Abstract
Mitochondrial dysfunction is a common hallmark in aging. In the female, reproductive senescence is characterized by loss of ovarian hormones, many of whose neuroprotective effects converge upon mitochondria. The functional integrity of mitochondria is dependent on membrane fatty acid and phospholipid composition, which are also affected during aging. The effect of long-term ovarian hormone deprivation upon mitochondrial function and its putative association with changes in mitochondrial membrane lipid profile in the hippocampus, an area primarily affected during aging and highly responsive to ovarian hormones, is unknown. To this aim, Wistar adult female rats were ovariectomized or sham-operated. Twelve weeks later, different parameters of mitochondrial function (O2 uptake, ATP production, membrane potential and respiratory complex activities) as well as membrane phospholipid content and composition were evaluated in hippocampal mitochondria. Chronic ovariectomy reduced mitochondrial O2 uptake and ATP production rates and induced membrane depolarization during active respiration without altering the activity of respiratory complexes. Mitochondrial membrane lipid profile showed no changes in cholesterol levels but higher levels of unsaturated fatty acids and a higher peroxidizability index in mitochondria from ovariectomized rats. Interestingly, ovariectomy also reduced cardiolipin content and altered cardiolipin fatty acid profile leading to a lower peroxidizability index. In conclusion, chronic ovarian hormone deprivation induces mitochondrial dysfunction and changes in the mitochondrial membrane lipid profile comparable to an aging phenotype. Our study provides insights into ovarian hormone loss-induced early lipidomic changes with bioenergetic deficits in the hippocampus that may contribute to the increased risk of Alzheimer’s disease and other age-associated disorders observed in postmenopause.
Introduction
Mitochondria are key organelles for cellular bioenergetics and survival. They are the main source of ATP production through oxidative phosphorylation by the mitochondrial respiratory chain and also the primary sites for cellular reactive oxygen species (ROS) generation. As an organ with a high demand of energy and low antioxidant capacity, the brain is particularly vulnerable to mitochondria dysfunction and oxidative stress, interdependent mechanisms that play a central role in brain aging (Chakrabarti et al. 2011). Aging is an inevitable biological process characterized by a progressive decline in physiological functions and increased susceptibility to disease. It is now accepted that normal aging and several aging-related diseases, such as Alzheimer’s and Parkinson’s, are related to mitochondrial dysfunction (Chakrabarti et al. 2011, Johri & Beal 2012). Sex steroid hormones, especially estrogens, have been widely investigated in relation to brain aging. Multiple lines of evidence point to brain mitochondria as targets of steroid hormone action (Jones & Brewer 2009, Arnold et al. 2012). In fact, a link between sex-dependent susceptibility and mitochondrial dysfunction has been identified in normal aging as well as in neurodegenerative diseases both in preclinical animal studies and in women after menopause (Rasgon et al. 2005, Yao et al. 2010, Johri & Beal 2012). Collectively, data from animal studies indicate that the decline in whole brain mitochondrial function associated with reproductive senescence is due to loss of ovarian hormones (Yao et al. 2009, 2010, 2012). Unlike primates, the loss of reproductive cycles during aging in rats does not occur concomitant with ovarian follicular atresia and its associated decline in estradiol levels. Rather, middle-aged acyclic rats are in a state of persistent estrus with chronically high estradiol levels and thus are not considered a good model for reproductive aging. Instead, ovariectomy has been widely used as a more appropriate model for reproductive aging in rats (Morrison et al. 2006). Detrimental effects of ovarian hormone deprivation upon mitochondrial bioenergetics include reduced respiration and ATP production rates, increased oxidative stress, and decreased expression and/or activity of metabolic enzymes within this organelle (Irwin et al. 2011, Shi et al. 2011, Yao et al. 2012, Gaignard et al. 2015). The vast majority of animal studies about steroid hormone modulation of mitochondrial function have been performed in the whole brain, without the dissection of different brain areas that have dissimilar response to ovarian hormones. Indeed, brain mitochondrial dysfunction in aged animals is more evident in specific brain areas, among which the hippocampus emerges as an early target of both aging and ovarian hormone loss (Navarro et al. 2008, Paradies et al. 2011, Hara et al. 2015).
The accumulation of oxidative products leading to changes in mitochondrial macromolecules has been proposed as a plausible mechanism underlying the aforementioned alterations induced by ovarian hormone loss and in aging (Paradies et al. 2011, Viña et al. 2011, López-Grueso et al. 2014). Mitochondrial membranes, mostly composed of phospholipids, are main targets of oxidative damage due to their physicochemical properties and the chemical reactivity of their fatty acid (FA) double bonds (Pamplona 2008, Paradies et al. 2011). In fact, membrane phospholipid unsaturation degree has been suggested to play a causal role in aging by modulating oxidative stress and molecular integrity of the membrane, which is particularly important for the proper activity of proteins involved in oxidative phosphorylation (Pamplona 2008, Schenkel & Bakovic 2014).
Similar to other subcellular membranes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the major phospholipids in mitochondrial membranes, comprising 40% and 30% of total mitochondrial phospholipids, respectively (Schenkel & Bakovic 2014). Unlike the plasma membrane, mitochondria contain high levels of cardiolipin (CL), a phospholipid localized almost exclusively in the inner mitochondrial membrane (IMM) that is essential for several mitochondrial processes such as oxidative phosphorylation, apoptosis, mitochondrial protein import and supercomplex formation (Claypool & Koehler 2012). Due to its location near the site of ROS production and to the fact that it is particularly rich in unsaturated FAs, brain CL is highly prone to oxidative attack (Pamplona 2008). During aging, both an increase in the levels of oxidized CL together with a reduction in CL content in brain mitochondria have been reported (Sen et al. 2007, Petrosillo et al. 2008, Jones & Brewer 2009), conditions that have been suggested to contribute to mitochondrial dysfunction in multiple aged tissues (Paradies et al. 2011). Studies on the putative association between ovarian hormone loss and alterations in mitochondrial membrane lipid profile, especially in CL, are still lacking.
