Unraveling sex differences in age-related hippocampal decline: differential mitochondrial dysfunction, Lonp1-dependent mitochondrial proteostasis and mtROS production in aged C57BL/6 mice methods
Aim. Evidence-backed execution summary for Unraveling sex differences in age-related hippocampal decline: differential mitochondrial dysfunction, Lonp1-dependent mitochondrial proteostasis and mtROS production in aged C57BL/6 mice methods from Unraveling sex differences in age-related hippocampal decline: differential mitochondrial dysfunction, Lonp1-dependent mitochondrial proteostasis and mtROS production in aged C57BL/6 mice.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Hormone levels
reagent used in the protocol.
- Use
- To determine the changes in sexual hormones in aging, we measured the levels of these hormones in adult and aged females and testosterone in adult and aged males. Blood samples were collected in the presence of an anticoagulant ( n = 5 per group). Serum estradiol levels were quantified in females and te...
Reagents and antibodies
reagent used in the protocol.
- Use
- The primary antibodies used were, rabbit anti-GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Inc. USA), mouse anti-4-HNE (H6275-02, US Biological, Life Sciences, 1:1,000), mouse anti-catalase (1:1000, sc- 271803, Santa Cruz Biotechnology, Inc. USA), mouse anti-SOD1 (1:1000, sc-271014, Santa Cruz Biotechnology, I...
Immunoblotting
reagent used in the protocol.
- Use
- The hippocampus of the 3- and 20-month-old male and female mice were dissected on ice and immediately processed as previously described [, ]. The hippocampal tissue was homogenized in HEPES buffer (25 mM Hepes, 125 mM NaCl, 25 mM NaF, 1 mM EDTA, 1 mM EGTA, 1% NP-40, pH = 7,...
Obtaining hippocampal slices
reagent used in the protocol.
- Use
- Animals were anesthetized and then perfused through the heart with 1x PBS, followed by fixation with 4% paraformaldehyde (PFA, 4896610S, Merck) in 0.1 M phosphate buffer for 30 min. Brains were postfixed overnight at 4 °C in 4% PFA and washed 10 min, 3 times in 1x PBS. A sucrose gradient...
Immunofluorescence (IF)
reagent used in the protocol.
- Use
- Slides were removed from -20 °C and left at room temperature; then, IF was performed. Briefly, they were hydrated in 1x PBS and incubated in blocking/permeabilization solution (Triton X-100 0.5%, BSA 5% in 1x PBS) for 1 h at room temperature. Primary antibodies were diluted in this solution and incu...
Isolation of an enriched-mitochondrial fraction from the hippocampus
reagent used in the protocol.
- Use
- As previously described, a fraction enriched in mitochondria was isolated from the hippocampus [, ]. Hippocampal tissue was homogenized in MSH buffer (230 mM mannitol, 70 mM sucrose, 5 mM Hepes, pH 7.4), supplemented with a protease and phosphatase inhibitor cocktail in a glass homogenizer. Homoge...
Measurement of ATP and mitochondrial membrane potential (MMP)
reagent used in the protocol.
- Use
- ATP concentration was measured in tissue lysates obtained with HEPES buffer (25 mM HEPES, 125 mM NaCl, 25 mM NaF, 1 mM EDTA, 1 mM EGTA, 1% NP-40, pH = 7,4) using a luciferin/luciferase bioluminescence assay kit (ATP determination kit no. A22066, Molecular Probes, Invitrogen,...
Measurement of ROS content and superoxide production
reagent used in the protocol.
- Use
- ROS content was measured using the fluorescent dye CM-H2DCFDA (DCF) (Thermo Fisher Scientific, USA). 25 ug of hippocampal protein samples were incubated with 25 µM of DCF. Fluorescence was measured in BioTek Synergy HT (485 nm, 530 nm) [ ]. The fluorescent dye MitoSox (Thermo Fisher Scie...
