Conceived and designed the experiments: SC GCO DBE. Performed the experiments: SC. Analyzed the data: SC. Wrote the paper: SC GCO DBE.
The authors have declared that no competing interests exist.
The trafficking of mitochondria within neurons is a highly regulated process. In an earlier study, we found that serotonin (5-HT), acting through the 5-HT1A receptor subtype, promotes axonal transport of mitochondria in cultured hippocampal neurons by increasing Akt activity, and consequently decreasing glycogen synthase kinase (GSK3β) activity. This finding suggests a critical role for neuromodulators in the regulation of mitochondrial trafficking in neurons. In the present study, we investigate the effects of a second important neuromodulator, dopamine, on mitochondrial transport in hippocampal neurons.
Here, we show that dopamine, like 5-HT, regulates mitochondrial motility in cultured hippocampal neurons through the Akt-GSK3β signaling cascade. But, in contrast to the stimulatory effect of 5-HT, administration of exogenous dopamine or bromocriptine, a dopamine 2 receptor (D2R) agonist, caused an inhibition of mitochondrial movement. Moreover, pretreatment with bromocriptine blocked the stimulatory effect of 5-HT on mitochondrial movement. Conversely, in cells pretreated with 5-HT, no further increases in movement were observed after administration of haloperidol, a D2R antagonist. In contrast to the effect of the D2R agonist, addition of SKF38393, a dopamine 1 receptor (D1R) agonist, promoted mitochondrial transport, indicating that the inhibitory effect of dopamine was actually the net summation of opposing influences of the two receptor subtypes. The most pronounced effect of dopamine signals was on mitochondria that were already moving directionally. Western blot analysis revealed that treatment with either a D2R agonist or a D1R antagonist decreased Akt activity, and conversely, treatment with either a D2R antagonist or a D1R agonist increased Akt activity.
Our observations strongly suggest a role for both dopamine and 5-HT in regulating mitochondrial movement, and indicate that the integrated effects of these two neuromodulators may be important in determining the distribution of energy sources in neurons.
It has been shown that axonal transport of mitochondria ensures proper neuronal function
Previously, we identified serotonin (5-HT) as an extracellular signal that can promote axonal mitochondrial movement in cultured hippocampal neurons, suggesting a possible relationship between neuronal activity and mitochondrial movement
Dopamine is an important neurotransmitter that is involved in many aspects of neural function, including motor activity, emotion, reward, sleep, and learning
With the foregoing considerations in mind, we decided to investigate the effect of dopamine on mitochondrial trafficking. Based on the analysis of data from time-lapse imaging of cultured hippocampal neurons, we report here that dopamine has a net inhibitory effect on mitochondrial movement. Specifically, whereas activation of the D2 receptor inhibited the movement of mitochondria, activation of the D1 receptor promoted the movement of mitochondria. Consistent with their effects on mitochondrial motility, dopamine agonists and antagonists also showed opposing effects on the Akt-GSK3β signaling cascade, the same pathway that is activated by 5-HT in modulating mitochondrial motility in hippocampal neurons
As described in our earlier study of 5-HT and mitochondrial transport
D1 receptors are shown in red (A); D2 receptors are shown in green (B); MAP2 expression (soma and dendrites) is shown in blue (C); the merged image (D) indicates colocalization (yellow).
A. A typical pyramidal neuron that was used in mitochondrial motility studies shows expression of both D1 and D2 receptors (A, top panel). D1 receptors (D1R) are shown in red (bottom, left panel); D2 receptors (D2R) are shown in green (bottom, middle panel); MAP2 expression (soma and dendrites) is shown in blue (bottom, right panel). The merged image (A) indicates colocalization (yellow). B–D. Detail of expression of D1R (B) and D2R (C) in axons (enlargement of rectangular region indicated by red dashed border in A); merged image of D1R and D2R labeling is shown in D (labeling of axon has been removed for clarity). Axons in B and C are immunolabeled with phospho-neurofilament (pNF) antibody (purple).
