Sex- and Age-Dependent Mitochondrial Dysfunction Links Familial Hypercholesterolemia to Cognitive Impairment

Sex- and Age-Dependent Mitochondrial Dysfunction Links Familial Hypercholesterolemia to Cognitive Impairment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sex- and Age-Dependent Mitochondrial Dysfunction Links Familial Hypercholesterolemia to Cognitive Impairment Nathasha M. C. Prado-Lopes, Daniel Fagundes, Letícia Tavares, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8561934/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Apr, 2026 Read the published version in Biology of Sex Differences → Version 1 posted 9 You are reading this latest preprint version Abstract Background. Familial hypercholesterolemia (FH) is a genetic disorder of cholesterol metabolism caused by loss-of-function variants in the low-density lipoprotein receptor (LDLR), resulting in persistently elevated LDL-cholesterol levels in plasma. Although hypercholesterolemia, especially the high levels of LDL, has been linked to an increased risk of dementia, the underlying mechanisms remain unclear. Here, we investigated the effects of sexual dimorphism and aging on metabolic and cognitive functions in a murine model of FH. Methods. Adult and middle-aged, male and female, C57BL/6 and LDLr −/− mice were used in this study. Behavioral assessments included locomotor activity, spatial memory, and anxiety-like behavior. Plasma lipid profiles were measured, and mitochondrial function in the hippocampus and brown adipose tissue (BAT) was assessed using high-resolution respirometry. Results. LDLr ⁻/⁻ mice of both sexes exhibited increased cholesterol and triglycerides levels. Male LDLr ⁻/⁻ mice displayed hyperlocomotion in the Open Field (OF) and Elevated Plus Maze (EPM) at both ages, whereas this phenotype emerged in middle-aged female LDLr ⁻/⁻ mice only in OF. Spatial memory impairments were observed in LDLr ⁻/⁻ mice regardless of sex or age. Hippocampal oxygen consumption was reduced in adult males and middle-aged female mice, whereas BAT respiration was impaired in both sexes at middle-aged animals, affecting distinct respiratory parameters. Correlation analyses revealed that elevated cholesterol levels were associated with memory deficits and hyperlocomotion, along with positive correlations between hippocampal and BAT mitochondrial function. Conclusions. Collectively, these findings demonstrate that FH induces sex- and age-dependent alterations in behavior and mitochondrial metabolism, providing mechanistic insights into the link between FH and neurodegenerative disease risk. aging sexual dimorphism behavior hypercholesterolemia mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights LDLr -/- mice exhibited hyperlocomotion; males were affected at both ages and females at middle-age. LDLr ⁻/⁻ mice exhibited spatial memory impairment, independent of sex and age. Adult male LDLr ⁻/⁻ mice exhibited higher susceptibility to hippocampal mitochondrial dysfunction; females were affected at middle-age. Correlation analyses indicated links between metabolic, mitochondrial, and behavioral outcomes. LDLr ⁻/⁻ mice showed age- and sex-dependent differences that may elucidate potential determinant factors in HF outcomes. Plain English Summary Familial hypercholesterolemia (FH) is a genetic condition in which cholesterol levels are high from an early age. Because the body cannot remove cholesterol efficiently, it can build up in the blood and in the arteries over time. FH is best known for increasing the risk of heart disease, but long-term high cholesterol may also affect brain function. In this study, we used a mouse model of FH to understand how high cholesterol affects memory, behavior, and mitochondrial metabolism, and whether these effects differ between males and females as they age. To do this, we studied male and female mice during adulthood and middle age. Mice with FH showed difficulties remembering the location of objects, indicating impaired spatial memory. This memory problem was observed in both sexes. However, other changes differed between males and females. Male mice with FH showed increased activity levels at both ages, while female mice showed this change only at middle age. We also examined how mitochondria function in the hippocampus, a brain region important for memory, and in brown fat, a tissue involved in metabolism. Mitochondrial function was altered in FH, with differences between males and females. Overall, our findings show that FH affects brain function and mitochondrial metabolism differently in males and females. Males were susceptible in adulthood, while females showed effects after middle age, emphasizing the role of sex and age in the long-term impact of high cholesterol. 1. INTRODUCTION Familial hypercholesterolemia (FH) is one of the most prevalent genetic disorders worldwide, particularly in the heterozygous form [ 1 , 2 ]. It is mainly caused by mutations in the low-density lipoprotein (LDL) receptor (LDLr) gene and is characterized by chronically elevated LDL-cholesterol levels, resulting in arterial deposition and substantially increased risk of atherosclerotic cardiovascular disease (ASCVD) [ 3 , 4 ]. Beyond cardiovascular consequences, increasing evidence suggests that FH also compromises brain health. Two aspects are particularly relevant: lifelong exposure to elevated cholesterol levels and LDLr dysfunction [ 5 ], which not only regulate cholesterol uptake but also contribute to synaptic function, neuronal plasticity and amyloid-β (Aβ) clearance [ 6 – 8 ]. In this context, the Lancet Commission on Dementia [ 9 ] highlights high LDL cholesterol as one of the main modifiable risk factors for dementia. Supporting this link, familial hypercholesterolemia (FH) has been associated with cognitive decline, mild cognitive impairment (MCI), and dementia [ 9 , 10 ], with memory deficits reported even in young adults aged 18 to 40 years [ 11 ]. Adding to this evidence, recent findings from the ELSA-Brazil cohort showed nonlinear associations between serum lipid levels and cognitive decline, particularly among individuals younger than 60 years and women [ 10 ] Sexual dimorphism adds further complexity to this scenario. Clinical data indicate a higher prevalence of FH in women, whereas men tend to develop ASCVD earlier and initiate treatment sooner [ 11 – 14 ]. While premenopausal women are relatively protected, menopause attenuates this advantage, raising cardiometabolic and cognitive risks to levels comparable to those observed in men [ 15 , 16 ]. In parallel, aging is accompanied by systemic metabolic alterations, including insulin resistance, dyslipidemia and chronic inflammation, which indirectly affect brain function and increase the risk of MCI and dementia across the lifespan [ 17 , 18 ]. As a major risk factor for neurodegenerative diseases, aging entails progressive biological changes that disrupt cholesterol homeostasis and compromise both physical and cognitive functions [ 19 – 22 ]. Therefore, the interplay between sex and aging may critically modulate metabolic and cognitive outcomes in FH. The LDLr ⁻/⁻ mice, generated by Ishibashi et al. [ 23 ], is a well-established model for studying the pathophysiology of FH. Consistent with clinical findings, LDLr ⁻/⁻ mice display elevated LDL-cholesterol levels and a broad spectrum of behavioral alterations, even at young ages, indicating early central nervous system vulnerability. These alterations include deficits in spatial, working and long-term memories [ 24 – 28 ]. Additionally, young and middle-aged LDLr ⁻/⁻ mice exhibit increased locomotor activity [ 27 , 29 ], as well as emotional dysregulation, characterized by heightened stress sensitivity and depressive-like behavior [ 30 ]. Underlying these cognitive impairments, several cellular and biochemical alterations have been reported in LDLr ⁻/⁻ mice. These include impaired adult hippocampal neurogenesis, enhanced glial reactivity, blood-brain barrier (BBB) disruption, hippocampal apoptosis, and neuronal and synaptic dysfunctions [ 25 , 30 – 32 ]. Among these pathological processes, mitochondrial dysfunction has emerged as a central mechanism, connecting cognitive and neuronal impairments with systemic metabolic alterations. In the brain, LDLr ⁻/⁻ mice exhibit oxidative stress, impaired respiratory chain function, and reduced coenzyme Q10 (CoQ10) levels, worsened by intracellular cholesterol accumulation driven by mevalonate pathway activation [ 33 – 35 ]. Peripheral tissues are also affected: white adipose tissue (WAT) shows oxidative stress and inflammation, promoting reactive oxygen species (ROS) accumulation, impairing insulin signaling, and contributing to insulin resistance, glucose intolerance, and metabolic dysfunction [ 36 , 37 ]. WAT from LDLr −/− mice also exhibits macrophage infiltration, increased proinflammatory cytokines, and altered adipokine secretion, establishing a chronic inflammatory state that disrupts energy homeostasis [ 38 , 39 ]. In contrast, brown adipose tissue (BAT), a thermogenic and mitochondria-rich depot essential for systemic metabolism, regulating thermogenesis, insulin sensitivity, and lipid metabolism [ 36 , 40 , 41 ], remains underexplored in the context of FH. Notably, most studies have focused on male LDLr ⁻/⁻ mice, leaving the influence of sex largely unresolved. However, sex is a critical determinant of both metabolic regulation and brain function. Given that aging also modulates cholesterol metabolism and hormonal status, FH may exert age- and sex-specific effects on cognition and mitochondrial function. Accordingly, the present study investigated the combined influence of genotype, sex, and age on behavioral and metabolic outcomes in LDLr ⁻/⁻ mice. We assessed cognitive, locomotor and anxiety-like behaviors, as well as mitochondrial function in the hippocampus and BAT, to elucidate mechanisms of vulnerability associated with FH. 2. MATERIAL AND METHODS 2.1. Animals Male and female C57Bl/6 wild-type (WT) and LDLr ⁻/⁻ (B6.129S7 LDLrtm1Her/J) mice, aged 6–8 months (adult group) and 12–14 months (middle-aged group), were originally purchased from the Jackson Laboratory and were bred in our own breeding colony at the University of Brasília. Mice were housed in acrylic cages under controlled conditions, with a filtered air system (Alesco; 3–4 mice/cage) at a controlled temperature (23–25°C), and 12h light/dark cycle (lights on at 6 a.m.). They had ad libitum access to food and water. All animal procedures complied with the National Institutes of Health (NIH) guidelines for animal care, the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, and were approved by the Animal Ethics Committee of the University of Brasília (SEI Protocol 23106.075489/2023-95). 2.2. Experimental design Mice were allocated according on sex, age, and genotype into eight groups of 6–12 animals. Mice weighed between 25­30 g, and were fed a standard rodent diet (SD; 70% carbohydrates, 20% protein, and 10% fat). Behavioral assessments included the open field (OF), object location (OL), and elevated plus maze (EPM) tests. Following this, the animals were anesthetized with ketamine and xylazine via intraperitoneal (i.p.) injection (80:8 mg/kg, respectively) until full analgesia and then were euthanized by cervical dislocation. Blood and tissues, including hippocampi and BAT, were collected immediately after euthanasia. 