Effects of long-term Rice Bran Extract supplementation on survival, cognition and brain mitochondrial function in aged NMRI mice
- Publikations-Art
- Zeitschriftenbeitrag (peer-reviewed)
- Autoren
- Hagl, S., Asseburg, H., Heinrich, M., Sus, N., Blumrich, E.-M., Dringen, R., Frank, J. & Eckert, G. P
- Erscheinungsjahr
- 2016
- Veröffentlicht in
- NeuroMolecular Medicine
- DOI
- doi: 10.1007/s12017-016-8420-z)
- Seite (von - bis)
- pp 347–363
Life expectancy has risen considerably over the last few decades and increased from 40 years in the early 1900s to around 80 years in the year 2000 in developed countries (Park 2010). Advanced age is a major risk factor for age-related diseases like cancer, cardiovascular and neurodegenerative diseases (Lagouge and Larsson 2013).
Mitochondrial dysfunction has been found to play a major role in brain aging and the development of neurodegenerative diseases like Alzheimer’s (AD) and Parkinson’s (PD) disease (Chistiakov et al. 2014; Müller et al. 2010; Navarro and Boveris 2010). Mitochondrial dysfunction occurs very early in brain aging and AD progression (Leuner et al. 2007) and has recently even been proposed to be the missing link between brain aging and sporadic AD (Grimm et al. 2015). Accordingly, mitochondrial dysfunction is a valuable target for the prevention of brain aging and AD (Eckert et al. 2012).
Plant components such as polyphenols as well as plant extracts and polyunsaturated fatty acids have already been shown to prevent mitochondrial dysfunction (Afshordel et al. 2015; Schaffer et al. 2012) and AD (Barnard et al. 2014; Howes and Perry 2011; Kim and Oh 2012). We recently reported that a stabilized rice bran extract containing vitamin E congeners and γ-oryzanol had beneficial effects on mitochondrial function in PC12 cells (Hagl et al. 2015a) and in the brains of young guinea pigs (Hagl et al. 2013). Furthermore, the extract improved mitochondrial dysfunction in a cell culture model of AD (Hagl et al. 2015b) as well as in the brains of aged NMRI mice (Hagl et al. 2016). Until now, the effects of rice bran extract have only been examined in short-term in vivo studies (feeding period 3–4 weeks) using oral gavage. To mimic the use of rice bran extract as food additive, we have now examined brain mitochondrial function in aged NMRI mice after long-term administration (feeding period 6 months) of a pelleted diet containing rice bran extract. Furthermore, we tested cognitive function of the study mice before and after the intervention to examine possible beneficial effects of improved brain mitochondrial function on cognition.
Materials and MethodsChemicals
Unless otherwise stated, chemicals were of highest available purity and purchased from Sigma (St. Louis, MO, USA) or Merck (Darmstadt, Germany). Heat-stabilized Egyptian rice bran extract (RBE) was obtained from IT&M SA (Giza, Egypt). After overnight maceration in ethanol, three successive extractions were performed under reflux at 40 °C. The extract was dried under vacuum at a temperature not exceeding 50 °C. The vitamin E profile of the RBE was quantified by HPLC as described elsewhere (Grebenstein and Frank 2012) and was as follows (all values in µg/g): α-tocopherol, 86; β-tocopherol, 71; γ-tocopherol, 288; δ-tocopherol, 93; α-tocotrienol, 55; β-tocotrienol, not detectable; γ-tocotrienol, 2226; δ-tocopherol, 266.
Animals and Treatment
Female NMRI (Naval Medical Research Institute) mice were purchased from Charles River (Sulzbach, Germany) and kept in the animal facility of the Pharmacological Institute until they reached the age of 12 months. Young (3 weeks old) and aged (12 months old) NMRI mice were divided into groups of 15 animals. The aged control group had ad libitum access to a standard pelleted diet (cat. no. 1324 with the addition of vitamin A (2500 IU/kg), vitamin E (20 mg/kg) and selenium (150 mcg/kg), Altromin, Lage, Germany) for 6 months. The young control group had ad libitum access to an identical pelleted diet for 3 months and the aged intervention group had ad libitum access to an identical pelleted diet spiked with ethanolic rice bran extract (RBE, 4 g/kg diet) for 6 months. All mice also had ad libitum access to drinking water. Feeding of young control mice started 3 months later than feeding of aged mice to ensure that the feeding periods ended at the same time. Behavioral testing was performed before the starting points and at the end of the feeding period. At the end of the feeding period, mice were killed by decapitation. The brain was quickly dissected on ice after removal of the cerebellum, the brain stem and the olfactory bulb.
Passive Avoidance Test
The passive avoidance test was conducted using a passive avoidance step through system (cat. no. 40553/mice, Ugo Basile, Gemonio, Italy) and a protocol similar to the protocol published by (Shiga et al. 2016). On day one of the trial, the mouse was put into the light chamber (light intensity 75 %). After 30 s, the door toward the dark chamber was opened and the time was recorded that the mouse needed to cross into the dark chamber. When the mouse entered the dark chamber, a light electric shock (0.5 mA, duration 1 s) was applied. If the mouse did not enter the dark chamber, the test was aborted after 180 s. 24 h after the first trial, the mouse was again put in the light chamber. After 5 s, the door toward the dark chamber was opened and the time was recorded that the mouse needed to enter the dark chamber. No electric shock was applied when the mouse entered the dark chamber. If the mouse did not enter the dark chamber, the test was aborted after 300 s.
One-Trial Y-Maze Test
The test was conducted in a custom-made Y-maze (material: polyvinyl chloride, length of arms: 36 cm, height of arms: 7 cm, width of arms: 5 cm, angle between arms: 120°) which was covered with a plate of acrylic glass. At the beginning of the test, the mouse was put into one of the three arms and the sequence of entries into the arms was recorded for 5 min. After the test, the alternation rate (an alternation is the successive entering into all three arms without entering one arm twice) is calculated using the formula [number of alternations/number of entries]/2 (Wolf et al. 2016). The alternation rate is correlated with cognitive function, while the number of entries into the arms is correlated with physical activity and curiosity.
