized users.


Introduction
Paraneoplastic cerebellar degeneration (PCD), caused by Purkinje cell (PC) death in the cerebellum, is a paraneoplastic neurological disorder with severe pancerebellar symptoms, such as ataxia, nystagmus, and dysarthria [72]. Cross-reactivity of onconeuronal Yo antibodies (Yo Abs) and T cells with antigens in the tumor tissue and cerebellum induce PCD [63]. Yo Abs bind to the cytoplasmic antigen of cerebellar degeneration-related protein 2 (CDR2, 50 kDa, RefSeq NM_001802.1) and 2-like (CDR2L, 62 kDa, RefSeq NM_014603.2) and are found in the serum and cerebrospinal fluid of patients with remote, non-metastatic ovarian and breast cancers [7,22,72].
The PCs are the sole projection neurons in the cerebellum and therefore proper morphological as well as physiological integrity of the PC dendrites is essential for cerebellar function [25,67]. Optimal intracellular Ca 2+ levels and Ca 2+ flux via cytoplasmic Ca 2+ -binding protein calbindin D 28K (CB) and the GoLoco domain protein, Purkinje cell-specific protein-2 (L7/Pcp-2), are essential for cellular and molecular mechanisms involved in neurotransmitter release, ion channel permeability, enzyme activity, and gene transcription [6,24,39,57]. With its four Ca 2+ -binding sites, CB is a Ca 2+ buffer and sensor; it regulates fast Ca 2+ influx by Ca 2+ binding [57] and is considered as a PC survival marker [28,38]. L7/Pcp-2 modulates P/Q-type voltage-gated calcium channels' (VGCC) function, whose dysfunction is implicated in ataxia [27,39,69]. Factors that modulate or disrupt CB and L7/Pcp-2 are expected to exert powerful physiological and pathological effects on PCs and are listed in Table S1.
In the present study, an ex vivo model of rat cerebellar organotypic slice culture (cOTSC) was used to study the neuropathological mechanisms underlying the antibodymediated PCD and to identify potential intracellular treatment targets which are elaborated in Table S1. Multiphoton imaging showed that CDR2 and CDR2L antibody internalization reduced the CB and L7/Pcp-2 immunoreactivity levels in PCs. This antibody-driven immunoreactivity loss was reduced by modifying the intracellular Ca 2+ transients and inhibiting the Ca 2+ -dependent protease calpain (Table 1). These findings suggest that widespread consequences of Ca 2+ homeostasis dysregulation can induce morphological changes and fatal alterations in the cell signaling pathways, thereby causing neurodegeneration.

Patients' sera
We used four female patients' sera that were antibody positive for CDR2 (hCDR2 +(PS1) ), CDR2L (hCDR2L +(PS2) ) or both CDR2 and CDR2L (hCDR2/2L +(PS3/PS4) ) [22] and negative for P/Q-type VGCC (RIA; DLD Diagnostika, #RA006/12). Patient data are listed in Table S2. The sera were collected before treatment took place and stored at −80 °C at the Paraneoplastic Neurological Diseases Biobank (#484) with the approval of the Regional Committee for Medical and Health Research Ethics in Western Norway, Diagnostic markers of cancer (188.05). As control serum, we pooled samples from 100 healthy blood donors without any known autoimmune disease (non-hCDR).

Primary antibodies
The antibodies used in immunohistochemical (IHC), Western blot (WB), and immunoprecipitation (IP) analyses are detailed in Table S3.

Cryostat sections
Anesthetized adult female rats were transcardially perfused with ice-cold 4 % PFA-PBS. The brains were post-fixed (

Paraffin-embedded sections
Six days after hCDR2/2L and non-hCDR internalization, slices were fixed (4 % PFA), embedded in paraffin, sliced into 4 µm thick sections, and stained with hematoxylin and  Figure S1b was created as 3D projections with orthogonal section from z-stacks using the Fiji plug-in 3D Viewer. Calbindin D 28K (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 (n E ) and group and projected to mm 3 (Fig. 1c)