In this context, the aim of this work was to assess bioenergetic alterations and mitochondrial membrane lipid profile induced by long-term ovarian hormone deprivation in the hippocampus, a highly hormone-responsive area primarily affected during aging. By doing so, we aimed to get a better understanding on how putative changes in mitochondrial phospholipid profile, especially in CL, are reflected in hippocampal mitochondria dysfunction usually associated with menopause.
Materials and methods
Drugs
All drugs and chemicals were obtained from Sigma Chemical, except for organic solvents (Carlo Erba, Milan, Italy), NAO and DiOC6 (Molecular Probes), phospholipid standards (Avanti Polar Lipids Inc., Alabama, USA) and the materials indicated below.
Animals
Adult female Wistar rats were kept in controlled conditions of light (12 h light–darkness cycles) and temperature (20–22°C). Rats were fed standard lab chow and water ad libitum and kept in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All the procedures were carried out in strict compliance with the ARRIVE and the Ethical Committee of the School of Medicine, University of Buenos Aires guidelines. Rats were ovariectomized (OVX) or sham-operated (SHAM) at three months of age under ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia and ketoprofen (5 mg/kg) for analgesia. Two weeks before the experiments, rats were monitored daily by vaginal smears for normal 4–5 day estrous cycles (SHAM) or continuous diestrus status (OVX). Twelve weeks after the surgery, rats were subjected to behavioral tests and then killed in a CO2 chamber followed by decapitation. Body and uteri weight were recorded. Brains were rapidly removed and hippocampi dissected on ice and immediately processed for mitochondrial isolation.
Behavioral tests
Open field test
The open field test (OFT) was performed to evaluate animal general locomotion and exploratory behavior. The arena consisted of a squared open field (70 × 70 cm) limited by a 40 cm high wall with the floor divided in squares (15 × 15 cm) by lines. Animals were individually placed in the center of the open field arena and were allowed to freely explore for 10 min. Locomotion was measured based on the number of lines crossed with all four paws (crossings). After each animal was tested, the open field was cleaned with a 10% v/v ethanol-damp cloth. Testing was performed between 10:00 and 14:00 h in a quiet room evenly illuminated with cool white lights situated above the center of the apparatus.
Forced swimming test
The forced swimming test (FST) was performed to evaluate animal depressive-like behaviors by quantifying their mobility, immobility and associated behaviors. The FST was performed 24–48 h after the animals were evaluated in the OFT. Each rat was individually placed for 5 min in a plastic cylinder (diameter, 40 cm; height, 35 cm) containing water (23–25°C) up to 25 cm from the bottom. At 5 s intervals throughout the test session, the predominant behavior was assigned to one of the followings categories: (1) immobility: lack of movement, except for the ones needed to keep the head above water; (2) swimming: swimming movement throughout the cylinder or (3) climbing: vigorous movements of the forepaws in and out of the water, usually directed against the walls. After each animal was tested, the water was changed and the cylinder rinsed with clean water. Following the session, each animal was dried and replaced to its housing cage in a temperature-controlled room. All swimming sessions were carried out between 10:00 and 16:00 h.
Mitochondria isolation
Hippocampal mitochondria purified fractions were obtained from tissue homogenates by differential centrifugation (Martino Adami et al. 2017). Briefly, hippocampi were homogenized by five strokes in a glass–Teflon homogenizer in a medium containing 250 mM sucrose, 5 mM Tris–HCl and 2 mM EGTA (pH 7.4) and centrifuged at 700 g for 10 min to discard nuclei and cell debris. Then, the supernatant was centrifuged at 8000 g for 10 min and the enriched mitochondria pellet was resuspended in a minimum volume (250 µL) of the same buffer. The whole procedure was carried out at 4°C. The purity of isolated mitochondria was assessed by determining lactate dehydrogenase activity; only mitochondrial preparations with less than 5% impurity were used (Vanasco et al. 2014).
Mitochondrial membrane preparation
Mitochondrial membranes were obtained by three freeze-thaw cycles of the mitochondrial preparation, followed by a homogenization step by passage through a 29 G hypodermic needle (Vanasco et al. 2014).
Mitochondrial function
O2 consumption
Mitochondrial O2 uptake was measured by high-resolution respirometry (Hansatech Oxygraph, Hansatech Instruments Ltd, Norfolk, England). Briefly, fresh isolated mitochondria at a concentration of 0.25–0.50 mg of protein/mL were incubated in an air-saturated respiration buffer ([O2] = 220 μM) containing 120 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 3 mM HEPES and 1 mg/mL fatty acid-free BSA (pH 7.4) at 30°C. State 4 respiration rates (resting or controlled respiration) were determined using 6 mM malate and 6 mM glutamate or 7 mM succinate as substrates of complex I or II, respectively; ADP (125 μM) was added to trigger state 3 (active respiration). Respiratory control ratio was calculated as the ratio of state 3/state 4 respiration rates. Then, 2 μM oligomycin (Fo–F1 ATP synthase inhibitor) was added to reach 4oligomycin (state 4o) respiration, followed by the protonophore carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) (2 μM) to evaluate mitochondrial uncoupled respiration (state 3u) (Brand & Nicholls 2011). Results were expressed as ng-at O/min mg protein.
