Cav2.3 channels contribute to dopaminergic neuron loss in a model of Parkinson’s disease

Benkert, Julia; Hess, Simon; Roy, Shoumik; Beccano-Kelly, Dayne; Wiederspohn, Nicole; Duda, Johanna; Simons, Carsten; Patil, Komal; Gaifullina, Aisylu; Mannal, Nadja; Dragicevic, Elena; Spaich, Desirée; Müller, Sonja; Nemeth, Julia; Hollmann, Helene; Deuter, Nora; Mousba, Yassine; Kubisch, Christian; Poetschke, Christina; Striessnig, Joerg; Pongs, Olaf; Schneider, Toni; Wade-Martins, Richard; Patel, Sandip; Parlato, Rosanna; Frank, Tobias; Kloppenburg, Peter; Liss, Birgit

doi:10.1038/s41467-019-12834-x

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Published: 08 November 2019

Cav2.3 channels contribute to dopaminergic neuron loss in a model of Parkinson’s disease

Julia Benkert¹,
Simon Hess²,
Shoumik Roy¹,
Dayne Beccano-Kelly³,
Nicole Wiederspohn¹,
Johanna Duda¹,
Carsten Simons ORCID: orcid.org/0000-0003-3936-5181¹,
Komal Patil¹,
Aisylu Gaifullina¹,
Nadja Mannal¹,
Elena Dragicevic¹,
Desirée Spaich¹,
Sonja Müller¹,
Julia Nemeth ORCID: orcid.org/0000-0002-9277-0736¹,
Helene Hollmann¹,
Nora Deuter¹,
Yassine Mousba ORCID: orcid.org/0000-0002-0676-332X³,
Christian Kubisch⁴,
Christina Poetschke¹,
Joerg Striessnig⁵,
Olaf Pongs⁶,
Toni Schneider ORCID: orcid.org/0000-0003-2816-2696⁷,
Richard Wade-Martins³,
Sandip Patel⁸,
Rosanna Parlato¹,
Tobias Frank⁹,
Peter Kloppenburg² &
…
Birgit Liss ORCID: orcid.org/0000-0002-6733-9207^1,10

Nature Communications volume 10, Article number: 5094 (2019) Cite this article

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Abstract

Degeneration of dopaminergic neurons in the substantia nigra causes the motor symptoms of Parkinson’s disease. The mechanisms underlying this age-dependent and region-selective neurodegeneration remain unclear. Here we identify Cav2.3 channels as regulators of nigral neuronal viability. Cav2.3 transcripts were more abundant than other voltage-gated Ca²⁺ channels in mouse nigral neurons and upregulated during aging. Plasmalemmal Cav2.3 protein was higher than in dopaminergic neurons of the ventral tegmental area, which do not degenerate in Parkinson’s disease. Cav2.3 knockout reduced activity-associated nigral somatic Ca²⁺ signals and Ca²⁺-dependent after-hyperpolarizations, and afforded full protection from degeneration in vivo in a neurotoxin Parkinson’s mouse model. Cav2.3 deficiency upregulated transcripts for NCS-1, a Ca²⁺-binding protein implicated in neuroprotection. Conversely, NCS-1 knockout exacerbated nigral neurodegeneration and downregulated Cav2.3. Moreover, NCS-1 levels were reduced in a human iPSC-model of familial Parkinson’s. Thus, Cav2.3 and NCS-1 may constitute potential therapeutic targets for combatting Ca²⁺-dependent neurodegeneration in Parkinson’s disease.

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Introduction

Parkinson’s disease is a complex movement disorder that affects millions of people worldwide^1,2. Its primary motor symptoms are caused by the progressive degeneration of dopaminergic midbrain neurons, particularly those within the substantia nigra (SN)^1,3. Aging is a major risk factor for the disease, and although most cases are sporadic, a not insignificant proportion show monogenic inheritance patterns^4,5. The pathogenic mechanisms underlying Parkinson’s disease remain unresolved and curative therapies are currently not available^6,7.

Dopaminergic midbrain neurons display autonomous pacemaker activity, which is crucial for somatodendritic and axonal striatal dopamine release and voluntary movement control^8,9,10,11. In SN dopaminergic neurons, this activity generates oscillatory increases in free cytosolic Ca²⁺ levels, which are thought to impart mitochondrial stress and render these neurons more vulnerable to degeneration by Parkinson’s disease stressors^12,13,14. This stressful Ca²⁺-driven mode of action distinguishes dopaminergic neurons in the SN (and other vulnerable neurons^3,15) from neighboring pacemaking dopaminergic neurons in the ventral tegmental area (VTA), which are spared in Parkinson’s disease^1,3,16. The metabolically demanding Ca²⁺ oscillations, particularly those in distal dendrites of SN dopaminergic neurons, are sensitive to inhibitors of L-type voltage-gated Ca²⁺ channels, such as isradipine^13,14.