Preparation of Dissociated Brain Cells and Measurement of MMP and ATP Concentrations
Dissociated brain cells (DBC) were prepared from one brain hemisphere according to a previously published protocol (Hagl et al. 2013). Measurement of mitochondrial membrane potential (MMP) and ATP concentrations was accomplished as described elsewhere (Hagl et al. 2013). Additionally to measurement of basal MMP and ATP levels, DBC were treated with sodium nitroprusside (SNP); this induction of nitrosative and oxidative stress should enhance conditions of increased oxidation found in aging and neurodegeneration. For MMP measurement, DBC were incubated with SNP (2 mM) for 3 h; for ATP measurements, DBC were incubated with SNP (0.1 mM) for 3 h.
Isolation of Brain Mitochondria and High-Resolution Respirometry
Half a brain hemisphere (the frontal part) was used to isolate mitochondria for high-resolution respirometry. The protocol for the isolation procedure is described elsewhere (Hagl et al. 2013). The pellet obtained from the last centrifugation step was dissolved in 250 µl MIRO5 [a mitochondrial respiration medium with protease inhibitor (Complete, Roche, Mannheim, Germany), see (Hagl et al. 2013)] and 80 µl was injected into the Oxygraph-2k chamber. To investigate the function of the respiratory system, a complex protocol (elaborated by Prof. Dr. Erich Gnaiger) was applied including different substrates, uncouplers and inhibitors. After adding mitochondria into the two chambers of the Oxygraph-2k, the capacity of oxidative phosphorylation (OXPHOS) was determined with complex I-related substrates (CI) pyruvate (5 mM), malate (2 mM) and ADP (2 mM) followed by addition of succinate (10 mM, CI + II). Mitochondrial integrity was examined via addition of cytochrome c (10 µM). Subsequently, oligomycin (2 µg/ml) was added to measure leak respiration (leak (omy)) and uncoupling was achieved by addition of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, injected stepwise up to 1–1.5 µM). Complex II respiration in the non-coupled state (CIIETS) was monitored after adding rotenone (0.5 µM) into the chambers. Residual oxygen consumption (ROX), oxygen consumption caused by enzymes which do not belong to the electron transfer system (ETS), was determined after inhibition of complex III by addition of antimycin A (2.5 µM) and was subtracted from all respiratory parameters. COX activity was measured after ROX determination by applying 0.5 mM tetramethylphenylenediamine (TMPD) as an artificial substrate of complex IV and 2 mM ascorbate to keep TMPD in the reduced state. Autoxidation rate was determined after the addition of sodium azide (≥100 mM), and COX respiration was additionally corrected for autoxidation.
Citrate Synthase Activity
Citrate synthase activity was determined photometrically in isolated brain mitochondria as recently described in Hagl et al. (2013).
Protein Quantification
Protein contents were quantified according to the BCA method using a Pierce™ BCA Protein Assay Kit (Fisher Scientific, Waltham, MA, USA).
Western Blot
Western blot analyses were performed using roughly 30 mg of the rear half of a mouse brain hemisphere. Brain homogenate was prepared in lysis buffer (1 mM EDTA, 0.5 % Triton X-100, 5 mM NaF, 6 M urea, 2.5 mM tetrasodium pyrophosphate, 1 mM sodium orthovanadate, 0.5 % sodium deoxycholate, 0.5 % sodium dodecyl sulfate in phosphate-buffered saline) with freshly added protease inhibitors (aprotinin 1.7 mg/ml, leupeptin 5 mg/ml, pepstatin 5 mg/ml, PMSF 100 µM). Samples were electrophoresed on self-casted polyacrylamide gels or ready-bought NuPAGE™ Novex™ 4–12 % Bis–Tris protein gels (Invitrogen, Carlsbad, USA) after application of 20 µg protein per lane and transferred to polyvinylidene fluoride (PVDF) membranes (Roth, Karlsruhe, Germany). Afterward, the membranes were incubated with the respective primary antibodies (MitoProfile Total OXPHOS (ab110411), Opa1 (ab42364), PGC1α (ab106814), Tubulin (ab6160), AMPK (ab80039), BDNF (ab72439), NRF1 (ab34682), CREB phosphor S133 (ab32096), CREB (ab31387), Citrate Synthase (ab96600), TFAM (ab131607), Abcam, Cambridge, UK; Mfn1 (ABC41), GAPDH (MAB374), Millipore, Billerica, USA; Drp1 (A303-410A), Bethyl, Montgomery, USA; Fis1 (A6-25B-0007), Adipogen, San Diego, USA) and secondary antibodies (Calbiochem, Germany) conjugated to horseradish peroxidase. Antibodies were diluted according to manufacturer’s instructions and processed for visualization by Luminata reagents (Millipore, Darmstadt, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or tubulin served as loading control. Band analysis was performed using BioRad’s Quantity One software.
Quantitative Real-Time PCR (qRT-PCR)
Brain tissue was stabilized with RNAlater (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at −80 °C. Total RNA was isolated from brain tissue (roughly 30 mg from the rear part of a brain hemisphere) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quantification of RNA and assessment of purity was conducted using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from total RNA (250 ng) using the iScript cDNA synthesis kit (BioRad, Munich, Germany) according to the manufacturer’s instructions. qRT-PCR was performed using SYBR Green technology on a CFX96 Touch™ real-time PCR detection system (BioRad, Munich, Germany).
