Paraneoplastic cerebellar degeneration: Yo antibody alters mitochondrial calcium buffering capacity

Aim Neurodegeneration is associated with dysfunction of calcium buffering capacity and thereby sustained cellular and mitochondrial calcium overload. Paraneoplastic cerebellar degeneration (PCD), characterized by progressive Purkinje neurone degeneration following paraneoplastic Yo antibody internalization and binding to cerebellar degeneration‐related protein CDR2 and CDR2L, has been linked to intracellular calcium homeostasis imbalance due to calbindin D28k malfunction. Therefore, we hypothesized that Yo antibody internalization affects not only calbindin calcium binding capacity, but also calcium‐sensitive mitochondrial‐associated signalling, causing mitochondrial calcium overload and thereby Purkinje neurone death. Methods Immunohistochemically, we evaluated cerebellar organotypic slice cultures of rat brains after inducing PCD through the application of Yo antibody‐positive PCD patient sera or purified antibodies against CDR2 and CDR2L how pharmacologically biased mitochondrial signalling affected PCD pathology. Results We found that Yo antibody internalization into Purkinje neurons caused depletion of Purkinje neurone calbindin‐immunoreactivity, cannabinoid 1 receptor over‐activation and alterations in the actions of the mitochondria permeability transition pore (MPTP), voltage‐dependent anion channels, reactive oxygen species (ROS) and Na+/Ca2+ exchangers (NCX). The pathological mechanisms caused by Yo antibody binding to CDR2 or CDR2L differed between the two targets. Yo‐CDR2 binding did not alter the mitochondrial calcium retention capacity, cyclophilin D‐independent opening of MPTP or activity of NCX. Conclusion These findings suggest that minimizing intracellular calcium overload toxicity either directly with cyclosporin‐A or indirectly with cannabidiol or the ROS scavenger butylated hydroxytoluene promotes mitochondrial calcium homeostasis and may therefore be used as future neuroprotective therapy for PCD patients.

Maintenance of intracellular calcium homeostasis, signal transduction regulation and ATP production are critical for normal cell metabolism and mitochondrial homeostasis, and if these processes are deregulated or dysfunctional, neurodegeneration eventually occurs [22][23][24][25]. Dysfunction of voltage-dependent anion channels (VDAC) or Na + /Ca 2+ exchangers (NCX) [26], deregulation of the mitochondrial calcium buffering capacity through mitochondria permeability transition pore (MPTP) opening [22,25], or the increase in cytochrome-C and reactive oxygen species (ROS) signalling and production [23,27] can all lead to excessive mitochondrial calcium overload, which in turn increases the intracellular calcium levels and can thereby induce cell death [24]. Calcium homeostasis is not only regulated by influx and efflux of calcium but also by the modulation of the availability of free intracellular calcium through calcium buffer molecules like calbindin D 28k [28]. Calbindin D 28k depletion has been correlated to neurodegeneration in PCD [29], Parkinson's disease [21] and Alzheimer's disease [30]. Furthermore, in Purkinje neurons, calbindin D 28k serves as a functional biomarker, as it modulates intracellular calcium and regulates Purkinje neurone motor coordination precision [28,31,32], which is affected in Yo-PCD patients. Recent experimental evidence suggests that Yo antibodies affect intracellular calcium homeostasis [20]; however, the detailed mechanisms are still largely unknown. As current antineoplastic and immunosuppressive PCD treatment is inadequate [33], greater understanding of the mechanisms behind the Yo driven calcium homeostasis imbalance could potentially provide crucial knowledge for development of effective neuroprotective therapies. We, therefore, investigated whether binding of autoantibody Yo to CDR2 and CDR2L affected mitochondrial homeostasis by investigating the activity of the MPTP, VDAC, NCX and ROS, and the deregulated activity of cannabinoid 1 receptor (CB1R) in Purkinje neurons by using a rat-based ex vivo PCD model of cerebellar organotypic slice culture (cOTSC). Clinically relevant compounds were used to modulate the assumed dysregulation of the calcium-sensitive mitochondrial-associated signalling.