2.3. Behavioral tasks For the behavioral tests, the animals were transferred to the testing room at least 1 hour before the beginning of the experiments, to allow acclimation. The testing room was maintained at controlled temperature and humidity, mirroring the conditions of their regular housing. The room was also free from extraneous odors, including those from the experimenter (i.e., perfume, deodorant, or lotion were avoided). All behavioral tests were recorded using AnyMaze® software (RRID:SCR_014289; version 7.1 for Windows) and conducted between 7 a.m. and 4 p.m., during the light phase of the animals’ light/dark cycle. 2.3.1. Open field (OF) OF was used to evaluate the spontaneous locomotor and exploratory activities induced by a novel environment. The OF apparatus was a 30 cm length cubic arena with a white background, containing spatial cues on the sidewalls. Animals were placed individually on the center of the apparatus to freely explore the arena for 5 min [ 42 ]. The arena was cleaned with ethanol 30% between each animal trial. Total distance traveled and time spent in the inner and peripheral quadrants (inner quadrant was drawn 7.50 cm away from the walls) were evaluated using ANY-maze tracking system (Stoelting Co., IL, USA). 2.3.2. Object location test (OL) OL test was performed to assess spatial reference memory, following the protocol previously described by Assini et al. [ 43 ]. During the training session, animals were placed in the arena for 5 minutes with two identical objects positioned parallel to each other, 5 cm away from the walls. After the training phase, the mice were removed from the arena for 90 minutes. Following the inter-trial interval, one object remained in the same location (nondisplaced object [ND]), and the other one was relocated to a new position (displaced object [D]). The animals were then reintroduced into the arena and allowed to explore for another 5 minutes. After each trial, the experimental apparatus was cleaned with 30% ethanol. Exploration time was recorded when the mice sniffed, looked at, touched, or smelled the object at least 1 cm away. A location index (LI) was calculated to evaluate location memory using the formula: LI = TD * 100 / (TD + TND), where TD and TND are the exploration times for the displaced and nondisplaced objects, respectively [ 44 ]. 2.3.3. Elevated plus maze (EPM) EPM test is currently performed to assess anxiety-like behavior in rodents [ 45 ]. The apparatus is elevated 60 cm from the floor, with four arms (18 cm long, 6 cm wide). Two opposite arms are surrounded by walls (6 cm high, enclosed arms), while the other two arms are devoid of enclosing walls (open arms). The four arms are connected by a central area (6 x 6 cm). The animals were individually placed in the central area and allowed to freely explore for 5 minutes [ 25 ]. The apparatus was cleaned with ethanol solution (30% v/v) and dried with paper towels after each trial, to avoid odor impregnation. During the test, the number of entries into each arm and the total distance traveled were recorded using ANY-maze tracking system (Stoelting Co., IL, USA). To normalize the number of entries into the open arms relative to the total number of arm entries, results were expressed as the percentage of entries into the open arms, calculated as follows: 100 * OA/(OA + CA), where OA corresponds to the number of entries into the open arms and CA to the number of entries into the closed arms. 2.4. Lipid profile analysis After euthanasia, approximately 500 µL of whole blood was collected from the animals. The blood was centrifuged at 3000 g for 10 minutes to obtain serum. Subsequently, cholesterol (Labtest, Cat# 76) and triglyceride (Labtest, Cat# 87) levels were measured in the serum using colorimetric assays, following the manufacturer’s instructions. 2.5. High-resolution respirometry Oxygen (O 2 ) consumption was assessed using high-resolution respirometry (HRR) with an Oroboros 2k Oxygraph (Oroboros Instruments, Innsbruck, Austria) at 37°C. The oxygraph system is a closed chamber that measures changes in O 2 concentration. Any variation in O 2 levels is attributed to the samples, which utilize the substrates or drugs added during the experiment, and consume O 2 , enabling the assessment of specific mitochondrial states [ 46 ]. 2.5.1. Hippocampal respirometry Hippocampi were collected and homogenized in 300 µL of reaction buffer [RB: 125 mM sucrose, 65 mM KCl, 2 mM KH 2 PO 4 , 2 mM MgCl 2 , 10 mM HEPES, 0.1 mM EGTA, 0.01% bovine serum albumin (BSA)] using a 5 mL glass-teflon homogenizer. The samples (protein ∼0.200 mg/mL) were added to a 2-mL chamber containing RB. Substrates (all purchased from Sigma-Aldrich) were added sequentially to assess O 2 flux as follows: pyruvate (P; 5mM) and malate (M; 2,5mM) were added to assess complex I activity. For complexes I + II, succinate (S; 10mM) was added. Oxidative phosphorylation (OXPHOS) was evaluated after the addition of 500 µM ADP, and state 4 (leak) was determined following the addition of oligomycin (OMY; 0.1 µg/mL). Uncoupling of the electron transport system (ETS) was induced with titrated carbonyl cyanide 3-chlorophenylhydrazone (CCCP, final concentration 1–3 µM). Complex II activity was assessed by adding 0.5 µM rotenone, and non-mitochondrial residual respiration was measured after the addition of Antimycin A (AA: 1 µM). ATP-linked respiration was calculated as the difference between oxygen consumption rate in OXPHOS and state 4. 2.5.2. BAT respirometry BAT was collected, weighted and cut into 1 mm³ pieces. Around 10 x the volume (µL) of RB corresponding to BAT weight was added to homogenize the tissue into a 5 mL glass-teflon homogenizer. The samples (protein ∼ 0.135 mg/mL) were added to a 2 mL chamber containing RB and 0.1% additional BSA. Initially, PM (5 and 2.5 mM, respectively) were added, followed by guanosine diphosphate (GDP: 1 mM) to evaluate uncoupling protein-1 (UCP-1) activity. Then, CCCP (3–7 µM final concentration) and rotenone (ROT: 0.5 µM) were sequentially added. 