Oligonucleotide primer sequences, product sizes and primer concentrations for quantitative real-time PCR are displayed in Table 1. qRT-PCR amplification was performed in duplicate or triplicate in a total reaction volume of 10 μL. qRT-PCR cycling conditions for Fis1 and Mfn1 were 95 °C for 3 min followed by 46 cycles of 95 °C for 10 s, 56 °C for 45 s and 72 °C for 29 s. qRT-PCR cycling conditions for Opa1 were 95 °C for 3 min followed by 37 cycles of 95 °C for 10 s, 56 °C for 45 s and 72 °C for 29 s. qRT-PCR cycling conditions for Drp1 were 95 °C for 3 min followed by 35 cycles of 95 °C for 10 s, 50 °C for 30 s and 72 °C for 29 s. qRT-PCR cycling conditions for BDNF, synaptophysin 1 and GAP43 were 95 °C for 3 min followed by 42 cycles of 95 °C for 10 s, 58 °C for 45 s and 72 °C for 29 s. qRT-PCR cycling conditions for all other genes were 95 °C for 3 min followed by 45 cycles of 95 °C for 10 s, 58 °C for 45 s and 72 °C for 29 s. Gene expression was analyzed using the 2(-ΔΔCq) method using BioRad CFX manager (BioRad, Munich, Germany) and Biogazelle qbase + (Biogazelle, Zwijnaarde, Belgium) and normalized to PGK1 and B2M expression levels.
Table 1
Oligonucleotide primer sequences, product sizes and primer concentrations for quantitative real-time PCR
|
Sequence |
Manufacturer |
Product size (bp) |
Primer conc. (µM) | |
|---|---|---|---|---|
|
AMPK (beta-subunit) |
5′-AGT ATC ACG GTG GTT GCT GT-3′ 5‘-CAA ATA CTG TGC CTG CCT CT-3‘ |
Biomol, Hamburg, Germany |
190 |
0.1 |
|
B2 M |
5′- GGC CTG TAT GCT ATC CAG AA -3′ 5′- GAA AGA CCA GTC CTT GCT GA -3′ |
Biomol, Hamburg, Germany |
198 |
0.4 |
|
BDNF |
5′GGT GCA GAA AAG CAA CAA GT-3‘ 5‘-GCA CAA AAA GTT CCC AGA GA-3‘ |
Biomol, Hamburg, Germany |
219 |
0.1 |
|
CI |
5′- CAC CTG TAA GGA CCG AGA GA -3′ 5′- GCA CCA CAA ACA CAT CAA AA -3′ |
Biomol, Hamburg, Germany |
227 |
0.1 |
|
CIV |
5′- CTG TTC CAT TCG CTG CTA TT -3′ 5′- GCG AAC AGC ACT AGC AAA AT -3′ |
Biomol, Hamburg, Germany |
217 |
0.1 |
|
CREB |
5′-TAG CTG TGA CTT GGC ATT CA-3′ 5‘-TTG TTC TGT TTG GGA CCT GT-3‘ |
Biomol, Hamburg, Germany |
184 |
0.5 |
|
CS |
5′- AAC AAG CCA GAC ATT GAT GC -3′ 5′- ATG AGG TCC TGC TTT GTC CT -3′ |
Biomol, Hamburg, Germany |
184 |
0.1 |
|
Drp1 |
5′-GCC CGT GAC AAA TGA AAT-3′ 5‘-CAG GCA TCA GCA AAG TCG-3‘ |
Biomers.net GmbH, Ulm, Germany |
87 |
0.1 |
|
fis1 |
5′-TAA AGT ATG TGC GAG GGC T-3′ 5‘-GCC TAC CAG TCC ATC TTT C-3‘ |
Biomers.net GmbH, Ulm, Germany |
104 |
0.1 |
|
GAP43 |
5‘-AGG GAG ATG GCT CTG CTA CT-3‘ 5‘-GAG GAC GGG GAG TTA TCA GT-3‘ |
Biomol, Hamburg, Germany |
190 |
0.15 |
|
Mfn1 |
5′-CGA AAA CTT GAA GCC ACT AC-3′ 5′-ACC GAA ACA CAA TGT CCT-3′ |
Biomers.net GmbH, Ulm, Germany |
140 |
0.1 |
|
Nrf1 |
5′-TCG GAG CAC TTA CTG GAG TC-3′ 5′-CTA GAA AAC GCT GCC ATG AT-3′ |
Biomol, Hamburg, Germany |
228 |
0.5 |
|
Opa1 |
5′-ATC AGA TAA GCA ACA GTG GG-3′ 5′-ACA TCC ACC TCT TTT TCC AG-3′ |
Biomers.net GmbH, Ulm, Germany |
125 |
0.1 |
|
PGC1α |
5′- TGT CAC CAC CGA AAT CCT -3′ 5′- CCT GGG GAC CTT GAT CTT -3′ |
Biomers.net GmbH, Ulm, Germany |
124 |
0.05 |
|
PGK1 |
5′- GCA GAT TGT TTG GAA TGG TC -3′ 5′- TGC TCA CAT GGC TGA CTTT TA -3′ |
Biomol, Hamburg, Germany |
185 |
0.4 |
|
SIRT1 |
5′-GTG AGA AAA TGC TGG CCT AA-3′ 5′-CTG CCA CAG GAA CTA GAG GA-3′ |
Biomol, Hamburg, Germany |
161 |
1 |
|
SIRT3 |
5′-CTG GAT GGA CAG GAC AGA TAA G-3′ 5′-TCT TGC TGG ACA TAG GAT GAT C-3′ |
Biomers.net GmbH, Ulm, Germany |
79 |
0.1 |
|
Synaptophysin 1 |
5‘-TTT GTG GTT GGT GAG TTC CT-3‘ 5‘-GCA TTT CCT CCC CAA AGT AT-3‘ |
Biomol, Hamburg, Germany |
204 |
0.1 |
|
Tfam |
5′-AGC CAG GTC CAG CTC ACT AA-3′ 5′-AAA CCC AAG AAA GCA TGT GG-3′ |
Biomol, Hamburg, Germany |
166 |
0.5 |
bp base pairs, conc. concentration
Quantification of Tocopherols and Tocotrienols
Tocopherols and tocotrienols were quantified in mouse brain (rear half of the brain hemisphere) using HPLC with fluorescence detection as previously described (Grebenstein and Frank 2012).
