Abnormal Ca²⁺ Dynamics in Transgenic Mice with Neuron-Specific Mitochondrial DNA Defects

Abstract

Maintenance of mitochondrial DNA (mtDNA) depends on nuclear-encoded proteins such as mtDNA polymerase (POLG), whose mutations are involved in the diseases caused by mtDNA defects including mutation and deletion. The defects in mtDNA and in intracellular Ca²⁺ ([Ca²⁺]_i) homeostasis have been reported in bipolar disorder (BD). To understand the relevance of the mtDNA defects to BD, we studied transgenic (Tg) mice in which mutant POLG (mutPOLG) was expressed specifically in neurons. mtDNA defects were accumulated in the brains of mutPOLG Tg mice in an age-dependent manner and the mutant mice showed BD-like behavior. However, the molecular and cellular basis for the abnormalities has not been clarified. In this study, we investigated Ca²⁺ regulation by isolated mitochondria and [Ca²⁺]_i dynamics in the neurons of mutPOLG Tg mice. Mitochondria from the mutant mice sequestered Ca²⁺ more rapidly, whereas Ca²⁺ retention capacity and membrane potential, a driving force of Ca²⁺ uptake, of mitochondria were unaffected. To elucidate the molecular mechanism of the altered Ca²⁺ uptake, we performed DNA microarray analysis and found that the expression of cyclophilin D (CyP-D), a component of the permeability transition pore, was downregulated in the brains of mutPOLG Tg mice. Cyclosporin A, an inhibitor of CyP-D, mimicked the enhanced Ca²⁺ uptake in mutant mice. Furthermore, G-protein-coupled receptor-mediated [Ca²⁺]_i increase was attenuated in hippocampal neurons of the mutant mice. These findings suggest that mtDNA defects lead to enhancement of Ca²⁺ uptake rate via CyP-D downregulation and alter [Ca²⁺]_i dynamics, which may be involved in the pathogenesis of BD.

Introduction

Defects in mitochondrial DNA (mtDNA) have been implicated in the pathophysiology of various neuropsychiatric diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) (Schapira, 1999), and bipolar disorder (BD) (Kato and Kato, 2000), in addition of hereditary mitochondrial diseases (MDs). Among MDs, chronic progressive external ophthalmoplegia (CPEO) is known to closely relate to mtDNA defects produced by mutations of nuclear genes (Kaukonen et al., 2000; Spelbrink et al., 2001; Van Goethem et al., 2001). Because CPEO was recently redefined as a multisystem disease including CNS involvement (Suomalainen et al., 1992; Luoma et al., 2004), we suspected that mtDNA defects were relevant to the pathogenic mechanisms in mood disorders. For mtDNA defects, causal mutations in the following three genes have been identified to date: mtDNA polymerase (POLG), adenine nucleotide translocator 1 (ANT1/Slc25a4), and mtDNA helicase (PEO1/Twinkle). We developed transgenic (Tg) mice expressing neuron-specific mutant POLG (mutPOLG) and have shown that mtDNA defects age-dependently increased in the forebrains of mutPOLG Tg mice. These animals exhibited several BD-like behavioral phenotypes (Kasahara et al., 2006). This implicates that the accumulation of mtDNA defects leads to the BD-like behavior of mutPOLG Tg mice, whereas the underlying molecular and cellular mechanisms are not clarified.

Mitochondria participate in regulation of Ca²⁺ homeostasis by sequestering and releasing Ca²⁺ in cooperation with endoplasmic reticulum (ER) (Missiaen et al., 1991; Pozzan and Rizzuto, 2000). The mitochondrial Ca²⁺ uptake is involved principally in inositol 1,4,5-triphosphate (IP₃)-mediated Ca²⁺ mobilization from the internal stores, which localize to the ER (Malli et al., 2003; Rizzuto et al., 2004). Ca²⁺ overload to mitochondria provokes opening of the permeability transition pore (PTP), disturbing the intracellular Ca²⁺ ([Ca²⁺]_i) homeostasis by Ca²⁺ flux through the PTP (Ichas et al., 1997; Bianchi et al., 2004). Perturbation of [Ca²⁺]_i homeostasis by mitochondrial Ca²⁺ dysregulation may be involved in pathogenesis of neurodegenerative diseases (Ghosh et al., 1999). For example, isolated mitochondria from brains of mice model for HD were prone to opening of the PTP and showed a slow rate of Ca²⁺ uptake (Panov et al., 2002), implying that mitochondrial Ca²⁺ dysregulation was associated with the pathophysiology of HD. In BD, altered [Ca²⁺]_i increase, which is dependent on both G-protein-coupled receptor (GPCR) and IP₃, has been found in blood cells (Dubovsky et al., 1989; Kusumi et al., 1994; Okamoto et al., 1995; Kato et al., 2003) and in neuronal cells (Hahn et al., 2005) of patients. These observations brought us a hypothesis that [Ca²⁺]_i homeostasis would be disturbed in the neurons of the mutant mice and that accumulation of mtDNA defects might be responsible for the altered [Ca²⁺]_i dynamics. In this study, we characterized Ca²⁺ retention capacity (CRC) and membrane potential of isolated mitochondria from the brains of Tg mice and investigated GPCR-mediated Ca²⁺ dynamics in CA1 pyramidal neurons of mutPOLG Tg mice by combining a whole-cell recording with a fluorometric Ca²⁺ imaging in the hippocampal slice preparation.

Materials and Methods

Chemicals and drugs.

All fluorescent dyes were purchased from Invitrogen (Grand Island, NY): 10-nonyl acridine orange (NAO), 5,5′,6,6′-tetracloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1), 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide [DiIC₁(5)], Calcium Green-5N (CaG-5N), bis-fura-2, and fura-6F. Percoll solution was purchased from GE Healthcare Bio-Science (Piscataway, NJ). Cyclosporin A (CsA), carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone (FCCP), atropine sulfate, carbamylcholine chloride [carbachol (CCh)], low molecular weight heparin (molecular weight, 3000 Da), and cyclopiazonic acid (CPA) were purchased from Sigma (St. Louis, MO). Amino-phosphonopentanoic acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), (S)-3,5-dihydroxyphenylglycine (DHPG), and α-methyl-4-carboxyphenylglycine (MCPG) were obtained from Tocris (Ellisville, MO). Chelex 100 resin was obtained from Bio-Rad (Richmond, CA).

Animals.

In the mutPOLG Tg mice, mutant mtDNA polymerase lacking proofreading activity because of a D181A substitution was attached to the promoter of calmodulin kinase II α. The mutPOLG Tg mice were always used as heterozygotes. Male mutant mice were used for mating to avoid possible transmission of mtDNA mutations from the maternal side. The genotyping was performed as described previously (Kasahara et al., 2006). Controls were wild-type littermates whenever possible. All experimental procedures involving animal preparation were approved by the Wako Animal Experiment Committee, RIKEN.

Isolation of mitochondria.