2.6. STATISTICAL ANALYSIS All statistical analyses were performed using R (version 4.3), utilizing key packages including tidyverse for data manipulation, car for ANOVA, emmeans for post-hoc comparisons, vegan for multivariate analysis, mediation for causal modeling, and caret for predictive modeling. Prior to analysis, outliers within each of the eight experimental groups were identified for each biological variable using the 1.5x interquartile range (IQR) rule and were excluded from the respective analyses. The assumption of normality of residuals was checked for all parametric tests. The primary statistical method to assess the main effects of sex, age, and phenotype, as well as their interactions, was a three-way analysis of variance (ANOVA). When significant main effects or interactions were detected, simple effects were assessed via pairwise post-hoc comparisons of the estimated marginal means, applying the Holm-Bonferroni method for p-value adjustment. Partial omega-squared (ω 2 p ) was calculated as an estimate of effect size for the ANOVA results. Graphs were generated using GraphPad Prism 9.0 (RRID:SCR_002798). All data are presented as mean ± SEM unless otherwise stated. A p-value of < 0.05 was considered statistically significant for all tests. 2.6.1. Behavioral and Metabolic Data Correlation To investigate the linear interrelationships among the phenotypic variables, we calculated Pearson's product-moment correlation coefficients (ρ). The resulting correlation matrix was visualized as a heatmap. To highlight statistically significant associations and facilitate interpretation, only correlation coefficients with an associated p-value of less than 0.05 were numerically displayed within the heatmap cells. Furthermore, we organized the variables a priori into three functional blocks — 'Metabolic & Behavioral', 'Hippocampal Bioenergetics', and 'BAT Thermogenesis'. 2.6.2. Data preprocessing and dimensionality reduction Our initial correlation analyses revealed strong collinearity within specific subsets of variables, particularly those related to mitochondrial respiration and locomotor activity. To mitigate the effects of multicollinearity in subsequent statistical models, and to derive robust, composite metrics for these biological domains, we employed Principal Component Analysis (PCA). We conducted three separate PCAs. The first consolidated five hippocampal mitochondrial respiration variables into a single 'Hippocampal Bioenergetics Score'. The second combined three BAT variables to create a 'BAT Thermogenic Score', and the third integrated two locomotor variables into a 'Locomotor Score'. We confirmed the suitability of the data for this approach using Bartlett’s test of sphericity and the Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy for each component. For each analysis, we retained the first principal component (PC1), as it explained the majority of the shared variance (84.3% for hippocampal, 52.5% for BAT, and 71.8% for locomotor variables). 2.6.3. PLS-DA Model We employed a Partial Least Squares Discriminant Analysis (PLS-DA), a supervised machine learning method, to identify the multivariate phenotypic signature capable of discriminating animals by genotype. The predictor set included the metabolic, behavioral, and principal component (PC) scores for bioenergetics; the individual variables comprising the PCs were excluded to prevent multicollinearity. Prior to model training, we autoscored all predictors (mean-centered and scaled to unit variance). We assessed the model's robustness and performance using a 10-fold cross-validation procedure. The optimal number of latent components for the final model was selected based on the maximization of the Area Under the Receiver Operating Characteristic (ROC) curve. We quantified each variable's importance in discriminating between genotypes using the Variable Importance in Projection (VIP) scores, where higher values indicate a greater contribution to group separation. 3. RESULTS 3.1. Lipid metabolism and BAT thermogenesis are differently affected in male and female LDLr -/- mice The experimental design included three variables — age (adult vs. middle-aged), genotype (WT vs. LDLr ⁻/⁻ ), and sex (female vs. male), resulting in eight experimental groups (Fig. 1 A). To assess the impact of sex and aging on lipid metabolism in the context of FH, we performed a comprehensive lipid profile analysis. Three-way ANOVA revealed a significant effect of genotype on cholesterol (F(1,51) = 51.57, p < 0.001, ω 2 p = 0.68; Fig. 1 B) and triglycerides (F(1,51) = 14.91, p < 0.001, ω 2 p 0.47; Fig. 1 C) levels, as well as a age:genotype interaction on cholesterol levels (F(1,51) = 4.38, p = 0.0414, ω 2 p = 0.01). Post-hoc Holm–Sidak test confirmed that both male (both ages: p < 0.0001) and female (adult: p < 0.0001; middle-aged: p = 0.0007) LDLr ⁻/⁻ mice displayed significantly elevated serum cholesterol compared with age-matched WT controls. For triglycerides levels, LDLr ⁻/⁻ females showed a significant increase at the adult stage compared with WT controls (p = 0.0032), whereas LDLr ⁻/⁻ males displayed elevated levels at both adult (p = 0.0012) and middle-aged (p = 0.0004) stages. Additionally, middle-aged males LDLr −/− exhibited higher triglyceride levels than females LDLr −/− of the same age (p = 0.0422). To evaluate mitochondrial thermogenic activity in BAT, we measured oxygen consumption associated with complex I (Fig. 1 D), the percentage of UCP-linked mitochondrial respiration (Fig. 1 E), and the maximal uncoupled respiratory