From the Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030
Received for publication, December 18, 2002, and in revised form, February 25, 2003
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ABSTRACT |
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Synaptic mechanisms of plasticity are
calcium-dependent processes that are affected by
dysfunction of mitochondrial calcium buffering. Recently, we observed
that mice deficient in mitochondrial voltage-dependent
anion channels, the outer component of the mitochondrial permeability
transition pore, have impairments in learning and hippocampal synaptic
plasticity, suggesting that the mitochondrial permeability transition
pore is involved in hippocampal synaptic plasticity. In this study, we
examined the effect on synaptic transmission and plasticity of blocking
the permeability transition pore with low doses of cyclosporin A and
found a deficit in synaptic plasticity and an increase in base-line
synaptic transmission. Calcium imaging of presynaptic terminals
revealed a transient increase in the resting calcium concentration
immediately upon incubation with cyclosporin A that correlated with the
changes in synaptic transmission and plasticity. The effect of
cyclosporin A on presynaptic calcium was abolished when
mitochondria were depolarized prior to cyclosporin A exposure, and the
effects of cyclosporin A and mitochondrial depolarization on
presynaptic resting calcium were similar, suggesting a mitochondrial
locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used an
in vitro assay of calcium handling by isolated brain
mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more
rapidly and subsequently triggered a more rapid mitochondrial
depolarization. Similarly, mitochondria lacking the
voltage-dependent anion channel 1 isoform depolarized more
readily than littermate controls. The data suggest a role for the
mitochondrial permeability transition pore and
voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity.
The mitochondrial permeability transition pore
(MPT)1 is a complex believed
to be composed of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, the adenine nucleotide transporter in the inner membrane, and cyclophilin-D in the
matrix and is found in mitochondria of all eukaryotic cells. Although the majority of research on the function of the MPT and its components has focused on apoptosis (1-3), the MPT has recently been shown to
play a role in learning and synaptic plasticity in mice (4) as well as
in other physiological cellular functions (5). In both pathological and
physiological functions, the induction of the MPT is mediated by
mitochondrial calcium influx above a certain threshold, and once
opened, the MPT conducts small substrates and ions out of the
mitochondrial matrix (6). It has therefore been proposed that following
physiologic calcium influx into the mitochondrial matrix, formation of
the MPT might be a physiological mechanism of mitochondrial calcium
release (5).
We are interested in determining the role of mitochondrial calcium
regulation in synaptic function. The most extensively characterized synapses in the mammalian central nervous system are the Schaffer collateral synapses between CA3 pyramidal axons and their postsynaptic targets on the dendrites of CA1 pyramidal neurons. As mitochondria are
typically found in CA3 presynaptic terminals as well as CA1 dendrites,
we hypothesized that CA3-CA1 hippocampal long term potentiation (LTP)
and paired pulse facilitation (PPF) would be sensitive to alterations
in mitochondrial calcium regulation by the MPT because both depend on
calcium-dependent processes within synaptic terminals.
In this study, we examined the effect of blocking the MPT on LTP and
PPF using low doses of cyclosporin A (CsA). In addition, we monitored
the changes in calcium dynamics caused by CsA by imaging presynaptic
terminals loaded with the fluorescent calcium indicator Fura-2. We
found that blocking the MPT caused a transient increase in the resting
calcium in presynaptic terminals, correlating with changes in synaptic
transmission and plasticity. The increase caused by CsA was abolished
by depolarizing mitochondria with tetraphenylphosphate
(TPP+) prior to CsA exposure indicating a common target,
i.e. mitochondria. In addition, the characteristics of the
effects of TPP+ and CsA on resting calcium were similar,
suggesting the possibility of a common mechanism of action.
To more precisely determine the effect of blocking the MPT on
mitochondrial calcium handling, we employed an in vitro
assay to study calcium handling in isolated brain mitochondria and
found that CsA induces earlier and more rapid mitochondrial
depolarization and subsequent calcium release, which may explain the
transient increase of calcium in presynaptic terminals. Similarly,
VDAC1-deficient mitochondria depolarized and subsequently released
their mitochondrial calcium stores more readily than wild-type
littermate controls, which may explain their similar
electrophysiological deficits (4). Based on these results we propose a
model to explain how the MPT serves to help prevent mitochondrial
calcium overload and subsequent depolarization. Overall, our studies
are consistent with an important role for synaptic mitochondria in
calcium regulation and specifically suggest a role for the MPT in
neuronal calcium regulation.