Following a method described previously
A, B. Mean speeds (µm/min) of individual mitochondria before (A) and after (B) treatment with dopamine (30 nM). Pie chart insets show the percentage of stationary (red), oscillatory (blue), and directionally moving (green) mitochondria in all pooled experiments in the initial (a) and final (b) 15 minutes of imaging. C. Changes in mean speeds of all directionally moving mitochondria over time following treatment with dopamine. “(−)” indicates retrograde movement; “(+)” indicates anterograde movement. The red dotted lines projecting from the highlighted region of the schematic axon to the Y-axis of each graph in A and B indicate the approximate location (∼100–150 µm from the soma) and extent (∼50–80 µm) of the axon segment that was imaged.
Given the observed inhibitory effect of dopamine on mitochondrial motility, we were concerned that the mitochondria in our cultured neurons had been directly impaired by exposure to exogenous monoamine. In other studies of mitochondrial motility, inhibitory effects on mitochondrial movement have generally been consistent with mitochondrial impairment, indicated by a loss of membrane potential and drastic changes in mitochondrial morphology
Administration of the dopamine 2 receptor (D2R) agonist, bromocriptine (5 µM), in the absence of added dopamine markedly inhibited both directional and oscillatory movement of mitochondria (compare
A–B and D–E. Mean speeds (µm/min) of individual mitochondria before and after treatment with Bromocriptine (5 µM; A,B) or Haloperidol (10 µM; D,E). Pie chart insets show the percentage of stationary (red), oscillatory (blue), and directionally moving (green) mitochondria in all pooled experiments in the initial (a) and final (b) 15 minutes of imaging. C,F. Changes in mean speeds of all directionally moving mitochondria over time following treatment with D2R agonists and antagonists. “(−)” indicates retrograde movement; “(+)” indicates anterograde movement. The red dotted lines projecting from the highlighted region of the schematic axon to the Y-axis of each graph indicate the approximate location (∼100–150 µm from the soma) and extent (∼50–80 µm) of the axon segment that was imaged.
To further investigate the role of the D2 receptor in regulating mitochondrial movement, we examined the effects of a D2R antagonist on cultured hippocampal neurons. In contrast to the effect of bromocriptine, administration of the D2R antagonist, haloperidol (10 µM), in the absence of added dopamine significantly stimulated mitochondrial movement; the majority of this movement was anterograde (e.g., toward the axonal terminal) (
D1 receptors are the second major class of dopamine receptors found in hippocampal neurons (
A–B and D–E. Mean speeds (µm/min) of individual mitochondria before and after treatment with SKF38393 (33 µM; A,B) or SCH23390 (55 µM; D,E). Pie chart insets show the percentage of stationary (red), oscillatory (blue), and directionally moving (green) mitochondria in all pooled experiments in the initial (a) and final (b) 15 minutes of imaging. C,F. Changes in mean speeds of all directionally mitochondria over time following treatment with D1R agonists and antagonists. “(−)” indicates retrograde movement; “(+)” indicates anterograde movement. The red dotted lines projecting from the highlighted region of the schematic axon to the Y-axis of each graph in A and B and E and F indicate the approximate location (∼100–150 µm from the soma) and extent (∼50–80 µm) of the axon segment that was imaged.