Data analysis and statistics
Data analysis and calculations were performed using the software Excel 2003 and Graph Pad Prism 4.0. Data are presented as mean ± SEM, and statistical significance was determined using the non-parametric two-tailed paired Mann-Whitney's U test. The level of significance is indicated with asterisks: *p < 0.05; **p < 0.01; ***p < 0.001.
To elucidate whether the observed pathology was induced by CDR antibodies, we treated the cOTSC with heat-inactivated affinity-purified polyclonal rabbit Abs against CDR2 (rCDR2 + ) and CDR2L (rCDR2L + ), or both (1:1 mixture, rCDR2/2L). We found that within 6 days both rCDR2 and rCDR2L were internalized and led to extensive loss of CB staining (400 ng/mL; Fig. 3a, merged). We observed three patterns of CB immunoreactivity modifications after rCDR treatment: (1) whole cell staining with neighboring cells unstained, (2) dendritic staining with only a weak remaining signal in the soma, and (3) soma staining with no dendritic staining (Fig. 3b). The remaining CB + -PCs showed morphological alterations in their dendritic arborizations with a loss of tertiary branches (Fig. 3c).
The Western blot analysis of rCDR-treated cOTSC lysates (6 days, 125 ng/mL) showed no effect on CB protein concentration compared to untreated naive control (n E = 10; Fig. 3f, g). It is therefore likely that the reduced CB immunoreactivity is caused by structural modifications of the CB Ca 2+ -binding site or misfolding of the protein itself [11,50]. To investigate this hypothesis, we replaced the rCDR-containing media (125 ng/mL, 6 days) with non-CDR media (Fig. 1b, washout, 7 days). The number of CB + -PCs in rIgG control was not affected by washout or by time (Fig. 3h, i). We found that the washout of rCDR improved the CB + -PC count in rCDR2L and rCDR2/2L -treated cOTSC within 48 h (D2wo) (rCDR2: p = 0.1383, rCDR2L: **p = 0.0018, rCDR2/2L: **p = 0.0010; Fig. 3i). All rCDR groups showed substantial recovery of CB + -PCs at day 7 after washout (***p < 0.0001; Fig. 3h, i; Table 1), but plateaued between days 4 and 7 at ~ 40 % CB + -PC loss before reaching the rIgG control count. This supports our hypothesis that the CB Ca 2+ -binding epitope is reversibly altered or blocked, or that the protein might be misfolded.
To analyze whether CDR proteins and CB interact at physiological expression levels, we assayed by reciprocal co-immunoprecipitation (Co-IP) CDR2, CDR2L, and CB proteins from cerebellar lysates. As shown in Fig. 4a, b, CB and CDR2 specifically Co-IPed, indicating endogenous CDR2-CB complex in PCs. Interestingly, CDR2L did not form any complex with CB (anti-CDR2L from three sources), but showed strong CDR2 Co-IP (Fig. 4a, c). These Co-IP results suggest that CDR2 antibody can substantially influence CB function by binding to an interacting protein.
This suggests that another important Ca 2+ homeostasis regulator is affected by CDR antibody internalization.
The results show that the CDR antibody-mediated pathology can be attenuated in the presence of VGCC, AMPA receptor, and PKC signaling inhibition.
These data suggest that the MAP kinase signaling cascade is not involved in the CB or L7/Pcp-2 immunoreactivity reduction induced by hCDR2/2L.