Mitochondria of the mutPOLG Tg mice and the littermates (70–75 weeks old) were isolated using a discontinuous Percoll gradient developed by Sims (1990). Briefly, a mouse was decapitated and the brain was transferred to an ice-cold isolation buffer [(in mm): 320 sucrose, 1 EGTA, and 10 MOPS (4-morpholinepropanesulfonic acid), pH 7.4]. The forebrain tissue was dissected and homogenized in 12% Percoll with a Dounce homogenizer. The homogenate was layered on a discontinuous gradient of 26 and 40% Percoll in the isolation buffer and centrifuged in a Beckman Coulter (Fullerton, CA) JA 25.15 rotor at 30,700 × g for 5 min at 4°C, followed by two washing steps in the isolation buffer at 16,700 and 7300 × g, respectively. Bovine serum albumin (final, 0.1%) was added at the last washing step. After the final sedimentation, the mitochondrial pellet was suspended in 0.3 ml of a Chelex-treated Ca²⁺-free buffer containing the following (in mm): 210 sucrose, 20 KCl, 3 glycylglycine, and 1 KH₂PO₄, pH 7.2. The suspension was divided into aliquots for measurements of CRC that were immediately performed after the isolation. Mitochondrial protein concentration was determined by the Bradford protein assay (usually 0.2–0.3 mg/ml). There was no difference in the averaged mitochondrial yield between genotypes.

Mitochondrial Ca²⁺ retention capacity and Ca²⁺ removal.

The mitochondrial pellet was resuspended in 150 μl of Ca²⁺-free buffer, and the suspension was added to 450 μl of buffer containing 20 mm glutamate plus 2 mm malate, or 10 mm succinate: the total solution was 0.6 ml in volume. Extramitochondrial free Ca²⁺ concentration ([Ca²⁺]_exm) was monitored with 25 nm Calcium Green-5N [excitation (Ex), 488 nm; emission (Em), 530 nm] in a stirred cuvette at 30°C in a spectrofluorometer (F2500; Hitachi, Tokyo, Japan). The time resolution of the [Ca²⁺]_exm measurement was set at 0.5 s. To evaluate the CRC, 1.5 μl of Ca²⁺ solution was added at 1 min intervals; the stock solutions of 0.625, 1.250, and 3.125 mm CaCl₂ gave final concentrations of 1.6, 3.1, and 7.8 μm, respectively, which approximately corresponded to 25, 50, and 100 nmol of Ca²⁺ per milligram of mitochondrial protein. When mitochondria were incubated in the buffer containing succinate, 3.1 μm Ca²⁺ solution was repeatedly added after four additions of 1.6 μm. However, when mitochondria were incubated in the buffer containing glutamate plus malate, 7.8 μm Ca²⁺ solution was repeatedly added after four additions of 1.6 μm Ca²⁺ solutions. To obtain representative parameters of Ca²⁺ uptake by mitochondria, the recovery phase of [Ca²⁺]_exm transients was normalized by the maximal peak amplitude and best fitted with the biexponential function. The percentage of change in fluorescence was calculated by ΔF/F_max × 100%, where ΔF = F − F₀ and F_max = F_peak − F₀. F is the fluorescence intensity at any time point after the addition of Ca²⁺, and F₀ is baseline fluorescence obtained from the average of 20 data points before the addition of Ca²⁺. The recovery time was measured from the peak to 37% (decay time) of peak [Ca²⁺]_exm transients using KyPlot software (version 4.0; Kyens, Tokyo, Japan).

Mitochondrial membrane potential.

We estimated mitochondrial membrane potential (ΔΨ_m) using JC-1 or DiIC₁(5) probe dye, of which the fluorescent intensity was analyzed by a fluorescence-activated cell sorter (Epics Elite ESP; Beckman Coulter). Flow cytometric analysis is useful to analyze mitochondrial function (Mattiasson et al., 2003), because a signal from each mitochondrion can be detected. Heteroplasmic mutations in mtDNA biochemically diversify individual mitochondrion as a result of the levels of mutations. Therefore, the population comparison is appropriate for evaluating the difference between groups. Mitochondria isolated from the brains of mutPOLG Tg or wild-type mice were suspended in an analysis buffer composed of 210 mm sucrose, 20 mm KCl, 3 mm glycylglycine, 1 mm KH₂PO₄, and 0.5 mm MgCl₂, pH 7.2, containing additive components of 10 mm succinate plus 0.1 μg/ml rotenone or 20 mm glutamate plus 2 mm malate. The mitochondrial suspension (7.5–12.5 μg of mitochondrial protein in 200 μl) was stained by 5 μg/ml JC-1 or 10 nm DiIC₁(5) at 37°C for 10 min in darkness. No stained samples were incubated in the buffer without a probe. Some samples were combined with 6.25 μm Ca²⁺ or 1 μm FCCP, a proton ionophore, before incubation at 37°C. The mitochondrial particles were gated based on light scattering properties in the side-scattering (SSC) and forward-scattering (FSC) modes, and 10,000 events per sample were collected. ΔΨ_m was assessed using a fluorescent potentiometric dye, JC-1, which displayed a green fluorescence of monomers (Ex, 490 nm; Em, 530 nm) and a red fluorescence of the dimer (Ex, 490 nm; Em, 590 nm). When dye was incorporated in mitochondria that retain a high membrane potential, JC-1 forms aggregates to exhibit the red fluorescence. Energized mitochondrial particle showed high emission intensity at both the wavelengths. The number of depolarized mitochondrial particles, defined as a ratio of red to green of <0.3, was counted. FCCP was applied as a negative control experiment to determine depolarized mitochondria. DiIC₁(5) was also used to detect ΔΨ_m changes (Ex, 635 nm; Em, 660 nm) by combining with 100 nm NAO, which selectively stains mitochondria (Ex, 488 nm; Em, 525 nm). ΔΨ_m was measured in NAO-positive particles that were also selected from background based on light-scattering properties (SSC and FSC). In every experiment, 98% of the events were NAO positive.

RNA sampling and DNA microarray analysis.

Male mutPOLG Tg and their wild-type littermates were killed at the age of 25–34 weeks old. The bilateral frontal cortices and hippocampi were rapidly dissected, and total RNA samples were extracted from the brains of six mutPOLG Tg or wild-type littermates using Trizol reagent (Invitrogen). The microarray analysis was conducted using RNA samples derived from five pairs of animals on the basis of the quality of mRNA. Five micrograms of total RNA of each sample was reverse-transcribed into cDNA, and biotinylated cRNA was synthesized from the cDNA by in vitro transcription. DNA microarray experiments were performed using MG_430A GeneChips (Affymetrix, Santa Clara, CA), which contained 42,153 probe sets. The hybridization signal on the chip was scanned by a GeneArray scanner and processed by GeneSuite software (Affymetrix). The raw data were initially analyzed using MAS5 (Affymetrix), and then imported into GeneSpring 6.1 software (Silicon Genetics, Redwood, CA). The fluorescence intensity of each spot on the microarray was divided by its median value using GeneSpring 6.1 software and normalized. The probe sets for mitochondria-related genes were chosen either from the NetAffx database on the distributor's web site with the keywords “mitochondria” or “mitochondrion” or from the gene list of “mitochondria” in the GeneSpring software, which classified probe sets based on the information from Gene Ontology. Only the probe sets that were called as being “present” in more than one-half of the samples were analyzed, and 1221 probe sets were identified. For statistical analysis, a two-tailed paired t test was performed between the mutPOLG Tg mice and their littermates, and a value of p < 0.05 was considered statistically significant.