ATP production rate and P/O ratio
Mitochondrial ATP production rate was determined using a chemiluminescence assay based on the luciferin–luciferase system. Freshly purified mitochondria were incubated in respiration buffer (150 mM KCl, 25 mM Tris–HCl, 2 mM EDTA, 0.1% (w/v) BSA and 100 μM MgCl2, pH 7.4) in the presence of 40 μM d-luciferin, 0.05 μg/mL luciferase and 150 μM di(adenosine) pentaphosphate. ATP production was triggered by the addition of 6 mM malate and 6 mM glutamate or 7 mM succinate and 125 mM ADP to the reaction well (Drew & Leeuwenburgh 2003). Chemiluminescence was measured at 30°C using a Labsystems Luminoskan RS Microplate Reader (Labsystem, Ramsey, Minnesota, USA) using ATP as standard. A negative control with 2 μM oligomycin was included to confirm that the emitted chemiluminescence was due to ATP synthesis by the Fo–F1 ATP synthase. Results were expressed as nmol ATP/min mg protein. In order to evaluate the efficiency of the oxidative phosphorylation process, the number of phosphorylated ADP molecules per oxygen atom (P/O ratio) was calculated as ATP production/state 3 O2 consumption rates (Brand & Nicholls 2011).
Membrane potential (ΔΨ m)
Freshly isolated mitochondria (100 μg protein/mL) were incubated with the potentiometric cationic probe 3,3′-dihexyloxacarbocyanine iodide (DiOC6, 30 nM) in respiration buffer in the dark at 37°C for 20 min. After the incubation period, mitochondria were analyzed by flow cytometry using a Partec PAS-III flow cytometer (Partec GmbH, Münster, Germany). Mitochondria were gated based on light-scattering properties and 30,000 events within this gate per sample were collected for analysis. 10-N-nonyl acridine orange (NAO, 100 nM) was used to selectively stain mitochondria through binding of cardiolipin and evaluate the purity of mitochondrial preparations. DiOC6 signal was analyzed after the addition of 6 mM malate and 6 mM glutamate (state 4) and 125 mM ADP (state 3) to the reaction mixture and the arithmetic mean values of the median fluorescence intensities (MFI) were obtained. Total depolarization induced by 2 μM m-CCCP was used as a positive control. Mitochondrial preparations that showed no changes in membrane potential under this condition were discarded. Analysis of the data was performed using Cyflogic software (Magnani et al. 2013).
Respiratory chain complex activity
NADH-cytochrome c reductase (complexes I–III) and succinate cytochrome c reductase (complexes II–III) activities were evaluated by a colorimetric assay following cytochrome c3+ reduction rate at 550 nm and 30°C using a spectrophotometer (Beckman DU 7400; ε = 19/mM/cm). Mitochondrial membranes (1.0 mg protein/mL) were added to 100 mM phosphate buffer (pH 7.2), supplemented with 0.2 mM NADH or 5 mM succinate, respectively, plus 25 μM cytochrome c3+ and 0.5 mM KCN. Results were expressed as nmol reduced cytochrome c3+/min mg protein. Cytochrome oxidase (complex IV) activity was determined at 30°C in 100 mM phosphate buffer containing freshly prepared 60 μM cytochrome c2+. The rate of cytochrome c2+ oxidation was calculated as the pseudo-first-order reaction constant k′/min mg protein.
Lipid analysis
Global FA composition of mitochondrial membranes and major phospholipid profile analysis
Total lipids from an aliquot of mitochondrial membranes containing 0.5 mg of protein were extracted by the method of Folch et al. (1957), dried under N2 and saponified with KOH 10% v/v in ethanol (30 min at 85°C). Unsaponified compounds were extracted with hexane. The aqueous phase was acidified with HCl 37% v/v and extracted twice with hexane. Both hexane phases were dried under N2 and FA methyl esters (FAMEs) were synthesized by incubating in BF3 10% v/v in methanol 1 h at 85°C. The resulting FAMEs were extracted with hexane and used to analyze the global FA composition of mitochondrial membranes.
In parallel, total lipids from an aliquot of mitochondrial membranes containing 2 mg of protein were extracted as described above. Phospholipids were separated by 2D high-performance thin layer chromatography (HPTLC) on pre-coated silica gel plates (10 × 20 cm) from Whatman Schleicher & Schuell (Maidstone, England). The mobile phase for the first-D was chloroform:methanol:water:amonium hydroxide (65:25:4:0.5, by volume) and the mobile phase for the second-D was chloroform:acetone:methanol:acetic acid:water (36:48:12:12:6, by volume). Spots were visualized by iodine vapor. CL, PC and PE were identified by comparison with commercial standards and quantified by densitometric analysis using ImageJ sofware (NIH). CL, PC and PE spots were scrapped off the plate and extracted from the silica with chloroform:methanol (1:2 v/v). The extracts were dried under N2, saponified and used to obtain FAMEs as described above to analyze the FA composition of each mitochondrial phospholipid.
FAMEs analysis was performed by gas–liquid chromatography using a capillary column (Omegawax 250) mounted on a Hewlett Packard HP 6890 Series GC System Plus (Avondale, PA, USA). FAMEs were identified by comparison of their relative retention times with authentic standards and mass distribution was calculated by quantification of the peak areas and expressed as relative quantity of each FAME from the total FAMEs in the sample. Peroxidizability index (PI) was calculated as (% monoenoic acids × 0.025) + (% dienoic acids × 1) + (% trienoic acids × 2) + (% tetraenoic acids × 4) + (pentaenoic acids × 6) + (hexaenoic acid × 8) (Pamplona 2008).
Cholesterol content
Cholesterol (CHOL) content was determined enzymatically using a commercial kit (Wienner Lab., Rosario, Argentina).