Patient sera
Sera were obtained from four patients with gynaecological cancer and PCD who had Yo autoantibodies against CDR2 and CDR2L (anti-Yo 1-4 ) [12] but lacked P/Q-type VGCC antibodies [20]. Four sex-and agematched nonparaneoplastic neurological syndrome sera (non-hCDR [1][2][3][4] ) and a pool of sera from 100 healthy donors (non-hCDR 100p ) were used as controls. Sera were not heat-inactivated before use. Control and anti-Yo patient sera were collected before patients were treated for cancer or PCD and were stored at the PND Biobank #133/2015 or the Biobank for diagnostic cancer marker #188.05 with approval of the regional ethics committee, Western-Norway.

Purkinje neurone-specific antibodies
As a control to evaluate the specificity of the cytotoxic nature of anti-Yo internalization in PCD, we studied two Purkinje neurone-specific antigens, GAD65 and PCP2 (also known as L7). cOTSC was treated with 1000 ng/ml mouse anti-GAD65 (#559931; BD Bioscience,  Franklin Lakes, NJ, USA) or 1000 ng/ml rabbit anti-PCP2 (#M194; Takara Bio Inc., Kusatsu, Shinga, Japan) for 6 days. These concentrations were 10-fold higher than the anti-rCDR concentration used in the rCDR PCD model.

Neuropharmacology
Approaches 1 and 2 ( Figure 1B): To evaluate the neuroprotective potential of compounds that modulate calcium-sensitive mitochondrial-associated signalling, we co-applied the compounds with anti-Yo/rCDR immediately at PCD onset (approach 1) or 4 days after PCD induction (approach 2); cultures were treated for 6 days. Compounds tested were AM281 [IC 50 50 : 700 nM (NCX rev ), 5.5 lM (MCU); #1244; Tocris]. As this PCD model system is based on an interface hydrophilic PTFE membrane-diffusion approach where the cOTSC is not submerged, the system behaves like an in vivo brain. Therefore, several test dosages were evaluated starting with 2.5-fold of the IC 50 value to determine effective, nontoxic doses if there was no literature guided initial dose selection available for this kind of culture system. Depending on the hydrophilic or hydrophobic nature of the used compounds, the effective dosage was determined between 2.5 to 200 times of the IC 50 value.
Imaging Z-stack images were collected at 0.5-1 lm intervals with a DMI6000-CS-TCS-SP5 microscope using a HC-PL-APO 20 9 0.75 IMM-CORR-CS2 objective to detect CF TM 488/ 594/647 dye emission and superimposed with LASAF software V2.5.1 (Leica Microsystems GmbH, Wetzlar, Germany). The fluorescence intensity was adjusted to 75% of the maximum in untreated naive control tissue for each experiment.

Quantification of Purkinje neurone pathology
The anti-Yo-induced pathological state was evaluated by assessment of functional biomarker calbindin, neurodegeneration marker Fluoro-Jade-C (0.001%, 30 min prior mounting; #AG325; Millipore), and cell death marker propidium iodide (2 lM, 48 h prior to fixation; #P3566; Invitrogen) as described by Brana and Noraberg [36,37]. The pathological progress was quantified by counting calbindin-immunoreactive Purkinje neurone somata (Calb + cells) manually (but blind) and automatically in 3-8 images of 750 9 750 9 50 lm in each slice for each treatment, experiment, and group with counts reported per mm 3 ( Figure 1C). The automatic count showed a discrepancy of approximately 5% compared to manual count; therefore, each experimental set was counted solely manually or automatically. The automatic count was performed with Fiji as previously described [20].

Data analysis and statistics
All treatments were performed in triplicate or quadruplicate. Data were evaluated using the nonparametric Mann-Whitney U-test (Graph-Pad-Prism 4.0 software, San Diego, CA, USA) with P < 0.05 considered statistically significant.