In contrast, an analysis of five paired time-lapse imaging experiments (five separately prepared cultures, each imaged for 2 h before and 2 h after treatment with SCH23390; a total of 287 mitochondria tracked) showed that administration of the D1R antagonist, SCH23390 (55 µM), inhibited virtually all directional movement of mitochondria within one hour, and dramatically decreased oscillatory movement (
As shown in
Kymographs of mitochondrial motility shown in supplementary movies (
Previously, using inhibitors of Akt or GSK3β and phospho-specific antibodies in a Western blot analysis, we showed that the Akt-GSK3β signaling pathway is explicitly involved in the modulation of mitochondrial movement in response to stimulation by 5-HT
A. Western blot analysis shows that treatment with dopamine (30 nM) for 60 minutes leads to the inactivation of Akt (i.e., decreased serine-473 phosphorylation) and the activation of GSK3β (i.e., decreased serine-9 phosphorylation). B. Quantification of Western blots (n>3) showing the effect of dopamine alone on the phosphorylation of Akt (top) and GSK3β (bottom). C. Treatment with a D2R agonist (Bromocriptine, 5 µM) or a D1R antagonist (SCH23390, 55 µM) for 60 minutes leads to the inactivation of Akt and the activation of GSK3β, whereas treatment with a D2R antagonist (Haloperidol, 10 µM) or a D1R agonist (SKF23383, 33 µM) for 60 minutes leads to the activation of Akt and inactivation of GSK3β. D. Quantification of Western blots (n>3) showing the effect of dopamine signals on the phosphorylation of Akt (top) and GSK3β (bottom). Whole cell extracts from hippocampal neuronal cultures (DIV 17) were used in Western blots. In B and D, intensity values were normalized to total Akt or total GSK3β values.
Given the importance of the cAMP pathway in dopamine signaling
A. Inhibition of mitochondrial movement by IBMX. Mean speed (µm/min) of all directionally moving mitochondria from pooled experiments before and after treatment with IBMX (100 µM; n = 5, paired
We have shown previously that exogenous 5-HT promotes mitochondrial trafficking in cultured hippocampal neurons
Mean speeds of directionally moving mitochondrial populations are shown at two hour intervals in A and C. Akt activity levels in cultures after treatment with DA and 5-HT are shown in B and D. Mitochondrial movement was measured following serial administration of dopamine or its receptor agonists, antagonists, and 5-HT. In these experiments, a 6 hour period of observation was divided into three two-hour intervals, each allocated for a different treatment, i.e., control (CTL), dopamine (DA), or 5-HT. A. Prior to treatment with 5-HT, hippocampal neurons were pretreated with dopamine receptor-specific agonists or antagonists, including DA, SKF38393, SCH2390, haloperidol, and bromocriptine. B. Western blot analysis shows that dopamine counteracts the effect of 5-HT on Akt activity in the presence of 5-HT. C. Prior to treatment with dopamine signals, hippocampal neurons were pretreated with 5-HT. Each mean speed value in the graph represents three repeat experiments, i.e., separately prepared cultures (n = 3, paired
Dopamine is an important neurotransmitter in the regulation of many aspects of neural function. Changes in levels of dopamine, as well as in the dynamic activity of mitochondria, have been implicated in the onset of symptoms in both Parkinson's disease and schizophrenia
A number of studies have reported that dopamine is toxic to neurons
We found that dopamine has a net inhibitory effect on mitochondrial movement in hippocampal neurons (
From studies of the striatum, it is known that D1 and D2 receptors have opposing effects on adenylyl cyclase activity via coupling to the different heterotrimeric G protein subunits
In our studies, stimulation of each of the two receptors subtypes appeared to have opposite effects on the Akt-GSK3β signaling pathway (
Axonal movement of mitochondria and other cargoes requires an association between the organelle and specific motor proteins, which, in turn, mediate movement of cargoes on cytoskeletal elements in an ATP-dependent manner
It has been noted that 5-HT and dopamine, as well as noradrenaline, interact in a highly intricate manner
A number of drugs designed to treat psychiatric disorders target the serotonergic or dopamineric systems. For example, fluoxetine, a selective serotonin reuptake inhibitor, is often prescribed for depression, and haloperidol, a D2R antagonist, has been used effectively in the treatment of schizophrenia. Together, the present study and our previous report
Primary cultures of rat hippocampal neurons were prepared according to previously described methods with minor modifications