capacity (ETS; Fig. 1 F). For complex I, three-way ANOVA revealed a significant effect of sex (F(1,51) = 9.50, p = 0.0033, ω 2 p = 0.22), and a sex:age interaction (F(1,51) = 6.95, p = 0.0111, ω 2 p = 0.08). Post-hoc analysis showed reduced oxygen consumption in middle-aged LDLr ⁻/⁻ males compared with controls (p = 0.0192), as well as an age effect in WT males, with middle-aged animals exhibited lower oxygen consumption than adult group (p = 0.0014). Sex differences were also detected in adult WT (p = 0.0298) and adult LDLr ⁻/⁻ mice (p = 0.0081), with males exhibiting higher oxygen consumption than females. For UCP1-linked respiration, three-way ANOVA revealed significant effects of sex (F(1,53) = 5.14, p = 0.0275, ω 2 p = 0.16), a sex:age:genotype interaction (F(1,53) = 7.23, p = 0.0096, ω 2 p = 0.09), and a trend toward a genotype effect (F(1,53) = 3.82, p = 0.0559). Post-hoc analysis indicated an age effect in LDLr ⁻/⁻ females, as middle-aged animals showed reduced UCP-1 activity compared with the adult group (p = 0.0085). Additionally, sex differences were observed in middle-aged WT (p = 0.0125) and LDLr ⁻/⁻ (p = 0.0097) mice, with males displaying higher UCP-1 activity than females. No significant differences were detected for ETS capacity. Ldlr -/- mice exhibit spatial memory impairment regardless of sex, while hyperactivity is more pronounced in males Spontaneous locomotor activity was evaluated in the OF test by measuring the total distance traveled for 5 minutes (Fig. 2 A). Three-way ANOVA revealed significant effects of sex (F(1,60) = 5.54, p = 0.0218, ω 2 p = 0.00) and age (F(1,60) = 25.13, p < 0.001, ω 2 p = 0.36), as well as a trend toward a genotype effect (F(1,60) = 3.51, p = 0.0657, ω 2 p = 0.54). Post hoc analysis showed that LDLr ⁻/⁻ males exhibited increased locomotion at both ages compared to age-matched WT controls (adult: p = 0.0005; middle-aged: p < 0.0001), whereas LDLr ⁻/⁻ females displayed this increase only at middle-age (p = 0.0072). An age effect was also detected in WT mice, with middle-aged males (p = 0.0247) and females (p = 0.0001) traveling shorter distances than their adult counterparts. Similarly, LDLr ⁻/⁻ females showed reduced locomotion at middle-age compared to adults. In addition, sex differences emerged in middle-aged LDLr ⁻/⁻ mice, with males traveling greater distances than females of the same age. The percentage of time spent on the periphery of the apparatus (Supplementary Fig. 1A) was also analyzed. Three-way ANOVA revealed significant effects of sex (F(1,56) = 19.64, p < 0.001, ω² p =0.10), age (F(1,56) = 14.87, p < 0.001, ω² p =0.05), and a sex:age:genotype interaction (F(1,56) = 14.52, p < 0.001, ω² p =0.17). Post hoc analysis indicated that LDLr ⁻/⁻ adult males (p = 0.0003) and LDLr ⁻/⁻ middle-aged females (p = 0.0003) spent more time in the periphery compared with their respective WT controls. Moreover, WT females at middle-age spent less time in the periphery than adult WT females (p = 0.0027). In addition, a sex effect was detected in adult WT mice, with males spending less time in the periphery than females of the same age (p = 0.0004). Spatial memory was evaluated using the OL test (Fig. 2 B). Only WT control groups of both sexes exhibited a location index significantly above the chance level of 50%, while LDLr ⁻/⁻ mice failed to discriminate the displaced object. Two-way ANOVA revealed a significant effect of age (F(1,61) = 6.50, p = 0.0133, ω² p =0.16), a sex:age:genotype interaction (F(1,61) = 6.74, p = 0.0118, ω² p =0.08), and a trend toward a genotype effect (F(1,61) = 3.98, p = 0.0505, ω² p =0.32). Anxiety-like behavior was assessed in the EPM test through the number of entries into open arms (Supplementary Fig. 1B), the percentage of open arm entries (Fig. 2 C), and the total distance traveled (Fig. 2 D) over 5 minutes. For the number of entries into open arms, three-way ANOVA revealed significant effects of sex (F(1,58) = 7.59, p = 0.0078, ω² p =0.00) and a sex:genotype interaction (F(1,58) = 6.67, p = 0.0123, ω² p =0.08), with a trend for an age effect (F(1,58) = 3.49, p = 0.0669, ω² p =0.00). Post hoc analysis showed that both adult (p = 0.0021) and middle-aged (p = 0.0025) LDLr ⁻/⁻ males displayed increased open arm entries compared to WT controls. To account for differences in overall locomotor activity, we also calculated the percentage of entries into the open arms relative to the total number of entries. Three-way ANOVA indicated significant effects of age (F(1,61) = 6.44, p = 0.0137, ω² p =0.00) and a sex:age interaction (F(1,61) = 6.85, p = 0.0112, ω² p =0.07), as well as a trend toward an age:genotype interaction (F(1,61) = 3.45, p = 0.0680, ω² p =0.01). Post hoc analysis revealed that middle-aged LDLr ⁻/⁻ females entered the open arms more frequently than WT females of the same age. Finally, total distance traveled in the EPM was analyzed to assess locomotor activity within the apparatus. Three-way ANOVA showed significant effects of sex (F(1,55) = 8.34, p = 0.0055, ω² p =0.00) and a sex:genotype interaction (F(1,55) = 18.72, p < 0.001, ω² p =0.26). Post hoc analysis demonstrated that LDLr ⁻/⁻ males traveled longer distances than their respective WT counterparts at both ages (adult: p < 0.0001; middle-aged: p = 0.0012). In addition, sex differences were detected in WT mice, with females traveling longer distances than males at both adulthood (p = 0.0442) and middle-age (p = 0.0382). A sex effect was also observed in adult LDLr ⁻/⁻ mice, with males covering more distance than females. 