Behavioral tests
All behavioral tests were monitored by Any-MAZE Behavioral software (Stoelting Co), using the chambers and instruments manufactured or recommended by the manufacturer. All behavioral tests were performed in the 12 h light phase of the animal's light/dark cycle.
- Use
- All behavioral tests were monitored by Any-MAZE Behavioral software (Stoelting Co), using the chambers and instruments manufactured or recommended by the manufacturer. All behavioral tests were performed in the 12 h light phase of the animal's light/dark cycle.
Morris water maze (MWM) test
The MWM test was used as a spatial memory behavioral test as previously described [,, ]. Mice were trained in a 1.2 m diameter circular pool (opaque water, 50 cm deep) filled with 19-21 °C water. A submerged 9 cm platform (1 cm below the surface, invisible to the animal) wa...
- Use
- The MWM test was used as a spatial memory behavioral test as previously described [,, ]. Mice were trained in a 1.2 m diameter circular pool (opaque water, 50 cm deep) filled with 19-21 °C water. A submerged 9 cm platform (1 cm below the surface, invisible to the animal) wa...
Novel object recognition (NOR) and novel object localization (NOL) tests
NOR and NOL tests were performed in a 38 × 38 × 32 cm acrylic box as previously described [ ]. The animals were habituated in the box for 5 minutes twice on the first day without any object. The next day, for the NOR test, each animal was placed in the box containing two...
- Use
- NOR and NOL tests were performed in a 38 × 38 × 32 cm acrylic box as previously described [ ]. The animals were habituated in the box for 5 minutes twice on the first day without any object. The next day, for the NOR test, each animal was placed in the box containing two...
FITC-Casein Lonp1 proteolytic activity assay
Immunoprecipitated Lonp1 samples were centrifuged at 3,000 rpm for 2 min at 4 °C, and the pellet was resuspended in activity buffer (150 mM NaCl, 50 mM HEPES-KOH pH 8.0, 10 mM MgCl₂, 0.1 mg/mL BSA) containing FITC-casein (0.1 mg/mL) and ATP (2 mM). The...
- Use
- Immunoprecipitated Lonp1 samples were centrifuged at 3,000 rpm for 2 min at 4 °C, and the pellet was resuspended in activity buffer (150 mM NaCl, 50 mM HEPES-KOH pH 8.0, 10 mM MgCl₂, 0.1 mg/mL BSA) containing FITC-casein (0.1 mg/mL) and ATP (2 mM). The...
Image analysis
Densitometry analysis for immunoblot images was performed using ImageJ (NIH) software, and the data were normalized to a specific loading control, as indicated in each figure legend. Confocal images were acquired under the same magnification, laser intensity, brightness, and gain. Images were processed using the Fij...
- Use
- Densitometry analysis for immunoblot images was performed using ImageJ (NIH) software, and the data were normalized to a specific loading control, as indicated in each figure legend. Confocal images were acquired under the same magnification, laser intensity, brightness, and gain. Images were processed using the Fij...
Statistical analysis
All data are expressed as mean ± standard error of the mean (SEM). We used two-way ANOVAs to determine the effects of sex, age, and sex-by-age interaction. Where appropriate, Tukey's post-hoc comparisons were performed to examine the individual main effect of sex and age. The assumptions of no...
- Use
- All data are expressed as mean ± standard error of the mean (SEM). We used two-way ANOVAs to determine the effects of sex, age, and sex-by-age interaction. Where appropriate, Tukey's post-hoc comparisons were performed to examine the individual main effect of sex and age. The assumptions of no...
Mitochondrial protein quality control failure in the aged hippocampus of mice
Maintaining protein homeostasis within mitochondria is crucial for cellular function and overall health. Key players in this intricate system include chaperones and proteases that collaborate to ensure proper protein folding, the degradation of damaged proteins, and overall mitochondrial quality control [ ]. Since w...
