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α-Synuclein-Dependent Calcium Entry Underlies Differential Sensitivity of Cultured SN and VTA Dopaminergic Neurons to a Parkinsonian Neurotoxin

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α-Synuclein-Dependent Calcium Entry Underlies Differential Sensitivity of Cultured SN and VTA Dopaminergic Neurons to a Parkinsonian Neurotoxin

Ori J Lieberman et al. eNeuro.

Abstract

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra (SN). Although mitochondrial dysfunction and dysregulated α-synuclein (aSyn) expression are postulated to play a role in PD pathogenesis, it is still debated why neurons of the SN are targeted while neighboring dopaminergic neurons of the ventral tegmental area (VTA) are spared. Using electrochemical and imaging approaches, we investigated metabolic changes in cultured primary mouse midbrain dopaminergic neurons exposed to a parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). We demonstrate that the higher level of neurotoxicity in SN than VTA neurons was due to SN neuron-specific toxin-induced increase in cytosolic dopamine (DA) and Ca2+, followed by an elevation of mitochondrial Ca2+, activation of nitric oxide synthase (NOS), and mitochondrial oxidation. The increase in cytosolic Ca2+ was not caused by MPP+-induced oxidative stress, but rather depended on the activity of both L-type calcium channels and aSyn expression, suggesting that these two established pathogenic factors in PD act in concert.

Keywords: MPP+; MPTP; Parkinson’s disease; calcium; dopamine; mitochondria; neurodegeneration; α-synuclein.

Figures

Figure 1.
Figure 1.
MPP+-induced toxicity in cultured SN and VTA DA neurons. A, Neurotoxicity following a 2-d exposure of primary cultures of mouse WT SN and VTA neurons to different concentrations of MPP+. DA neurons were tallied following fixation and immunostaining for TH; p < 0.001 by two-way ANOVA (n = 9-23 dishes in each group from 12 independent experiments). Basal electrophysiological characteristics of cultured mouse midbrain neurons are shown in Extended Data Figure 1-1. MPP+-mediated toxicity in neuronal cultures from different mouse strains is shown in Extended Data Figure 1-2. B, Time course of toxicity in cultures treated with 10 µM MPP+; p < 0.001 by two-way ANOVA (n = 3-6 dishes from two independent experiments). C, Representative images of midbrain cultures treated with 5 µM APP+ for 10 min and then fixed and stained for TH. All APP+-positive cells were TH-positive, whereas 95% of TH-positive neurons were APP+-positive (n = 17). Inset demonstrates punctate APP+ staining in neuronal somas and processes. Scale bar: 10 µm. D, Concentration dependence of APP+ uptake into SN and VTA neurons, as well as non-DA cell in the same cultures. Lineweaver-Burk plot of the data are shown in Extended Data Figure 1-3. E, Average nomifensine-sensitive (10 µM; 30 min) part of DA uptake into SN and VTA neurons pretreated for 30 min with MAO and VMAT inhibitors pargyline (10 µM) and reserpine (2 µM), correspondingly, and then treated with 10 µM DA for 1 h (p < 0.05 by t test). DAcyt concentrations were determined by IPE in the cyclic voltammetric mode of detection (Extended Data Fig. 1-4). F, Dependence of APP+ uptake (left axis) and survival of MPP+-treated SN neurons (right axis) on nomifensine concentration. For DAT activity assay, cells were preincubated with nomifensine for 30 min, and then exposed to 10 µM APP+ for another 10 min. Fluorescence intensity of the cell bodies was quantified, background subtracted and normalized to that in nomifensine-untreated SN neurons. The IC50 of the DAT inhibition is ∼250 nM nomifensine. Toxicity was measured following cell incubation with indicated concentrations of nomifensine and 10 µM MPP+. Zero toxicity rescue corresponds to the levels of MPP+ toxicity in the absence of nomifensine, and 100% to that in untreated SN neuronal cultures. The IC50 of the nomifensine-mediated rescue of SN neurons is ∼2 µM. Left and right diamonds represent DAT activity and toxicity in VTA neurons, respectively.