Consistent with a role for activity-related Ca²⁺ signals in triggering neuronal demise in Parkinson’s disease is epidemiological evidence, correlating use of blood–brain barrier permeable L-type voltage-gated Ca²⁺ channel blockers with a reduced risk for developing the disease later in life^17,18. These blockers are well-established drugs for treating high blood pressure¹⁷. But a recent phase-III clinical trial with isradipine to test its potential for neuroprotection in Parkinson’s disease patients (ClinicalTrials.gov NCT02168842)¹⁹ was negative²⁰. Indeed, in Parkinson’s disease animal models, there is not full agreement regarding the extent of neuroprotection by L-type voltage-gated Ca²⁺ channel inhibition²¹. Notably, recent studies have identified T-type voltage-gated Ca²⁺ channels to be important for stressful activity-related Ca²⁺ oscillations particularly in proximal dendrites of SN dopaminergic neurons, and also for their vulnerability to degenerative stressors^{14,22,23,24,25}. Whether other voltage-gated Ca²⁺ channels contribute to degeneration of SN dopaminergic neurons in Parkinson’s disease is unclear^26,27.

L-type voltage-gated Ca²⁺ channels in the brain and in SN dopaminergic neurons in particular are built from Cav1.2 and Cav1.3 pore-forming α₁-subunits²⁸. These channels are emerging as key regulators of dopaminergic excitability. On the one hand, they stabilize pacemaker robustness thus sustaining metabolic demand^13,22,29,30. But on the other, they can also inhibit spontaneous activity in an indirect feedback loop^22,30. This feedback mechanism involves Cav1.3, which serves as a Ca²⁺ source for the neuronal Ca²⁺ sensor, NCS-1. NCS-1 is a Ca²⁺-binding protein that has been linked to a variety of neuronal functions in health and disease^31,32,33. In SN dopaminergic neurons, NCS-1 binds in a Ca²⁺-dependent fashion to inhibitory dopamine D2-autoreceptors^30,34,35. This interaction prevents D2-autoreceptor desensitization thereby promoting dopamine autoinhibition^30,32. NCS-1 is therefore suggested to protect SN dopaminergic neurons from activity-related Ca²⁺ overload, and degeneration^31,36 but direct supporting evidence is currently lacking.

Here, we combine single-cell molecular techniques, brain slice patch-clamp recordings and Ca²⁺ imaging with pharmacological and genetic tools to analyze the role of voltage-gated Ca²⁺ channels and NCS-1 for dopaminergic neuronal viability in Parkinson’s disease. We identify R-type voltage-gated Ca²⁺ channels (Cav2.3) as crucial contributors to somatic activity-related Ca²⁺ signaling in mature SN dopaminergic neurons, and to their selective degeneration in an in vivo model of Parkinson’s disease. Moreover, we identify a neuroprotective role for NCS-1.

Results

Cav2.3 is the most abundant Cav subtype in SN dopaminergic neurons

To probe the role of voltage-gated Ca²⁺ channels in the vulnerability of SN dopaminergic neurons, we performed quantitative RNAScope analysis to compare numbers of mRNA molecules encoding Cav α₁-subunits in adult mice (Fig. 1a and Supplementary Table 1). mRNA for L-type (Cav1.2 and Cav1.3), P/Q-type (Cav2.1), N-type (Cav2.2), R-type (Cav2.3) and T-type (Cav3.1-3.3) voltage-gated Ca²⁺ channels were readily detected. Unexpectedly, mRNA for Cav2.3 R-type voltage-gated Ca²⁺ channels, which have yet to be studied in the context of neurodegeneration, were significantly higher than those for the other isoforms in adult SN dopaminergic neurons (Cav2.3 > Cav2.1 ≥ Cav2.2 > Cav3.1 > Cav1.3 > Cav1.2 > Cav3.3 > Cav3.2).