3.3. Sex-specific differences in hippocampal mitochondrial dysfunction in LDLr -/- mice We evaluated hippocampal mitochondrial bioenergetics using HRR to measure oxygen consumption under different stimuli. For complex I–related oxygen consumption (Fig. 3 C), three-way ANOVA revealed significant effects of sex (F(1,52) = 8.99, p = 0.0042, ω² p =0.00), age (F(1,52) = 5.02, p = 0.0294, ω² p =0.25), and a sex:age:genotype interaction (F(1,52) = 29.44, p < 0.001, ω² p =0.32), with a trend toward a genotype effect (F(1,52) = 3.91, p = 0.0533, ω² p =0.09). Post hoc analysis indicated that middle-aged LDLr ⁻/⁻ females exhibited reduced oxygen consumption compared with WT females (p < 0.0001). Age effects were observed in WT males (p < 0.0001) and LDLr ⁻/⁻ females (p = 0.0001), with middle-aged animals showing lower mitochondrial activity than adults of the same genotype. Sex differences were also detected in WT mice: adult males displayed higher Complex I–linked respiration than adult females (p = 0.0333), whereas middle-aged males exhibited lower activity than females (p = 0.0004). For complex I + II respiration (Fig. 3 D), three-way ANOVA revealed significant effects of sex (F(1,53) = 10.59, p = 0.0020, ω² p =0.02) and a sex:age:genotype interaction (F(1,53) = 10.45, p = 0.0021, ω² p =0.13). Post hoc analysis indicated reduced mitochondrial respiration in adult LDLr ⁻/⁻ males compared with WT males (p = 0.0143). An age effect was observed in WT males, with middle-aged animals displaying lower oxygen consumption than adults (p = 0.0143). Additionally, adult WT males exhibited higher respiration than adult females (p = 0.0198). Regarding OXPHOS capacity (Fig. 3 E), three-way ANOVA revealed significant effects of sex (F(1,55) = 8.74, p = 0.0046, ω² p =0.006) and a sex:age:genotype interaction (F(1,55) = 11.84, p = 0.0011, ω² p =0.15). Post hoc analysis showed that middle-aged LDLr ⁻/⁻ females exhibited reduced OXPHOS capacity compared with WT females (p = 0.0208). Middle-aged WT males also showed a trend toward reduced capacity compared with adults (p = 0.0712), and a similar trend was observed for sex differences in adult WT mice, with males showing higher values than females (p = 0.0503). For ETS capacity (Fig. 3 F), three-way ANOVA revealed significant effects of sex (F(1,54) = 7.99, p = 0.0066, ω² p =0.00) and a sex:age:genotype interaction (F(1,54) = 10.37, p = 0.0022, ω² p =0.13). Post hoc analysis indicated reduced maximal uncoupled respiration in adult LDLr ⁻/⁻ males (p = 0.0401) and middle-aged LDLr ⁻/⁻ females (p = 0.0019) compared with their WT counterparts. Age effects were also observed in WT males and LDLr ⁻/⁻ females, as middle-aged animals displayed lower oxygen consumption than adults. Additionally, a trend toward sex differences was observed in adult WT mice (p = 0.0527). Finally, for ATP-linked respiration (Fig. 3 G), three-way ANOVA revealed significant effects of sex (F(1,52) = 6.21, p = 0.0160, ω² p =0.02) and a sex:age:genotype interaction (F(1,52) = 7.58, p = 0.0081, ω² p =0.10), with a trend toward an age effect (F(1,52) = 3.67, p = 0.0608, ω² p =0.04). Post hoc analysis demonstrated that middle-aged LDLr ⁻/⁻ females exhibited reduced ATP-linked respiration compared with WT females (p = 0.0119). 3.4. PCA–based functional scores for locomotor, hippocampal, and BAT domains Following the correlogram analysis (Supplementary Fig. 2), to condense correlated variables into meaningful biological indices, we performed three separated principal component analyses (PCAs). In all analyses, the first principal component (PC1) accounted for the majority of the variance, a finding visually confirmed by the scree plot analysis, where PC1 was the only component across all three domains with an eigenvalue greater than 1.0 (Kaiser’s criterion) (Supplementary Fig. 3). Locomotor Score: the two locomotor variables (OF and EPM) were also condensed into a single component (Bartlett's χ2 (1) = 12.31, p < 0.001; KMO = 0.50). PC1 explained 71.8% of the combined variance, with both distance variables contributing equally (loadings = 0.707). The "Locomotor Score" thus effectively represents an overall measure of locomotor activity. Hippocampal Bioenergetics Score: the five variables related to hippocampal mitochondrial respiration were highly suitable for PCA, as indicated by a significant Bartlett's test of sphericity (χ2 (10) = 373.57, p < 0.001) and a good KMO value of 0.74. PC1, designated the "Hippocampal Bioenergetics Score," explained 84.3% of the total variance. All five input variables demonstrated strong, negative loadings on this component (loadings from − 0.41 to -0.47), indicating that PC1 represents a robust, integrated measure of overall mitochondrial respiratory function. BAT Thermogenic Score: similarly, the three variables measuring BAT mitochondrial function were appropriate for PCA (Bartlett's χ2 (3) = 12.2, p = 0.007; KMO = 0.62). The resulting PC1, termed the "BAT Thermogenic Score", accounted for 52.5% of the shared variance. The variables for complex I-linked, maximal, and uncoupled respirations all contributed positively and with similar weights (loadings from 0.56 to 0.59), reflecting a composite index of thermogenic potential. 3.5. Correlational Analysis Reveals Links Between Metabolic, Mitochondrial, and Behavioral Genotypes To elucidate the interrelationships between the observed alterations, we performed a comprehensive Spearman's correlational analysis (Fig. 4 ). Critically, metabolic dysregulation was tightly linked to the behavioral genotype: the composite locomotion score was strongly and positively correlated with cholesterol levels (ρ = 0.620, p < 0.001). Conversely, cognitive performance was inversely related to metabolic health, with the spatial memory index showing significant negative correlations with serum cholesterol levels (ρ = −0.421, p = 0.001). Furthermore, a novel positive correlation was found between overall hippocampal bioenergetic and BAT thermogenic scores (ρ = 0.392, p = 0.006), which may reflect an association between central and peripheral bioenergetic impairment. 