Figure 2.
Figure 2.
Difference in mechanisms of MPP+-induced toxicity in SN and VTA neurons. A, Schematics of possible MPP+ toxicity pathways. Abbreviations, concentrations of drugs, and preincubation times used for pharmacological analysis: AADC, aromatic l-amino acid decarboxylase; Arg, arginine; ADP, adenosine diphosphate; Bsrz, benserazide (10 µM, 48 h); Cav1.3, voltage-gated L-type calcium channel; DHBP, 1,1'-diheptyl-4,4'-bipyridinium dibromide (100 µM, 30 min); DOPAL, 3,4-dihydroxyphenylacetaldehyde; Isr, isradipine (5 µM, 30 min); l-DOPA, l-3,4-dihydroxyphenylalanine (100 µM, 30 min); LNM, NG-nitro-l-arginine methyl ester (l-NAME; 100 µM, 1 h); mPTP, mitochondrial permeability transition pore; Nmdp, nimodipine (5 µM, 30 min); Res, reserpine (10 µM, 24 h); ROS, reactive oxygen species; Ru360 (10 µM, 30 min); RuR, ruthenium red (20 µM, 24 h); Succ, succinate (Complex II substrate; 1 mM, 1 h); TH, tyrosine hydroxylase. Channels are shown in turquoise, enzymes and transporters in yellow and protein complexes in green. B, Pharmacological analysis of known mechanisms of MPP+ toxicity in VTA and SN DA neurons. Cultures were treated with various pharmacological agents as described above followed by 10 µM MPP+; surviving TH-positive neurons were tallied 48 h later. None of the compounds were neurotoxic when applied without MPP+; p < 0.05 from MPP+ (*) or both MPP+ and control (**) by one-way ANOVA with Tukey’s post hoc test (n = 6-16 for VTA and 7-17 for SN dishes from 12 independent experiments). C, Dependence of survival of SN and VTA DA neurons on the concentration of mitochondrial Complex I inhibitor pierecidin A. Following 2 d of exposure, neurons were fixed with paraformaldehyde, immunostained for TH and tallied; p < 0.001 by two-way ANOVA (n = 5-16 dishes in each group from six independent experiments). D, Representative images of TH-mito-roGFP SN neuronal cell bodies (left) and axons (right) before and after 2 h of treatment with 50 µM MPP+. Scale bar: 5 µm. E, Changes in mitochondria circularity in SN and VTA neurons treated with 50 µM MPP+ for 2 h; *p < 0.05 from untreated cells by one-way ANOVA with Tukey’s post hoc test (n = 14-22 cells from two independent experiments). F, Average roGFP 410/470 fluorescence ratios in the somas of neurons either before (live) or after treatment with 1 mM DTT (middle) followed by 2 mM H2O2 (right; n = 18-21 cells from three independent experiments). G, Relative oxidation of VTA and SN neurons before and after treatment with 50 µM MPP+ for 2 h; p < 0.05 from control VTA (*) or control SN (**) by one-way ANOVA with Tukey’s post hoc test (n = 29-106 cells from 14 independent experiments). Horizontal bars represent means and SDs. H, Time-dependent changes in mitochondrial oxidation in SN and VTA neurons treated with 50 µM MPP+; p < 0.001 by two-way ANOVA (n = 22-70 cells from 11 independent experiments). Neurotoxin was added at time 0.
Figure 3.
Figure 3.