We also compared expression of α₁-subunits in an independent approach by reverse transcription quantitative polymerase chain reaction (RT-qPCR) of single microdissected SN dopaminergic neurons. In these experiments, we used both juvenile and adult neurons. As shown in Fig. 1b (and Supplementary Table 4A), the expression of Cav1.2, Cav1.3 and Cav2.3 was similar in SN dopaminergic neurons from juvenile mice (PN13). In adult mice, transcripts for Cav1.3 and Cav1.2 were modestly downregulated as previously described³⁷. In contrast, Cav2.3 expression was markedly upregulated during aging, such that it was the most abundant in adult neurons, consistent with the RNAScope analyses.

To extend Cav2.3 expression analysis to the protein level, we performed immunocytochemical analysis of Cav2.3 in adult mice (Fig. 1c and Supplementary Table 5A). We confirmed the specificity of the Cav2.3 antibody using Cav2.3 knockout mice in western blots and midbrain sections (Supplementary Fig. 6b/d and Supplementary Table 5B). Cav2.3 protein-derived immunofluorescence signals in the plasma membranes of SN dopaminergic neurons were readily detected. Notably, signal intensities were significantly higher in vulnerable SN dopaminergic neurons compared to that in VTA dopaminergic neurons, which are much less affected in Parkinson’s disease. Together these data prompted us to investigate the role of R-type voltage-gated Ca²⁺ channels, built by Cav2.3 pore-forming α₁-subunits, in SN dopaminergic neuron function and viability.

Cav2.3 contributes to activity-related Ca²⁺ oscillations

Previous work has linked Ca²⁺ entry upon autonomous pacemaking to Ca²⁺-dependent stress and degeneration of SN dopaminergic neurons, and showed that activity-related Ca²⁺ transients in dendritic compartments of SN dopaminergic neurons are inhibited by L-type and also T-type voltage-gated Ca²⁺ channel blockers^13,14. We combined ratiometric Ca²⁺ imaging with perforated patch-clamp electrophysiology in brain slices to examine the role of Cav2.3 in activity-related Ca²⁺ dynamics in somata of dopaminergic midbrain neurons. We took two approaches.

In the first approach, we compared somatic Ca²⁺ signals and the electrical properties of SN dopaminergic neurons in brain slices of adult wildtype and Cav2.3 knockout mice. Knockout of Cav2.3 was confirmed by western blotting and immunocytochemical analysis (Supplementary Fig. 6b/d and Supplementary Table 5B). As shown in Fig. 2, SN dopaminergic neurons recorded from wildtype mice displayed spontaneous action potentials as expected. These action potentials were associated with transient rises in cytosolic Ca²⁺ concentration in the soma. In Cav2.3 knockout animals, spontaneous electrical activity of SN neurons was largely unaffected (although we did note small changes in spike waveform, Supplementary Fig. 1 and Supplementary Table 6B). However, the amplitude of the activity-related Ca²⁺ oscillations (at a frequency of ~1.5 Hz) was significantly reduced by ~50% (Fig. 2a, b, d, e and Supplementary Table 6A). VTA dopaminergic neurons from wildtype animals displayed much smaller somatic Ca²⁺ oscillations (Fig. 2c–e and Supplementary Table 6A), in agreement with previous work^13,14. Ca²⁺-dependent action potential after-hyperpolarizations (AHPs), which are driven in SN dopaminergic neurons by Ca²⁺-sensitive small conductance K⁺ channels (SK)^38,39,40, were also significantly reduced in SN dopaminergic neurons of Cav2.3 knockout mice, consistent with the reduction in the Ca²⁺ signals (Fig. 2e, Supplementary Fig. 1c and Supplementary Table 6B).

In a second approach, we examined the effect of acutely inhibiting Cav2.3 with SNX-482. The use of SNX-482 is complicated as it also potently inhibits A-type K⁺ channels in SN dopaminergic neurons^41,42,43 and at higher concentrations probably also other Ca²⁺ channels⁴⁴. We therefore blocked K⁺ channels with 4-AP (4 mM), and limited the concentration of SNX-482 to 100 nM, which causes only a partial block of Cav2.3 ⁴³. In line with the reduced Ca²⁺ transients upon Cav2.3 deletion, bath application of SNX-482 reduced evoked Ca²⁺ transients by about 25% in wildtype animals (Fig. 2f and Supplementary Table 6C). Like Cav2.3 deficiency, SNX-482 also reduced Ca²⁺-dependent AHPs (Fig. 2f, Supplementary Fig. 2 and Supplementary Table 6D). These SNX effects were specific to Cav2.3 channel inhibition, because SNX-482 was without effect in SN dopaminergic neurons of Cav2.3 knockout mice (Fig. 2f and Supplementary Table 6C/D). In whole-cell voltage-clamp experiments, SNX-482 (100 nM) inhibited voltage-gated Ca²⁺ currents in wildtype mouse SN dopaminergic neurons by ~30% (Supplementary Fig. 3 and Supplementary Table 7).