Rat cOTSC-based ex vivo PCD model
Yo antibody-positive sera from four PCD patients, and purified rabbit polyclonal antibodies against the Yo antigens CDR2 and CDR2L were used to induce Purkinje neurone degeneration. The antibody-induced Purkinje neurone pathology was evaluated by visualizing the targeted neurons with the functional biomarker calbindin D 28k (Calb). Confocal imaging 6 days after application of Yo antibody showed that the tested Yo + sera reduced the Calb + cell count by 70.7 AE 7.8% (Yo 1 ) and 67.1 AE 7.0% (Yo 2 ) relative to naive controls (Figure 2A). This effect was specific to anti-Yo as none of the control sera (non-hCDR 1-4 and non-hCDR 100p ) caused depletion of calbindin-immunoreactive cells (Figure 2A). Similar results were obtained when purified rabbit polyclonal antibodies against CDR2 and CDR2L were used. Antibody against CDR2 reduced the Calb + cell count by 62.4 AE 5.5% and antibody against CDR2L by 67.7 AE 5.7%, whereas antibodies against other Purkinje neurone-specific proteins such as anti-PCP2 or ataxiarelated anti-GAD65 showed no loss of calbindin immunostaining ( Figure 2B). To verify that the depletion of calbindin-immunoreactive cells reflects neurodegeneration, we employed the marker Fluoro-Jade-C. After Yo antibody treatment, the few remaining Calb + Purkinje neurons were neither positive for Yo incorporation nor the neurodegeneration marker Fluoro-Jade-C (Figure 2C). In contrast, the calbindin depleted Purkinje neurons showed strong Yo internalization, Jade-C staining and shrunken cell bodies ( Figure 2C1), but were negative for cell death marker propidium iodide ( Figure 2C2), indicating that neurodegeneration had begun but that cell death had not yet occurred.
Yo antibody binding to CDR2/2L leads to deregulation of cyclophilin D-dependent MPTP opening and increased ROS production Immunohistochemical staining revealed that CsA (1 lM), which inhibits the cyclophilin D-dependent opening of the MPTP [22], abolished the CDR antibody associated loss of calbindin immunoreactivity when cultures were co-treated at PCD onset ( Figure 3A,B) and after PCD was established ( Figure 3C; post-PCD). CsA showed similar neuroprotective effects whether PCD was induced by human Yo + PCD sera or by purified CDR antibodies and did not affect the counts of Calb + cells in the control slices ( Figure 3A-C) nor the Yo antibody internalization ( Figure 3D). CsA co-treatment reduced the numbers of shrunken Purkinje neurone cell bodies that showed high Yo antibody internalization. When cultures were treated with anti-Yo and CsA simultaneously, most Yo + cells had normal cell body size and were positive for calbindin ( Figure 3D). BHT (10 lM), a ROS scavenger [38], was tested to confirm that Yo antibody internalization leads to deregulation of the mitochondrial calcium buffering capacity, indicated by the CsA data. At PCD onset, BHT completely prevented the depletion of calbindin-immunoreactive cells caused by anti-Yo 2-4 and anti-rCDR2/2L internalization but was less effective against anti-Yo 1 , anti-rCDR2 and anti-rCDR2L-induced pathology ( Figure 3A,B). After PCD was established (post-PCD), BHT treatment was able to reduce the loss of calbindin immunoreactivity caused by anti-Yo 1-2 and anti-rCDR2/2L internalization, almost to baseline (Figure 3C). BHT had no impact on control tissue (Figure 3A-C) and did not prevent the internalization of Yo antibodies ( Figure 3D). Under BHT treatment, the cell count of Purkinje neurons positive for both calbindin and anti-Yo increased and Yo-positive Purkinje neurons did not display shrunken cell bodies. In support of the results obtained with CsA, these data indicate that Yo antibody binding to CDR2 and CDR2L induces ROS overproduction potentially through calcium overload and effect on respiratory chain complex I [27].

Yo antibody binding reduces mitochondrial membrane potential and increases ROS activity
To support the pharmacological data, we performed a semiquantitative fluorescence intensity analysis of the mitochondrial membrane potential by using Mito-Tracker deep red (MTDR) and also ROS activity by using MitoTracker Red CM-H2xROS (MTROS) (Figure 5A,C).

