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.

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 Ca2+ 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.

Neuronal Ca2+ sensor proteins (NCS) transduce changes in Ca2+ homeostasis into altered signaling and neuronal function. NCS-1 activity has emerged as important for neuronal viability and pathophysiology. The progressive degeneration of dopaminergic (DA) neurons, particularly within the Substantia nigra (SN), is the hallmark of Parkinson’s disease (PD), causing its motor symptoms. The activity-related Ca2+ homeostasis of SN DA neurons, mitochondrial dysfunction, and metabolic stress promote neurodegeneration and PD. In contrast, NCS-1 in general has neuroprotective effects. The underlying mechanisms are unclear. We analyzed transcriptional changes in SN DA neurons upon NCS-1 loss by combining UV-laser microdissection and RT-qPCR-approaches to compare expression levels of a panel of PD and/or Ca2+-stress related genes from wildtype and NCS-1 KO mice. In NCS-1 KO, we detected significantly lower mRNA levels of mitochondrially coded ND1, a subunit of the respiratory chain, and of the neuron-specific enolase ENO2, a glycolytic enzyme. We also detected lower levels of the mitochondrial uncoupling proteins UCP4 and UCP5, the PARK7 gene product DJ-1, and the voltage-gated Ca2+ channel Cav2.3 in SN DA neurons from NCS-1 KO. Transcripts of other analyzed uncoupling proteins (UCPs), mitochondrial Ca2+ transporters, PARK genes, and ion channels were not altered. As Cav channels are linked to regulation of gene expression, metabolic stress and degeneration of SN DA neurons in PD, we analyzed Cav2.3 KO mice, to address if the transcriptional changes in NCS-1 KO were also present in Cav.2.3 KO, and thus probably correlated with lower Cav2.3 transcripts. However, in SN DA neurons from Cav2.3 KO mice, ND1 mRNA as well as genomic DNA levels were elevated, while ENO2, UCP4, UCP5, and DJ-1 transcript levels were not altered. In conclusion, our data indicate a possible novel function of NCS-1 in regulating gene transcription or stabilization of mRNAs in SN DA neurons. Although we do not provide functional data, our findings at the transcript level could point to impaired ATP production (lower ND1 and ENO2) and elevated metabolic stress (lower UCP4, UCP5, and DJ-1 levels) in SN DA neurons from NCS-1 KO mice. We speculate that NCS-1 is involved in stimulating ATP synthesis, while at the same time controlling mitochondrial metabolic stress, and in this way could protect SN DA neurons from degeneration.

In dopaminergic (DA) substantia nigra (SN) neurons Cav2.3 R-type Ca2+-currents contribute to somatodendritic Ca2+-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 Ca2+-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 Ca2+-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 Ca2+ signals contributing to the (patho)physiological role of Cav2.3 channels in PD.

No abstract

Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify numbers of neuronal subtypes in defined areas, and of fluorescence signals, derived from RNAscope probes or immunohistochemistry, in defined cellular compartments. As proof-of-principle, we utilized DLAP to analyse subtypes of dopaminergic midbrain neurons in mouse and human brain-sections. These neurons modulate complex behaviour like voluntary movement, and are differentially affected in Parkinson's and other diseases. DLAP allows the analysis of large cell numbers from different species, and facilitates the identification of small cellular subpopulations, based on differential mRNA- or protein-expression, and anatomical location. Using DLAP, we identified a small subpopulation of dopaminergic midbrain neurons (~5%), mainly located in the very lateral Substantia nigra (SN), that was immunofluorescence-negative for the plasmalemmal dopamine transporter (DAT). These results have important implications, as DAT is crucial for dopamine-signalling, and its expression is commonly used as marker for dopaminergic SN neurons.

Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify numbers of neuronal subtypes in defined areas, and of fluorescence signals, derived from RNAscope probes or immunohistochemistry, in defined cellular compartments. As proof-of-principle, we utilized DLAP to analyse subtypes of dopaminergic midbrain neurons in mouse and human brain-sections. These neurons modulate complex behaviour like voluntary movement, and are differentially affected in Parkinson’s and other diseases. DLAP allows the analysis of large cell numbers from different species, and facilitates the identification of small cellular subpopulations, based on differential mRNA- or protein-expression, and anatomical location. Using DLAP, we identified a small subpopulation of dopaminergic midbrain neurons (~5%), mainly located in the very lateral Substantia nigra (SN), that was immunofluorescence-negative for the plasmalemma dopamine transporter (DAT). These results have important implications, as DAT is crucial for dopamine-signalling, and its expression is commonly used as marker for dopaminergic SN neurons.

Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify numbers of neuronal subtypes in defined areas, and of fluorescence signals, derived from RNAscope probes or immunohistochemistry, in defined cellular compartments. As proof-of-principle, we utilized DLAP to analyse subtypes of dopaminergic midbrain neurons in mouse and human brain-sections. These neurons modulate complex behaviour like voluntary movement, and are differentially affected in Parkinson’s and other diseases. DLAP allows the analysis of large cell numbers from different species, and facilitates the identification of small cellular subpopulations, based on differential mRNA- or protein-expression, and anatomical location. Using DLAP, we identified a small subpopulation of dopaminergic midbrain neurons (~5%), mainly located in the very lateral Substantia nigra (SN), that was immunofluorescence-negative for the plasmalemma dopamine transporter (DAT), with ~30% smaller cell-bodies, and a ~4-fold higher likelihood of calbindin-d28k co-expression. These results have important implications, as DAT is crucial for dopamine-signalling, and its expression is commonly used as marker for dopaminergic SN neurons.