3.6. Predictive Modeling Identifies Dyslipidemia and Hyperactivity as the Core Genotypic Signature To move beyond identifying group differences and instead determine the core "genotypic signature" of the LDLr ⁻/⁻ model, we employed a supervised machine learning approach, Partial Least Squares Discriminant Analysis (PLS-DA), to classify animals based on their complete biological profile. Remarkably, the PLS-DA model distinguished LDLr ⁻/⁻ from control mice with perfect accuracy in cross-validation (ROC-AUC = 1.00), indicating that the measured variables created a highly consistent and separable biological fingerprint for the genotype. An analysis of Variable Importance in Projection (VIP) scores provided a crucial insight into this signature: the most powerful and reliable predictors were serum cholesterol (VIP = 100.00), the composite locomotor score (VIP = 93.55), and serum triglycerides (VIP = 91.33) (Supplementary Fig. 4). 4. DISCUSSION Epidemiological studies over the past decades have consistently shown that high cholesterol is linked to a greater risk of dementia. This connection is even stronger in familial hypercholesterolemia (FH), a condition long known for its cardiovascular complications and now increasingly associated with cognitive problems. In this regard, LDLr⁻/⁻ mice are widely used to explore how chronically elevated cholesterol affects tissues that are sensitive to metabolic stress, including the brain, providing an important model for interpreting our findings [ 23 , 47 ]. In our study, both adult and middle-aged male and female LDLr ⁻/⁻ mice, fed a standard diet, exhibited impaired cholesterol clearance and significantly elevated plasma cholesterol levels compared to age- and sex-matched WT controls. The increase was approximately two-fold, consistent with the well-characterized hypercholesterolemic phenotype of the model. However, we did not detect sex differences in cholesterol levels at either age. Previous studies have reported varying results: Ishibashi et al. (1993,1994) reported higher cholesterol in female LDLr ⁻/⁻ mice at 56 days and 6 months [ 23 , 47 ], while Marsh et al. (1999) observed higher levels in male LDLr ⁻/⁻ mice at 4 months [ 48 ], and Ghosh et al. (2020) described no sex differences at 6 months in LDLr −/− mice on a standard diet [ 49 ]. Similarly, Rinninger et al. (2014) reported that both male and female LDLr ⁻/⁻ mice showed markedly elevated and triglycerides compared to WT [ 50 ]. The discrepancies between studies may stem from methodological and physiological variables, including fasting status, diet composition, age, and hormonal influences, all of which can markedly affect lipid metabolism and mask potential sex-related differences. Conversely, sex- and age-dependent differences in triglyceride (TG) levels were observed in our study. Adult female LDLr ⁻/⁻ mice displayed elevated TG compared to WT, while male LDLr ⁻/⁻ mice exhibited increased TG at both ages. While some studies did not observe differences between LDLr ⁻/⁻ and WT [ 23 , 31 ], others described pronounced TG elevations in male LDLr ⁻/⁻ mice, even under standard diet conditions [ 51 , 52 ]. The Jackson Laboratory also notes that TG increases can be detected in LDLr ⁻/⁻ mice on chow diet, though they become more evident under high-fat feeding. A similar profile has been reported in LDLr ⁻/⁻ rats, with increased plasma cholesterol and TG under standard diet [ 53 ]. This aligns with the classification of genetic dyslipidemias, in which this form of hypercholesterolemia may include higher TG levels, as LDL particles—while mainly cholesterol-rich—also carry a smaller TG fraction. Given that elevated serum TG reflects atherogenic lipoproteins and adds cardiovascular risk [ 56 ], these observations further underscore the systemic metabolic burden caused by LDLr dysfunction, paralleling features seen in FH patients [ 2 ] Thermogenic adipocytes substantially contribute to systemic lipid clearance by accelerating the uptake, oxidation and re-esterification of circulating fatty acids, thereby lowering plasma triglyceride and cholesterol levels [ 54 , 55 ]. White adipocytes are unilocular with a single large lipid droplet and relatively few elongated mitochondria, whereas brown adipocytes are multilocular and densely packed with mitochondria that are rich in iron and cytochromes, features that underlie BAT’s characteristic coloration and high oxidative capacity [ 54 , 55 ]. BAT is also highly vascularized and extensively sympathetically innervated, structural attributes that support rapid fuel delivery and heat generation [ 54 ]. Mechanistically, BAT thermogenesis is mediated by uncoupling protein 1 (UCP-1), which dissipates the proton motive force across the inner mitochondrial membrane and uncouples electron transport from ATP synthesis, converting chemical energy into heat [ 56 , 57 ]. In our oximetry analyses, middle-aged male LDLr ⁻/⁻ mice exhibited reduced oxygen consumption linked to complex I substrates, while middle-aged female LDLr ⁻/⁻ mice showed a selective reduction in UCP-1–related oxygen consumption, indicating age-dependent declines in thermogenic function in both sexes. We also identified sex-related differences in BAT bioenergetics: adult WT males displayed higher respiration rates linked to complex I substrates compared with adult females, and middle-aged LDLr ⁻/⁻ males showed higher respiration rates than LDLr ⁻/⁻ females of the same age. Functionally, such BAT dysfunction is expected to impair thermogenic capacity and lipid clearance, thereby contributing to the exacerbated dyslipidemia observed in LDLr ⁻/⁻ mice. These experimental