Taken together, these genetic and pharmacological data identify a significant contribution of Cav2.3 to action potential-associated Ca²⁺ oscillations in the somata of adult SN dopaminergic neurons and to Ca²⁺-dependent AHPs. The established link between activity-related Ca²⁺ oscillations and vulnerability of SN dopaminergic neurons to Parkinson’s disease stressors^13,14,21 raises the possibility that Cav2.3 contributes to their preferential degeneration.

Cav2.3 KO protects SN dopaminergic neurons in a Parkinson’s model

We next explored the role of Cav2.3 in neurodegeneration using a neurotoxin-based Parkinson’s disease mouse model. To do this, we compared neuronal viability in wildtype and Cav2.3 knockout mice, subjected to chronic low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/probenecid treatment, which is still the standard model for preclinical testing of neuroprotective Parkinson’s disease therapies in animals^45,46. We compared the patterns and degrees of neurotoxin-induced loss of SN and VTA dopaminergic midbrain neurons and their axonal striatal terminals by densitometry, unbiased stereology and automated cell counting after tyrosine hydroxylase (as marker for dopaminergic neurons) and hematoxylin immunostaining (Fig. 3, Supplementary Fig. 4a/b/c, and Supplementary Table 8A/B). Knockout of Cav2.3 in SN dopaminergic neurons was confirmed by western blotting and immunocytochemical analysis (Supplementary Fig. 6b/d and Supplementary Table 5B).

We first performed immunostaining of tyrosine hydroxylase and stereology in Cav2.3 knockout mice and wildtype littermates. As shown in Fig. 3a, MPTP treatment resulted in about 40% loss of tyrosine hydroxylase-positive SN dopaminergic neurons from wildtype animals when compared to saline controls. This loss was selective, as MPTP had little effect on numbers of VTA dopaminergic neurons (Fig. 3a and Supplementary Table 8A). In stark contrast, we observed no loss of SN dopaminergic neurons in Cav2.3 knockout animals after MPTP treatment (Fig. 3a and Supplementary Table 8A), thus uncovering a prominent neurodegenerative role for Cav2.3 in SN dopaminergic neurons.

In an independent approach, we performed hematoxylin staining and automated counting of the total neuronal population in the SN (vulnerable dopaminergic and unaffected GABAergic neurons). This analysis confirmed a significant reduction in SN neuron number in the wildtype but not the Cav2.3 knockout animals after MPTP treatment (Fig. 3b, Supplementary Fig. 5b and Supplementary Table 8A).

We also performed densitometric analysis⁴⁷ of tyrosine hydroxylase expression in the dorsal and ventral striatum. These regions harbor the axonal projections of dopaminergic neurons from the SN and VTA, respectively. As shown in Fig. 3c (and Supplementary Table 8B), the tyrosine hydroxylase signal was significantly higher in the dorsal striatum from the Cav2.3 knockout mice compared to wildtype controls after MPTP treatment. In contrast, no such differences were found in the ventral striatum, consistent with Cav2.3 deficiency driving selective protection of highly vulnerable nigro-striatal SN dopaminergic neurons upon neurotoxin insult.

To determine whether the neuroprotective effects of Cav2.3 knockout on SN dopaminergic neurons might be secondary to changes in the functional expression of L-type or T-type voltage-gated Ca²⁺ channels that contribute to dendritic Ca²⁺ oscillations, we quantified Cav1.2, Cav1.3 and Cav3.1 expression in SN dopaminergic neurons (Fig. 3d and Supplementary Table 4B). mRNA levels of all three Cav isoforms were unchanged upon Cav2.3 knockout. In marked contrast, mRNA levels of the neuronal Ca²⁺ sensor NCS-1 were elevated by ~40% in SN dopaminergic neurons from adult but not juvenile Cav2.3 knockout mice (Fig. 3d and Supplementary Table 4C).

Taken together, these data identify Cav2.3 as mediator of SN dopaminergic neuron vulnerability to a degenerative stressor.

NCS-1 KO enhances SN dopaminergic neuron loss in a Parkinson’s model

The increase in NCS-1 levels in Cav2.3 knockout mice, coupled with our previous study identifying NCS-1 as the functional linker between activity-related Ca²⁺ entry and sensitization of inhibitory dopamine D2-autoreceptors³⁰, prompted us to test whether NCS-1 might protect SN dopaminergic neurons from degeneration. We therefore applied the same chronic MPTP Parkinson’s disease model to NCS-1 knockout mice and wildtype controls.