Real-time quantitative PCR.

To quantify mRNA levels of several interesting genes, we performed real-time quantitative PCR analysis (TaqMan technology) using commercially available probe–primer sets (Applied Biosystems, Foster City, CA) as described previously (Kakiuchi et al., 2003). The relative levels of each mRNA were calculated by 2^−CT (CT standing for the cycle number at which the signal reached the threshold) and normalized to the corresponding β-actin mRNA level. Each CT value used for these calculations was the mean of four values obtained when each reaction was performed in quadruplicate.

Electrophysiology and intracellular Ca²⁺ imaging.

Fluorometric measurement of intracellular Ca²⁺ ([Ca²⁺]_i) was performed according to the method of Nakamura et al. (1999) with slight modification. Transverse hippocampal slices (300 μm thick) were prepared from age-matched 5- to 7-week-old mutPOLG Tg and wild-type mice as described previously (Kubota et al., 2001). Each mouse was anesthetized under ether vapor anesthesia and quickly decapitated. The brain was chilled in ice-cold cutting solution, which is a modified artificial CSF (aCSF) containing the following (in mm): 120 choline-Cl, 26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 2.5 MgCl₂, 0.5 CaCl₂, and 20 glucose. Slices were made in the cutting solution using a tissue slicer (VT1000S; Leica, Wetzlar, Germany) and transferred to aCSF, which was composed of the following (in mm): 124 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 10 glucose. The slice was mounted on a recording chamber on an upright microscope (BX50WI; Olympus, Tokyo, Japan) and superfused with the aCSF during experiments at a rate of 1.5 ml/min. The cutting solution and aCSF had an osmolarity of 280 mOsm and a final pH of 7.4 after it was saturated with 95% O₂ and 5% CO₂. The recording chamber composition was changed by 90% in 3 min. The whole-cell recordings were made in CA1 pyramidal neurons using patch pipettes pulled from 1.5 mm outer diameter, thick-walled borosilicate glass tubing (GC-150 F-10; Harvard, Kent, UK) using an electrode puller (P-97; Sutter Instruments, Novato, CA). The patch pipette contained an internal solution of the following (in mm): 140 K-gluconate, 4 NaCl, 4 Mg-ATP, 0.3 Na-GTP, and 10 HEPES, pH 7.4 with KOH; and the osmolarity was adjusted to 280 mOsm with sucrose. This solution was supplemented with 300 μm of a calcium indicator, bis-fura-2, in the experiments of imaging for action potential (AP)-induced [Ca²⁺]_i transient. In the experiments to see the enhancing effects of DHPG on AP-induced [Ca²⁺]_i transient, a low-affinity Ca²⁺ indicator, fura-6F, was used. A tight seal on CA1 pyramidal cell soma was made under visual control using a 40× water immersion lens and video-enhanced differential interference contrast (DIC) optics, and the electrical recordings were performed in current-clamp mode at room temperature. The backpropagating APs were induced by depolarizing pulses (1 ms duration), which were evoked from the patch electrode (resistance, 3–5 MΩ) with a patch-clamp amplifier (Axoclamp 2B; Molecular Devices, Union City, CA). The signals were sampled at 100 μs intervals and digitized at 10 kHz (Digidata 1322A; Molecular Devices). Holding potentials, data acquisition, and analysis were controlled by pCLAMP 7.0 software (Molecular Devices). Time-lapse [Ca²⁺]_i imaging was acquired at 30 ms intervals for 6.4 s using a CCD camera (C4742-95; Hamamatsu Photonics, Hamamatsu, Japan). The fluorescence images were analyzed with AQUACOSMOS system (version 2.0; Hamamatsu Photonics). The regions of interests (ROIs) (3 × 3 pixels) were put on the cell body and dendrites of each neuron. Relative [Ca²⁺]_i levels associated with electrical activities were measured with single-wavelength imaging (Ex, 380 nm; Em, 530 nm) and were expressed as fluorescence change [−ΔF/F = (F_i − F_b)/(F_b − F_a)], where F_i is the average fluorescence of the ROI in each image, F_b is the baseline fluorescence averaged over at least 10 images before stimulation, and F_a is the autofluorescence. Normalized in this way, F is the fluorescence intensity when the cell was rest, and ΔF is the change in fluorescence during activity. In some cases, background intensity was defined as fluorescence in each ROI that was located outside the neuron of interest. Determination of absolute Ca²⁺ concentration by a ratiometric measurement cannot catch up with rapid [Ca²⁺]_i transient in neurons, which makes it difficult to compare the [Ca²⁺]_i levels between Tg mice and wild-type mice. Thus, we used the ratio of ΔF/F after DHPG application to the averaged ΔF/F before drug application. [Ca²⁺]_i change was assessed by measuring peak amplitude and area under the curve (AUC) for each set of responses, to reflect actions on both the amplitude and the duration of the [Ca²⁺]_i response. To detect the effect of a metabotropic glutamate receptor (mGluR) agonist, 20 μm CNQX and 50 μm APV were added to block ionotropic glutamate receptors in some experiments (see Figs. 4, 5).

Figure 4.

Effect of various pharmacological agents on GPCR-mediated [Ca²⁺]_i increase. A, Effect of mGluR agonist or antagonist on AP-induced [Ca²⁺]_i increase at the soma of hippocampal pyramidal neuron filled with the low-affinity indicator fura-6F (K_d, 6 μm) in wild-type mice. Time-lapse imaging of fluorescence changes (top) and voltage responses induced by APs at 30 Hz (bottom) were taken before (−DHPG) and during (+DHPG) bath application of 30 μm DHPG, a group I mGluR agonist. DHPG-induced [Ca²⁺]_i increase in the presence of 1 mm MCPG, a mGluR antagonist (MCPG+DHPG), after washing out of MCPG (+DHPG), and without the presence of any agents (wash). B, Effect of a cholinergic agonist, CCh, on AP-induced [Ca²⁺]_i increase. Time-lapse imaging of fluorescence changes (top) and voltage responses induced by APs at 30 Hz (bottom) were taken before (−CCh) and during (+CCh) bath application of 30 μm CCh. After application of a muscarinic receptor antagonist, atropine (1 μm), for 5 min, CCh was applied (atropine+CCh). C, Dependence of DHPG-induced [Ca²⁺]_i increases on intracellular Ca²⁺ stores. Time-lapse imaging of fluorescence changes (top) and voltage responses induced by APs at 30 Hz (bottom) were taken before (−DHPG) and during (+DHPG) bath application of 30 μm DHPG. An endoplasmic reticulum Ca²⁺/ATPase blocker, CPA, is known to cause depletion of the stores. After application of 20 μm CPA for 7.5 min, DHPG was applied (CPA+DHPG). D, Dependence of DHPG-induced [Ca²⁺]_i increases on IP₃ receptor activation. Fluorescence changes (top) were taken before (−DHPG) and during (+DHPG) bath application of 30 μm DHPG. Recordings were made with a pipette containing solution with an impermeable IP₃ receptor blocker, 2 mg/ml heparin (right), or no additives (left). Voltage responses induced by APs at 30 Hz at the middle are shown on an expanded timescale at the bottom. The arrows indicate GPCR-mediated membrane depolarization during AP firing. A set of measurements was done using slices derived from an animal. The perfusate contained 20 μm CNQX and 50 μm APV in all of the experiments.