Yo antibody binding to CDR2L deregulates NCX signalling
As there is a tight coupling of intracellular calcium homeostasis and mitochondrial-associated signalling, we evaluated whether Yo antibody internalization alters the function of NCX. NCX exchange of Ca 2+ for Na + is essential for neurone survival and associated with plasma and mitochondrial membranes [25,26,42]. Under certain pathological conditions, NCX goes into reverse mode (NCX rev ) and increases calcium entry into the cell by 30% [26] with deleterious consequences for cellular homeostasis [25]. We found that at PCD onset NCX rev antagonist KB-R7943 (KB-R, 10 lM) [43,44], or NCX antagonists CGP37157 (CGP, 10 lM) or benzamil (Benz, 250 nM) [42], reduced depletion of calbindin-immunoreactive cells by up to 60% when co-applied with Yo + PCD sera or antibody against CDR2L ( Figure 6A,B). Interestingly, the depletion of calbindin-immunoreactive cells induced by CDR2 antibody binding was not affected by KB-R7943 and CGP37157. In control slices, however, blocking NCX rev with KB-R7943 did cause strong depletion of calbindin-immunoreactive cells (53.2 AE 6.2%; P < 0.001), whereas the blockage of NCX with CGP37157 or benzamil did not affect the Calb + cell count ( Figure 6B).