Knockout of NCS-1 in SN dopaminergic neurons was confirmed by western blotting and immunocytochemical analysis (Supplementary Fig. 6a/c and Supplementary Table 5B). As shown in Fig. 4a, b (Supplementary Fig. 4d/e/f and Supplementary Table 8C/D), MPTP-induced loss of SN dopaminergic neurons was significantly greater in the NCS-1 knockout mice compared to controls. Similar results were obtained when analyzing axonal projections in the dorsal striatum. In contrast, no differences to MPTP treatment were observed in the VTA or the ventral striatum. These data uncover a region-selective protective effect of NCS-1 on SN dopaminergic neuron viability.

Given the reciprocal effects of Cav2.3 and NCS-1 deficiency on SN dopaminergic neurodegeneration, and the elevated NCS-1 levels in adult SN dopaminergic neurons from Cav2.3 knockout mice, we quantified Cav2.3 mRNA levels in individual SN dopaminergic neurons of NCS-1 knockout and wildtype mice. Intriguingly, Cav2.3 mRNA levels were significantly lower (~50%) in SN dopaminergic neurons of NCS-1 knockout mice compared to those of control mice, suggesting that expressions of Cav2.3 and NCS-1 are linked (Fig. 4c and Supplementary Table 4D). No differences were observed in NCS-1 mRNA levels between young and adult mice (Fig. 4d and Supplementary Table 4E), and NCS-1 protein levels between SN and VTA dopaminergic neurons (Fig. 4e and Supplementary Table 5A).

In our final analysis, we compared NCS-1 and Cav2.3 protein levels in a human model of Parkinson’s disease. For these experiments, we derived dopaminergic neurons from induced pluripotent stem cells (iPSCs) that were obtained from Parkinson’s disease patients heterozygous for a point mutation (N370S) in the GBA gene coding for a lysosomal glucocerebrosidase (Supplementary Table 9A)⁴⁸. Mutations in this gene constitute the highest genetic risk factor associated with idiopathic Parkinson’s disease^49,50. Differentiated neurons were positive for class III beta-tubulin and for tyrosine hydroxylase and showed the typical pacemaker-activity of dopaminergic neurons (Supplementary Fig. 7)^51,52,53. Both, NCS-1 and Cav2.3 proteins were readily detectable in these neurons by western blotting (Fig. 5a). Specificity of antibodies was confirmed using the respective knockout mice (Supplementary Fig. 6a and refs. ^54,55,56). For Cav2.3, no significant difference in protein expression was detected between human dopaminergic neurons from healthy controls and Parkinson’s disease patients (Fig. 5b and Supplementary Table 9B). In contrast, NCS-1 protein levels were about 40% lower in the diseased neurons (Fig. 5b and Supplementary Table 9B).

NCS-1 thus emerges as protective factor during SN dopaminergic degeneration, of likely relevance to Parkinson’s disease.

Discussion

Here, we identify Cav2.3 as mediator of SN dopaminergic neuron loss in an in vivo model of Parkinson’s disease. Together with an identified protective role of NCS-1 for these neurons, our data highlight de-regulated Ca²⁺ signaling as a culprit in the disease. Cav2.3 and NCS-1 thus emerge as potential targets for neuroprotective therapy.

Voltage-gated Ca²⁺ entry is a key determinant of neuronal function. Ca²⁺ entry during pacemaking in vulnerable but not in resistant dopaminergic neurons is coupled to Ca²⁺ uptake by mitochondria, and to ATP production, particularly under increased metabolic demand^21,36,57. Such homeostasis sustains neuronal activity, dopamine release and thus movement, but it comes at a metabolic cost. Thus, SN dopaminergic neurons, with their activity-related Ca²⁺ oscillations, and their large, arborized axonal structures, are energetically living on the edge^36,58,59. Parkinson’s disease stressors such as aging, mitochondrial complex-I dysfunction, or mutations in PARK genes likely disrupt this delicate balance and tip them over the edge^1,36,58. Such a scenario might explain the higher vulnerability of SN dopaminergic neurons to degeneration compared to those in the VTA in Parkinson’s disease^1,36. Thus, understanding the exact sources of activity-related Ca²⁺ load and the downstream effectors in dopaminergic neurons is key to understanding how their viability is maintained.