- Use
- Maintaining protein homeostasis within mitochondria is crucial for cellular function and overall health. Key players in this intricate system include chaperones and proteases that collaborate to ensure proper protein folding, the degradation of damaged proteins, and overall mitochondrial quality control [ ]. Since w...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All data are expressed as mean ± standard error of the mean (SEM). We used two-way ANOVAs to determine the effects of sex, age, and sex-by-age interaction. Where appropriate, Tukey's post-hoc comparisons were performed to examine the individual main effect of sex and age. The assumptions of no...
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Hormone levels
To account for the high variability typical of hormone measurements, we analyzed age-related differences using geometric means (GM) derived from log10-transformed values with 95% confidence intervals (CI). Comparisons between age groups (Adult vs. Old) were performed using Welch's t-test. The fold-change was calculated as the ratio of the geometric means. The use of geometric means is particularly appropriate for skewed biological data, as it reduces the influence of extreme values and provides a more robust measure of central tendency.
Morris water maze (MWM) test
The MWM test was used as a spatial memory behavioral test as previously described [,, ]. Mice were trained in a 1.2 m diameter circular pool (opaque water, 50 cm deep) filled with 19-21 °C water. A submerged 9 cm platform (1 cm below the surface, invisible to the animal) was used for training, with a maximum trial duration of 60 s and 2 s on the platform at the end of the trials. Each animal was trained to locate the platform. The test was performed with three trials per day for 10 days except for weekends, and a probe test was carried out on day 11, when the platform was removed. The latency time (in seconds) required for the animal to reach the platform and the time spent in each quadrant (in seconds) was measured. After testing, the mouse was gently removed from the maze and returned to its cage. Spatial acuity was calculated u...
Obtaining hippocampal slices
Animals were anesthetized and then perfused through the heart with 1x PBS, followed by fixation with 4% paraformaldehyde (PFA, 4896610S, Merck) in 0.1 M phosphate buffer for 30 min. Brains were postfixed overnight at 4 °C in 4% PFA and washed 10 min, 3 times in 1x PBS. A sucrose gradient (10% and 20%, 2 h each, at room temperature and 30% overnight at 4 °C) was performed. Then, the whole brain was immersed in OCT Compound (4583, Sakura). Coronal sections 25 µm thick were collected from anterior to posterior on a Leica cryostat in a 24-well plate with 1x PBS and kept at 4 °C until use. Representative sections from each animal were mounted on Premium Micro Slide (PC2-302-16, PR PorLab) in 1x PBS, allowed to dry at room temperature, and stored at -20 °C until use [, ].
Immunofluorescence (IF)
Slides were removed from -20 °C and left at room temperature; then, IF was performed. Briefly, they were hydrated in 1x PBS and incubated in blocking/permeabilization solution (Triton X-100 0.5%, BSA 5% in 1x PBS) for 1 h at room temperature. Primary antibodies were diluted in this solution and incubated at 4 °C overnight. After 3 washes in 1x PBS, 10 min each, the secondary antibodies were diluted in the same solution, incubated for 2 h at room temperature, and washed again 10 min, 3 times in 1x PBS. The slides were coated with Fluoromount-G (00-4958-02, Invitrogen, USA). Antibodies used were: mouse anti-4HNE (1:300; 298112, USBiological), mouse anti Lonp1 (1:500; sc-293244, Santa Cruz Biotechnology, Inc. USA) and anti-mouse Alexa 488 (1:500; A-21202, Invitrogen, USA) [, ].
ATP deficiency is more severe in aged male mice
The primary mitochondrial function involves the production of ATP, and it is known that during aging, mitochondrial function and ATP formation are reduced in the hippocampus [, ]. However, whether sex-dependent differences emerge during aging remains unclear. First, we evaluated the bioenergetic state of the hippocampus. To this end, whole hippocampal lysate, mitochondria-free cytoplasmic, and mitochondrial-enriched fractions were tested using a bioluminescence detection kit to measure the ATP content. The two-way ANOVA analyses revealed a significant effect of sex [F (1,16) = 6.00; p = 0.03] and a significant effect of age [F (1,16) = 30.542; p < 0.01], with no significant interaction between the two factors ( p = 0.12). Both aged groups exhibited a reduction in ATP content compared to adult mice, with a more pronounce...