To allow visualization of mitochondria during live-cell imaging by fluorescence microscopy, cells were infected at 14 DIV with a Feline Immunodeficiency Virus (FIV)-based vector lentivirus, designated Flx1.8/CMVMitoEYFP, which encodes a MitoEYFP transgene (Clontech, Mountain View, CA). A second lentivirus was made, designated Flx1.8/CMVMitoTurboRFP, in order to label mitochondria with a red fluorescent protein (Axxora, San Diego, CA). Pseudotyped virus was generated by co-transfecting 293T cells with the transfer vector together with plasmids encoding the vesicular stomatitis virus G-glycoprotein (VSVG) and FIV gag-pol genes
To perform live-cell imaging experiments, we used a microscope stage-top incubator that we had previously designed and built to enclose a 35mm glass-bottom culture dish
Fluorescence microscopy was used to observe axonal transport of EYFP-labeled mitochondria in live hippocampal neurons. Time-lapse image series were acquired under high magnification (63× PLAN APO oil immersion objective; numerical aperture = 1.32; Leica, GmbH, Germany) using a Leica DMI-6000B inverted fluorescence microscope (Leica GmbH, Germany) equipped with a Sutter Lamda 10-2 emission filter wheel, Sutter DG-4 xenon light source (Sutter Instruments, Novato, CA), and a Cooke Sensiscam qe™ cooled CCD camera (Cooke Corporation, Romulus, MI). Microscope control, image capture, post-processing, and particle tracking of mitochondria were all accomplished using the Slidebook™ image acquisition and analysis software package (Intelligent Imaging Innovations, Inc., Denver, CO). In each imaging session, individual frames of mitochondria within an axon segment were acquired every 30 seconds (except where noted) for a total recording time of two hours. Cells were imaged for two hours before and two hours after administration of drugs. For masking purposes, each individual mitochondrion within a given axon segment of interest was distinguishable by its morphology (e.g., size, shape, position, and signal intensity) and behavior (e.g., pattern of movement), due in large measure to the intensity and stability of the EYFP signal.
Using Slidebook™, masks of individual mitochondria were generated, and the center of area (centroid) of each mask was determined. Masks for stationary and oscillatory mitochondria were generated by Slidebook™; masks of directionally moving mitochondria were generated manually. The ID tag of each individual mitochondrion was verified by carefully reviewing movie files frame-by-frame to ensure continuity over the course of the image series, and ambiguous labels were discarded. Manual masking was performed in a double-blind procedure. The individual executing this procedure did not know whether a given time-lapse image series was acquired before treatment or after treatment. Coordinates of the centroids of masked mitochondria were then obtained using the particle tracking module within Slidebook™. The parameters used for characterizing the movement of each mitochondrion (e.g., individual label assignment, displacement, speed) were calculated from recorded changes in their X-Y coordinates. The displacement plots shown in
EYFP-labeled hippocampal neurons cultured on glass-bottom culture dishes were fixed in 4% paraformaldehyde (pH 7.4) for 20 minutes. The cells were then permeabilized in PBT buffer (0.01% Tween-20, 0.02% BSA in PBS) for 40 minutes at room temperature. Following this, the cells were first incubated with mouse monoclonal antibody (primary antibody) for 1 hour. The cells were then incubated with a secondary fluorescein-labeled antibody for 1 hour. Finally, cells were covered in ProLong Gold® antifade reagent (Invitrogen, Carlsbad, CA) before a coverslip was applied and images were aquired. Primary antibodies used for staining were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Chemicon (Temecula, CA). Secondary antibodies were purchased from Invitrogen (Carlsbad, CA).
Western blotting was performed to determine levels of kinase activation. Cells were harvested in RIPA buffer (Tris-HCl: 50 mM, pH7.4, NP-40: 1%, Na-deoxycholate: 0.25%, NaCl: 150 mM, EDTA: 1 mM, PMSF: 1 mM, Aprotinin, leupeptin, pepstatin: 1 ug/ml each, Na3VO4: 1 mM, NaF: 1 mM). Samples were run on 10% SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA). Western blotting was performed using primary antibodies to phospho-ser9-GSK3β, total GSK3β, phospho-ser473-Akt, and total Akt (Cell Signaling Technology, Danvers, MA). The secondary antibodies used for Western blotting were horseradish peroxidase (HRP)-conjugated (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and blots were developed using ECL (GE Healthcare,Piscataway, NJ). For quantification, Western blots were scanned and intensity values were determined using Scion Image (Scion Corporation, Frederick, MD).