Protein measurements
Protein content was determined by the Bradford protein assay (BioRad Laboratories) or the method of Lowry et al. (1951), using bovine serum albumin as standard.
Statistical analysis
Results are expressed as mean ± s.e.m. and evaluated by unpaired Student’s t-test. The ‘n’ in each experiment corresponds to the number of animals used. Differences were considered significant if P < 0.05.
Results
Ovariectomy validation parameters
Previous reports have shown that ovarian hormone depletion induces depression-like effects in both young and middle-aged rats (Diz-Chaves et al. 2012, Kiss et al. 2012, da Silva Moreira et al. 2016). We thus evaluated animal performance in the FST as an independent indicator for the efficacy of experimental ovariectomy in our model. Twelve-week ovarian hormone deprivation increased despair behavior in OVX rats, evidenced by an increase in the time spent immobile in the FST (Fig. 1A). Analysis of the animals’ performance in the OFT did not show any difference in spontaneous locomotion between OVX and SHAM groups, indicating that the increased immobility time was not due to changes in locomotor activity (Fig. 1B). Uterine weight was also used as a bioassay to verify the efficacy of long-term ovarian hormone deprivation induced by ovariectomy. As expected, OVX rats showed a decrease in uterine weight relative to body weight when compared with SHAM rats (Fig. 1C).
Evaluation of ovarian hormone depletion parameters in adult rats. Adult Wistar female rats were ovariectomized (OVX) or sham-operated (SHAM). Twelve weeks after surgery, the animals were subjected to a 10-min open field session followed by a 5-min forced swimming session 24 h later. Then, the animals were killed and body and uteri were weighed. (A) Forced swimming test: every 5 s, a time-sampling technique was used to score the presence of immobility, swimming, or climbing behavior. (B) SHAM or OVX rats were placed individually in the center of a field marked with a grid of 12 equal squares for 10 min. The number of times the animal crossed each line was registered. (C) Animals’ body and uterine weight were recorded. Each column represents the mean ± s.e.m. of (A) the number of counts or (B) the number of crossings per session; (C) uterine weight relative to body weight (n = 6–9 animals/group). *P < 0.05, ***P < 0.001, Student’s t-test.
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0451
Mitochondrial function
Mitochondrial O2 uptake and ATP production rates
Increasing evidence indicates that brain mitochondria are targets of gonadal steroid action and, consequently, ovarian hormone loss during reproductive senescence has been associated with whole brain energetic deficits (Yao et al. 2012, Velarde 2014). We evaluated different parameters of mitochondrial function in isolated hippocampal mitochondria from long-term OVX rats. We first measured oxygen consumption using NADH-dependent substrates malate–glutamate. Representative traces are shown in Fig. 2. While ovariectomy did not affect the rate of resting respiration, it significantly decreased mitochondrial respiration in the active state (i.e. the respiratory activity that sustains ATP formation) (Table 1). We further assessed mitochondrial respiration in the presence of the Fo–F1 ATP synthase inhibitor oligomycin and the protonophore m-CCCP. Inhibition of ATP synthesis by oligomycin (state 4o) yielded a slower respiration rate while uncoupling by m-CCCP (state 3u) accelerated the rate of oxygen consumption (Table 1). No differences between groups were observed in state 4o respiration rate, which mainly reflects proton leak. Also, since state 3u is exclusively dependent on substrate oxidation and electron transfer rates, no differences between groups may indicate lack of inhibition at the mitochondrial electron transport chain. On the other hand, ATP production rate was lower in hippocampal mitochondria from OVX rats (Fig. 3). Calculation of the P/O ratio showed no differences between groups, indicating that the lower chemical energy production in mitochondria from OVX rats is due to the observed decreased state 3 oxygen uptake rather than a hindered oxidative phosphorylation (Table 1).
Representative traces obtained during the assessment of mitochondrial O2 uptake by hippocampal mitochondria from SHAM (thick, black line) and OVX (thin, gray line) rats. O2 consumption was measured in resting (state 4) and active (state 3) metabolic states using malate–glutamate as complex I substrates and after the addition of 2 μM oligomycin (state 4o) and 2 μM m-CCCP (state 3u) to the reaction chamber.
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0451
ATP production rates of freshly isolated hippocampal mitochondria. ATP production rates were evaluated in isolated hippocampal mitochondria from SHAM and OVX rats by chemiluminescence. ATP production was triggered by the addition of malate–glutamate as complex I substrates or succinate as complex II substrate and ADP to the reaction chamber. Results are expressed as mean ± s.e.m. (n = 3–5 animals/group). *P < 0.05, Student’s t-test.
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0451
Oxygen consumption rates and P/O ratios of freshly isolated hippocampal mitochondria.
| SHAM | OVX | |
|---|---|---|
| NADH-linked respiration (substrate malate–glutamate) | ||
| State 4 | 22.0 ± 1.0 | 21.0 ± 2.0 |
| State 3 | 69.0 ± 3.0 | 49.0 ± 7.0* |
| RCR | 3.1 ± 0.4 | 2.6 ± 0.7 |
| State 4o | 43.0 ± 5.0 | 38.0 ± 5.0 |
| State 3u | 61.0 ± 2.0 | 63.0 ± 3.0 |
| P/O | 2.1 ± 0.3 | 2.0 ± 0.3 |
| FADH2-linked respiration (substrate succinate) | ||
| State 4 | 43.0 ± 2.0 | 43.0 ± 2.0 |
| State 3 | 68.0 ± 5.0 | 59.0 ± 6.0 |
| RCR | 1.6 ± 0.1 | 1.4 ± 0.2 |
| State 4o | 49.0 ± 10.0 | 41.0 ± 12.0 |
| State 3u | 76.0 ± 10.0 | 54.0 ± 10.0 |
| P/O | 1.2 ± 0.1 | 1.0 ± 0.1 |
Oxygen uptake rates were evaluated in isolated hippocampal mitochondria from SHAM and OVX rats by high-resolution polarography. Resting respiration (state 4) rate was determined with malate–glutamate as complex I substrates or succinate as complex II substrate; active respiration (state 3) rate was determined by adding ADP in both cases. RCR was calculated as state 3/state 4 respiration rates. State 4o was induced by oligomycin (2 μM), while m-CCCP (2 μM) was used to establish state 3u respiration. Oxygen uptake is expressed as ng-at O/min mg protein. P/O ratios were calculated as ATP production/state 3 O2 consumption rates. Results are expressed as mean ± s.e.m. (n = 3–5 animals/group). *P < 0.05, Student’s t-test.