Primary Neural Cell Cultures
Cerebellar granule neuron-rich primary cultures were prepared from the brains of 7-day-old Wistar rats as recently described (Tulpule et al. 2014). 750,000 cells were seeded in wells of 24-well plates. The cells were cultured in a humidified atmosphere of a cell incubator (Sanyo, Osaka, Japan) with 5 % CO2 and used for experiments at a culture age between 7 and 9 days. Astrocyte-rich primary cultures were prepared from the brains of newborn Wistar rats as recently described (Tulpule et al. 2014). 300,000 cells were seeded in wells of 24-well plates and incubated in the humidified atmosphere of a Sanyo incubator with 10 % CO2. The culture medium was renewed every seventh day, and the confluent cultures were used for experiments at an age between 14 and 25 days. Neuron- and astrocyte-rich primary cultures contain predominately neurons and astrocytes, respectively, and only low number of other types of brain cells (Tulpule et al. 2014; Petters and Dringen 2014).
Experimental Incubations of Cultured Cells
The cultures in wells of 24-well dishes were washed once with 1 mL pre-warmed (37 °C) incubation buffer (IB; 145 mM NaCl, 5.4 mM KCl (for astrocytes) or 30.4 mM KCl (for neurons), 1.8 mM CaCl2, 1 mM MgCl2, 0.8 mM Na2HPO4, 20 mM HEPES, 5 mM glucose, adjusted at 37 °C to pH 7.4 with NaOH) and incubated at 37 °C in 200 µL IB in the absence or the presence of RBE in the final concentrations of 0.3 or 1 mg/mL. 1 % (v/v) ethanol was applied as solvent control since this corresponds to the amount of ethanol applied with 1 mg/mL RBE. After an incubation period of 4 h at 37 °C, the incubation media were harvested and used to determine extracellular LDH activity (as indicator for a loss of membrane integrity), the extracellular concentrations of glucose and lactate, and the extracellular and cellular concentrations of total glutathione (GSx, amount of GSH plus twice the amount of GSSG) and GSSG. The cells were washed once with 1 mL ice-cold phosphate-buffered saline (PBS: 10 mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl) and lysed with 200 µL 1 % (w/v) sulfosalicylic acid to determine cellular contents of GSx and GSSG.
Determination of Cell Viability and Protein Content of Cultured Cells
To test for a potential loss in cell membrane integrity of cultured primary brain cells after a given treatment, the release of the cytosolic enzyme LDH was determined as previously described (Dringen et al. 1998; Tulpule et al. 2014) using 10 µL samples of the harvested incubation media. To determine the initial cellular LDH activity of non-incubated cells, the cultures were lysed in 200 µL 1 % (v/v) Triton X-100 in IB and 10 µL of the lysate was used in the assay. The protein content of the cultured cells was determined by the Lowry method (Lowry et al. 1951) using bovine serum albumin as standard protein.
Determination of Glucose Consumption, Glutathione Content, Lactate Release and Glutathione Release
The concentration of extracellular glucose was determined in 10 µL media samples by coupled enzymatic assays as previously described (Tulpule et al. 2014). Glucose consumption was calculated as difference of the glucose concentrations in the media before and after cell incubation. Glucose consumption and lactate production were normalized to the protein content of the respective cultures.
Total glutathione contents (GSx = amount of GSH plus twice the amount of glutathione disulfide (GSSG)) and GSSG contents in the cells and incubation media were determined by the colorimetric Tietze cycling assay as previously described (Tulpule et al. 2014; Hohnholt and Dringen 2014). The GSx and GSSG values for cells and media were normalized to the protein content of the cultures.
Statistics
Data obtained from the mouse feeding study are presented as mean ± standard error of the mean (SEM). Data from primary neural cell cultures are presented as mean ± standard deviation (SD) of values obtained from experiments on three independently prepared cultures. Statistical analyses were performed by applying one-way ANOVA or two-way ANOVA with Bonferroni post-tests (Prism 5.0 GraphPad Software, San Diego, CA, USA). For analysis of the survival curve a log-rank (Mantel–Cox) test was used (Prism 5.0 GraphPad Software, San Diego, CA, USA). A P value of <0.05 was considered statistically significant.
Results
Female NMRI mice (aged 12 months) were fed with a standardized pelleted diet (aged control) or a pelleted diet supplemented with rice bran extract (aged + RBE, 4 g extract/kg diet) for 6 months. Female NMRI mice (aged 3 weeks) were fed with a standardized pelleted diet for 3 months (young control). The body weight of all mice was monitored during the feeding period. There was no significant difference in body weight between aged control group and aged intervention group mice (see Fig. 10 in Supplementary Material). Young control mice constantly gained body weight during the study period since they were still growing (see Fig. 10 in Supplementary Material). At the end of the study period, survival rates, cognitive function and brain mitochondrial function of the study mice were assessed.