Our data show that Cav2.3 is the most abundantly expressed voltage-gated Ca²⁺ channel subtype in adult SN dopaminergic neurons. This channel has not yet been linked with metabolic stress in these neurons or with their degeneration in Parkinson’s disease. Our data using both, Cav2.3 knockout mice and the Cav2.3 inhibitor SNX-482, indicate that Cav2.3 contributes significantly to Ca²⁺ fluxes and Ca²⁺ currents upon pacemaking in the somata of SN dopaminergic neurons. SNX-482 inhibits voltage-gated A-type K⁺ channels with similar affinity as to Cav2.3⁴³, and these channels are functionally expressed in SN dopaminergic neurons^41,42. But A-type channel blockade is unlikely to contribute to observed inhibition of Ca²⁺ signals by SNX-482, because (i) experiments were performed in the presence of 4-amino-pyridine, which fully inhibits A-type K⁺ channels and (ii) we observed no inhibition of Ca²⁺ signals by SNX-482 in SN dopaminergic neurons from Cav2.3 knockout animals. Therefore, the congruent effects of knockout and pharmacological block of Cav2.3 leads us to conclude that the observed effects in the Cav2.3 knockout mice are rather caused by the loss of the functional channel than compensatory mechanisms.

Previous work showed that Ca²⁺ transients in SN dopaminergic neurons were inhibited by the L-type voltage-gated Ca²⁺ channel blocker isradipine and Cav1.3 shRNA by about 50%¹⁴. This is similar to the degree of inhibition we observed here upon interfering with Cav2.3. T-type Ca²⁺ channels also contribute to Ca²⁺ fluxes in SN dopaminergic neurons¹⁴ and to rotenone-induced cell death in iPSC-derived dopaminergic neurons^23,25. Notably, these previous Ca²⁺ recordings were made in dendrites^13,14 whereas our measurements were made in the soma, pointing strongly to subcellular differences in Ca²⁺ channel activity in SN dopaminergic neurons. Indeed, somatic Ca²⁺ transients are only modestly sensitive to isradipine³⁷. Of relevance, L-type Ca²⁺ channel blockers failed to protect neurons in the latter model of Parkinson’s²³, and they also failed in modifying disease progression in human Parkinson’s patients²⁰.

SNX-482-sensitive currents have recently been described in juvenile rat SN dopaminergic neurons²⁷ contributing to ~10% of voltage-gated Ca²⁺ currents. In adult mouse SN dopaminergic neurons, the relative contribution of Cav2.3 remains unclear. But our experiments showing an age-dependent upregulation of Cav2.3 expression (Fig. 1) coupled with the reported downregulation of L-type expression³⁷ and reduction of L-type currents in SN dopaminergic neurons⁶⁰ suggest a significant role for R-type currents. Currently, it remains also unclear to which extent Cav-mediated Ca²⁺ oscillations^13,14,21 in dopaminergic neurons are coupled and amplified by Ca²⁺-induced Ca²⁺ release from intracellular Ca²⁺ stores⁶¹. Cav2.3 and RyR3 are functionally coupled in pancreatic delta-cells⁶², raising the possibility that this may also occur in neurons.

Independent evidence that Ca²⁺ fluxes in SN dopaminergic neurons are de-regulated upon Cav2.3 knockout or pharmacological Cav2.3 inhibition stems from our observations that Ca²⁺-dependent AHPs are correspondingly reduced. These AHPs in SN dopaminergic neurons are sensitive to inhibition of small conductance K⁺ channels^40,63. Our data strongly suggest functional coupling with Cav2.3 channels. This is similar to coupling of Cav2.3 and big conductance K⁺ channels underlying AHPs in hippocampal pyramidal neurons⁶⁴.

Pacemaking in VTA dopaminergic neurons was associated with only modest Ca²⁺ signals in the somata (similar to that described for dendrites¹⁴) and small AHPs. Thus, loss or blockade of Cav2.3 shifts the properties of dopaminergic neurons in the SN that are vulnerable in Parkinson’s disease toward those in the VTA which are resistant^13,40,65.

We used the chronic MPTP/probenecid Parkinson’s disease model (injection interval 3.5 days) to avoid acute toxicity, described for other (acute/subacute) MPTP protocols. The toxicity mechanism of MPTP and MPP⁺ (the neurotoxic metabolite) has been extensively studied^66,67. MPP⁺ inhibits the complex I of the mitochondrial respiratory chain and thus mimics the complex I deficiency, described for mitochondria in human SN dopaminergic neurons from Parkinson’s patients^66,68.