Unlike what was observed for NADH-associated respiration, there were no differences in either active or basal respiration or ATP production rates between the experimental groups when FADH2-linked respiration was promoted by using succinate as a substrate for complex II (Table 1 and Fig. 3).
Mitochondrial membrane potential (ΔΨ m)
We then evaluated mitochondrial membrane potential (ΔΨm), the charge gradient across the IMM that ultimately regulates ATP production. Isolated hippocampal mitochondria were selected from background by light-scattering and NAO-binding properties (Fig. 4A and B). As it is shown in the overlaid histograms and DIOC6 MFI quantification, hippocampal mitochondria from OVX rats showed no changes in ΔΨm during resting respiration (Fig. 4C and D). However, when active respiration was established, OVX ΔΨm was lower than SHAM ΔΨm, indicating a significant depolarization in isolated mitochondria from long-term ovarian hormone-deprived rats (Fig. 4C and D).
Assessment of hippocampal mitochondria membrane potential (ΔΨm) by flow cytometry. Mitochondrial membrane potential in states 4 and 3 was evaluated in isolated hippocampal mitochondria from SHAM and OVX rats using the potentiometric cationic probe DiOC6 and flow cytometry. (A) Representative dot blots when selecting hippocampal mitochondria from SHAM and OVX rats based on light scattering properties. (B) Representative histograms showing fluorescence of the events in the selected gate in (A) for NAO stained mitochondria (black line) compared to an unstained mitochondrial sample (gray line) in SHAM and OVX samples. (C) Representative histograms of DiOC6 fluorescence from gated hippocampal mitochondria from SHAM and OVX rats. (D) DiOC6 median fluorescence intensity (MFI) quantification of state 4 and state 3 in hippocampal mitochondria from SHAM and OVX rats. Each column represents the mean ± s.e.m. of DiOC6 MFI in resting (state 4) or active (state 3) respiration (n = 4–6 animals/group). *P < 0.05, Student’s t-test.
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0451
Mitochondrial respiratory chain complex activity
In order to study the source for this mitochondrial dysfunction with shortage of energy supply, we evaluated the enzymatic activity of respiratory complexes in mitochondrial membranes. We found no differences in the activity of any of the complexes, as shown in Table 2.
Colorimetric assessment of hippocampal mitochondria respiratory chain complex activities.
| SHAM | OVX | |
|---|---|---|
| Complexes I–III (nmol/min mg protein) | 106.0 ± 4.0 | 103.0 ± 4.0 |
| Complexes II–III (nmol/min mg protein) | 35.8 ± 2.8 | 33.3 ± 2.2 |
| Complex IV (k′/min mg protein) | 3.1 ± 0.1 | 3.2 ± 0.2 |
The enzymatic activity of NADH-cytochrome c reductase (complexes I–III), succinate cytochrome c reductase (complexes II–III) and cytochrome oxidase (complex IV) was evaluated spectrophotometrically in hippocampal mitochondria membranes from SHAM and OVX rats. Results are expressed as mean ± s.e.m. (n = 6 animals/group). Ns, Student’s t-test.
Mitochondrial lipid analysis
Lipid composition and FA profile of mitochondrial membranes
The observed bioenergetics deficits in hippocampal mitochondria from OVX rats may be related to an altered lipid composition of mitochondrial membranes. Thus, we analyzed lipid components of mitochondrial membranes, including cholesterol (CHOL), FA composition as well as content and FA composition of main individual phospholipids. Total lipids were extracted from hippocampal mitochondrial membranes of OVX and SHAM rats and CHOL content was determined. As shown in Fig. 5A, CHOL levels were similar between the two groups (Fig. 5A). On the other hand, we found that long-term ovarian hormone deprivation altered the FA profile of mitochondrial membranes by decreasing the proportion of saturated palmitic (16:0) and unsaturated oleic (18:1 n-9) acids together with increasing saturated arachidic (20:0) and polyunsaturated (PUFA) arachidonic (20:4 n-6) acids (Fig. 5B). This FA composition led to a higher PI in hippocampal mitochondrial membranes from OVX rats (Fig. 5C).
Cholesterol content, fatty acid composition and peroxidizability index of hippocampal mitochondrial membranes. Total lipids from mitochondrial membranes from SHAM and OVX rats were extracted by the method of Folch et al., saponified and converted to their corresponding fatty acid methyl esters (FAMEs). FAMEs were used to analyze the global fatty acid composition of hippocampal mitochondrial membranes by gas–liquid chromatography (GLC). Cholesterol (CHOL) content was determined spectrophotometrically in mitochondrial membranes from SHAM and OVX rats. Each column represents the mean ± s.e.m. of (A) CHOL content, (B) relative quantity of each FAME, or (C) peroxidizability index (PI) (n = 7–8 animals/group). *P < 0.05; **P < 0.01, Student’s t-test.