Figure 5.

GPCR-mediated [Ca²⁺]_i increase in the neurons of wild-type and Tg mice. A, Hippocampal pyramidal neurons were filled with the low-affinity indicator fura-6F (K_d, 6 μm). DIC images of the neurons are shown with regions of interest located at the soma (black rectangles) and the dendrites (white rectangles) in wild-type (WT) and mutPOLG Tg (Tg) mice, respectively. Pseudocolor images of spatiotemporal [Ca²⁺]_i distribution reflect the basal level of [Ca²⁺]_i before AP firing (panels 1 and 4), the peak of [Ca²⁺]_i increase during AP firing (panels 2 and 5), and the recovered level of [Ca²⁺]_i after AP firing (panels 3 and 6), respectively. Images taken before (−DHPG) and during (+DHPG) bath application of 30 μm DHPG are shown. B, [Ca²⁺]_i increases at the soma (thick lines) and the dendrites (thin lines) were superimposed for the wild-type (WT) and mutPOLG Tg (Tg) mice, respectively. [Ca²⁺]_i increase was induced by APs and observed by time-lapse imaging of fluorescence changes (top). The distinct time points (1–6) over traces correspond to the [Ca²⁺]_i images of panels 1–6 in A. Traces of membrane potential at the middle are shown on an expanded timescale at the bottom. The arrows indicate GPCR-mediated membrane depolarization during AP firing. C, Averaged traces of [Ca²⁺]_i increases were given at the soma (thick lines) and the dendrite (broken lines) in wild-type (WT) and mutPOLG Tg (Tg) mice, respectively. The AP-induced [Ca²⁺]_i increases before (−DHPG) and during (+DHPG) bath application of DHPG are shown. Data at each point are expressed as mean ± SEM, which is indicated by the gray line. D, Statistical analysis of DHPG-induced [Ca²⁺]_i changes. The amplitude of peak (peak) and AUC of fluorescence changes at the soma are compared with the wild-type (WT) and mutPOLG Tg (Tg) mice (n = 11 and 15, respectively). Data were given as the ratio of during application of DHPG to before application of drugs. [Ca²⁺]_i increase during application of DHPG was attenuated in the neurons of mutPOLG Tg mice (peak, *p < 0.05; AUC, ^#p < 0.1). Data are expressed as mean ± SEM.

Statistics.

Statistical calculation was performed using KyPlot software (version 4.0; Kyens) or SPSS software (version 10.0; SPSS, Chicago, IL). Results were presented as mean ± SEM and were evaluated using a paired or a two-sample Student's t test when appropriate. Paired t test was used to test for difference in the presence or absence of drug. Interaction between CsA treatment and Ca²⁺ addition in the experiment of isolated mitochondria was statistically evaluated by two-way repeated-measures ANOVA with the factors of Ca²⁺ addition and CsA treatment. Degree of freedom for repeated-measures ANOVA was adjusted for sphericity by Greenhouse–Geisser method. Post hoc analysis of the difference between recovery time with or without CsA was conducted by the Mann–Whitney U test. Values of p < 0.05 were considered statistically significant.

Results

Ca²⁺ retention capacity and Ca²⁺ uptake kinetics in isolated mitochondria

Mitochondrial Ca²⁺ uptake through Ca²⁺ uniporter is associated with the activity of the mitochondrial respiratory chain, which consists of a set of five complexes (I to V). None of the proteins of complex II are encoded by mtDNA (Larsson and Clayton, 1995). To assess the impact of accumulated mtDNA defects on mitochondrial Ca²⁺ transport, we performed two experiments; in one experiment, we used the substrate for complex I (glutamate plus malate), and the substrate for complex II (succinate) was used in another experiment. We added aliquots of Ca²⁺ solution to mitochondrial suspension and determined the CRC by monitoring [Ca²⁺]_exm. In the experiment using a buffer containing glutamate plus malate, four additions of 1.6 μm final concentration of Ca²⁺ (short arrows) induced small [Ca²⁺]_exm transients, and 7.8 μm final concentration of Ca²⁺ (long arrows) induced larger changes (Fig. 1A). The [Ca²⁺]_exm increased at first and was completely restored to the basal level by mitochondrial uptake. At the point marked by asterisks (Fig. 1A), PTP opened and Ca²⁺ was released from the matrix to the outside of mitochondria. Finally, a similar number of additions was required to open the PTP in mitochondria from wild-type and mutPOLG Tg mice. The point of PTP opening was defined according to the previous studies (Ichas et al., 1997; Fontaine et al., 1998). Next, we measured the CRC of mitochondria incubated in a buffer containing succinate, in which the mitochondria are energized through complex II components independent of mtDNA involvement. By additions of 7.8 μm Ca²⁺ in the buffer containing succinate, PTP opening was induced more readily than was done in the buffer containing glutamate plus malate (data not shown). Thus, we added final 1.6 and 3.1 μm Ca²⁺ solution to measure the CRC. Four additions of 1.6 μm final concentration of Ca²⁺ (short arrows) and several additions of 3.1 μm final concentration of Ca²⁺ (long arrows) induced [Ca²⁺]_exm transients in a dose-dependent manner (Fig. 1C). There was no clear distinction in the CRC of mitochondria between mutPOLG Tg mice and wild-type mice (Fig. 1E, left, n = 6, p = 0.81; right, n = 6, p = 0.85, Student's t test), whereas we noticed a difference in [Ca²⁺]_exm kinetics in response to the first addition of 7.8 μm Ca²⁺ solution. The traces of [Ca²⁺]_exm shown in Figure 1B were obtained from six independent experiments in the substrate for complex I and superimposed. In mitochondria of mutPOLG Tg mice, the [Ca²⁺]_exm were rapidly restored back toward the basal level, and the recovery time was significantly shorter than in mitochondria from wild-type mice (Fig. 1F, left, n = 6, p < 0.05, Student's t test). No significant difference was observed between genotypes in the buffer containing the substrate for complex II (Fig. 1D,F, right, n = 6, p = 0.27, Student's t test).

Figure 1.