SurvivalSurvival rates of young and aged control mice were 93 and 46 % (P < 0.01), while the survival rate of aged mice administered RBE was 79 % (see Fig. 1). Thus, RBE administration significantly increased survival rates of aged NMRI mice over a 6-month period (P < 0.05). Open image in new window Fig. 1
Survival rates of NMRI mice after feeding without or with rice bran extract (RBE). Aged (12 months old) mice were fed with a standardized pelleted diet (aged) or a pelleted diet containing RBE (4 g extract/kg diet, aged + RBE) for 6 months. As further control, young mice (3 months old, starting point of the analysis: 90 days) were fed with a pelleted standard diet for 3 months (young); n = 15–25; mean without SEM; log-rank (Mantel-Cox) test; *P < 0.05; **P < 0.01
Behavioral TestingA passive avoidance test was used to assess the cognitive function of the studied mice. The passive avoidance test is conducted in a box which consists of an illuminated and a dark chamber. On day one (1st repeat), the mouse is put in the light part and the time is recorded that the mouse needs to enter the dark chamber. After entering the dark chamber, the mouse receives a mild electric shock (0.5 mA). On day two (2nd repeat), the mouse is again put in the illuminated chamber and the time needed to enter the dark chamber is recorded. If the mouse remembers the electric shock, it will need a longer time to re-enter the dark chamber or it might not re-enter it at all. In comparison with young control mice, aged control mice required significantly longer times to enter the dark chamber on day one (see Fig. 2, P < 0.01), probably because they were less mobile and less curious than their younger counterparts. On day two, aged control mice re-entered the dark chamber significantly faster than young control mice (see Fig. 2, P < 0.05), indicating that they did not remember the electric shock as well as young control mice. Aged mice fed with RBE behaved very similar to young control mice since they entered the dark chamber significantly faster than aged control mice on day one and stayed in the light chamber significantly longer than aged control mice on day two (see Fig. 2, P < 0.05). Open image in new window Fig. 2
Passive avoidance test with young and aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1); 1st repeat: session on day one in which the time is recorded that the mouse needs to enter the dark chamber; upon entering the dark chamber, the mouse receives a mild electric shock (0.5 mA); 2nd repeat: session on day two (24 h after the first session) in which the time is recorded that the mouse needs to re-enter the dark chamber; n = 11–19; mean ± SEM; one-way ANOVA with Bonferroni post-test; *P < 0.05; **P < 0.01
In the one-trial Y-maze spontaneous alternation test, the alternation rate was not significantly different between the three study groups (see Fig. 3c). Nevertheless, we observed a decreased number of alternations as well as a decreased number of entries into the arms in the aged control group compared to the young control group (see Fig. 3a, b, P < 0.001), indicating less mobility and curiosity in aged mice. RBE administration significantly increased both the number of alternations and entries into the arms in aged mice (see Fig. 3a, b, P < 0.01), indicating increased need for movement and curiosity in these mice. Open image in new window Fig. 3
Number of alternations (a) and entries (b) as well as alternation rate (c) of young and aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1) during a 5-min time span in the Y-maze spontaneous alternation test; n = 12–19; mean ± SEM; one-way ANOVA with Bonferroni post-test; **P < 0.01; ***P < 0.001
Brain Mitochondrial FunctionIn comparison with young control mice, basal ATP concentrations of dissociated brain cells (DBC) of aged control mice were significantly decreased (see Fig. 4a, P < 0.05). RBE administration slightly but not significantly increased ATP concentrations in DBC of aged mice (see Fig. 4a). To examine the resistance of DBC against nitrosative stress, cells were incubated with sodium nitroprusside (SNP) for 3 h. This treatment caused a severe ATP loss in DBC isolated from mice belonging to the three study groups (see Fig. 4b). However, compared to cells from young control mice, no significant differences in ATP concentrations were detected after SNP incubation although a slight increase in ATP concentrations was observed in DBC from aged control mice and aged RBE-fed mice (see Fig. 4b), which did not reach the level of significance. Neither age nor RBE administration significantly affected basal mitochondrial membrane potential (MMP) or MMP after SNP incubation in DBC of any of the three study groups (data not shown). Open image in new window Fig. 4
Basal ATP concentrations (a) and ATP concentrations after insult with sodium nitroprusside (SNP, 3 h, 0.1 mM; b) of dissociated brain cells (DBC) isolated from young and aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1); basal ATP concentrations served as control for normalization in b; n = 10–11; mean ± SEM; one-way ANOVA with Bonferroni post-test; *P < 0.05
Brain mitochondria isolated from aged control mice exhibited significantly decreased activity of respiratory system complex IV (CIV, see Fig. 5, P < 0.001). The activity of respiratory system complexes I, CI + CII (OXPHOS) and uncoupled CII was numerically though not significantly decreased (see Fig. 5). RBE administration to aged mice significantly increased the activity of respiratory system complexes CI + CII (P < 0.05) and CIV (P < 0.001). Furthermore, RBE administration numerically though not significantly increased the activities of respiratory system complexes CI and uncoupled CII (see Fig. 5). Open image in new window Fig. 5
Protein-normalized respiration of brain mitochondria isolated from young and aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1); respiration was measured using an Oxygraph-2k (Oroboros, Innsbruck, Austria); the addition of a substance into the Oxygraph chamber is indicated with a plus sign; n = 10–11; mean ± SEM; two-way ANOVA with Bonferroni post-test; *P < 0.05; ***P < 0.001
Respiratory control ratio (RCR) which is calculated as ratio between CI + CII respiration and leak respiration after addition of oligomycin (leakomy) gives information about the coupling of the respiratory system (Hagl et al. 2013). RCR was not significantly changed by either age or RBE administration (data not shown).