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0451
Mitochondrial phospholipid content and phospholipid FA profile
In parallel, phospholipids were isolated from lipid extracts by HPTLC fractionation and the relative abundance of CL, PC and PE were assessed. Long-term ovarian hormone deprivation promoted a significant decrease (20%) in CL content without changes in the levels of PC or PE (Fig. 6A). The analysis of the FA profile for each phospholipid showed that CL from hippocampal mitochondria from OVX rats contained an increased proportion of saturated palmitic acid and a PUFA profile that led to a lower PI for this phospholipid. A similar result was observed for PC. On the other hand, PE showed a higher proportion of docosatetraenoic (22:4) and docosahexaenoic (22:6) acids leading to a higher PI (Fig. 6B and C). These results show that long-term ovarian hormone deprivation alters the FA composition within major mitochondrial phospholipids and suggest that PE accounts for the higher global PI of hippocampal mitochondrial membranes.
Content, fatty acid composition and peroxidizability index of main phospholipids from hippocampal mitochondrial membranes. Total lipids from mitochondrial membranes from SHAM and OVX rats were extracted and phospholipids were separated by HPTLC. Cardiolipin (CL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified by comparison with commercial standards and quantified by densitometric analysis using ImageJ software. CL, PC and PE spots were scrapped, extracted and converted to their corresponding FAMEs and analyzed by GLC. Each column represents the mean ± s.e.m. of (A) the relative change in each phospholipid content respect to SHAM, (B) relative quantity of each FAME, or (C) peroxidizability index (PI) (n = 7–8 animals/group). *P < 0.05; **P < 0.01, Student’s t-test.
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0451
Discussion
The brain consumes 20% of the body fuel to sustain its high demand of energy in the form of ATP and thus is particularly susceptible to a bioenergetic decline if mitochondrial function is impaired (Rettberg et al. 2014). Multiple epidemiologic studies reported that women are more prone to develop dementia and Alzheimer’s disease than men (Fratiglioni et al. 1997, Gao et al. 1998, Andersen et al. 1999, Zandi et al. 2002), and suggested a strong link with the drop in circulating ovarian hormones during menopause and reduced brain bioenergetics (Maki & Resnick 2000, Rasgon et al. 2005). Studies in animals undergoing natural or surgical reproductive senescence have also shown a decline in the bioenergetic system of the brain, from decreased glucose metabolism to impaired mitochondrial function (Yao et al. 2009, 2010, 2012). However, most (if not all) of these studies have been performed in the whole brain. Considering the variety and complexity of metabolic conditions in different brain areas, we chose to perform this study in the hippocampus for its exceptional vulnerability to the detrimental effects of aging and loss of ovarian hormones (Navarro et al. 2008, Paradies et al. 2011, Hara et al. 2015). Evidence supporting this fact comes from several studies showing synaptic decline in this brain area associated with cognitive impairment and increased risk of neurodegeneration after experimental endocrine senescence in animal models and menopause in women (Morrison et al. 2006, Brinton 2009, Velarde 2014, Hara et al. 2015). Also, a more marked mitochondrial dysfunction is observed in the hippocampus than in brain cortex or whole brain, and oxidative damage is higher in the hippocampus than in whole brain in male rodents during aging (Navarro et al. 2008). Moreover, ovariectomy induces a sharp decrease in the activity of the antioxidant enzyme superoxide dismutase in the hippocampus but not in the cortex of young rats (Huang & Zhang 2010).
Our results show that long-term ovarian hormone deprivation induces a decline in mitochondrial bioenergetics in the hippocampus, specifically a reduction in active respiration and ATP production rates without changes in the rate of basal respiration with NADH-dependent substrates. Also, membrane depolarization was only observed during active respiration, which could account for the decrease in ATP production rate in hippocampal mitochondria from OVX rats. This lower membrane potential may indicate a defective formation of a proper proton gradient across the IMM during active respiration in mitochondria from OVX rats. Our results are in line with those obtained in isolated mitochondria from whole brain in OVX mice, in which ovariectomy induced a deficit in state 3 respiration without altering state 4 respiration both in wild-type mice and in the 3xTg-AD mouse model of Alzheimer’s disease (Yao et al. 2012). On the other hand, FADH2-linked respiration, either active or basal, was not affected by ovarian hormone loss, suggesting the involvement of complex I alterations in mitochondrial dysfunction. One possible explanation for altered complex I function could be a reduction in its activity. Indeed, mitochondrial dysfunction in terms of reduced complex I and IV enzymatic activities and decreased mitochondrial respiration with NADH-dependent substrates has been consistently observed in brain mitochondria from aged rats and mice (Navarro & Boveris 2004, Cocco et al. 2005, Navarro et al. 2005). Also, decreased bioenergetics in synaptic mitochondria from whole brain is mainly associated with a decay in complex I activity in 3xTg-AD mice at an early, pre-symptomatic stage of the disease (Monteiro-Cardoso et al. 2015). However, our results show no changes in the enzymatic activity of any of the respiratory complexes and are in agreement with those obtained in a recent report in whole brain mitochondria from OVX mice (Gaignard et al. 2015). The apparent discrepancy between decreased mitochondrial respiration in state 3 when using NADH-related substrates in intact mitochondria and lack of differences in complex I–III enzymatic activity evaluated in mitochondrial membrane preparations could be methodological in nature. It has been reported that freeze-thawing cycles to obtain preparations of mitochondrial membranes alter the organization of the IMM complexes, allowing detection of the activity of only individual complexes or smaller aggregates (Parenti et al. 1987). These smaller aggregates would exhibit less efficient collision-based electron transport behavior instead of stoichiometric behavior, characterized by kinetically advanced substrate/electron channeling, observed in the presence of supercomplexes in intact mitochondria (Lenaz & Genova 2007, Genova & Lenaz 2014). It can thus be hypothesized that, when assessing complex I–III activity in mitochondrial membrane preparations, NADH oxidation is less efficient and thus no changes are found in complex I–III activity between experimental groups.