Hippocampal Slice Physiology--
Hippocampal slices (400 µm)
were prepared as previously described (7). Hippocampal slices were
bathed (1 ml/min) with artificial cerebrospinal fluid (125 mM NaCl, 2.5 mM KCl, 1.24 mM
NaH2PO4, 25 mM NaHCO3,
10 mM D-glucose, 2 mM
CaCl2, 1 mM MgCl2, and 10 µM cyclosporin A as indicated) in an interface chamber
maintained at 30 °C bubbled with 95% O2, 5%
CO2. The Schaffer collateral axons were stimulated with a
bipolar electrode, and the field excitatory postsynaptic potential
(fEPSP) was recorded in the stratum radiatum of area CA1. Responses
were monitored every 20-30 s for 20 min to ensure a stable base line.
Measurements are shown as the average slopes of the fEPSP from four to
six individual traces and are normalized to 10 min of base-line
recordings. Base-line stimulus intensities were adjusted to produce an
fEPSP at 50% of the maximal response. 100 Hz LTP was induced by
stimulating twice at high frequency (100 Hz) at base-line intensity for
1 s each, 20 s apart. Theta burst stimulation (TBS) consisted
of three trains of stimuli delivered at 20-s intervals, each train composed of 10 stimulus bursts delivered at 5 Hz, with each burst consisting of four pulses at 100 Hz. PPF was induced by two single stimuli at base-line intensity, 30 milliseconds apart. CsA (Alexis Pharmaceuticals) was dissolved in 99% ethanol (final ethanol
concentration, 0.1%) and added to artificial cerebrospinal
fluid while stirring for 10 min to increase solubilization. Thorough
rinsing of all plastic tubes and parts exposed to CsA with ethanol and
water prevented contamination of control slices.
Hippocampal Calcium Imaging--
The hippocampal slices were
prepared as described above, submersed in artificial cerebral spinal
fluid, and maintained with 95% O2, 5% CO2
bubbling at 30 °C. As described in detail in Wu and Saggau (8),
Fura-2 AM (Molecular Probes) dissolved in 80:20 Me2SO:pluronic acid was pressure-injected in the stratum
radiatum of area CA1 where the AM-ester dye is taken up by CA3 axons,
the AM-ester was cleaved, and the cell-impermeable Fura-2 was
transported to the presynaptic terminals. An area in stratum radiatum
of CA1 about 1 mm away from the injection site was illuminated at a
single excitation wavelength, 340 or 380 nm as indicated. Fluorescence was collected with a 50× objective, filtered by a long pass filter (490 nm), and converted into an electrical signal by a single photodiode. The resting presynaptic calcium was determined by ratiometric measurements of fluorescence excited at both 340 and 380 nm
in the absence of CA3 stimulation. For PPF measurements, two traces
recorded 1 min apart were averaged to improve the signal-to-noise ratio.
Western Blotting--
Hippocampal slices were prepared and
incubated in an interface chamber as described above. High doses of CsA
(250 µM), low doses of CsA (10 µM), or no
CsA in 0.5% Tween 20, 0.5% ethanol was added to the artificial
cerebrospinal fluid for 30 min. The slices were removed from the
interface chamber and homogenized in buffer containing 20 mM Tris, 1 mM EGTA, 1 mM EDTA, 1 mM Na4P2O7, protease
inhibitor mixture (Sigma), 1 mM p-nitrophenyl
phosphate, 0.25 mM Na3VO4, pH 7.5, at 4 °C. The homogenates were run on a 12% SDS-PAGE, transferred to
a nylon membrane (Millipore), and probed with phosphospecific
antibodies to phosphorylated synapsin I ( Hippocampal ATP Concentration--
Mouse hippocampal slices were
prepared and maintained as previously described for
electrophysiological experiments. The slices were exposed to 50 mM KCl for 10, 2, or 0 min before perchlorate extraction. A
luciferase-based assay from Molecular Probes was used to quantify the
ATP concentration and normalized to protein concentration, as
determined by BCA protein assay (Pierce).