Citrate synthase (CS) activity is a marker for the determination of mitochondrial content (Larsen et al. 2012). CS activity was significantly decreased in brain mitochondria isolated from aged control mice (see Fig. 6, P < 0.05). RBE administration significantly increased CS activity in aged mice, indicating increased mitochondrial content in the brains of these animals (see Fig. 6, P < 0.05). Normalizing respiration to CS activity gives information about the respiration of a single mitochondrion (Rabøl et al. 2010). Neither age nor RBE administration had an effect on CS-normalized respiration (data not shown). Open image in new window Fig. 6
Citrate synthase (CS) activity of isolated brain mitochondria from young and aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1); CS activity was measured photometrically in isolated brain mitochondria; IU international units; n = 10–11; mean ± SEM; two-way ANOVA with Bonferroni post-test; *P < 0.05
To further examine the mechanism of action of RBE on a molecular level, we determined mRNA as well as protein expression of proteins associated with mitochondrial biogenesis, mitochondrial respiration, mitochondrial dynamics and synaptic plasticity (Fig. 7). Open image in new window Fig. 7
Relative normalized mRNA (a, c, e, g) and protein (b, d, f, h) expression of cAMP response element-binding protein 1 (CREB1; a, b), AMP-activated protein kinase subunit β (AMPK β-subunit; c, d), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; e, f) and mitochondrial transcription factor A (Tfam; g, h) in brain homogenate from aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1) determined using quantitative real-time PCR and western blot; mRNA and protein expression of young control mice is 100 %; b, d, f, h: representative images of western blots are depicted above each graph (1: young control, 2: aged control, 3: aged + RBE); n = 8; mean ± SEM; two-way ANOVA with Bonferroni post-test; *P < 0.05; **P < 0.01; ***P < 0.001
Mitochondrial biogenesis is mediated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) (Fernandez-Marcos and Auwerx 2011). PGC1α itself is activated by deacetylation via sirtuins (SIRT) and phosphorylation via AMP-activated protein kinases (AMPK). Furthermore, phosphorylated cAMP response element-binding protein (CREB) can induce gene expression of PGC1α. PGC1α facilitates the expression of transcription factors nuclear respiratory factor 1 (Nrf1) and mitochondrial transcription factor A (Tfam) which in turn induces mitochondrial biogenesis (Fernandez-Marcos and Auwerx 2011; Canto and Auwerx 2009). Compared to young mice, mRNA expression of all proteins involved in this pathway was significantly decreased in aged control mice (see Table 2; Fig. 7a, c, e, g). Compared to young mice, protein expression of CREB, PGC1α and AMPKβ was significantly decreased in aged control mice and protein expression of phospho-CREB and Tfam was numerically though not significantly decreased (see Table 3; Fig. 7b, d, f, h). RBE administration to aged mice significantly increased mRNA levels of CREB and Tfam and numerically though not significantly increased mRNA levels of AMPKβ in comparison with aged control mice (see Table 2; Fig. 7a, c, e, g). Protein levels of PGC1α were significantly increased after RBE administration to aged mice, and protein levels of CREB, phospho-CREB, AMPKα, Nrf1 and Tfam were numerically though not significantly increased (see Table 3; Fig. 7b, d, f, h).Table 2
Relative normalized mRNA expression in brain homogenate from aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1) determined using quantitative real-time PCR; mRNA expression of young control mice is 100 %; n = 8; mean without SEM; two-way ANOVA with Bonferroni post-test
|
Aged |
Aged + RBE | |
|---|---|---|
|
cAMP response element-binding protein (CREB) |
58.9*** |
83.7a |
|
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) |
80.0* |
78.7* |
|
Sirtuin 1 (SIRT1) |
80.7** |
71.2*** |
|
Sirtuin 3 (SIRT3) |
77.1* |
83.0 |
|
AMP-activated protein kinase (AMPK beta) |
76.2* |
90.9 |
|
Nuclear respiratory factor 1 (Nrf1) |
76.4*** |
73.9*** |
|
Mitochondrial transcription factor A (Tfam) |
60.4** |
90.3a |
|
Complex I (CI) |
63.2*** |
62.5*** |
|
Complex IV (CIV) |
77.9*** |
68.8*** |
|
Citrate synthase (CS) |
83.3 |
172.3*,a |
|
Dynamin-related protein 1 (Drp1) |
76.2*** |
72.4*** |
|
Fission 1 (fis1) |
84.2 |
80.3* |
|
Mitofusin 1 (Mfn1) |
68.6*** |
77.0** |
|
Optic atrophy 1 (Opa1) |
75.9*** |
73.7*** |
|
Brain-derived neurotrophic factor (BDNF) |
39.2*** |
56.2* |
|
Synaptophysin 1 (Syp1) |
65.2*** |
65.4*** |
|
Growth-associated protein 43 (GAP43) |
64.0** |
75.5* |
* indicates significant differences with respect to young control mice; “a” indicates significant differences with respect to aged control mice; *,a P < 0.05; **P < 0.01; ***P < 0.001
Open image in new window Fig. 8
Relative normalized mRNA (a) and protein (b) expression of citrate synthase in brain homogenate from aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1) determined using quantitative real-time PCR and western blot; mRNA and protein expression of young control mice is 100 %; representative images of western blots are depicted above b (1: young control, 2: aged control, 3: aged + RBE); n = 8; mean ± SEM; two-way ANOVA with Bonferroni post-test; *P < 0.05; **P < 0.01; ***P < 0.001
Table 3
Protein expression in brain homogenate from aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1) determined using western blot analysis and normalized to protein expression of young control mice (100 %); n = 8; mean without SEM; two-way ANOVA with Bonferroni post-test
|
Aged |
Aged + RBE | |
|---|---|---|
|
cAMP response element-binding protein (CREB) |
77.9* |
101.1 |
|
Phosphorylated CREB (phospho-CREB) |
77.2 |
82.9 |
|
pCREB/CREB |
67.8 |
82.6 |
|
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) |
59.8*** |
81.8a |
|
AMP-activated protein kinase (AMPK (alpha 1 + 2)) |
53.5*** |
75.1* |
|
Nuclear respiratory factor 1 (Nrf1) |
107.1 |
121.0** |
|
Mitochondrial transcription factor A (Tfam) |
77.5 |
99.6 |
|
Complex I (CI) |
84.0 |
111.0 |
|
Complex II (CII) |
106.9 |
106.0 |
|
Complex III (CIII) |
101.6 |
88.6 |
|
Complex IV (CIV) |
87.2 |
78.7* |
|
Complex V (CV) |
76.4** |
86.1 |
|
Citrate synthase (CS) |
94.2 |
116.4a |
|
Dynamin-related protein 1 (Drp1) |
121.0 |
123.5 |
|
Fission 1 (fis1) |
95.6 |
108.7 |
|
Mitofusin 1 (Mfn1) |
75.9 |
95.7 |
|
Optic atrophy 1 (Opa1) |
85.4 |
131.8*,aaa |
|
Brain-derived neurotrophic factor (BDNF) |
91.1 |
99.9 |
* indicates significant differences with respect to young control mice; “a” indicates significant differences with respect to aged control mice; *,a P < 0.05; **P < 0.01; ***P < 0.001
Compared to young control mice, mRNA expression of mitochondrial respiratory system complexes I and IV was significantly decreased in aged control mice (see Table 2). Protein expression of respiratory system complexes I and IV was numerically though not significantly decreased in aged control mice, and protein expression of complex V was significantly decreased (see Table 3). mRNA and protein expression of citrate synthase was slightly decreased in aged control mice in comparison with young control mice (see Tables 2, 3; Fig. 8). RBE administration did not significantly alter mRNA or protein expression of the respiratory system complexes but significantly increased mRNA and protein expression of citrate synthase in comparison with aged control mice (see Tables 2, 3; Fig. 8).