Mitochondrial CRC and uptake kinetics. A, CRC of mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice in a buffer containing 20 mm glutamate plus 2 mm malate, which is a substrate for complex I. Changes in extramitochondrial Ca²⁺ concentration ([Ca²⁺]_exm) were monitored by a fluorescent Ca²⁺-sensitive dye, Calcium Green-5N. Ca²⁺ solution was applied by four additions at a final concentration of 1.6 μm (short arrows) and subsequent additions at a final concentration of 7.8 μm (long arrows) at 60 s intervals. An asterisk on the trace indicates the point at which Ca²⁺-overloaded mitochondria released Ca²⁺. A closed circle indicates the trace that was used for the analysis in B. The representative data of six experiments are shown. B, Kinetics of [Ca²⁺]_exm responses in a buffer containing 20 mm glutamate plus 2 mm malate. The fluorescence changes in response to the first additions of 7.8 μm Ca²⁺ in six independent experiments were superimposed on an expanded timescale after normalization by maximal peak amplitude. C, CRC of mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice in a buffer containing 10 mm succinate, a substrate for complex II. Ca²⁺ solution was applied by four additions of 1.6 μm Ca²⁺ (short arrows) and subsequent additions of 3.1 μm Ca²⁺ (long arrows) at 60 s intervals. An asterisk on the trace indicates the point at which Ca²⁺-overloaded mitochondria released Ca²⁺. A closed circle indicates the trace that was used for the analysis in D. The representative data of seven independent experiments are shown. D, Kinetics of [Ca²⁺]_exm responses in the buffer containing 10 mm succinate. The fluorescence changes in response to the first additions of 3.1 μm Ca²⁺ in six independent experiments were superimposed on an expanded timescale after normalization by maximal peak amplitude. E, Statistical analysis of results from the CRC assays in the buffer containing glutamate plus malate or succinate. Total amount of Ca²⁺ (micromoles of Ca²⁺ per milligram of protein) necessary to induce Ca²⁺ release was compared between mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice (n = 6). Data are expressed as mean ± SEM. F, Statistical analysis of the recovery times for Ca²⁺ uptake in two different conditions. Mitochondria from mutPOLG Tg (Tg) mice sequestered Ca²⁺ more rapidly than did those from wild-type (WT) mice (n = 6; *p < 0.05) when a CRC assay was performed in the buffer containing glutamate plus malate. Data are expressed as mean ± SEM.

Mitochondrial membrane potential in isolated mitochondria

As a mechanism underlying the enhanced Ca²⁺ uptake of mitochondria from mutPOLG Tg mice, it was assumed that a higher ΔΨ_m across the mitochondrial inner membrane would provide a higher driving force for the Ca²⁺ uptake. We stained mitochondria with a ΔΨ_m-sensitive dye, JC-1, including the substrate for complex I and assessed ΔΨ_m (Fig. 2A); a depolarized mitochondria particle was defined on the basis of a ratio of the red-to-green signals (R/G). The mitochondria of mutant mice did not display higher R/G than those from wild-type mice under the steady-state condition (Fig. 2B, control, p = 0.64, Student's t test). A treatment with Ca²⁺ corresponding to the level of one addition in the above CRC assay (100 nmol of Ca²⁺ per milligram of mitochondrial protein) decreased the fluorescence intensity of the red signal of JC-1, indicating that mitochondria were depolarized in both genotypes (Fig. 2A, +Ca²⁺). The addition of Ca²⁺ resulted in a clear shift in the peak of histogram to a lower R/G value (Fig. 2B, +Ca²⁺). A remarkable loss of fluorescence was induced after exposure to the protonophore FCCP, indicating the ΔΨ_m collapse (Fig. 2C, +FCCP). There were no differences between genotypes in ΔΨ_m or ΔΨ_m response under any of the conditions (Fig. 2C, control, p = 0.64; +FCCP, p = 0.94; +Ca²⁺, p = 0.48, Student's t test). These observations were confirmed when samples were incubated with the complex II substrate (data not shown). However, the aggregate formation of JC-1 may provide a higher estimate in a population of polarized mitochondria, which interfere with detection of the difference in genotypes. Therefore, we verified our result using another ΔΨ_m-dependent dye, DiIC₁(5), that exhibits a 630 nm excitability of emission spectrum, which is also used with a mitochondria-targeted dye, NAO. By the double staining with NAO and DiIC₁(5), mitochondria particles were identified. The NAO-labeled particles showing higher intensity of DiIC₁(5) signal were determined to be more “energized” mitochondria incubated in the complex I substrate (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). No significant differences were detected in the mean intensities between genotypes under any of the conditions (steady state, p = 0.66; FCCP, p = 0.10; Ca²⁺, p = 0.21, Student's t test). Neither was there any difference in ΔΨ_m response between genotypes. These results were confirmed by a measurement taken in the buffer containing the complex II substrate (data not shown).

Figure 2.

Mitochondrial membrane potential. A, Measurement of mitochondrial membrane potential (ΔΨ_m) in isolated mitochondria using a ΔΨ_m-sensitive dye, JC-1. Isolated brain mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice were subjected to flow-cytometric analysis in a buffer containing 20 mm glutamate plus 2 mm malate. A potentiometric dye, JC-1, accumulates in mitochondria. The emission fluorescence exhibited green and red, which were plotted on the x- and y-axes, respectively. Each point indicates fluorescence intensity from each mitochondrion under steady-state condition (control) and treatment with 1 μm FCCP (+FCCP) or 6.25 μm Ca²⁺ (+Ca²⁺). A negative shift in red signals along the y-axis indicated a loss of ΔΨ_m. B, Frequency distributions of red-to-green fluorescence ratio (R/G) of JC-1. Depolarized mitochondria particles were counted by defining that the R/G was <0.3 (a gray broken line indicates the threshold). A loss of ΔΨ_m was evaluated by decreased R/G. Ca²⁺ exposure increased the population of depolarized mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice. C, Quantifications of depolarized mitochondria. The population of depolarized mitochondria was averaged and compared with wild-type (WT) and mutPOLG Tg (Tg) under steady-state condition (control) and treatment with Ca²⁺ (+Ca²⁺) or FCCP (+FCCP). There were no significant differences between genotypes (n = 6) in ΔΨ_m or ΔΨ_m response under any of the conditions. Data are expressed as mean ± SEM.

Differentially expressed genes in brains of mutPOLG Tg mice

Our findings were not attributable to respiratory failure but caused by some unknown mechanisms related to Ca²⁺ transport. To clarify the molecular basis of the abnormality in mitochondria of mutPOLG Tg mice, we performed DNA microarray analysis of the brains from male mutPOLG Tg and wild-type littermates of 34–35 weeks of age (n = 5 for each genotype). The expression of four genes related to mitochondria was significantly affected commonly in the two regions of the brain in mutPOLG Tg mice; two genes were downregulated and the other two genes were upregulated (Table 1). We performed quantitative real-time RT-PCR to validate the microarray results and confirmed the downregulation of Ppif and Glud1 genes in the hippocampal tissues of mutPOLG Tg mice compared with wild-type mice (Table 1) (n = 6 for each genotype). The level of Ppif [cyclophilin D (CyP-D)] and Glud1 (glutamate dehydrogenase) genes were significantly decreased in mutPOLG Tg mice. Because the Ppif gene encodes CyP-D, which is a critical component of PTP and regulates Ca²⁺ sensitivity to PTP opening, we considered that the downregulated expression of the Ppif gene would be relevant to the enhanced Ca²⁺ uptake in mutPOLG Tg mice. To determine whether several genes encoding proteins required for the opening of PTP was entirely suppressed as well as the Ppif gene, we monitored mRNA levels of the genes encoding the other proteins constituting the PTP components [e.g., adenine nucleotide translocator (ANT1/Slc25a4), voltage-dependent anion channel (Vdac1), hexokinase (Hk1), creatine kinase (Ckmt1), and peripheral benzodiazepine receptor (Bzrp)]. The relative amounts of mRNAs for these genes were not significantly changed in the brains of mutPOLG Tg mice (data not shown). Our experimental and analytical procedures were validated by a higher expression level of the mutPolg gene in mutant mice than wild-type mice. The mutPolg gene expression is likely to be engaged in upregulation of mtDNA topoisomerase I gene (Top1mt) expression. Because Top1mt protein has been known to be involved in the mtDNA replication (Moraes, 2001; Zhang et al., 2001), this upregulation was thought to be the influence of the transgene, mutPolg.

