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. 2018 Jun 1;128(6):2266-2280.
doi: 10.1172/JCI95898. Epub 2018 Apr 30.

Systemic Isradipine Treatment Diminishes Calcium-Dependent Mitochondrial Oxidant Stress

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Free PMC article

Systemic Isradipine Treatment Diminishes Calcium-Dependent Mitochondrial Oxidant Stress

Jaime N Guzman et al. J Clin Invest. .
Free PMC article

Abstract

The ability of the Cav1 channel inhibitor isradipine to slow the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons and the progression of Parkinson's disease (PD) is being tested in a phase 3 human clinical trial. But it is unclear whether and how chronic isradipine treatment will benefit SNc DA neurons in vivo. To pursue this question, isradipine was given systemically to mice at doses that achieved low nanomolar concentrations in plasma, near those achieved in patients. This treatment diminished cytosolic Ca2+ oscillations in SNc DA neurons without altering autonomous spiking or expression of Ca2+ channels, an effect mimicked by selectively knocking down expression of Cav1.3 channel subunits. Treatment also lowered mitochondrial oxidant stress, reduced a high basal rate of mitophagy, and normalized mitochondrial mass - demonstrating that Cav1 channels drive mitochondrial oxidant stress and turnover in vivo. Thus, chronic isradipine treatment remodeled SNc DA neurons in a way that should not only diminish their vulnerability to mitochondrial challenges, but to autophagic stress as well.