To assess the effect of age and RBE administration on mitochondrial dynamics, mRNA and protein levels of fusion-associated proteins mitofusin 1 (Mfn1) and optic atrophy-1 (Opa1) and fission-associated proteins fission-1 (fis1) and dynamin-related protein-1 (Drp1) were examined. In comparison to young control mice, mRNA expression of Drp1, Mfn1 and Opa1 was significantly reduced in aged control mice and fis1 mRNA expression was numerically though not significantly reduced (see Table 2). Protein expression of Drp1 was numerically increased, and protein expression of Mfn1 was numerically decreased in aged control mice (see Table 3). RBE administration did not influence mRNA expression of Mfn1, Opa1, fis1 and Drp1 (see Table 2) but significantly increased protein expression of Opa1. Additionally, protein expression of Mfn1 was numerically though not significantly increased (see Table 3).
mRNA expression of the synaptic markers brain-derived neurotrophic factor (BDNF), synaptophysin 1 and growth-associated protein 43 (GAP43) was significantly decreased in aged control mice (see Table 2). Additionally, protein expression of BDNF was slightly though not significantly decreased (see Table 3). RBE administration numerically though not significantly increased mRNA expression of BDNF and GAP43 and protein expression of BDNF (see Tables 2, 3).
Vitamin E Concentrations in the BrainConcentrations of vitamin E congeners were determined in blood plasma and brain tissue of the studied mouse groups. Only α-tocopherol was detectable in blood plasma. In brain tissue, α-tocopherol and very small amounts of δ-tocopherol (<0.15 nmol/g tissue) were detectable. Since δ-tocopherol concentrations in brain tissue were very low, only α-tocopherol concentrations are discussed here. α-tocopherol concentrations were similar in blood plasma from young and aged control mice (2.7 and 2.6 µmol/L), while RBE administration significantly increased α-tocopherol concentrations in blood plasma of aged mice (4.0 µmol/L; see Fig. 9a, P < 0.01). In brain tissue, α-tocopherol concentrations were not significantly changed by either age or RBE administration (young: 6.1 nmol/g; aged: 6.3 nmol/g; aged + RBE: 7.7 nmol/g) although a slightly but not significantly enhanced concentration was measured after RBE administration (see Fig. 9b). Open image in new window Fig. 9
α-Tocopherol concentrations in blood plasma (a) and brain tissue (b) of young and aged NMRI mice (for more information about the treatment of mice see labeling of Fig. 1) determined using HPLC with fluorescence detection; n = 11–19 (plasma); n = 3–9 (brain tissue); mean ± SEM; two-way ANOVA with Bonferroni post-test; **P < 0.01
Discussion
NMRI mice have frequently been used as a well-established mouse model to study brain aging (Afshordel et al. 2015; Schindowski et al. 2001; Hagl et al. 2016). To establish the effects of long-term feeding of rice bran extract (RBE) on brain mitochondrial function in aged female NMRI mice, a group of 12-month-old mice was fed the extract (4 g/kg pelleted diet) for 6 months. A group of 12-month-old mice that received a pelleted control diet for 6 months and a group of 3-week-old mice that received a pelleted control diet for 3 months served as control groups. Translated to human studies, the administered RBE dose equals 28 mg RBE/kg body weight/day (Reagan-Shaw et al. 2008). No effects of RBE administration on body weight of mice were detected during the feeding period. To assess toxicity of rice bran extract prior to starting the intervention study, we determined viability, glucose metabolism and glutathione contents in cultured primary neurons and astrocytes isolated from rat brain and incubated with RBE (0.3 and 1 mg RBE/mL) for 4 h. None of the tested concentrations of RBE had any effects on the above-mentioned parameters, indicating that RBE is not toxic for cultured rat neurons or astrocytes (see Tables 1 and 2 in Supplementary Material).