Effect of MPP+ on DAcyt and NO. A, Time course of changes in DAcyt following treatment of SN or VTA neurons with 10 or 50 µM MPP+. All curves are statistically different from each other by two-way ANOVA (p < 0.01; n = 6-34 cells from eight independent experiments). Cells were pretreated with 100 µM l-DOPA for 30 min before the recordings. B, Average DAcyt concentrations (top) and representative voltammogram (bottom) in SN and VTA neurons treated with 50 µM MPP+ for 2 h. No l-DOPA was added before IPE measurements. Detection threshold of IPE in CV mode is ∼50 nM; *p < 0.01 by t test (n = 6 and 8 cells from two independent experiments). C, Representative images of control and MPP+ (50 µM for 2 h)-treated SN neurons from TH-mito-roGFP mice stained with live NO indicator DAR-4M-AM. Scale bar: 10 µm. D, Time-dependent changes in NO concentration in SN and VTA neurons treated with 50 µM MPP+. DAR-4M-AM was added during the last 10 min of MPP+ exposure; p < 0.001 by two-way ANOVA (n = 24-71 cells from seven independent experiments).
Figure 4.
Figure 4.
MPP+-induced changes in neuronal Ca2+. A, Representative images of SN neurons from TH-mito-roGFP mice exposed to 50 µM MPP+ for 2 h and stained with live Ca2+ indicator Rhod2-AM. Scale bar: 10 µm. No difference in intracellular fluorescence of Calcein Blue was observed between SN and VTA neurons (Extended Data Fig. 4-1). B, Time-dependent changes in Ca2+ signal in SN and VTA neurons treated with 50 µM MPP+. Rhod2-AM was always added during the last 30 min of MPP+ exposure; p < 0.001 by two-way ANOVA (n = 9-51 cells from four independent experiments). C, Effect of LTCC inhibitor isradipine (5 µM), Complex II substrate succinate (1 mM), NOS inhibitor l-NAME (100 µM), RyR blocker DHBP (100 µM), or IP3R antagonist 2-APB (50 µM) on MPP+-induced Ca2+ elevation in SN and VTA DA neurons. Cultures were preincubated with inhibitors for 30-60 min (Fig. 2, legend) and then exposed to 50 µM MPP+ for 2 h; p < 0.01 from corresponding control (*; white bars) or MPP+ (**) by one-way ANOVA with Tukey’s post hoc test (n = 11-100 cells from 14 independent experiments). D, Fluorescence intensity ratios at 340 and 380 nm excitation and 510 nm emission of fura-2 AM-treated SN and VTA neurons from TH-GFP mice exposed to 50 µM MPP+ for 2 h. LTCC inhibitor nimodipine (5 µM) was added 30 min before the toxin; *p < 0.05 from all other groups by one-way ANOVA with Tukey’s post hoc test (n = 37-68 cells from three independent experiments).
Figure 5.
Figure 5.
Changes in cytosolic and mitochondrial Ca2+. A, Representative live images of SN and VTA neurons from DAT-GCaMP3 mice exposed to 50 µM MPP+ for 2 h. Scale bar: 10 µm. B, Examples of live GCaMP3 recordings from MPP+-treated SN and VTA neurons. At 0, 30, and 120 min of toxin exposure, a series of 50 images at 2 Hz frequency was taken from the same cells. See Extended Data Figure 5-1A for an example of untreated cells and Extended Data Figure 5-1B for the analysis of the SD of each 30-s recording. Recordings were done at 37°C. C, Changes in GCaMP3 fluorescence intensity in the cell bodies of VTA and SN neurons exposed to MPP+ (50 µM, 2 h) in the presence or in the absence of 5 µM isradipine or 100 µM DHBP (see Extended Data Fig. 5-1C for the effect of isradipine in VTA DA neurons). The blockers were added either 30 min before MPP+ or during the last 30 min of toxin exposure; p < 0.01 from control (*) or MPP+ (**) by one-way ANOVA with Tukey’s post hoc test (n = 20-75 cells from eight independent experiments). Extended Data Figure 5-2 shows analysis of Rhod2 fluorescence in SN neurons treated with isradipine, DHBP, and MPP+ the same way. D, Schematics of the TH-2mt-GCaMP6m vector and representative images of untreated and MPP+-treated SN neurons infected with AAV9-TH-2mt-GCaMP6m. ITR, inverted terminal repeat; TH, rat tyrosine hydroxylase promoter. E, Ratio of background-subtracted fluorescence intensities (410 and 470 nm excitation, 535 nm emission) of the mitochondria in SN and VTA neurons expressing 2mt-GCaMP6m; *p < 0.01 from all other groups by one-way ANOVA with Tukey’s post hoc test (n = 18-29 cells from three independent experiments).