Table 1.

Differentially expressed genes in the mutPOLG Tg mice brains

Gene				DNA microarray				RT-PCR
				Hippocampus		Frontal cortex		Hippocampus
Symbol	Description	Accession no.	Affymetrix ID	Fold changea	p value*	Fold changea	p value*	Fold changea	p value*
Ppif	Cyclophilin D	NM_134084	1416940_at	0.91	0.038	0.93	0.008	0.84	0.021
Glud1	Glutamate dehydrogenase 1	BI329832	1416209_at	0.93	0.049	0.97	0.040	0.84	0.020
Top1mt	mtDNA topoisomerase 1	AF362952	1460370_at	1.12	0.005	1.05	0.002	Not tested
Polg	mtDNA polymerase	BG064799	1423272_at	1.49	0.045	1.32	0.007	Not tested

^aFold changes were given as ratio of signals of mutPOLG Tg mice relative to those of wild-type mice.

*Student's t test for paired comparison (two-tailed).

Effect of the cyclophilin D inhibitor on rate of Ca²⁺ uptake in isolated mitochondria

We speculated that the decrease of CyP-D would be responsible for the abnormal Ca²⁺ uptake in mitochondria from mutPOLG Tg mice. A CyP-D inhibitor, CsA, prevents PTP from opening at the adenine nucleotide translocator (ANT)-binding site of CyP-D (Hansson et al., 2003). So, we tested whether CyP-D inhibition would enhance Ca²⁺ uptake in mitochondria from wild-type mice (n = 6). PTP opening occurred after six additions of 7.8 μm Ca²⁺ in the absence of CsA (Fig. 3A, −CsA). In the presence of 2 μm CsA, over 20 additions of 7.8 μm Ca²⁺ were required to open the PTP (Fig. 3A, +CsA). This indicated that CsA inhibited PTP opening and significantly increased the CRC (Fig. 3A, inset) (p < 0.05, paired t test). We then measured the recovery time of [Ca²⁺]_exm to evaluate the rate of Ca²⁺ uptake. Shortening of the recovery time by CsA became evident after repeated addition of Ca²⁺ (Fig. 3B, a–c, +CsA and −CsA). This finding was confirmed in five independent experiments. The recovery time was almost unchanged in the presence of CsA even when Ca²⁺ solution was added repeatedly (Fig. 3C, +CsA), which was similarly observed in mutPOLG Tg mice (Fig. 3, compare C, D).

Figure 3.

Effect of the cyclophilin D inhibitor, cyclosporin A, on Ca²⁺ uptake in mitochondria. A, CRC was measured in the absence (−CsA) or presence (+CsA) of 1 μm CsA in a buffer containing glutamate plus malate. Changes in [Ca²⁺]_exm were monitored by the fluorescent Ca²⁺-sensitive dye, Calcium Green-5N. Ca²⁺ solution was subsequently applied at a final concentration of 7.8 μm for untreated mitochondria (−CsA) or for CsA-treated mitochondria (+CsA) at 60 s intervals. The traces marked by a–c were used for analysis in B. Inset, The CRC was determined from three independent experiments (n = 6; *p < 0.05). Data are expressed as mean ± SEM. B, Kinetics of [Ca²⁺]_exm changes. Representative traces of [Ca²⁺]_exm marked by a–c in A were expanded. The traces were normalized by maximal peak amplitude and compared between the absence (−CsA) and presence (+CsA) of CsA. After the repeated addition of Ca²⁺, the recovery time became shorter in the presence of CsA than in the absence of CsA. As shown in C, CsA apparently decreased the recovery time of Ca²⁺. C, Changes in recovery time with sequential additions of Ca²⁺, plotted by the amount of Ca²⁺ added in the absence (−CsA) or presence (+CsA) of CsA. Each circle corresponds to a Ca²⁺ addition, and each trace of [Ca²⁺]_exm is from one experiment. Two-way repeated-measures ANOVA in the first four additions revealed a significant interaction between the number of Ca²⁺ addition and CsA treatment (F = 4.78; df = 1.08; p < 0.05, repeated two-way ANOVA, Greenhouse–Geisser correction). Although there was no statistically significant difference of recovery time after the first addition of Ca²⁺ (p = 0.35, Mann–Whitney U test), recovery time in the presence of CsA (+CsA) became shorter than that in the absence of CsA at the fourth addition of Ca²⁺ (p < 0.05, Mann–Whitney U test). D, Changes in recovery time with the subsequent additions of Ca²⁺. Each circle corresponds to a Ca²⁺ addition and each trace of [Ca²⁺]_exm is from one experiment using mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice in the absence of CsA.

Electrical membrane properties and action potential-induced Ca²⁺ transient in hippocampal neurons

To examine the effect of enhanced Ca²⁺ uptake of isolated mitochondria of mutPOLG Tg mice on the [Ca²⁺]_i dynamics, hippocampal slice preparation was used. Whole-cell recordings were made from the soma of pyramidal neurons in area CA1. Basic neuronal excitability of hippocampal pyramidal neurons of mutPOLG Tg mice was similar to that of wild-type mice (see supplemental Table 1, available at www.jneurosci.org as supplemental material) (n = 33 for each genotype). Because mitochondria participate in regulating [Ca²⁺]_i according to the level of [Ca²⁺]_i change induced by a varying frequency of AP generation (David et al., 1998; Peng, 1998), we elicited a repetitive AP in pyramidal neurons of the mutant mice and compared the induced [Ca²⁺]_i changes with those in wild-type mice (supplemental Fig. 2A,B, available at www.jneurosci.org as supplemental material). The magnitude of [Ca²⁺]_i transient was measured using two parameters, the amplitude or the time of peak, and revealed no significant difference between genotypes (supplemental Fig. 2C, available at www.jneurosci.org as supplemental material).