Mitochondrial Isolation from Mouse Brain--
Mouse brain
mitochondria were isolated by Percoll centrifugation as outlined in
Ref. 9. One mouse forebrain was minced on ice and homogenized in a
small glass homogenizer with 3 ml of a 12% Percoll solution in
isolation buffer composed of 0.32 M sucrose, 1 mM K-EGTA, 10 mM Tris, pH 7.1. The Percoll
homogenate was carefully added on top of 3.5 ml of 26% Percoll, which
was layered over 3.5 ml of 40% Percoll in a tube and centrifuged at 30,700 × g for 5 min at 4 °C. The cloudy layer at
the interface of the lower two Percoll solutions was aspirated, washed
1:4 (v/v) with isolation buffer, and centrifuged at 16,700 × g for 10 min at 4 °C. The pellet was resuspended in 1%
bovine serum albumin (in isolation buffer) and spun at 8,000 × g for 12 min at 4 °C. The pellet was then washed in 900 µl of bovine serum albumin-free isolation buffer, centrifuged at
8,000 × g for 12 min at 4 °C and finally
resuspended in 750 µl of bovine serum albumin-free isolation buffer.
Isolated mitochondria were tested for respiration using an oxygraph
(biological oxygen monitor, YSI model 5300) and a Clark-type electrode
(oxygen probe, YSI model 5331) as described in Ref. 10. Only
mitochondria with a respiratory ratio of >2 on succinate were used for
the in vitro assays. In addition, only mitochondria that
possessed an intact outer membrane, as tested indirectly by the
cytochrome c assay (11), were used. Isolated mitochondria
stored on ice can be reliably used for experiments for up to 3 h.
In Vitro Assay for Mitochondrial Calcium Handling--
300 µl
of buffer (125 mM mannitol, 75 mM sucrose, 10 mM KCl, 10 mM Tris-HCl, 5 mM
KH2PO4, 5 mM glutamic acid, 5 mM pyruvic acid, 5 mM succinic acid, 5 mM malic acid; pH adjusted with KOH to 7.3; osmolarity = 300 mosmol) was prewarmed in a stirring measurement chamber
for 1 min at 32 °C. To the warming buffer, ADP was added to a final
concentration of 100 µM and CsA (dissolved in 99% EtOH), or an equivalent volume of EtOH for control was added to a final concentration of 1 µM as indicated. Mitochondria were
added to a final protein concentration of 60-70 µg/ml (~50 µl of
mitochondrial preparation) and incubated for 2 min to warm up to
32 °C. Extramitochondrial calcium was monitored using a
calcium-selective electrode (World Precision Instruments) and recorded
every 20 s. A CaCl2 bolus was added to reach the
desired calcium concentration as indicated. CsA and other drugs were
added either 2 min before the calcium bolus or after calcium was
maximally buffered, defined as 40 s of zero net flux. Ruthenium
red, an inhibitor of the mitochondrial calcium uniporter, carbonyl
cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial proton
uncoupler, and TPP+ were purchased from Sigma.
In these experiments, we observed that there is a range of calcium
release rates that varies between mitochondrial preparations and
depends partly on the initial calcium bolus, the mitochondrial concentration, the background strain of mouse, and the degree of
mitochondrial depolarization. The traces we show represent the average
release rates for one set of experiments, where each set was run at
least three times with different mitochondrial preparations.
Approximately half of all CA3 presynaptic terminals contain at
least one mitochondrion (26), and mitochondria are also typically found
in dendrites of CA1 pyramidal neurons (Fig.
1). We investigated the role of these
mitochondria in synaptic transmission and plasticity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Ser603 and
Ser62/67 phosphospecific antibodies were generous gifts
from the lab of Paul Greengard;
Ser553 was purchased
from Santa Cruz Biotechnology) and total synapsin (
-synapsin was
purchased from Cell Signaling Technology). The blots were developed
using the ECL system (Amersham Biosciences). Each experiment was
repeated at least three times.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Electron micrograph of hippocampus area
CA1. In this electron micrograph of a rat hippocampus area CA1
(previously published (26); copyright 1999 by the Society for
Neuroscience), mitochondria can be seen packed into presynaptic
terminals (gray arrows) and dendrites (black
arrows).
