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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. Voltage-gated Ca 2+ channels are thought to contribute to neurodegeneration of dopaminergic neurons. Here the authors find that the R-type channel Cav2.3 in substantia nigra dopaminergic neurons may contribute to neurodegeneration in a model of Parkinson’s disease, in contrast to the neuroprotective action of the neuronal Ca 2+ sensor NCS-1.

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. Significance Patients with schizophrenia suffer from cognitive and negative deficits that are largely resistant to current therapeutic strategies. Here, using a genetic mouse model that displays phenotypes similar to these cognitive and negative symptoms, we found that increased postsynaptic D2 receptor (D2R) activity in the striatum leads to changes in the firing pattern of presynaptic dopamine (DA) neurons of the midbrain. These alterations occur in the ventral tegmental area (VTA) of the midbrain, but not in the substantia nigra, suggesting that DA pathways may be differently regulated by striatal D2R hyperactivity. The changes in neuron firing patterns were accompanied by a reduction in NMDA receptor subunits selectively in dopaminergic VTA neurons, providing a potential new target for the treatment of schizophrenia symptoms.

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.

Neuronal Ca sensor proteins (NCS) transduce changes in Ca 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 (SN), is the hallmark of Parkinson's disease (PD), causing its motor symptoms. The activity-related Ca 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 Ca -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 Ca channel Cav2.3 in SN DA neurons from NCS-1 KO. Transcripts of other analyzed uncoupling proteins (UCPs), mitochondrial Ca 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.

Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify cell numbers, and fluorescence signals within cellular compartments, derived from RNAscope or immunohistochemistry. We utilised DLAP to analyse subtypes of tyrosine hydroxylase (TH)-positive dopaminergic midbrain neurons in mouse and human brain-sections. These neurons modulate complex behaviour, and are differentially affected in Parkinson’s and other diseases. DLAP allows the analysis of large cell numbers, and facilitates the identification of small cellular subpopulations. Using DLAP, we identified a small subpopulation of TH-positive neurons (~5%), mainly located in the very lateral Substantia nigra (SN), that was immunofluorescence-negative for the plasmalemmal dopamine transporter (DAT), with ~40% smaller cell bodies. These neurons were negative for aldehyde dehydrogenase 1A1, with a lower co-expression rate for dopamine-D2-autoreceptors, but a ~7-fold higher likelihood of calbindin-d28k co-expression (~70%). These results have important implications, as DAT is crucial for dopamine signalling, and is commonly used as a marker for dopaminergic SN neurons. A deep learning-based image analysis platform quantifies cell numbers and mRNA or protein derived fluorescence signals, and is used to identify a unique population of dopamine transporter-negative dopaminergic neurons in the lateral Substantia nigra.

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) (SN) neurons Cav2.3 R-type Ca -currents contribute to somatodendritic Ca -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 -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 -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 signals contributing to the (patho)physiological role of Cav2.3 channels in PD.

Disturbances in Ca(2+) homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS), a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca(2+) homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs) from a common ALS mouse model, the endstage superoxide dismutase SOD1(G93A) transgenic mouse, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca(2+) uptake capacity and elevated Ca(2+) extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca(2+) transporters in individual, choline-acetyltransferase (ChAT) positive hMNs from wildtype (WT) and endstage SOD1(G93A) mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1(G93A) mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca(2+) transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca(2+) transporters MCU/MICU1, Letm1, and UCP2 in remaining hMNs from endstage SOD1(G93A) mice. These higher expression-levels of mitochondrial Ca(2+) transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na(+)/Ca(2+) exchanger NCX1 were also about 2-fold higher in hMNs from SOD1(G93A) mice. Thus, pharmacological stimulation of Ca(2+) transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS.

Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS), a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs) from a common ALS mouse model, endstage superoxide dismutase SOD1G93A transgenic mice, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT) positive hMNs from wildtype (WT) and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1 and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na2+/Ca2+exchanger NCX1 was also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a novel neuroprotective strategy for ALS.

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.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Figure 7 extended, new Figure 8 and 9. New/revised Supplemental Figure S5 and Table S14. Text modified accordingly.