Discussion
CDR antibodies, also known as onconeuronal Yo antibodies, have attracted increasing interest due to their suggested pathological role in the progressive loss of PCs seen in PCD after binding to CDR2 and CDR2L antigens [22,31,33,70]. Because the mechanisms underlying PCD, in particular the effects of CDR antibodies, are largely unknown, we investigated whether they are internalized into PCs and how that affects PC physiology. : n E = 4) or rCDR-induced pathology (125 ng/mL, 10 μM, n E = 6) in a concentration-dependent manner and more beneficially for rCDR2L than rCDR2 (CB: **p = 0.0071; L7/Pcp-2: **p = 0.0011). Data are mean ± SEM. Non-parametric two-tailed paired Mann-Whitney's U test. *p < 0.05; **p < 0.01; ***p < 0.001; # p < 0.005; Table 1: CDR antibody effects in percentage PKCγ expression after rCDR/K252a co-treatment. h K252a co-treatment reduces the rCDR-induced PKCγ expression rise in the rCDR2 and rCDR2/2L group (125 ng/mL; n E = 5) after K252a co-treatment. Investigated samples: 6 days of CDR internalization; data in mean ± SEM; non-parametric two-tailed paired Mann-Whitney's U test. *p < 0.05; **p < 0.01; ***p < 0.001; # p < 0.003; Table 1: CDR antibody effects in percentage  (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; n E = 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. OTSC can be used to study neurochemical, structural, and physiological changes linked to diseased in vivo brains [13,44]. Hence, we created an ex vivo antibody-mediated PCD model by applying CDR-positive patients' sera and affinity-purified rabbit CDRs to the culture medium of cerebellar rat OTSC. These slices offer unique advantages because the tissue architecture is preserved, synaptic circuitries are maintained, and various treatments can be evaluated without the influence of activated immune cells and the bloodbrain barrier (BBB) [13,21,[42][43][44]. The mechanisms of autoimmunity in paraneoplastic neurological diseases are complex and not yet understood. It is suggested that brain epitope-specific serum Abs can access the brain only if the integrity of the BBB is compromised [18,20]. However, brain epitope-specific cerebrospinal fluid Abs can be produced by intrathecal synthesis due to activated T and B cells that can cross the BBB [51,62]. Data from anti-Yo/CDR2-Ab positive PCD patients are contradictory in terms of cytotoxic T cell involvement [2,3,10,56,61,65]. Therefore, it is of interest that PCs can internalize IgGs and that anti-Yo Abs can lead to cell injury and non-apoptotic death without the influence of activated immune cells [31,33].
Here, we demonstrate that both hCDR and rCDR can cause similar PC pathology. By using rCDR we exclude cytotoxic T cell involvement, since the cOTSC was not exposed to related peptides which could activate brainnaive resident T cells [42]. Therefore, our data are most likely immune cell independent. We found that both CDR2 Fig. 9 Cell Ca 2+ homeostasis regulating signaling pathways influenced by CDR antibody internalization in Purkinje cells. The flowchart clarifies how the cell Ca 2+ homeostasis is affected by CDR2 and CDR2L antibody internalization. In our CDR antibody-mediated PCD model, we found that (1) CDR2 interacts with calbindin D 28K , (2) both CDR2 and CDR2L antibodies attenuate the Ca 2+ -buffering activity of calbindin D 28K , (3) modify the VGCC modulator L7/Pcp-2, and (4) cause increase protein levels of VGCC and PKC. These events dramatically increase the levels of unbound cytoplasmic Ca 2+ to the millimolar range and thereby induce over-activation of calpain-2. (5) It is known that calpain over-activation causes rapid modifications of synaptic membrane-associated proteins (VGCC, AMPAR), cytoskeleton proteins, major postsynaptic density scaffolding proteins, and synaptic protein kinases (PKC) and phosphatases.
To prevent the enhanced Ca 2+ influx through VGCC and free the CB binding capability by lowering the unbound cytoplasmic Ca 2+ could be a putative neuroprotective treatment strategy. Antagonists to VGCC (ω-agatoxin), AMPA receptor (CNQX), PKCγ (K252a) and calpain (MDL28170) showed satisfactory neuroprotective impacts. Our data suggest that CDR2 are more likely interacting with cytosolic components and therefore responsible for calpain activation (K252a and MDL28170 treatment), whereas CDR2L are more likely to modify the postsynaptic density structure and physiology (ω-agatoxin and CNQX treatment). Therefore, both CDR antibodies can cause fatal alterations in the Purkinje cell signaling pathways and membrane structures by affecting actin remodeling, membrane reorganization, and protein trafficking, thereby cause neurodegeneration and CDR2L are internalized independently and their removal can partly reverse the calpain-2-dependent, but caspase-3-independent, pathology. We found equal effects of CDR2 and CDR2L on PC survival, although only a combination of CDR2/2L is associated with PCD [22]. However, CDR2/2L pathology was stronger, e.g., PKCγ results, and reciprocal Co-IP showed a CDR2-CDR2L complex in PCs, which supports the hypothesis that increased Ab avidity enhances the pathological effects [71]. Our CDR-induced pathology followed a similar time frame as described by Greenlee and colleagues [31], but the CDR internalization mechanisms remain to be explored. A recent study by Congdon et al. [14] demonstrated antibody internalization via IgG-FcγII receptor endocytosis. IgG-FcγII receptors are found in PC and regulate cerebellar function [47].