Although our transgenic mouse model identifies a clear phenotype upon Cav2.3 loss, possible compensation is important to consider particularly for global knockouts. Indeed, we had described that loss of Cav1.3 upon knockout in mice is functionally compensated by upregulation of Cav3.1 in SN dopaminergic neurons²². Here, we found no such evidence for compensatory increases in Cav3.1, Cav1.2 or Cav1.3 mRNA levels in SN dopaminergic neurons of Cav2.3 knockout mice. Thus, the neuroprotection we observe in these animals is unlikely to be explained by changes in expression of other Ca²⁺ channels.

We did however detect an increase in levels of NCS-1 mRNA in SN dopaminergic neurons from Cav2.3 knockout mice. Notably, this increase in Cav2.3 knockout mice was manifest only in SN dopaminergic neurons from adult mice and thus age-dependent. In contrast, NCS-1 levels remained stable in wildtype mice, both age-dependent and region-selective. Ca²⁺-dependent regulation of NCS-1 expression has been described in yeast⁶⁹. We hypothesize that functional expression of protective NCS-1 in SN dopaminergic neurons might get upregulated in response to Parkinson’s disease stressors. This view is supported, as NCS-1 stimulates mitochondrial function and neuronal survival promotion in general^70,71, and as NCS-1 mRNA levels are elevated in remaining human SN dopaminergic neurons from post mortem Parkinson’s disease brains³⁰. In contrast, the lower NCS-1 levels detected in iPSC-derived dopaminergic neurons from GBA-1 Parkinson’s disease patients, offers an explanation for their accelerated degeneration.

Previous work suggested that NCS-1 function in SN dopaminergic neurons could have neuroprotective effects^30,31,32. Consistent with this, knockout of NCS-1 resulted in more severe dopaminergic neuronal loss in our Parkinson’s disease model. Importantly, this elevated loss was restricted to the SN, despite global NCS-1 knockout. Thus, elevated levels of NCS-1 in SN dopaminergic neurons of adult Cav2.3 knockout mice might contribute to the observed neuroprotection.

We also found that SN dopaminergic neurons from NCS-1 knockout mice displayed lower Cav2.3 mRNA levels than wildtype mice. We speculate that the Cav2.3 downregulation reflects a compensatory response to combat Cav2.3-mediated degeneration in NCS-1-depleted SN neurons. In this context, Cav2.3 mRNA levels are also lowered upon overexpression of mutant human α-synuclein (PARK1) in dorsal motor nucleus tissue of the vagus nerve⁷². Cholinergic neurons in this area display Ca²⁺-associated pacemaker activity, similar to SN dopaminergic neurons, and Lewy-body protein aggregates in Parkinson’s disease. But for unknown reasons, these neurons are less vulnerable to degeneration^3,72,73. Based on our findings, downregulation of Cav2.3 in response to Parkinson’s disease stressors offers a feasible explanation.

Collectively, our data strongly suggest opposing roles for Cav2.3 and NCS-1 in Parkinson’s disease. Cav2.3 is neurodegenerative whereas NCS-1 is protective for SN dopaminergic neurons. Whether this involves any direct functional or molecular interactions between the two proteins must be clarified in future experiments.

Importantly, NCS-1 expression is reduced in iPSC-derived dopaminergic neurons from familial Parkinson’s disease patients heterozygous for the N370S mutation in the GBA1 gene. Previous studies using iPSC-derived dopaminergic neurons from Parkinson’s patients with GBA1 mutations identified disrupted Ca²⁺ homeostasis and increased vulnerability to stress responses that were rescued by isogenic correction⁷⁴. Age-dependent dysregulation of Ca²⁺ homeostasis has also been described in patient fibroblasts carrying the N370S mutation in the GBA gene⁴⁸. Notably, NCS-1 levels are higher in the surviving dopaminergic neurons from post mortem idiopathic Parkinson’s disease patients³⁰. We suggest that the relative activity of Cav2.3 and NCS-1 within the complex Ca²⁺ signaling and PARK gene network in SN dopaminergic neurons may contribute to define their viability during stress.

Although epidemiological evidence links use of L-type Ca²⁺ channel blockers to reduced risk of Parkinson’s disease, isradipine afforded no protection in Parkinson’s patients in a recently concluded phase III clinical trial²⁰. Plasma levels reached during treatment with a maximal tolerable dose of isradipine might have been insufficient to fully inhibit L-type voltage-gated Ca²⁺ channels in SN dopaminergic neurons^21,37. Alternatively, inhibition of L-type voltage-gated Ca²⁺ channels might preferentially be protective only under distinct conditions, e.g. before motor symptoms manifest, or in response to transiently elevated dopamine levels³⁰ during dopamine replacement therapy^6,21. Inhibition of Cav2.3—alone or in combination with inhibition of L-type and/or T-type voltage-gated Ca²⁺ channels^14,23—could form the basis of a neuroprotective strategy for Parkinson’s disease in the future. However, the only available Cav2.3 inhibitor (SNX-482) is unsuitable for neuroprotection in a clinical setting due to off target effects^28,43,75. Thus, development of high affinity, brain-permeable, and selective Cav2.3 channel blockers is warranted^28,75.