Involvement of Ca²⁺ store in GPCR-mediated [Ca²⁺]_i increase in hippocampal neurons

Because IP₃-mediated Ca²⁺ response was reported to be enhanced in platelets from BD patients (Dubovsky et al., 1989; Yamawaki et al., 1996), we had hypothesized that reduced mitochondrial Ca²⁺ uptake may result in increased cytosolic [Ca²⁺]_i response in BD (Kato and Kato 2000). Contrary to our expectation, however, mitochondrial Ca²⁺ uptake was enhanced in mutant mice. To test how the enhanced Ca²⁺ uptake in mitochondria affects cytosolic [Ca²⁺]_i response of mutPOLG Tg mice, we studied the agonist-stimulated [Ca²⁺]_i response in hippocampal neurons. Although it is difficult to assess this impact quantitatively in neurons, previous studies showed that GPCR activation paired by backpropagating APs could induce Ca²⁺ mobilization from internal stores (Nakamura et al., 1999). To apply this experimental paradigm for investigating the molecular basis of altered Ca²⁺ signaling in the neurons of Tg mice, we first pharmacologically characterized this response. After group I mGluR agonist, DHPG (30 μm), was applied, all of the neurons tested were slightly depolarized within the first few minutes as described previously (Rae et al., 2000). In the presence of DHPG, the membrane potential gradually depolarized during the AP firing (Figs. 4D, 5B, bottom, +DHPG, indicated by arrows), which was often followed by a spontaneous bursting discharge and excessive [Ca²⁺]_i increase in some neurons. Because this Ca²⁺ change hampered an evaluation of IP₃-mediated Ca²⁺ mobilization, these cells were excluded from the analysis. When DHPG was applied, the same number of APs (30 Hz) as before elicited a larger [Ca²⁺]_i increase (+DHPG) compared with before addition of DHPG (−DHPG) in hippocampal CA1 pyramidal neurons of wild-type mice (Fig. 4A). DHPG binds to mGluR, stimulates Gq protein, and produces IP₃ to activate IP₃ receptor (IP₃R), releasing Ca²⁺ from internal store in neurons (Berridge, 1998). The mGluR receptor antagonist, MCPG (1 mm), significantly inhibited this enhancement (26.6 ± 12.4%; n = 4; p < 0.01, paired t test) (Fig. 4A, MCPG+DHPG). The enhanced[Ca²⁺]_i increase was restored by washing out of MCPG (+DHPG), supporting the role of mGluR in this reaction as suggested by previous studies (Nakamura et al., 2000; Rae et al., 2000). Application of a cholinergic receptor agonist, 30 or 300 μm CCh, also caused a similar enhancement (Fig. 4B), which was abolished by a muscarinic receptor antagonist, atropine (1 μm) (25.9 ± 2.3%; n = 3; p < 0.01, paired t test). Two agonists acting on different receptors but affecting the common intracellular signaling pathway, Gq–PLCβ (phospholipase Cβ)–IP₃ (Shirasaki et al., 1994; Power and Sah, 2002), showed similar Ca²⁺ enhancement, suggesting the role of this pathway in this enhancement. To further verify the source of [Ca²⁺]_i, the effect of an ER Ca²⁺/ATPase blocker, CPA, was examined. DHPG-induced [Ca²⁺]_i increase was attenuated in the presence of CPA (Fig. 4C). An IP₃R blocker, heparin (2 mg/ml), also abolished the enhancement of DHPG- or CCh-induced [Ca²⁺]_i increase (Fig. 4D or data not shown). These results further confirmed that the enhancement of [Ca²⁺]_i increase by GPCR stimulation is mediated by Ca²⁺ release from the ER through IP₃R.

Effect of GPCR agonist on AP-induced [Ca²⁺]_i transient in hippocampal neurons of wild-type and mutPOLG Tg mice

[Ca²⁺]_i transient induced by a train of APs was not different in both genotypes. Additionally, DHPG effect on AP-induced [Ca²⁺]_i increase was observed in neurons of mutPOLG Tg mice. In the absence of DHPG, a train of 20 APs similarly induced cytosolic and dendritic [Ca²⁺]_i transients both in the neurons of wild-type and mutPOLG Tg mice (Fig. 5A,B, −DHPG). The magnitude of membrane depolarization after DHPG application did not differ between genotypes (−3.5 ± 1.4 mV in neurons of wild-type mice and −3.5 ± 1.7 mV in neurons of mutPOLG Tg mice; p = 0.40, Student's t test). The different population of cells showing the spontaneous bursting in the presence of DHPG did not found between wild-type (4 of 19 cells) and mutPOLG Tg mice (8 of 19 cells) (p = 0.14, Fisher's exact test). In the presence of DHPG, [Ca²⁺]_i increase was enhanced in neurons of both genotypes (Fig. 5C, +DHPG). The impact of DHPG was estimated by a change in the peak amplitude or the AUC of the [Ca²⁺]_i increase at the soma. Ratio of the [Ca²⁺]_i increase after DHPG application to that before the application was significantly smaller in mutPOLG Tg mice (n = 11) compared with that in wild-type mice (n = 15) (Fig. 5D, peak, *p < 0.05; AUC, ^#p < 0.1, Student's t test). The modulatory effect of DHPG on AP-induced [Ca²⁺]_i increase was less in the neurons of mutPOLG Tg mice than that of wild-type mice.

Discussion

We investigated Ca²⁺ dynamics in mutPOLG Tg mice harboring neuron-specific defects in mtDNA age dependently. We proposed that the mutant mice would be a model of BD, because it not only fulfills construct validity based on mitochondrial dysfunction hypothesis of BD but also fulfills face and predictive validities: its BD-like phenotypes such as periodic activity change were improved by a mood stabilizer. The present study demonstrated that the rate of Ca²⁺ uptake was enhanced in isolated mitochondria from the mutant mice and GPCR-mediated [Ca²⁺]_i increase was attenuated in the hippocampal neurons of the mutPOLG Tg mice. These findings might shed light on the pathophysiology of BD.

Molecular basis of enhanced rate of Ca²⁺ uptake in mitochondria from mutPOLG Tg mice

The recovery time of [Ca²⁺]_exm in isolated mitochondria from mutPOLG Tg mice was shorter than that in wild-type mice (Fig. 1B), although mitochondrial CRC was not altered (Fig. 1A,E). This result could be explained by two functional states of PTP, persistent and brief openings (Ichas and Mazat, 1998). Persistent PTP opening with a large conductance is thought to associate with a disruption of mitochondrial outer membrane and a subsequent release of cytochrome c to the cytosol (Weiss et al., 2003), and translocation of cytochrome c is often accompanied with apoptosis and a loss of ΔΨ_m (Schild et al., 2001). In contrast, brief PTP opening with a small conductance is induced by binding of CyP-D in the matrix to ANT in the inner membrane of mitochondria and enables Ca²⁺ to be released. A role of CyP-D in brief PTP opening has been well established by forming the pore that associates with the conformational change of inner membrane (Crompton, 1999).

In this study, we found downregulation of Ppif encoding CyP-D in the forebrains of mutPOLG Tg mice (Table 1). Moreover, application of CyP-D inhibitor, CsA, facilitated Ca²⁺ uptake (Fig. 3B) and mimicked the altered Ca²⁺ uptake kinetics in mitochondria from mutPOLG Tg mice (Fig. 3C,D) in addition to marked increase of CRC (Fig. 3A) as shown in a previous study (Fontaine et al., 1998). Thus, we can speculate that downregulation of CyP-D inhibited the brief PTP opening, which resulted in the enhanced mitochondrial Ca²⁺ uptake in mutPOLG Tg mice. This finding is consistent with the reports that CsA facilitates mitochondrial Ca²⁺ sequestration in brain mitochondria (Levy et al., 2003) or chromaffin cells (Montero et al., 2001). Mitochondria from CyP-D-deficient mice caused accumulation of a larger amount of Ca²⁺ (Baines et al., 2005; Basso et al., 2005; Nakagawa et al., 2005). The downregulation of CyP-D, however, may be a consequence of the pathological Ca²⁺ homeostasis caused by the accumulation of mtDNA defects. Additional studies will be required for elucidating the causal relationship.