CsA-induced Changes in Synaptic Transmission and
Plasticity--
As previously reported in Weeber et al.
(4), we found that 10 µM CsA significantly attenuates
hippocampal 100 Hz LTP at Schaffer collateral synapses in area CA1 of
the mouse hippocampus (Fig.
2A). We sought to investigate
the basis of this effect by assessing acute changes caused by CsA.
Immediately upon the addition of CsA, synaptic transmission began to
increase and reach a plateau at ~25% above base line (Fig.
2B, also evident in the base lines of panels A
and C). Interestingly, although CsA attenuated LTP induced
by a 100 Hz stimulating protocol (Fig. 2A), it did not appear to attenuate LTP induced by TBS (Fig. 2C), even when
the increase in base-line synaptic transmission is subtracted from the
theta burst-induced potentiation (139% potentiation in controls versus 128% in CsA-exposed slices, p = 0.11). 100 Hz stimulation (Fig. 2A) and TBS (Fig.
2C) are believed to differ in their mechanism of LTP
induction because of differences in the characteristics of calcium
influx into synaptic terminals (12), which may affect different
biochemical pathways. For example, theta burst LTP involves the MAPK
pathway in the mouse hippocampus, whereas 100 Hz LTP does not (27).
Thus our studies suggest that the effect of CsA on synaptic plasticity
affects biochemical pathways that are important for 100 Hz LTP but not
theta burst LTP; however, it is important to note that the CsA-elicited
increase in base-line synaptic transmission complicates interpretation
of our TBS-induced LTP result.
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One question that arises from our CsA experiments is whether the locus of action is mitochondrial as opposed to other potential sites of action. TPP+ has been shown to attenuate post-tetanic potentiation at the crayfish neuromuscular junction by causing presynaptic mitochondrial depolarization (22). In the mouse hippocampal area CA1, exposure to TPP+ 20 min prior to a 100 Hz LTP simulation paradigm caused a marked reduction in LTP induction, not unlike CsA exposure (Fig. 2D). Although the mechanisms of action of TPP+ and CsA are different, the similarity of the effect of TPP+ and CsA on early LTP insinuates a common final effect on mitochondrial calcium handling. Unlike CsA, however, sustained mitochondrial depolarization by TPP+ eventually causes swelling and alterations in metabolic activity (see Fig. 5), resulting in a loss of synaptic activity (Fig. 2D). Overall, however, our data indicating that TPP+ application blocks LTP induction in a fashion reminiscent of CsA is consistent with our interpretation that CsA acts at a mitochondrial locus.
We also assessed the effects of MPT inhibition on shorter term forms of
synaptic plasticity, in particular PPF. We particularly followed PPF
because it is generally held to involve presynaptic calcium handling.
The effect of CsA exposure on PPF, becoming evident ~20 min after the
addition of CsA, was a reduction in the second fEPSP compared with the
first fEPSP such that PPF was attenuated (Fig.
3). These findings are consistent with
our previous observations of the effect of CsA on hippocampal synaptic
plasticity (4).
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In summary, our electrophysiologic studies indicate that CsA has both an immediate effect to increase synaptic transmission, followed by a delayed attenuation of two forms of synaptic plasticity, PPF, and 100 Hz LTP. We hypothesized that the effect of CsA in synaptic terminals is in part caused by inhibition of the MPT and subsequent alterations in mitochondrial calcium handling.
Low Doses of CsA Did Not Inhibit Calcineurin-- One specific concern with the use of CsA to block the MPT is the possibility of inhibition of the protein phosphatase, calcineurin. Typically, the concentrations of CsA used to block calcineurin are at least 10-25-fold greater than used in this study (13, 14). In addition, several considerations suggest that calcineurin is not inhibited in our studies. Hippocampal slices from mice lacking calcineurin exhibit enhanced PPF (15), and FK506, another calcineurin inhibitor, does not affect PPF (16). FK506 also reduces base-line synaptic transmission (16). None of these effects of calcineurin inhibition are observable in our experiments with low doses of CsA.