Detailed information for all methodological approaches are given in the Supplementary Methods.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

All data generated or analyzed during this study are included in this published article and Supplementary Information file, or available from the authors upon request. Source data underlying Figs. 1–5 are available as a Source Data file.

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Acknowledgements

This work was supported by the German DFG (SFB 497, Graduate school CEMMA, and LI-1745/1 to B.L., Po144/1 to O.P., SFB 1218/TP B07 to P.K.), the Austrian Science Fund (FWF: P27809 to J.S., F4412 to B.L.), the Alfried Krupp foundation (B.L.), a Parkinson’s UK grant K-1802 (S.P.), and a Monument Trust Discovery Award J-1403 to D.B.K., R.W.-M.). We particularly thank the human donors from OPDC Discovery cohort. We thank Frank Kirchhoff for access to the Zeiss LSM 710 microscope. We thank Annette Dolphin, Antony Galione, Dennis Kätzel, Terry Snutch and Jim Surmeier for discussion/critically reading the manuscript. We thank Elsevier for the permission to align injection site reconstruction pictures in Figs. 1b, 3d and 4c with illustration 24 from the mouse brain atlas, published in “The Mouse Brain in Stereotaxic Coordinates“, Second Edition, ISBN: 0-12-547636-1/0-12-547637-X; Authors: George Paxinos & Keith B.J. Franklin, Copyright Elsevier, 2001.

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Institute of Applied Physiology, University of Ulm, Ulm, Germany
Julia Benkert, Shoumik Roy, Nicole Wiederspohn, Johanna Duda, Carsten Simons, Komal Patil, Aisylu Gaifullina, Nadja Mannal, Elena Dragicevic, Desirée Spaich, Sonja Müller, Julia Nemeth, Helene Hollmann, Nora Deuter, Christina Poetschke, Rosanna Parlato & Birgit Liss
Institute for Zoology, Biocenter, CECAD, University of Cologne, Cologne, Germany
Simon Hess & Peter Kloppenburg
Oxford Parkinson’s Disease Centre, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
Dayne Beccano-Kelly, Yassine Mousba & Richard Wade-Martins
Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Christian Kubisch
Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria
Joerg Striessnig
Institute of Physiology, CIPMM, University of the Saarland, Homburg, Germany
Olaf Pongs
Institute for Neurophysiology, University of Cologne, Cologne, Germany
Toni Schneider
Department of Cell and Developmental Biology, UCL, London, UK
Sandip Patel
Department of Neurology, University Medicine Göttingen, Göttingen, Germany
Tobias Frank
New College, University of Oxford, Oxford, UK
Birgit Liss

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Julia Benkert
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Contributions

J.B., J.D., C.S., D.S., N.D., N.W.: molecular biology experiments; C.P., E.D., N.M.: mouse breeding and help with initial electrophysiology; C.P., J.B., J.D.: retrograde tracing; P.K., S.H.: Ca²⁺ imaging; A.G.: whole-cell voltage-clamp experiments; D.B.K., R.W.-M., Y.M.: iPSC analysis; C.K.: human genetic analysis; T.F.: MPTP injections; D.S., H.H., J.B., J.D., J.N., K.P., R.P., S.M., S.R.: immunohistology and stereology; O.P. provided NCS-1 knockout mice; T.S. provided Cav2.3 knockout mice; B.L. designed the study; B.L., S.P., J.S. and J.B. wrote the manuscript, all authors revised the manuscript.

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Correspondence to Birgit Liss.

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A patent application has been filed by B.L. (PCT/EP2017/064644). The remaining authors declare no competing interests.

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Benkert, J., Hess, S., Roy, S. et al. Cav2.3 channels contribute to dopaminergic neuron loss in a model of Parkinson’s disease. Nat Commun 10, 5094 (2019). https://doi.org/10.1038/s41467-019-12834-x

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Received: 31 October 2017
Accepted: 27 September 2019
Published: 08 November 2019
DOI: https://doi.org/10.1038/s41467-019-12834-x

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