CDR internalization affects postsynaptic signaling and thereby Ca 2+ homeostasis: AMPAR-VGCC-PKCcalpain In Fig. 9 we show the complexity, by which h/rCDR internalization changes PC physiology by targeting postsynaptic signaling factors and thereby modifies Ca 2+ homeostasis. We described these factors along with related neurological diseases in Table S1. We found that the immunoreactivity of Ca 2+ homeostasis regulator CB and VGCC modulator L7/Pcp-2 was reduced. These proteins are functionally linked, since CB shapes the post-tetanic potentiation induced by the Ca 2+ influx through AMPAR and VGCC and can act as an activity-dependent sensor [32,57]. When endogenous CB levels are reduced, PC dendritic outgrowth and differentiation is inhibited, and motor coordination and sensory integration fail [1,4,37,74]. We found that the total CB protein levels are not affected by CDR internalization. However, Co-IP reveals that CDR2-CB forms a strong protein complex and therefore CDR2 antibody binding to CDR2 protein in the cell may disrupt CB function by sequestering CDR2 and thus hijacking CB's interacting partner. "CDR antibody washout" restored the CB immunoreactivity only partially, indicating irreversible modifications of the CB Ca 2+ -binding mechanism or protein structure, as seen in other neurodegenerative diseases [11,50].
CB dysfunction or deletion can result in drastic fast intracellular Ca 2+ -transient elevation which greatly influences Ca 2+ homeostasis [4]. In PC signaling, intracellular Ca 2+ microdomain variations due to AMPAR, VGCC, or PKC activity play a crucial role [24,27,34,57,68]. AMPAR activation by glutamate binding and site-specific PKC and receptor tyrosine kinases' phosphorylation provides the depolarization necessary for opening VGCCs and therefore increases Ca 2+ influx [19,32,57,73]. We found that the P/Q-type VGCC modulator L7/Pcp-2 immunoreactivity is reduced and Cav2.1 (P/Q-type VGCC) protein concentration is increased by twofold after CDR internalization. L7/Pcp-2 modulates VGCC function in a concentration-dependent manner and enhances or dampens VGCC kinetics to shape fast Ca 2+ influx in synaptic Ca 2+ microdomains [39]. Inhibiting VGCC activity directly (ω-agatoxin) or indirectly (CNQX) prevented or reduced dramatically the CDR-induced CB and L7/Pcp-2 loss, respectively. CDR2L-induced pathology was more effectively reversed than CDR2 during AMPAR inhibition. Data from primary PC culture and synaptoneurosome preparation (data not shown) support that CDR2L is synaptic and could thereby be involved in membrane-associated signaling cascades. CB and L7/Pcp-2 dysfunction and VGCC up-regulation will promote excessive Ca 2+ influx into PCs, which can activate cell death pathways by enhancing PKC and Ca 2+ -dependent protease activity [41].
PKC is associated with apoptosis in ischemia and stroke [8,29,60]. In PCs, enhanced PKC activity will reduce dendritic differentiation, whereas reduced PKC activity increases it [45,46]. We found that the up-regulation of PKCγ expression under CDR internalization was accompanied by reduced dendritic arborizations and loss of tertiary branches. In retina, L7/Pcp-2 interacts with PKC [64] and we found that the increased PKC activity led to reduced L7/Pcp-2 expression (data not shown). Inhibition of PKCγ activity prevented the CDR-induced morphological changes, the loss of CB as well as L7/Pcp-2 immunoreactivity and blocked the increased PKCγ protein expression. However, the increase in PKCγ expression was only seen for CDR2 and CDR2/2L, but not for CDR2L internalization, although K252a rescued the CB and L7/Pcp-2 immunoreactivity in all rCDR-treated cOTSCs. We therefore hypothesize that CDR internalization enhances PKC activity, which: first, reduces L7/Pcp-2 expression; second, increases Cav2.1 expression; third, increases intracellular Ca 2+ , which activates Ca 2+ -dependent proteases such as calpain-1 and calpain-2 and thereby induces neuronal death.
We hypothesize that CDR internalization affects activitydependent modifications of synaptic integrity, stability, and function of target proteins, which modulates the synaptic Ca 2+ transient by mediating over-activation of calpain-2 [5,17]. As calpain-mediated truncation of substrates is regulated by their phosphorylation state, there is a possibility of cross talk between calpain activation and activation of mitogen-activated protein (MAP) kinase. MAP kinase signaling cascades are associated with PKC-dependent synaptic depression and declustering of receptors, inflammation, death receptors' activation, apoptosis, and oxidative stress [12,23,35]. Althought PKC inhibition prevented CDRinduced CB and L7/Pcp-2 loss, blockage of MAP kinase activity did not show any rescue effects.

Conclusion
PCD pathogenesis is largely unknown. We propose a twostep pathology mechanism. First, internalization of CDR antibodies modifies important regulatory factors of Ca 2+ homeostasis, which lead to the silencing of the PCs. Second, cytototoxic T cells and microglia mediate the clearing of diseased cells, as known from autopsy studies [61].
CDR antibodies play an important role in the PCD pathogenesis by inducing PC loss, which causes severe ataxia, dysarthria, diplopia, and vertigo in PCD patients. In line with previous results, we have shown that such cerebellar findings can be linked to imbalance in intracellular Ca 2+ homeostasis due to alterations of CB, VGCC, or PKC [1,27,40,58,69]. Therefore, lowering the intracellular Ca 2+ levels by inhibition, the VGCC-AMPAR-PKC signaling pathway (Fig. 9) during the progress of PCD will beneficially modulate Ca 2+ homeostasis by stabilizing the Ca 2+ -binding capability of CB and prevent the induction of the critical calpain response cascade. The strong neuroprotective effect of antagonizing VGCC-AMPAR-PKC signaling pathway during CDR antibody internalization may therefore be of potential clinical relevance.