Ex vivo PCD model
The ex vivo PCD model used, makes it possible to study in vivo Yo antibody-induced pathology [45,46] without the complexities of the blood-brain barrier and an active immune system [45,47]. The Yo antibody-positive sera contained active complement factors, whereas the affinity-purified rabbit CDR antibodies (rCDR) were used to perform neutral IgG antibody assay without exposing the cOTSC to any related peptides which could activate brain naive-resident T cells [47]. Both of these approaches led to identical depletion of the functional biomarker calbindin [28,29,31,32,48,49] in Purkinje neurons indicating that PCD is predominantly an antibody-driven neurodegenerative disease. The Yo/CDR antibodies were clearly internalized by Purkinje neurons and caused a loss of calbindin immunoreactivity, whereas age-/sex-matched sera without Yo antibody or antibodies against Purkinje cell-specific protein 2 (PCP2) or ataxia-related GAD65 were not internalized and did not affect the immunoreactivity of biomarker calbindin. Yo antibody-induced pathology can be linked to the disturbance of calcium-sensitive mitochondrialassociated signalling Calbindin is the major modulator and buffer of Purkinje neurone intracellular calcium [28,32], and calbindin depletion has been linked to neurodegeneration in PCD [29], Parkinson's disease [21] and Alzheimer's disease [30]. We have shown that calbindin co-immunoprecipitated with CDR2, but not with CDR2L [20], which suggests that calbindin could stabilize both CDR2 expression and function. This has been recently confirmed by others [21]. We have also demonstrated that Yo antibody binding to CDR2 and CDR2L leads to dysregulation of the calcium-dependent VGCC-PKC-calbindin signalling pathway and calpain-2 over-activation, suggesting that cellular calcium overload plays an important role in Purkinje neurone degeneration and cerebellar atrophy in PCD [20]. In Parkinson's disease, CDR2 expression is modulated by calpain-and ROSactivity [21]. PKC-related kinase 1, an important factor in Alzheimer's disease and amyotrophic lateral sclerosis [50], interacts with CDR2 by phosphorylation [51].
As intracellular calcium homeostasis and mitochondrial-associated signalling are strongly interdependent, sustained perturbations in this network can have devastating consequences to the cell. As neurons have high energy requirements, they are especially vulnerable to mitochondrial dysfunction, and we have found that Purkinje neurons display the highest mitochondria density of cells in the cerebellum (unpublished results). In the present study, we found that anti-Yo internalization enhanced ROS production and lowered the mitochondrial membrane potential (DΨm) to similar levels as the protonophore uncoupler FCCP and BAM15. The rate of ROS production, DΨm and the activity of the electron transport chain complex are highly interdependent [27]. We believe that anti-Yo internalization not only downregulates DΨm and increases ROS generation through inhibition of respiration, it is also affecting the cation inward and anion outward transport of mitochondrial and plasma membrane to counteract calcium overload [52]. Protonophore uncoupler FCCP does depolarize both mitochondrial and plasma membranes and thus causes and rapid collapse of calcium homeostasis within neurons [34,35], as was indicated by the increased MTROS fluorescence levels under FCCP in our study. The specific depolarization of only the mitochondrial membrane by BAM15 did not show such an effect on the ROS output.
In the present study, we used the ex vivo PCD model to evaluate clinically used compounds that could possibly modulate the Purkinje neurone calcium homeostasis, as mitochondrial calcium overload, ROS signalling/production and MPTP opening are several key steps in neurodegeneration [22,23,25,26]. Mitochondrial calcium overload can be caused by increased VDAC and NCX rev mitochondrial calcium influx, reduced NCX mitochondrial calcium efflux, and changes in mitochondrial calcium buffering capacity through MPTP opening [23]. Prolonged opening of MPTP, a voltage and calcium-dependent, CsA-sensitive, high conductance channel is associated with increased inner mitochondrial membrane permeability. Depolarization of the inner mitochondrial membrane leads to respiratory chain dysfunction and is associated with Figure 7. Calcium flux regulation in Purkinje neurons. Schematic diagram illustrates important calcium flux regulators in Purkinje neurone calcium homeostasis and how the six used antagonists modulate calcium flux and reveal neuroprotective capacity in antibodydependent manner. Some of the used antagonists have multiple targets, and a total of eight calcium flux regulating processes were subdue: (1) mitochondria permeability transition pore opening by cyclosporin-A (cyclophilin D-dependent) and KB-R7943 (cyclophilin Dindependent, grey), (2) voltage-dependent anion channels (VDAC) activity by cannabidiol (CBD), (3) respiratory chain (RC) regulation indirectly by butylated hydroxytoluene (BHT) and KB-R7943 (grey), (4) cytochrome-c (Cyt C) production by CBD, (5) reactive oxygen species production by BHT and CBD, (6) Na + /Ca 2+ exchanger (NCX) activity by CGP37157 and benzamil, (7) NCX rev activity by KB-R7943 and (8) voltage-gated calcium channels (VGCC) activity by CGP37157 and CBD. Balancing these eight calcium-sensitive targets resulted in moderate (single arrow) to high (triple arrows) neuroprotective effects during anti-Yo/rCDR internalization, with one exception. In Purkinje neurone degeneration caused by anti-rCDR2, inhibition of NCX (process 6) or its reverse mode (process 7) showed no beneficial effect (ø). Indirect inhibitory effects are marked in grey. increased ROS production [27] as we have recorded with MTDR and MTROS after anti-Yo was internalized into Purkinje neurons.