Also, defects in complex I substrate transport could account for the involvement of complex I alterations in mitochondrial dysfunction induced by hormone deprivation. It is well established that the respiratory rate in state 3 is limited by not only the activity of the individual complexes of the respiratory chain, but also substrate permeation and adenine nucleotide translocation. The decreased state 3 respiration rate with malate–glutamate in brain mitochondria from aged rats has been ascribed to glutamate uptake impairment that could involve the glutamate-OH antiporter or the glutamate/aspartate exchanger (Vitorica et al. 1985). Also, the low activity of the phosphate carrier, one of the transporters involved in the uptake of some of the Krebs-cycle intermediates such as malate, has been suggested to account for the decreased energy metabolism typically found in liver mitochondria during aging (Paradies et al. 1991). On the other hand, state 3 respiration rate when using succinate as a substrate was not significantly modified, indicating that succinate permeation and oxidation were not altered in brain mitochondria from old rats (Vitorica et al. 1985). Although the ADP/ATP carrier is also a major regulator of state 3 respiration, the fact that succinate-induced active oxygen uptake is not altered in hormone-deprived animals makes very unlikely for impaired ADP uptake as the cause of decreased NADH-linked bioenergetic capacity. Further experiments are needed to test whether ovarian hormone loss impairs the aforementioned complex I substrate transporters.
A further explanation for the observed ovariectomy-related complex I alterations leading to mitochondrial dysfunction is that the lipid milieu of the IMM may be altered due to ovarian hormone loss. An altered IMM is expected to influence the stability and activity of the proteins embedded in it, both respiratory complexes and substrate carriers (Vitorica et al. 1985, Gómez & Hagen 2012). Our results show that long-term ovarian hormone deprivation induces changes in the FA profile of mitochondrial membranes that renders them more prone to peroxidation, indicated by a higher PI. This fact may alter membrane fluidity while cholesterol levels remain the same (Choe et al. 1995). Although a relative increase in the PUFA content of a membrane would be expected to render it more fluid, the opposite has been systematically reported during aging; membrane fluidity decreases while PI increases with age (Hulbert et al. 2007, Naudí et al. 2013). A possible explanation for this paradox is that PUFAs, being more vulnerable to oxidative attack, experience greater lipid oxidative damage and the resulting endoperoxide reactive intermediates have been reported to significantly contribute to membrane rigidity and loss of membrane function (Naudí et al. 2013). Indeed, it has been suggested that the age-related decrease in mitochondrial membrane fluidity can have a considerable impact on the activity of the respiratory complexes as well as the generation of the proton gradient (Kwong & Sohal 2000). Age-induced mitochondrial transporter deficiencies have been suggested to be due to either a general alteration of membrane lipid composition/fluidity or to a more confined change in the lipid milieu surrounding carrier molecules in the IMM, such as a lower content of cardiolipin (Paradies et al. 1991, Vitorica et al. 1985). Age-related modifications in the IMM lipid content and composition have been broadly reported as causing a deleterious effect in membrane structural organization, which in turn contributes to mitochondrial dysfunction (Gómez & Hagen 2012). Particularly, membrane unsaturation degree has been extensively related to aging. An increase in membrane PI and lipid peroxidation during aging in an organ-dependent manner has also been reported (Pamplona 2008). Thus, our results suggest that ovarian hormone loss induces a mitochondrial phenotype similar to an aging-related one in terms of higher susceptibility to membrane peroxidation concomitant with impaired mitochondrial bioenergetic capacity.
Among a variety of factors, both ovarian hormones and aging have been shown to regulate the expression of elongases and desaturases, which together with the peroxisomal beta-oxidation pathway are involved in the production of all the diversity of FAs present in a cellular membrane (Stark et al. 2003, Xu et al. 2007, Marks et al. 2013, Naudí et al. 2013). The expression of elongase 6, which preferentially elongates saturated FAs such as 16:0 palmitic acid, is increased specifically in the hippocampus of old mice and is considered an aging-associated gene (Xu et al. 2007). In addition, the expression of this enzyme increases in the liver of OVX animals (Marks et al. 2013). Postmenopausal women have a lower percentage of 16:0 in serum phospholipids as compared with premenopausal women and postmenopausal women receiving hormone therapy (Stark et al. 2003). In addition, the protein expression of stearoyl-CoA desaturase 1 (SCD1), involved in the generation of 18:1 n-9 by delta-9 desaturation of 18:0, is higher in the liver of estradiol and progesterone-treated rats, suggesting that ovarian hormones are implicated in MUFA biosynthesis through elongase 6 and SCD1 (Marks et al. 2013). Interestingly, we observed decreased levels of 16:0 and 18:1 n-9 in mitochondria membranes from OVX rats, suggesting that ovarian hormones may also be involved in MUFA biosynthesis through these enzymes in the hippocampus. Regarding PUFA biosynthesis, ovariectomy has been reported to increase the transcript levels of hepatic Δ6- and Δ5-desaturases (Alessandri et al. 2011). Notably, we observed that ovarian hormone deprivation increased the levels of 20:4 and 22:6 FAs, which can be generated as the products of Δ6- and Δ5-desaturases. It can therefore be speculated that ovarian hormone loss may promote a higher PI in mitochondria membranes through the modulation of the expression and/or activity of specific elongases and desaturases in the hippocampus.