Relationship between enhanced Ca²⁺ uptake rate in isolated mitochondria and attenuated DHPG-induced Ca²⁺ increase in hippocampal neurons of mutPOLG Tg mice

Regarding synaptic Ca²⁺ transients, mitochondrial Ca²⁺ uptake is known to contribute to Ca²⁺ clearance at the presynaptic terminals (Billups and Forsythe, 2002) and postsynaptic sites (Pivovarova et al., 2002). The enhanced rate of Ca²⁺ uptake in mitochondria of the mutPOLG Tg mice should result in attenuated Ca²⁺ signaling in the neurons. As we expected, GPCR-mediated [Ca²⁺]_i increase, which is primarily coupled to the IP₃ mobilization, was influenced in mutant mice (Fig. 5). Previous studies demonstrated that GPCR-mediated [Ca²⁺]_i increase was not affected by a ryanodine receptor (RyR) inhibitor and was triggered by photolysis of caged IP₃ (Nakamura et al., 1999; Yamamoto et al., 2000). The present result that IP₃-mediated [Ca²⁺]_i increase was sensitive to the store depletion and IP₃R blockade (Fig. 4C,D) is consistent with those previous findings. However, [Ca²⁺]_i transient by AP-induced depolarization was not altered in neurons of mutPOLG Tg mice (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). One possible explanation for the lack of influence on this component is that [Ca²⁺]_i increase by GPCR activation is much larger than that by AP-induced depolarization. When [Ca²⁺]_i reaches 1–2.5 μm, mitochondria exert their function for Ca²⁺ clearance in synaptic terminals (Billups and Forsythe, 2002; Kim et al., 2005). In pyramidal neurons, [Ca²⁺]_i level induced by depolarization is ∼1 μm, whereas if the depolarization is evoked by paired with an activation of GPCR, peak [Ca²⁺]_i level augments to ∼3 μm (Nakamura et al., 1999). The Ca²⁺ concentration applied to mitochondrial suspension in this study (3.1 or 7.8 μm) may be comparable with the latter situation. Close contact of mitochondria with ER at microdomain may cause even higher Ca²⁺ level, which may further enhance mitochondrial Ca²⁺ uptake (Rizzuto et al., 1998).

It is reasonable to attribute the attenuation of agonist-induced [Ca²⁺]_i enhancement to accelerated mitochondrial Ca²⁺ uptake based on previous observations (Hajnóczky et al., 1999; Pivovarova et al., 2004). It should be noted, however, that a possible role of adaptive changes secondary to mitochondrial dysfunction cannot be ruled out. For example, reduced Ca²⁺ levels in ER or a promoted Ca²⁺ extrusion through the plasma membrane, a decrease in Ca²⁺ influx from plasma membrane can also explain the present findings. Ca²⁺-induced Ca²⁺ release mediated by RyR or IP₃R may also be involved.

Additionally, adenine nucleotides including cAMP (Snyder and Supattapone, 1989; Shimizu et al., 1993; Giovannucci et al., 2000), interactions with other proteins (Gee et al., 2003; Tozzi et al., 2003; Wanaverbecq et al., 2003), or phosphorylation by various kinases (Wagner et al., 2003; Skeberdis et al., 2006) can modulate IP₃R function. Because both serotonin- and thrombin-induced Ca²⁺ mobilizations in platelets are reportedly enhanced in BD patients (Yamawaki et al., 1998), we anticipated that altered mitochondrial Ca²⁺ regulation may enhance agonist-induced [Ca²⁺]_i increase in neurons of mutPOLG Tg mice. Although our results are in the opposite direction to the finding in platelets from patients (Fig. 5), they are compatible with a recent study demonstrating that GPCR-mediated [Ca²⁺]_i increase was diminished in the olfactory neurons from BD patients (Hahn et al., 2005).

Possible role of mitochondrial Ca²⁺ transport in cell death and/or respiratory failure

The enhanced Ca²⁺ uptake in mitochondria from mutPOLG Tg mice was seen in the buffer containing a substrate for complex I (Fig. 1B,F), but not in the buffer containing a substrate for complex II (Fig. 1D,F). Many of the complex I subunits are encoded by mtDNA, whereas all of the complex II subunits are by nuclear DNA. This fact supports the association between the mtDNA defects and the enhanced mitochondrial Ca²⁺ uptake. Similar discrepancy between substrates has been discussed in studies using cybrids from patients with mtDNA mutations (Moudy et al., 1995; Brini et al., 1999) and those from patients with AD or PD (Sheehan et al., 1997a,b). A slower rate of Ca²⁺ uptake in these cybrids was improved by a treatment with succinate, which is the substrate for complex II. This is in accord with our finding that the rate of Ca²⁺ uptake was not different between genotypes when we used the buffer containing succinate.

Unlike mitochondria of the model mice for HD, we found no difference between genotypes in the ΔΨ_m as measured by flow cytometric analysis (Fig. 2; supplemental Fig. 1, available at www.jneurosci.org as supplemental material), and the CRC was not altered in mitochondria of the mutPOLG Tg mice. Preliminary observation showed no different histological features and no marked cell death in the brains of mutPOLG Tg mice. It would be interesting to study how amounts of mutations could enhance Ca²⁺ uptake rate in mitochondria from mutPOLG Tg mice without causing energy dissipation. Another group reported that transgenic mice expressing heart-specific mutPOLG suffered from a cardiomyopathy attributable to an accumulation of mtDNA defects (Zhang et al., 2000). Nevertheless, neither an abnormality of mitochondrial respiration nor a loss of ATP could be detected in the hearts of these mice (Mott et al., 2001; Zhang et al., 2003). More studies are still needed to elucidate the mechanisms by which mtDNA defects alter mitochondrial Ca²⁺ uptake. It will be interesting to see the cellular phenotypes in different lines of mice with accumulation of mtDNA defects, such as knock-in mice of POLG mutation (Trifunovic et al., 2004; Kujoth et al., 2005) and mutant mice with defect in Twinkle (Tyynismaa et al., 2005).

Relationship of altered Ca²⁺ dynamics in behavioral phenotypes of mutPOLG Tg mice

The mutPOLG Tg mice apparently exhibited a different pattern in wheel-running activity from wild-type mice, but were not impaired in a comprehensive series of learning and memory tests (Kasahara et al., 2006). In this study, it was difficult to provide a precise explanation with respect to the cause of behavioral phenotypes in mutPOLG Tg mice. However, the mtDNA defects in neurons might underlie BD-like behavioral phenotypes of mutPOLG Tg mice through the attenuation of GPCR-mediated [Ca²⁺]_i increase and the subsequent intracellular events.