Figure 6.
Figure 6.
Dependence of DAcyt and NO on intracellular Ca2+ levels. A, Changes in DAcyt in SN and VTA neurons exposed to 50 µM MPP+ for 2 h in the presence and in the absence of nimodipine (5 µM) or DHBP (100 µM) added 30 min before MPP+; p < 0.01 from corresponding control (*) or MPP+-treated (**) neurons by one-way ANOVA with Tukey’s post hoc test (n = 9-28 cells from four independent experiments). Incubations with all drugs were done at 37°C, followed by imaging at RT. Dependence of DAcyt on nimodipine concentration in naïve SN cultures is shown of Extended Data Figure 6-1. B, DAcyt levels in SN and VTA neurons from WT (+/+) and Cav1.3 knock out (-/-) mice; p < 0.01 from corresponding +/+ (*) or both +/+ and -/- groups (**) by one-way ANOVA with Tukey’s post hoc test (n = 20-64 cells from seven independent experiments). C, Normalized fluorescence intensity of NO indicator DAR-4M in SN neurons pretreated with indicated inhibitors and then exposed to 50 µM MPP+ for 2 h; p < 0.001 from control (*) or MPP+ (**) groups by one-way ANOVA with Tukey’s post hoc test (n = 15-80 cells from 14 independent experiments). Quantification of DAR-4M fluorescence in VTA neurons treated with some of the same drugs is shown in Extended Data Figure 6-2. D, Fluorescence intensity in the somas of SN and VTA neurons treated with 10 or 50 µM APP+ for 10 min in the presence and the absence of 5 µM isradipine (30-min preincubation). N = 16-20 cells.
Figure 7.
Figure 7.
Rescue of DA neurons from MPP+-induced mitochondrial oxidation and toxicity. A, B, Relative oxidation of cultured SN neurons from TH-mito-roGFP mice in the absence (A) and in the presence (B) of 50 µM MPP+ for 2 h. Neurons were preincubated with metabolic effectors as indicated in the text and Figure 2A, legend; p < 0.001 from control (*) or MPP+ (**) by one-way ANOVA with Tukey’s post hoc test (n = 12-44 cells from 20 independent experiments). C, Rescue of mitochondria morphology in SN neurons treated with 50 µM MPP+ for 2 h in the presence of 5 µM isradipine (30-min preincubation); *p < 0.05 from untreated cells by one-way ANOVA with Tukey’s post hoc test (n = 14-23 cells from three independent experiments). D, MPP+-mediated toxicity in cultures pretreated with nimodipine (5 µM), ruthenium red (20 µM) or Ru360 (10 µM). None of the drugs were toxic to DA neurons in the absence of MPP+; *p < 0.001 from both control and MPP+ by one-way ANOVA with Tukey’s post hoc test (n = 3-23 dishes from seven independent experiments). E, Representative images of SN neurons immunostained for TH and ATF6. Scale bar: 10 µm. F, Quantification of the ratios of nuclear to cytosolic (perinuclear) ATF6 fluorescence in SN and VTA neurons treated with MPP+ for 24 h. Isradipine was added 30 min before MPP+; *p < 0.001 from control SN neurons by one-way ANOVA with Tukey’s post hoc test (n = 16-26 cells from two independent experiments). G, Toxicity in SN cultures treated with both isradipine and MPP+. Dotted line and gray bar represent the level of survival (mean ± SEM) of SN neurons treated with MPP+ only. In the presence of the LTCC blocker, the sensitivity of SN neurons to different metabolic effectors became similar to that of VTA neurons (compare to Fig. 2B). Note, however, that none of the inhibitors combinations provided complete rescue of SN neurons from neurotoxicity. “All” designates a group pretreated with isradipine, Bsrz, succinate, and l-NAME simultaneously; *p < 0.01 from both control and MPP+ by one-way ANOVA with Tukey’s post hoc test (n = 6-28 dishes from 13 independent experiments). H, Effect of the LTCC blockade on the survival of pierecidin A-treated SN and VTA neurons; p < 0.001 from control (*) or pierecidin A (**) by one-way ANOVA with Tukey’s post hoc test (n = 7-13 cells from four independent experiments).