We found that targeting the MPTP with CsA and KB-R7943 [22,44], the respiratory chain with CBD and KB-R7943 [41,43], VDAC and cytochrome-C with CBD [41] and ROS over-production with BHT [38] provided graded neuroprotective capacity during exposure and accumulation of Yo antibodies in Purkinje neurons (Figure 7) albeit these compounds did not prevent the internalization of Yo antibody into Purkinje neurons.
Although CsA is used clinically, there are caveats to its use especially as it does not efficiently cross the blood-brain barrier [53]. It is unlikely therefore to be an effective neuroprotective agent in the cerebellum in vivo. However, we show that CsA has a beneficial effect on the mitochondrial membrane potential without affecting the ROS release when anti-Yo was internalized in Purkinje neurons. Furthermore, because of its immunosuppressive properties and ability to induce necrosis in breast cancer cells by downregulating PKM2 expression and ATP synthesis [54], CsA may still have a potential for use in PCD therapy.
The seconded tested clinical compound, CBD, is not only a low potency CB1R inverse agonist, but potentially targets multiple calcium signalling modulators such as the respiratory chain, VDACs and cytochrome-C [41,55,56]. Furthermore, it has anti-inflammatory and antioxidant properties, and interferes with tumour neovascularization, cancer cell migration, adhesion and invasion [57]. We found that CBD can minimize the downgrading of the mitochondrial membrane potential if Yo pathology occurs; however, CBD also potentiated the anti-Yo-induced ROS production boost probably due to its modulatory property towards the respiratory chain. Furthermore, CBD can easily cross the bloodbrain barrier because of its lipophilic nature. CB1Rs are highly expressed in the cerebellum [40] and their activation reduces neurotransmitter release at all major classes of Purkinje neurone synapses [58]. The results obtained with CBD and AM281 treatment suggest that Yo antibody binding mainly to CDR2 causes a reduced neurotransmitter release [58] and sparse neural activity due to CB1R over-activation [59]. Under normal physiological conditions, blocking CB1Rs can, however, disrupt neuronal activity and cause neurodegeneration [59] as we observed in the experimental controls.
Currently, an oromucosal spray containing CBD is in clinical use to treat moderate to severe spasticity in multiples sclerosis [60] which might be used as a first step towards a CBD-based PCD therapy (Figure 7).
The effect of CsA and CBD both support mitochondrial dysfunction as a major factor in PCD, which is also supported by the effect of ROS scavenger BHT data. Similar to observations in Parkinson's disease, where CDR2 expression is modulated by calpain and ROS activity [21] we showed earlier that Yo antibody internalization alters the activity of calpain [20] and now the ROS output. These findings indicate that binding of Yo antibodies to CDR2 and CDR2L potentially affects the complex I of the respiratory chain, a known site for increased ROS production [27].
Targeting the calcium efflux regulator NCX that regulates not only cytosolic calcium efflux, but also mitochondrial calcium homeostasis with CGP37157 [26,42] or KB-R7943 [43,44] revealed deregulation after anti-Yo internalization and a significant difference in the neuropathological processes induced by Yo antibody binding to CDR2 and CDR2L, respectively. NCX blockage failed to block the loss of calbindin immunoreactivity induced by Yo antibody binding to CDR2 but did reduce the depletion of calbindin-immunoreactive cells induced by Yo binding to CDR2L. This supports our previous findings that functionally linked CDR2L to membrane-associated signalling [20]. Although KB-R7943 is mainly known as an NCX rev antagonist, it is also reported to modulate calcium flux by modulating the opening of MPTPs independently of cyclophilin D, L-type VGCC, ryanoidine receptors, store-operated calcium entry [44] and mitochondrial complex I [43]. KB-R7943 influences mitochondrial calcium handling by increasing the calcium retention capacity, which can protect mitochondrial function from pathological calcium overload [44]. Therefore, the nonresponse of anti-CDR2 pathology to KB-R7943 indicates that CDR2 binding does not alter the mitochondrial calcium retention capacity or the cyclophilin D-independent opening of MPTPs.
We show that the binding of the Yo antibody (ies) to the assigned antigens CDR2 or CDR2L initiates different pathological cascades in the cerebellum, especially in Purkinje neurons. Our data indicate that not so much the antibody concentration [20] more like the ratio of Yo binding to CDR2 or CDR2L influences the effectiveness of each antagonistic neurodegenerative treatment strategy.

Conclusion
Taken together, our data indicate that Yo antibody incorporation into Purkinje neurons causes multiple alterations regarding mitochondrial and cytosolic calcium influx/efflux regulation: CB1R activity, MPTP opening, mitochondrial membrane potential, ROS output or NCX calcium influx/efflux regulation. Thus, anti-Yo-induced pathology can be beneficially modulated by clinical used compounds such as CsA, CBD and ROS scavenger BHT.
However, our study is solely based on neuropharmacological manipulation where the used compounds can interfere with more than one signalling pathway, for that reason we have provided only a first indication that mitochondrial dysfunction occurs if autoantibody Yo is internalized into Purkinje neurons. Further molecular biological studies have to be done to characterize the precise mechanistic alterations, for example, alteration of the complex I of the respiratory chain, to deepen our knowledge on mitochondrialassociated autoimmune-mediated neurodegeneration.
As current immunomodulatory therapies for Yo antibody-mediated PCD are not very effective [33,61], additional therapeutic strategies addressing in particular the Purkinje neurone degeneration are important for future PCD therapy and our findings demonstrate that treatment addressing calcium homeostasis imbalance and ROS over-production seems to provide neuroprotective potential.