Nevertheless, to confirm that low doses (10 µM) of CsA do
not inhibit calcineurin in the hippocampal slice to the same extent as
high doses (250 µM) of CsA do, we used phosphospecific
antibodies to detect the phosphorylation state of a calcineurin
substrate, synapsin I. Hippocampal slices incubated under the same
conditions as for electrophysiology, in the presence or absence of high
or low doses of CsA, were harvested for Western blotting with
phosphospecific antibodies to synapsin I phosphorylated at
Ser603, Ser553, and Ser62/67, all
of which have been shown to be sensitive to calcineurin (17).
Incubation in 250 µM CsA resulted in dramatically
enhanced phosphorylation of all three synapsin I phosphorylation sites, which is likely due to inhibition of dephosphorylation by calcineurin (Fig. 4). Equally dramatic is the lack of
phosphorylation in slices exposed to 10 µM or no CsA
(Fig. 4). Taken together with our electrophysiological observations,
these findings indicate that the low doses of CsA used in this study do
not cause calcineurin inhibition and suggest that low doses of CsA are
acting via a different mechanism. Among the known targets of CsA,
mitochondrial cyclophilin-D has one of the lowest Ki
values in vitro (18) and has been shown to mediate the
functional inhibition of the MPT by CsA in isolated mitochondria (19).
In addition, our prior studies of hippocampal slices from
VDAC-deficient mice showed a similar electrophysiological phenotype to
CsA-exposed wild-type slices. Thus, it is likely that the effect of CsA
on hippocampal electrophysiology results from inhibition of the
MPT.
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Hippocampal ATP Was Reduced by TPP+ but Unaltered by
CsA--
Inhibition of the MPT by CsA may perturb the mitochondrial
proton gradient important in ATP production (20). To address the
possibility that CsA may alter hippocampal ATP concentrations, we
measured total ATP concentration in hippocampal slices in the presence
and absence of 10 µM CsA and found no significant change in ATP concentration caused by 10 µM CsA (Fig.
5). To induce synaptic calcium influx as
might occur during synaptic activity, hippocampal slices were exposed
to 50 mM KCl for 2-10 min and then harvested for ATP
quantification. Interestingly, ATP levels dropped to the same extent
after KCl depolarization in both CsA-exposed slices and controls and
stayed constant throughout KCl exposure (Fig. 5A). Thus, CsA
did not affect ATP concentration before or after KCl depolarization,
which suggests that changes in synaptic function caused by CsA are not
due to altered ATP availability.
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Sustained exposure to 100 µM TPP+ caused a significant decline in levels of ATP in the hippocampal slice after 40 min of incubation (Fig. 5B). The decline in ATP correlated with the degradation of electrophysiological signals in hippocampal slices continuously exposed to 100 µM TPP+ (Fig. 2D). In contrast, hippocampal slices exposed to 10 µM CsA did not show altered ATP levels and accordingly maintained electrophysiological integrity (Fig. 5B).
Effects of CsA on Presynaptic Calcium--
The effect of CsA on
synaptic function suggested a two-phase mechanism: an immediate
increase in base-line synaptic transmission, followed ~20 min later
by an attenuation in synaptic plasticity (Fig. 2). We hypothesized that
the changes in synaptic function might be due to secondary changes in
mitochondrial calcium handling. To determine the effect of CsA on
calcium handling, we monitored cytosolic calcium within presynaptic
terminals exposed to CsA in acute hippocampal slices using an
adaptation of our previously published protocol (8). Briefly, the
calcium indicator Fura-2 was applied to the axons of the Schaffer
collateral pathway and transported to presynaptic terminals in CA1.
This allowed us to measure relative changes in fluorescence caused by
calcium binding to Fura-2 (Fig.
6A).
