Unraveling sex differences in age-related hippocampal decline: differential mitochondrial dysfunction, Lonp1-dependent mitochondrial proteostasis and mtROS production in aged C57BL/6 mice methods
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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...
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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...
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