Measurement outputs
What raw and processed outputs should exist?
The analysis revealed a significant effect of sex [F (1,16) = 11.49; p < 0.003], no significant effect of age ( p = 0.07), and a significa...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Adult and aged C57BL/6 J and SAMP8 mice (Senescence-Accelerated Mouse Prone 8) were obtained from Fundación Ciencia and Vida. These animals were housed and maintained...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
For a 2 × 2 design, considering a main effect with two levels (e.g., females vs. males) and collapsing across the other factor (age), the analysis reduces to a...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The MWM test was used as a spatial memory behavioral test as previously described [,, ]. Mice were trained in a 1.2 m diameter circular pool (opaque water, 50 cm d...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
The analysis revealed a significant effect of sex [F (1,16) = 11.49; p < 0.003], no significant effect of age ( p = 0.07), and a significant interaction between both factors [F (1,16) = 5.15, p = 0.04].
from paperScoring or quantification
Quantify the primary readouts for this experiment: The analysis revealed a significant effect of sex [F (1,16) = 11.49; p < 0.003], no significant effect of age ( p = 0.07), and a significa...; Adult and aged C57BL/6 J and SAMP8 mice (Senescence-Accelerated Mouse Prone 8) were obtained from Fundación Ciencia and Vida. These animals were housed and maintained...; For a 2 × 2 design, considering a main effect with two levels (e.g., females vs. males) and collapsing across the other factor (age), the analysis reduces to a...; The MWM test was used as a spatial memory behavioral test as previously described [,, ]. Mice were trained in a 1.2 m diameter circular pool (opaque water, 50 cm d....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
The analysis revealed a significant effect of sex [F (1,16) = 11.49; p < 0.003], no significant effect of age ( p = 0.07), and a significa...; Adult and aged C57BL/6 J and SAMP8 mice (Senescence-Accelerated Mouse Prone 8) were obtained from Fundación Ciencia and Vida. These animals were housed and maintained...; For a 2 × 2 design, considering a main effect with two levels (e.g., females vs. males) and collapsing across the other factor (age), the analysis reduces to a...; For the Sex × Age interaction in the 2 × 2 ANOVA (4 cells total): n = 5 per condition ( N = 20 total) → detectable Cohen...
from paperReporting output
Report representative outputs alongside summary comparisons for The analysis revealed a significant effect of sex [F (1,16) = 11.49; p < 0.003], no significant effect of age ( p = 0.07), and a significa..., Adult and aged C57BL/6 J and SAMP8 mice (Senescence-Accelerated Mouse Prone 8) were obtained from Fundación Ciencia and Vida. These animals were housed and maintained..., For a 2 × 2 design, considering a main effect with two levels (e.g., females vs. males) and collapsing across the other factor (age), the analysis reduces to a..., The MWM test was used as a spatial memory behavioral test as previously described [,, ]. Mice were trained in a 1.2 m diameter circular pool (opaque water, 50 cm d....
inferred from protocolStructured statistical methods
The analysis revealed a significant effect of sex [F (1,16) = 11.49; p < 0.003], no significant effect of age ( p = 0.07), and a significa...; Adult and aged C57BL/6 J and SAMP8 mice (Senescence-Accelerated Mouse Prone 8) were obtained from Fundación Ciencia and Vida. These animals were housed and maintained...; For a 2 × 2 design, considering a main effect with two levels (e.g., females vs. males) and collapsing across the other factor (age), the analysis reduces to a...; For the Sex × Age interaction in the 2 × 2 ANOVA (4 cells total): n = 5 per condition ( N = 20 total) → detectable Cohen...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
To account for the high variability typical of hormone measurements, we analyzed age-related differences using geometric means (GM) derived from log10-transformed values with 95% confidence intervals (CI). Comparisons between age groups (Adult vs. Old) were performed using Welch's t-test. The fold-change was calculated as the ratio of the geometric means. The use of geometric means is particularly appropriate for skewed biological data, as it reduces the influence of extreme values and provides a more robust measure of central tendency.