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Using this approach, we were able to monitor the real time effects of CsA on presynaptic calcium. Immediately upon exposure to 10 µM CsA, resting presynaptic calcium was rapidly increased, followed by a more gradual return to base line by 30 min (Fig. 6B). Application of the vehicle, ethanol, had no effect (Fig. 6C). Thus, the increase in base-line synaptic transmission that we observed in Fig. 2B correlates with a transient increase in resting calcium over the same time period. Interestingly, the return of resting calcium to base-line levels occurs at about the same time that the PPF deficit began (Fig. 3).
To confirm the target of CsA on mitochondrial calcium stores, we performed the same experiment using TPP+ to depolarize mitochondria. By depolarizing mitochondria, we effectively remove the ability of mitochondria to take up calcium. As expected, exposure to 100 µM TPP+ immediately caused mitochondrial depolarization and consequent calcium release (Fig. 6D) caused by loss of the mitochondrial proton gradient. When CsA was added to the TPP+ bath 30 min later, there was no effect (Fig. 6D), suggesting that the target of CsA and TPP+ are the same, i.e. mitochondrial calcium stores. When the TPP+ was washed out for 10 min, mitochondria recovered, and CsA regained its ability to cause an increase in resting calcium (Fig. 6E).
CsA did not measurably affect stimulus-evoked calcium transients using the paired pulse protocol (Fig. 6F), suggesting that rather than participating in the dynamic handling of calcium during paired pulse stimulation, the MPT is more involved in regulating resting presynaptic calcium concentration. Thus, we can now correlate changes in resting presynaptic calcium handling with alterations in synaptic function in hippocampal slices exposed to CsA.
Surprisingly, the effect of TPP+ on resting presynaptic calcium remarkably resembled the effect of CsA (Fig. 6E), again suggesting a common mechanism. Because TPP+ is known to depolarize mitochondria as part of its mechanism of causing calcium release, we used an in vitro assay of calcium handling in isolated mitochondria to determine whether CsA might be affecting mitochondrial calcium handling as well.
Calcium Buffering in Isolated Mitochondria Is Altered by
CsA--
Intact and functional isolated brain mitochondria rapidly
buffered extramitochondrial calcium via the mitochondrial calcium uniporter, which could be blocked by ruthenium red (Fig.
7A). Calcium influx is driven
by a respiration-dependent electrochemical gradient across
the mitochondrial inner membrane. CCCP, which dissipates the
mitochondrial electrochemical gradient, also blocked calcium influx
and, in addition, induced mitochondrial depolarization and subsequent
calcium release when added at the point of maximal buffering (Fig.
7A). Overwhelmed by the amount of calcium used in this
in vitro assay, control mitochondria eventually depolarized and subsequently released their stores of calcium. These observations are consistent with previously published studies on mitochondrial calcium handling (21) and indicate that our isolated mitochondrial preparation contains intact and functional mitochondria capable of
sequestering calcium.
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Exposure of mitochondria to CsA prior to a 5-10 µM calcium bolus resulted in an increased rate of calcium influx (Fig. 7B) and an earlier and more rapid mitochondrial depolarization and subsequent calcium release (Fig. 7C). CsA exposure to at least three different mitochondrial preparations consistently resulted in an increased influx rate and more rapid calcium release (1.6-2.0-fold faster influx and release in each preparation) and consequently greater extramitochondrial calcium. Thus, the transient increase in cytosolic calcium observed in presynaptic terminals exposed to CsA (Fig. 6B) may have been due to triggering of mitochondrial depolarization and subsequent calcium release.
Mitochondrial depolarization by CsA is not a direct effect, as with TPP+ and CCCP, because CsA added after maximal calcium buffering did not induce mitochondrial depolarization and subsequent calcium release (Fig. 7D). For CsA to depolarize mitochondria, it had to be present before calcium influx. One interesting possibility is that the increased rate of calcium influx caused by CsA triggered an earlier and more rapid mitochondrial depolarization and subsequent calcium release.