Among membrane lipids, CL and PE are crucial for mitochondrial functions (Genova & Lenaz 2014). Due to their particular conical shape, they facilitate membrane bending and can provide order to surrounding lipids (Zeczycki et al. 2014). Modifications in the content, structure or acyl chain composition of CL and PE are expected to directly impact on the properties of the membrane and on the efficiency of the respiratory complexes. CL is almost exclusively located in the IMM, where it is critical for the proper folding and functioning of a number of proteins playing an important role in mitochondrial bioenergetic processes (Raja & Greenberg 2014). A significant decline in CL content, alterations in its acyl chain composition and/or CL peroxidation have been associated with mitochondrial dysfunction in multiple tissues during aging and in a variety of pathological conditions, including ischemia, hypothyroidism, heart failure and Alzheimer’s disease (Gómez & Hagen 2012, Paradies et al. 2014, Monteiro-Cardoso et al. 2015). Our present data from HPTLC fractionation of mitochondrial phospholipids showed a specific decrease in CL content in hippocampal mitochondria from OVX rats, suggesting that ovarian hormones may be involved in the maintenance of CL levels. In fact, in vitro 17β-estradiol treatment prevents age-related loss of CL and improves neuronal respiration in response to glutamate in cultured aging neurons (Jones & Brewer 2009). Alterations in normal CL biosynthesis and remodeling have been associated with numerous pathological situations. It has been reported that failures in CL remodeling leads to decreased CL content due to reduced stability or increased degradation of CL with abnormal side chain composition, which is shifted toward more saturated species (Lu & Claypool 2015). Interestingly, we observed that ovariectomy induced an altered CL fatty acid profile characterized by increased levels of saturated palmitic acid and reduced levels in PUFAs, suggesting that ovarian hormone loss may alter CL remodeling and/or increase CL oxidation resulting in the observed FA profile. Similarly, PC FA profile showed decreased PUFA levels, resulting in a lower PI. On the other hand, the opposite was observed for PE FA profile, with increased levels of PUFAs resulting in a higher PI, which could account for the general higher PI of hippocampal mitochondria membranes from OVX rats. It can be speculated that alterations in CL remodeling, which uses PC or PE as substrates for FA transacylation, could explain the altered phospholipid FA profiles found in long-term hormone-deprived animals. Despite that sequences of estrogen-responsive elements have been found in the promoters of enzymes involved in CL remodeling (Acaz-Fonseca et al. 2017), further analysis of these pathways should be performed to test this hypothesis.
Extensive evidence indicates that, in addition to affecting the stability and catalytic activity of individual complexes, CL stabilizes respiratory supercomplexes, quaternary supramolecular structures that favor efficient electron transfer by directly transferring electrons among individual complexes without its diffusion in the bulk medium (Gómez & Hagen 2012, Genova & Lenaz 2014). Moreover, it has been reported that the stability of complex I is highly dependent on its assembly within the respiratory supercomplex (Genova & Lenaz 2014). The interactions between membrane lipids and proteins in the IMM are key to maintaining supercomplex stability. It has been suggested that altered CL acyl chain content induces age-related destabilization of brain cortical supercomplexes (Frenzel et al. 2010). Therefore, alterations induced by ovarian hormone deprivation in CL content and/or its FA composition could compromise the stability of respiratory supercomplexes that, in turn, would affect the stability of complex I leading to decreased active respiration with NADH-linked substrates in hippocampal mitochondria. In fact, it has been reported that supercomplexes are dispensable for maintaining basal respiration but essential for supporting active respiration and bioenergetic reserve capacity (Gómez & Hagen 2012). Since complex I needs to be assembled into the NADH oxidase supercomplex for its optimal redox activity (Stroh et al. 2004), it can be speculated that reduced complex I redox energy leads to lower proton translocation across the IMM, resulting in lower membrane potential and decreased respiration with NADH-dependent substrates. Moreover, mitochondrial dysfunction and deficiency of respiratory supercomplexes are correlated to the mitochondrial aging phenotype and in the etiopathology of several diseases like Parkinson’s and Alzheimer’s (Seelert et al. 2009, Frenzel et al. 2010, Kuter et al. 2016). On the other hand, complex II does not form part of respiratory supercomplexes, which is in agreement with lack of alterations in FADH2-linked respiration and further supports the putative involvement of supercomplex destabilization induced by loss of ovarian hormones.
Taken together, our data support the hypothesis that hippocampal mitochondria dysfunction in terms of bioenergetic decay and altered mitochondria membrane phospholipid composition are interconnected players contributing to the early bioenergetic decay during menopause. It has been previously suggested that loss of ovarian hormones precipitates the decline in mitochondrial bioenergetics promoting an accelerated aging phenotype eventually leading to the development of brain hypometabolism, which is clinically observed in menopausal women and prodromal Alzheimer’s disease brains (Yao et al. 2009). Our data agrees with and further expands this hypothesis in terms of the aging effects of ovarian hormone loss in mitochondrial membrane composition. In line with this, the maintenance of membrane properties emerges as a putative therapeutic target worth exploring to avoid early impairments in mitochondrial energy expenditure that affects the high-energy-demanding brain after natural or surgical loss of ovarian hormones.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by ANPCYT (PICT 2014-1549 and PICT 2014-0334), CONICET (PIP 0159 and PIP 0321) and UBA (UBACyT (2013–2016) 20020120300064BA and UBACYT (2014–2017) 20020130100786). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgements
We thank Dr Daniel Pisera for critical reading of the manuscript and Dr Martin Codagnone for his helpful assistance with behavioral tests.
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