The MWM test was used as a spatial memory behavioral test as previously described [,, ]. Mice were trained in a 1.2 m diameter circular pool (opaque water, 50 cm deep) filled with 19-21 °C water. A submerged 9 cm platform (1 cm below the surface, invisible to the animal) was used for training, with a maximum trial duration of 60 s and 2 s on the platform at the end of the trials. Each animal was trained to locate the platform. The test was performed with three trials per day for 10 days except for weekends, and a probe test was carried out on day 11, when the platform was removed. The latency time (in seconds) required for the animal to reach the platform and the time spent in each quadrant (in seconds) was measured. After testing, the mouse was gently removed from the maze and returned to its cage. Spatial acuity was calculated using the time mice spent in the quadrant after the platform was removed on the probe day and the time mice spent in the platform area. Both times were multiplicated to obtain the spatial acuity score. Data was plotted as the sum of escape latency (from the 10 days of learning phase) in the X axis, v...
Animals were anesthetized and then perfused through the heart with 1x PBS, followed by fixation with 4% paraformaldehyde (PFA, 4896610S, Merck) in 0.1 M phosphate buffer for 30 min. Brains were postfixed overnight at 4 °C in 4% PFA and washed 10 min, 3 times in 1x PBS. A sucrose gradient (10% and 20%, 2 h each, at room temperature and 30% overnight at 4 °C) was performed. Then, the whole brain was immersed in OCT Compound (4583, Sakura). Coronal sections 25 µm thick were collected from anterior to posterior on a Leica cryostat in a 24-well plate with 1x PBS and kept at 4 °C until use. Representative sections from each animal were mounted on Premium Micro Slide (PC2-302-16, PR PorLab) in 1x PBS, allowed to dry at room temperature, and stored at -20 °C until use [, ].
Slides were removed from -20 °C and left at room temperature; then, IF was performed. Briefly, they were hydrated in 1x PBS and incubated in blocking/permeabilization solution (Triton X-100 0.5%, BSA 5% in 1x PBS) for 1 h at room temperature. Primary antibodies were diluted in this solution and incubated at 4 °C overnight. After 3 washes in 1x PBS, 10 min each, the secondary antibodies were diluted in the same solution, incubated for 2 h at room temperature, and washed again 10 min, 3 times in 1x PBS. The slides were coated with Fluoromount-G (00-4958-02, Invitrogen, USA). Antibodies used were: mouse anti-4HNE (1:300; 298112, USBiological), mouse anti Lonp1 (1:500; sc-293244, Santa Cruz Biotechnology, Inc. USA) and anti-mouse Alexa 488 (1:500; A-21202, Invitrogen, USA) [, ].
The primary mitochondrial function involves the production of ATP, and it is known that during aging, mitochondrial function and ATP formation are reduced in the hippocampus [, ]. However, whether sex-dependent differences emerge during aging remains unclear. First, we evaluated the bioenergetic state of the hippocampus. To this end, whole hippocampal lysate, mitochondria-free cytoplasmic, and mitochondrial-enriched fractions were tested using a bioluminescence detection kit to measure the ATP content. The two-way ANOVA analyses revealed a significant effect of sex [F (1,16) = 6.00; p = 0.03] and a significant effect of age [F (1,16) = 30.542; p < 0.01], with no significant interaction between the two factors ( p = 0.12). Both aged groups exhibited a reduction in ATP content compared to adult mice, with a more pronounced decrease in aged male mice than in the female ones (Fig. ). More importantly, in the cytoplasmic fraction, the analysis showed a significant effect of sex [F (1,16) = 4.96; p = 0.04], a significant effect of age [F (1,16) = 24.42; p < 0.01], and...
Machine-readable layer
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