Christina Poetschke

Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca(2+) channels both contribute to Parkinson's disease pathology. L-type Ca(2+) channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca(2+) channel activity, internal Ca(2+), and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca(2+) channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca(2+) channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca(2+) channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.

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 Ca 2+ 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 Ca 2+ signals and Ca 2+ -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 Ca 2+ -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 Ca 2+ -dependent neurodegeneration in Parkinson’s disease.

There is strong evidence that the core deficits of schizophrenia result from dysfunction of the dopamine (DA) system, but details of this dysfunction remain unclear. We previously reported a model of transgenic mice that selectively and reversibly overexpress DA D2 receptors (D2Rs) in the striatum (D2R-OE mice). D2R-OE mice display deficits in cognition and motivation that are strikingly similar to the deficits in cognition and motivation observed in patients with schizophrenia. Here, we show that in vivo, both the firing rate (tonic activity) and burst firing (phasic activity) of identified midbrain DA neurons are impaired in the ventral tegmental area (VTA), but not in the substantia nigra (SN), of D2R-OE mice. Normalizing striatal D2R activity by switching off the transgene in adulthood recovered the reduction in tonic activity of VTA DA neurons, which is concordant with the rescue in motivation that we previously reported in our model. On the other hand, the reduction in burst activity was not rescued, which may be reflected in the observed persistence of cognitive deficits in D2R-OE mice. We have identified a potential molecular mechanism for the altered activity of DA VTA neurons in D2R-OE mice: a reduction in the expression of distinct NMDA receptor subunits selectively in identified mesolimbic DA VTA, but not nigrostriatal DA SN, neurons. These results suggest that functional deficits relevant for schizophrenia symptoms may involve differential regulation of selective DA pathways.

The preferential degeneration of Substantia nigra dopamine midbrain neurons (SN DA) causes the motor-symptoms of Parkinson's disease (PD). Voltage-gated L-type calcium channels (LTCCs), especially the Cav1.3-subtype, generate an activity-related oscillatory Ca(2+) burden in SN DA neurons, contributing to their degeneration and PD. While LTCC-blockers are already in clinical trials as PD-therapy, age-dependent functional roles of Cav1.3 LTCCs in SN DA neurons remain unclear. Thus, we analysed juvenile and adult Cav1.3-deficient mice with electrophysiological and molecular techniques. To unmask compensatory effects, we compared Cav1.3 KO mice with pharmacological LTCC-inhibition. LTCC-function was not necessary for SN DA pacemaker-activity at either age, but rather contributed to their pacemaker-precision. Moreover, juvenile Cav1.3 KO but not WT mice displayed adult wildtype-like, sensitised inhibitory dopamine-D2-autoreceptor (D2-AR) responses that depended upon both, interaction of the neuronal calcium sensor NCS-1 with D2-ARs, and on voltage-gated T-type calcium channel (TTCC) activity. This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA. Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults. This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.

In dopaminergic (DA) Substantia nigra (SN) neurons Cav2.3 R-type Ca 2+ -currents contribute to somatodendritic Ca 2+ -oscillations. This activity may contribute to the selective degeneration of these neurons in Parkinson’s disease (PD) since Cav2.3-knockout is neuroprotective in a PD mouse model. Here, we show that in tsA-201-cells the membrane-anchored β2-splice variants β2a and β2e are required to stabilize Cav2.3 gating properties allowing sustained Cav2.3 availability during simulated pacemaking and enhanced Ca 2+ -currents during bursts. We confirmed the expression of β2a- and β2e-subunit transcripts in the mouse SN and in identified SN DA neurons. Patch-clamp recordings of mouse DA midbrain neurons in culture and SN DA neurons in brain slices revealed SNX-482-sensitive R-type Ca 2+ -currents with voltage-dependent gating properties that suggest modulation by β2a- and/or β2e-subunits. Thus, β-subunit alternative splicing may prevent a fraction of Cav2.3 channels from inactivation in continuously active, highly vulnerable SN DA neurons, thereby also supporting Ca 2+ signals contributing to the (patho)physiological role of Cav2.3 channels in PD.