Mitochondrial Dysfunction in Aged Mice
In comparison with young control mice, mitochondrial function was impaired in aged control mice. Mitochondrial respiration was decreased whereby complex IV respiration was especially affected. Decreased respiratory system activity results in impaired ATP production which is likely to explain the decreased basal ATP concentrations in the brains of aged control mice. Citrate synthase (CS) activity was also reduced in brains of aged control mice. Since CS activity is an accurate marker of mitochondrial content (Larsen et al. 2012) we hypothesize that mitochondrial content is decreased in brains of aged control mice. To validate our hypothesis, we examined mRNA and protein levels of CREB, SIRT1, AMPK, PGC1α, Nrf1 and Tfam which are involved in mitochondrial biogenesis (Canto and Auwerx 2009; Fernandez-Marcos and Auwerx 2011). Mitochondrial biogenesis is mediated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). PGC1α itself is activated by deacetylation via sirtuins (SIRT) and phosphorylation via AMP-activated protein kinases (AMPK). Furthermore, phosphorylated cAMP response element-binding protein (CREB) can induce gene expression of PGC1α. PGC1α facilitates the expression of transcription factors nuclear respiratory factor 1 (Nrf1) and mitochondrial transcription factor A (Tfam) which in turn induces mitochondrial biogenesis (Fernandez-Marcos and Auwerx 2011; Canto and Auwerx 2009). mRNA expression of all of the above-mentioned proteins as well as protein concentrations of CREB, PGC1α and AMPK was significantly decreased in brains of aged mice compared to young mice, consistent with the view that mitochondrial biogenesis is impaired in aged NMRI mice. Additionally, mRNA expression of respiratory system complexes I and IV as well as protein expression of complexes I, IV and V was numerically or significantly decreased which might contribute to impaired mitochondrial respiration in the brains of aged NMRI mice. Our findings about impaired brain mitochondrial function in aged mice very well match existing literature data about brain mitochondrial dysfunction in aged mice (Afshordel et al. 2015; Navarro et al. 2002, 2005, 2011) as recently discussed in detail (Hagl et al. 2016).
We additionally found that mRNA and protein expression of proteins associated with fission and fusion processes were changed in aged NMRI mice. mRNA expression of fusion-associated proteins optic atrophy 1 (Opa1) and mitofusin 1 (Mfn1) and of fission-associated protein dynamin-related protein 1 (Drp1) were decreased. On the other hand, protein expression of Drp1 was increased in aged mice. According to Seo and coworkers, mitochondrial dynamics are changed during the aging process (Seo et al. 2010). This is in accordance with our own findings and might be part of a compensatory mechanism with which the cells try to ameliorate mitochondrial function.
Rice Bran Extract Ameliorates Mitochondrial Dysfunction in Aged NMRI Mice
Administration of rice bran extract (RBE) increased respiration in isolated brain mitochondria whereby the effect on complex IV and on combined complex I + II respiration was most distinct. Increased mitochondrial respiration then led to numerically although not significantly increased ATP concentrations in brains of aged mice fed with RBE. CS activity was significantly increased in brains of aged mice fed with RBE which indicates increased mitochondrial content. These results are very similar to results recently obtained in a short-term feeding study with RBE in aged NMRI mice. In this study, we found increased mitochondrial respiration and numerically increased basal ATP concentrations in brains of aged NMRI mice administered RBE for 3 weeks. These beneficial effects on mitochondrial function were mediated by increased mitochondrial content (Hagl et al. 2016). Similarly, RBE administration to young guinea pigs ameliorated brain mitochondrial function by increasing mitochondrial content and by protecting cells from nitrosative stress (Hagl et al. 2013). Navarro and coworkers reported that α-tocopherol, which is a key component of RBE, increased brain mitochondrial function (respiration, ATP production, mitochondrial content) in aging rats (Navarro et al. 2011) and mice (Navarro et al. 2005).
To further evaluate the mechanism of action of RBE, we determined mRNA and protein expression of mitochondria-related proteins. mRNA levels of CREB and Tfam were significantly increased, and mRNA levels of AMPK were numerically increased by RBE administration. Additionally, protein levels of PGC1α were significantly increased and protein levels of CREB, p-CREB, AMPK and Tfam were numerically increased by RBE administration. These findings are in accordance with earlier findings from our group and confirm that RBE exerts beneficial effects on mitochondria by increasing mitochondrial content via a PGC1α-dependent mechanism (Hagl et al. 2013, 2015a, b, 2016). RBE administration significantly increased protein concentrations of Opa1, but did not influence mRNA or protein expression of the fission- and fusion-associated proteins fis1, Drp1 and Mfn1. Accordingly, RBE does not seem to have major impact on mitochondrial dynamics.
Behavioral Testing
We found that in comparison with young control mice (aged 3 months), 18-month-old control mice performed significantly worse in the passive avoidance test and were less mobile and less curious in the one-trial Y-maze test. These results indicate impaired memory and motor behavior which might be caused by decreased mitochondrial function and impaired ATP production in brains of aged mice. To examine the effect of age on neuronal plasticity, mRNA expression of brain-derived neurotrophic factor (BDNF), synaptophysin 1 (Syp1) and growth-associated protein 43 (GAP43) was determined in brains of aged mice. BDNF is a key molecule involved in the control of synapse formation as well as neuronal differentiation and survival (Leal et al. 2015; Patterson 2015). Syp1 is an abundant presynaptic vesicle protein which has been found to decrease with age in the hippocampus and various cortical regions in human and mouse brain. Additionally, Syp1 has been reported to be increased by environmental enrichment in the brains of aged mice (Frick and Fernandez 2003; Tarsa and Goda 2002). GAP43 is important for axonal remodeling and neuronal morphology in the adult brain and has been found to be a valid marker for axonal sprouting (Grasselli and Strata 2013). Compared to young mice, mRNA expression of BDNF, Syp1 and GAP43 was significantly decreased in aged mice, indicating reduced synaptic plasticity and survival (Marlatt et al. 2012; Schmoll et al. 2005; Mattson et al. 2004; Frick and Fernandez 2003) which might contribute to impaired motor behavior and memory function.
RBE administration ameliorated brain mitochondrial function in aged mice. Additionally, aged mice administered RBE for 6 months were more mobile and more curious in the one-trial Y-maze test and significantly slower in re-entering the dark chamber in the second session of the passive avoidance test. The beneficial effects of RBE administration on brain mitochondrial function might very well explain the ameliorated performance of mice in the passive avoidance and one-trial Y-maze test. Additional