Dopamine has a sustained inhibitory effect on the activity of Akt. Western blot analysis shows that administration of dopamine (30 nM) reduced Akt activity in hippocampal neurons (as represented by the phosphorylation of serine-473 of Akt) over time.
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D1 and D2 receptors exert opposing effects on each other. Western blot analysis shows that D1 and D2 receptor activation has opposing effects on Akt activity (as represented by the phosphorylation of serine-473) in hippocampal neurons. In these experiments, hippocampal neurons were treated with the first drug for one hour. Following washout of the first drug (receptor-specific agonist or antagonist), the second drug was applied, and after one hour, total cell lysates were collected.
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Mitochondrial motility before treatment with Dopamine. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours before administration of dopamine.
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Mitochondrial motility after treatment with Dopamine. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours after administration of dopamine.
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Mitochondrial membrane potential after treatment with Dopamine. Time-lapse series showing stability of mitochondrial membrane potential in mitochondria labeled with the vital dye JC-1 after administration of dopamine.
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Mitochondrial membrane potential after treatment with FCCP. Time-lapse series showing changes in mitochondrial membrane potential in mitochondria labeled with the vital dye JC-1 after administration of FCCP.
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Mitochondrial motility before treatment with Bromocriptine, a D2R agonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours before administration of Bromocriptine, a D2R agonist.
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Mitochondrial motility after treatment with Bromocriptine, a D2R agonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours after administration of Bromocriptine, a D2R agonist.
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Mitochondrial motility before treatment with Haloperidol, a D2R antagonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours before administration of Haloperidol, a D2R antagonist.
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Mitochondrial motility after treatment with Haloperidol, a D2R antagonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours after administration of Haloperidol, a D2R antagonist.
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Mitochondrial motility before treatment with SKF38393, a D1R agonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours before administration of SKF38393, a D1R agonist.
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Mitochondrial motility after treatment with SKF38393, a D1R agonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours after administration of SKF38393, a D1R agonist.
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Mitochondrial motility before treatment with SCH23390, a D1R antagonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours before administration of SCH23390, a D1R antagonist.
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Mitochondrial motility after treatment with SCH23390, a D1R antagonist. Time-lapse series showing motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours after administration of SCH23390, a D1R antagonist.
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Mitochondrial motility before treatment with Bromocriptine. Time-lapse series showing basal motility of EYFP-labeled mitochondria in cultured hippocampal neurons for two hours before administration of Bromocriptine.
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Mitochondrial motility following washout - first hour. Time-lapse series showing mitochondrial motility in the first hour following the washout of bromocriptine and subsequent treatment with SKF38393, a D1 agonist.
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Mitochondrial motility after treatment with Bromocriptine. Time-lapse series showing mitochondrial motility following treatment with bromocriptine.
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Mitochondrial motility following washout - second hour. Time-lapse series showing mitochondrial motility in the second hour following washout of Bromocriptine and treatment with SKF38393.
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Mitochondrial motility before treatment with IBMX. Time-lapse series showing mitochondrial motility before treatment with IBMX.
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Mitochondrial motility after treatment with IBMX. Time-lapse series showing mitochondrial motility following treatment with IBMX.
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The authors are grateful to Drs. Gerald Edelman, Joseph Gally and Frederick Jones for their critical reading of the manuscript, as well as for helpful suggestions during the course of our research. We would like to thank Lara Pickle for invaluable assistance with tissue culture and Donald Hutson for his technical expertise in the construction and ongoing maintenance of the microscope stage-top incubator used in our live imaging experiments. We would also like to thank Dr. Adelheid Junger for her excellent drawing of a hippocampal neuron, which appears in