Figure 8.
Figure 8.
Rescue of MPP+-treated SN neurons by aSyn deletion. A, Toxicity in SN cultures from WT and aSyn null mice treated with nimodipine and MPP+; p < 0.001 from corresponding control (*), both corresponding control and MPP+ (**) or WT and aSyn KO MPP+-treated groups (***) by one-way ANOVA with Tukey’s post hoc test (n = 6-16 dishes from three independent experiments). B, Effect of aSyn deletion on MPP+-mediated toxicity in VTA DA cultures; p < 0.01 from corresponding control (*) or WT and aSyn KO MPP+-treated groups (***) by one-way ANOVA with Tukey’s post hoc test (n = 6-23 dishes from two independent experiments). C, DAT-mediated uptake of APP+ is unchanged in aSyn-deficient SN neurons (n = 16-24 cells from two independent experiments). D, MPP+ does not induce Ca2+ surge in SN neurons from aSyn null mice. WT and sSyn KO SN neurons were treated with 50 µM MPP+ for 2 h and changes in Ca2+ were accessed with Rhod2 (n = 41-124 neurons from five independent experiments). E, Deletion of aSyn prevented the increase in mitochondrial Ca2+ in MPP+-treated SN neurons (n = 18-26 neurons from three independent experiments). F, Mitochondria morphology did not change significantly in toxin-exposed SN neurons from aSyn null mice (n = 7-15 neurons from three independent experiments).
Figure 9.
Figure 9.
aSyn overexpression exacerbates the effect of MPP+ on Ca2+ in SN but not VTA neurons. A, Representative images of WT, aSyn KO, and aSyn-overexpressing SN neurons immuostained against TH and aSyn with antibodies that react with both mouse and human protein. To overexpress aSyn, cultures were infected with AAV2 carrying human WT aSyn under the control of CBA promoter. Immunostaining was done 5 d after the infection. Insets show the same images at different brightness range to allow seeing the pattern of exogenous aSyn expression. Scale bar: 10 µm. Note that nuclear staining that is present in both WT and AAV-aSyn neurons is absent in aSyn KO neuorns. B, Quantification of endogenous cytosolic aSyn immunolabel in cultured SN and VTA DA neurons; *p < 0.0001 from WT SN and VTA neurons (n = 83-127 neurons from two independent experiments). C, Comparison of endogenous and exogenous aSyn levels in SN and VTA neurons treated with AAV2-haSyn; p < 0.01 from either WT (*) or both WT and SN AAV-aSyn groups (**) by one-way ANOVA with Tukey’s post hoc test (n = 38-104 cells from three independent experiments). D, E, Effect of human aSyn overexpression on cytosolic GCaMP3 fluorescence in SN (D) and VTA (E) DA neurons. One-week-old cultures of DAT-GCaMP3 neurons were infected with AAV2 carrying either mKate (red fluorescent tag) or human WT aSyn under the control of CBA promoter. GCaMP3 fluorescence in the presence and in the absence of MPP+ (50 µM MPP+ for 2 h) was monitored 5 d after the infection; p < 0.001 from corresponding control (*), corresponding MPP+ (**), or between indicated MPP+-treated groups (***) by one-way ANOVA with Tukey’s post hoc test (n = 20-75 cells for SN and 21-64 cells for VTA from four independent experiments). F, Possible mechanism of neurotoxic interactions between Ca2+, DAcyt, and NO in MPP+-treated SN neurons.

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