Role of VDACs in Calcium Buffering in Isolated
Mitochondria--
We interpreted the effect of CsA on mitochondrial
calcium handling as being due to inhibition of the MPT. To confirm this interpretation, we evaluated the effects of genetic deletion of an
outer pore component of the MPT, VDAC1, using the in vitro method above. Similar to CsA-exposed wild-type mitochondria,
VDAC1-deficient mitochondria depolarized and released calcium more
rapidly compared with littermate controls (Fig. 7E), which
ultimately resulted in greater extramitochondrial calcium. This common
feature of mitochondrial depolarization and increased
extramitochondrial calcium may explain the electrophysiological
similarity of CsA exposed wild-type hippocampal slices and
VDAC-deficient hippocampal slices in synaptic plasticity (4).
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DISCUSSION |
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Our data along with other recent reports suggest that mitochondrial calcium buffering is an important regulator of synaptic function (22, 23). Extracellular field recordings in hippocampal slices exposed to low doses of CsA, which functionally inhibits the mitochondrial permeability transition pore, showed significant deficits in PPF and 100 Hz LTP and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals loaded with Fura-2 demonstrated that CsA induces an increase in resting calcium that may account for the acute change in synaptic transmission. Although the changes in presynaptic calcium and base-line synaptic transmission were acute effects of CsA, the deficits in short term synaptic plasticity began as the resting presynaptic calcium returned to base line, ~20 min after CsA exposure. This delayed outcome of the synaptic plasticity deficit might be due to a secondary effect of the transient calcium increase or to prolonged mitochondrial depolarization. The former possibility is supported by previous studies showing that elevated presynaptic calcium can activate calcium-calmodulin kinases that phosphorylate the vesicle-associated protein synapsins; synapsin phosphorylation may result in impaired presynaptic plasticity (17). However, as indicated in Fig. 4, 10 µM CsA exposure does not result in phosphorylation of synapsin I at any of the sites tested, suggesting that synapsin phosphorylation is not likely mediating the changes in synaptic plasticity caused by CsA. Nevertheless, there are numerous other calcium-sensitive pathways within synaptic terminals that may impinge on mechanisms important in synaptic plasticity. Alternatively, the transient increase in calcium may represent a more generalized mitochondrial malfunction resulting from prolonged depolarization. In support of this idea are recent studies showing that respiratory chain uncouplers affecting mitochondrial calcium handling disrupt synaptic plasticity in the calyx of Held (23) and the crayfish neuromuscular junction (22).
The location of mitochondria in presynaptic terminals does not lend itself well to buffering calcium influx from the extracellular space during single synaptic depolarizations (24) because this type of calcium influx may cause calcium elevations restricted to a smaller submembranous space. Rather, mitochondria appear to be more important in regulating the overall resting calcium level. To understand the role of the MPT and VDACs in regulating the resting calcium level, we employed an in vitro assay of mitochondrial calcium buffering. Studies of calcium buffering in isolated mitochondria are useful for understanding the roles of channels, exchangers, and pores in calcium influx and/or efflux, but their behavior in vivo might differ from their function in vitro. For example, although a calcium bolus of low micromolar concentration is necessary to observe the effect of CsA in isolated mitochondria, resting calcium levels in presynaptic terminals do not likely reach these levels. In addition, isolated mitochondria include those from both presynaptic and postsynaptic compartments as well as glia. For these reasons, this assay only serves as a model for the role of the MPT in calcium regulation, which might help to explain the function of mitochondrial buffering in the intact presynaptic terminal in synaptic transmission and plasticity.
It appears that in vitro mitochondrial calcium handling can be functionally divided into two stages, the uptake phase and the release phase. The uptake phase is dominated by calcium uptake by the uniporter. Blocking the MPT with CsA resulted in an increased net uptake during this phase. There are two workable hypotheses to explain this effect of CsA. First, if CsA hyperpolarizes the mitochondrial membrane, the drive for calcium influx would be greater and would thus cause increased calcium influx. Alternatively, one could suppose that the MPT conducts calcium release and, if blocked by CsA, would lead to a greater net influx. Either mechanism is plausible to explain the results of this assay but more importantly, the upshot is the same, that is, excessive calcium influx caused by blockage of the MPT results in calcium overload and subsequent mitochondrial depolarization. The result of mitochondrial depolarization is rapid calcium release from isolated mitochondria, as observed with CCCP. Consistent with this mechanism, adding CsA at the point of maximal calcium buffering did not have any effect (Fig. 5), indicating that the effect of CsA is dependent on calcium influx specifically.