Keywords: Neuroscience; Parkinson’s disease.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Combined patch clamp and Fura-2 Ca2+ imaging from SNc DA neurons revealed large oscillations in cytosolic [Ca2+] during pacemaking.
(A) Schematic coronal section of the midbrain, positioned 3.5 mm posterior to bregma. At the bottom, magnified ventral part of the midbrain showing sampled region of SNc (highlighted in red). (B) Whole-cell recording from a SNc DA neuron shown at left as a representative reconstruction of a Fura-2 –filled cell. At the bottom, 2PLSM measurement of Fura-2 fluorescence at a proximal dendritic location is shown (~15 μm from the soma). (C) Somatic recording during imaging at a distal dendritic location (~100 μm from the soma from the projection image of a SNc DA neuron). Note the increase in Ca2+ transient at the distal imaging site. (D) Ca2+ transients increased along the dendrite of shown SNc DA neuron ranging from 15 to 200 μm from the soma.
Figure 2
Figure 2. Both Cav1 and Cav3 Ca2+ channels contributed to dendritic Ca2+ oscillations in SNc DA neurons.
(A) Projection image of a SNc DA neuron recorded from with a somatic patch electrode containing Fura-2 showing Ca2+ transient in distal dendrite. From left, control condition (black trace), with bath incubation of either 1 μM isradipine (green trace) or 1 μM TTA-P2 (blue trace). Note diminished dendritic Ca2+ transient with 1 μM isradipine. (B and C) Box plots summarizing the average and the peak [Ca2+], respectively, of the control, isradipine, and TTA-P2 treatment for both proximal and distal dendrites. Sample sizes for proximal dendrites were as follows: control group, n = 28 neurons from 23 mice; isradipine-treated group, n = 6 neurons from 4 mice; and TTA-P2–treated group, n = 9 neurons from 7 mice. In distal dendrites, sample sizes were as follows: control group, n = 31 neurons from 22 mice; isradipine-treated group, n = 7 neurons from 6 mice; and TTA-P2–treated group, n = 11 neurons from 7 mice. Data were analyzed using 1-tailed Mann-Whitney U test with Dunn’s correction for multiple comparisons. *P < 0.05.
Figure 3
Figure 3. Selective knockdown of Cav1.3 mRNA diminished both proximal and distal Ca2+ oscillations in SNc DA neurons.
(A) Cav1.3 shRNA–infected SNc DA neurons (red) and patched neurons filled with Fura-2 dye (yellow). Scale bar: 40 μm. (B) Cav1.3 knockdown decreased Cav1.3 mRNA, but didn’t change Cav1.2, Cav3.1, and Cav3.2 mRNA (n = 4 tissue samples from 3 mice for Cav1.2, Cav3.1, and Cav3.2; n = 5 tissue samples from 3 mice for Cav1.3). (C) Whole-cell recording from a SNc DA neuron with scrambled shRNA (black trace) and Cav1.3 shRNA (green trace). At the bottom, Ca2+ transients in distal dendrites were diminished with Cav1.3 shRNA but not scrambled shRNA. (D) Summary of peak [Ca2+] in proximal and distal dendrites of experiments as in C. (Proximal dendritic locations: scrambled shRNA, n = 5 neurons from 4 mice; Cav1.3 shRNA, n = 7 neurons from 5 mice; distal dendrites, scrambled shRNA, n = 8 neurons from 4 mice; Cav1.3 shRNA, n = 8 neurons from 6 mice). (E) Upper panel shows average spike trajectory; lower panel shows average Ca2+ transients in the distal dendrite. (F) Upper panel shows average Ca2+ transients with injected scrambled shRNA (black trace) or Cav1.3 shRNA (green trace) in distal dendrites; lower panel shows average distal dendritic Ca2+ transients in the presence of 1 μM isradipine (green trace) and control (black trace). (G) Box plots summarizing the ramp [Ca2+] in control (n = 8 neurons from 6 mice), Cav1.3 shRNA–injected (n = 5 neurons from 5 mice), and 1 μM isradipine-treated (n = 8 neurons from 4 mice) SNc DA neurons. Note that Cav1.3 shRNA and isradipine suppressed slow increase in [Ca2+] that preceded the spike. Data were analyzed using 1-tailed Mann-Whitney U test with Dunn’s correction for multiple comparisons. *P < 0.05.
Figure 4
Figure 4. Chronic administration of isradipine reduced dendritic Ca2+ oscillations without inducing compensations in channel expression.
(A) qPCR revealed no significant change in the mRNA expression of Cav1.3, Cav1.2, Cav3.1, and Cav3.2 Ca2+ channels after chronic isradipine treatment (n = 6 tissues from each group os 3 mice for Cav1.2 and Cav1.3; n = 7 tissues from each group of 3 mice for Cav3.1 and Cav3.2). (B) Perforated-patched recordings from vehicle-treated (black) and isradipine-treated (green) neurons. (C) Pacemaking rates in SNc DA neurons from control and isradipine-treated mice were unchanged (vehicle, 9 neurons from 4 mice; isradipine, 11 neurons from 4 mice). (D) Whole-cell somatic recording (left) and distal dendritic Ca2+ transients; chronic isradipine treatment (green trace, upper middle) reduced dendritic Ca2+ oscillations. Removal of the pumps (washout) led to restoration of the oscillation (lower right, black traces). (E) Peak and average proximal dendritic Ca2+ measurements under control (from Figure 2, B and C), isradipine-treated, and isradipine-washout conditions (control, 28 neurons from 23 mice; chronic isradipine, 5 neurons from 5 mice; washout, 5 neurons from 5 mice). (F) Summary of data from distal dendrites (control, 31 neurons from 22 mice; chronic isradipine, 8 neurons from 8 mice; acute 10 nM isradipine, 14 neurons from 6 mice; washout, 5 neurons from 5 mice). (G) Average Ca2+ transients in control (black trace) and isradipine-treated (green trace) distal dendrite of an SNc DA neuron. (H) Box plots summarizing the ramp [Ca2+] in control (n = 8 neurons from 6 mice, from Figure 3G), Cav1.3 shRNA–injected (n = 5 neurons from 5 mice, from historical Cav1.3 shRNA in Figure 3G), and chronic isradipine (n = 9 neurons from 8 mice) SNc DA neurons. Data were analyzed using 1-tailed Mann-Whitney U test with Dunn’s correction for multiple comparisons. *P < 0.05.
Figure 5
Figure 5. Chronic isradipine treatment decreased mitochondrial oxidant stress and mitochondrial turnover, resulting in increased mitochondrial density.
(A) Top: low-magnification image of a midbrain slice from transgenic TH-mito-roGFP mouse showing fluorescence in SNc and VTA. Bottom: higher-magnification image of a SNc DA neuron showing mitochondrial labeling, with a white dashed line at cell membrane. (B) Mito-roGFP measurements from a SNc DA neuron before (control, black trace) and after application of dithiothreitol (DTT, blue trace) and aldrithiol (red trace). (C) Left: Mito-roGFP measurements in SNc DA neuron after chronic systemic administration of isradipine showing diminished mitochondrial oxidation. Right: box plots summarizing median redox measurements in control (7 neurons from 4 mice) and isradipine-treated mice (8 neurons from 4 mice). Dashed line indicates published data (14). (D) Left: drawing showing the pH-dependent fluorescent properties of mito-Keima, which allowed rapid determination as to whether the protein was in the mitochondria (pH 8.0) or the lysosome (pH 4.5). Mito-Keima fluorescence signal from 488 nm laser excitation (neutral pH) is shown in green and the signal from 561 nm laser excitation (acidic) is shown in red. Note the difference in mito-Keima fluorescence between VTA and SNc DA neuron. (E) Left: schematic presentation of chronic isradipine treatment and mito-Keima experiments. Right: chronic isradipine treatment changed the ratio of mito-Keima in SNc DA neurons compared with control. (F) Box plots summarizing the rate of mitophagy in GPe (10 neurons from 4 mice), VTA DA neurons (15 neurons from 6 mice), and control (21 neurons from 8 mice) and isradipine-treated SNc DA neurons (40 neurons from 7 mice). Note that isradipine significantly decreased the rate of mitophagy in SNc DA neurons. Data were analyzed using 1-tailed Mann-Whitney U test with Dunn’s correction for multiple comparisons. *P < 0.05. Scale bars: 10 μm (A, top); 5 μm (A, bottom); 10 μm (D, E).
Figure 6
Figure 6. Mitochondrial mass in SNc DA neurons rises after chronic isradipine treatment.
(A) Mitochondria in VTA DA, LC, and SNc DA neurons shown with mito-roGFP fluorescence. (B) Note the increased mitochondrial mass in SNc DA neurons after chronic isradipine treatment. (C) Relative mitochondrial mass in cytosol of VTA DA (5 neurons from 4 mice) and of LC neurons (5 neurons from 4 mice) was significantly higher than that in SNc DA (8 neurons from 5 mice) neurons. Isradipine did not change mitochondrial mass in VTA DA neurons (5 neurons from 4 mice), but did significantly increase it in SNc DA neurons (6 neurons from 5 mice). Removal of isradipine for 4 weeks (washout) showed return of mitochondrial mass to low, control values in SNc DA neurons (8 neurons from 6 mice). Scale bars: 10 μm (A); 8 μm (B). Data were analyzed using 1-tailed Mann-Whitney U test with Dunn’s correction for multiple comparisons. *P < 0.05.

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