It is not yet known how calcium exits the mitochondria during the release phase, but based on our studies, it is not likely to be via the MPT or the Na+/Ca2+ exchanger (data not shown) because inhibitors of these pathways did not affect calcium release during this phase. The trigger for calcium release appears to be mitochondrial depolarization by cation overload. Although there is a correlation between increasing the rate of calcium uptake by adding CsA and inducing earlier calcium release, mitochondria can buffer anywhere from 5 to 75 µM total calcium before releasing it. Thus, it is not the total calcium uptake, but the rate of calcium uptake, that appears to play a role in triggering calcium release in our in vitro preparation.
The increase in resting calcium stimulated by CsA strongly resembles the increase stimulated by TPP+, suggesting at least a common locus of action and possibility a common mechanism of action in the presynaptic terminal. TPP+, a lipophilic cation, works by carrying its positive charge across mitochondrial membranes into the negatively charged matrix and depolarizing it by virtue of overloading the matrix with positive charges. CsA is believed to work by binding to cyclophilin-D, the matrix component of the MPT, and functionally blocking the pore. The in vitro assay results suggest that blockage of the MPT leads to calcium (or other cation) overload and subsequent mitochondrial depolarization, complete with rapid calcium release. Thus, we can now speculate why CsA and TPP+ have the same effect on resting presynaptic calcium. They both cause mitochondrial depolarization leading to calcium release; TPP+ does it by virtue of loading positive charges into the matrix, whereas CsA does it by overloading the matrix with calcium or other cations such as potassium, sodium, and/or protons. Mitochondria lacking VDAC1 were also more likely to depolarize and subsequently release their calcium stores. VDAC1-deficient mitochondria may be more vulnerable to calcium overload, resulting in more rapid calcium release as observed in in vitro assays of VDAC1-deficient isolated mitochondria.
Although previously known mitochondrial calcium-specific transporters and channels have only been observed on the mitochondrial inner membrane, the mechanism of calcium transport across the mitochondrial outer membrane is not known. VDACs are the only known channels that may be able to conduct calcium into the intermitochondrial space (25) and therefore may play an important function in calcium influx into the mitochondrial matrix. Although calcium flux into the mitochondrial matrix has hitherto been attributed solely to the calcium uniporter of the mitochondrial inner membrane, we think it is interesting that VDAC-1-deficient mitochondria were impaired in mitochondrial calcium uptake in this assay (Fig. 7F). This finding is consistent with the growing notion that the mitochondrial outer membrane is not simply a molecular sieve but rather a regulated barrier that is involved in calcium flux into the intermitochondrial space, the source of calcium for the uniporter. Thus, the role of VDACs in calcium flux may not only be as a component of the MPT but also as calcium regulators across the mitochondrial outer membrane.
Putting all the clues together, our study defines the roles of VDACs
and the MPT in synaptic function. Based on the in vitro assay results, the MPT and VDACs serve to regulate the release of
cations from mitochondria to prevent overload and subsequent depolarization. VDAC deficiency is detrimental for the proper function
of the MPT because the absence of VDAC1 resulted in an increased
likelihood of mitochondrial depolarization and subsequent calcium
release as well. The overall result of mitochondrial depolarization in
presynaptic terminals, induced by TPP+, CsA, or the absence
of VDACs, is dysregulation of resting calcium, which may impinge on
signal transduction pathways important in synaptic transmission and plasticity.
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FOOTNOTES |
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* This work was supported by National Research Service Award F31NS42488 (to M. L.) and National Institutes of Health Grants MH57014 and HD24064 (to J. D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M212878200
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ABBREVIATIONS |
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The abbreviations used are: MPT, mitochondrial permeability transition pore; VDAC, voltage-dependent anion channel; LTP, long term potentiation; PPF, paired pulse facilitation; CsA, cyclosporin A; TPP+, tetraphenylphosphate; fEPSP, field excitatory postsynaptic potential; TBS, theta burst stimulation; CCCP, carbonyl cyanide 3-chlorophenylhydrazone.
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