observations are consistent with interventional studies in the LDLr ⁻/⁻ model: pharmacological activation of thermogenic adipocytes with the selective β3-adrenergic agonist CL316,243 reduced plasma lipids and induced regression of atherosclerotic plaques in LDLr ⁻/⁻ mice [ 58 ]. Conversely, surgical removal of adipose depots, including BAT, aggravated metabolic disturbances in LDLr ⁻/⁻ mice under high-fat feeding [ 59 ], demonstrating the protective, lipid-clearing role of brown and beige fat. More recently, β3-adrenoceptor agonists used clinically, such as mirabegron, were reported to lower plasma triglycerides in LDLr ⁻/⁻ mice, further supporting the translational relevance of BAT activation for lipid control [ 55 ]. Notably, however, most of these studies do not disaggregate outcomes by sex, and evidence specifically addressing UCP-1 activity in female LDLr ⁻/⁻ mice remains limited. Beyond systemic metabolism, the LDLr ⁻/⁻ mice has also been used to study neurocognitive function, including behavioral outcomes and brain cellular homeostasis [ 28 ]. In the OF test, we found that adult male LDLr ⁻/⁻ mice exhibited hyperlocomotion at both ages, traveling significantly greater distances than WT males (Fig. 2 A), whereas female LDLr ⁻/⁻ mice displayed hyperlocomotion only at the middle-age. Aging also influenced female behavior, with middle-aged females showing reduced locomotion compared to adults of the same genotype. These findings extend prior studies that reported hyperlocomotion in male LDLr ⁻/⁻ mice even under standard diet [ 27 , 29 , 60 ], but which did not include females or assess the role of age. In the object location (OL) task, both male and female LDLr ⁻/⁻ mice, independent of age, failed to discriminate the displaced object, unlike WT controls, that explored the relocated object more than 50% of the time. Spatial memory impairment in OL and cognitive decline has been previously reported in male LDLr ⁻/⁻ mice, even when fed a standard diet [ 25 , 27 , 61 , 62 ]. In the present study we demonstrated that female exhibited this memory impairment like males. Furthermore, LDLr ⁻/⁻ mice have been shown to be particularly susceptible to cognitive disturbances triggered by various stimuli that induce memory deficits, such as intracerebroventricular Aβ₁–₄₀ injection, a peptide implicated in Alzheimer’s disease pathophysiology. These mice display impairments in both spatial and working memory even in the absence of exogenous Aβ, as reflected by poor performance in the object location and spontaneous alternation tasks. In addition, only LDLr⁻/⁻ mice injected with Aβ₁–₄₀ exhibited impaired experience-dependent avoidance behavior in the EPM retest, a form of learning in which prior exposure to the apparatus normally promotes increased open-arm avoidance [ 25 ]. These findings align with recent human studies showing that FH contributes to cellular dysfunction in the brain and cognitive decline. For instance, a clinical study comparing Heterozygous FH (HeFH) patients with age-matched controls with no history of cognitive-affecting disorders over 50 years old reported that 21.3% of HeFH patients exhibited MCI, compared with only 2.9% of controls, indicating a significant difference. The authors proposed that early exposure to high cholesterol levels or LDLr dysfunction may constitute a risk factor for MCI [ 5 ]. Similarly, another study found that HeFH patients aged 18–40 performed worse on tasks assessing verbal memory and executive function compared with controls, and impairments in executive function correlated with higher serum LDL-C levels in the HeFH group [ 63 ]. Considering other behavioral assessments, we employed the EPM to investigate the impact of FH on anxiety-like behavior, particularly given the lack of literature on male and female LDLr ⁻/⁻ mice in the context of aging and sexual dimorphism. In this study, LDLr ⁻/⁻ males exhibited a higher number of open-arm entries in the EPM at both ages compared with WT controls (Supplementary Fig. 1B). However, when the percentage of open-arm entries was analyzed, no differences were observed in males, whereas middle-aged LDLr ⁻/⁻ females entered the open arms more frequently than WT females of the same age. To clarify whether this pattern reflected changes on behavior or locomotor activity, we further examined distance traveled in the EPM. This analysis confirmed hyperlocomotion in LDLr ⁻/⁻ males, consistent with findings from the OF test. Therefore, the increased number of open-arm entries in LDLr ⁻/⁻ males may reflect disinhibited behavior, but it should not be interpreted as altered anxiety-like behavior, since this effect is likely driven by hyperlocomotion in the absence of pharmacological or dietary interventions. Moreover, we identified sex-related differences, in which WT females displayed higher locomotor activity than WT males, whereas adult LDLr ⁻/⁻ males traveled greater distances than LDLr ⁻/⁻ females. To date, no studies have specifically examined EPM performance in the LDLr ⁻/⁻ model. However, previous work has reported depressive-like behavior in these animals [ 30 ]. In line with this, a study using C57BL/6 mice showed that a high-cholesterol diet promotes both depressive- and anxiety-like behaviors [ 64 ], further supporting a potential link between cholesterol dysregulation and emotional alterations. Nevertheless, further studies are required to better elucidate the nature and mechanisms of these behavioral changes in the context of LDLr dysfunction. The hippocampus is essential for learning, memory, and mood regulation [ 65 ]. Previous studies indicate that FH can impact brain regions involved in cognitive processes, such as the hippocampus[ 30 ] and the prefrontal cortex [ 27 ], independent of dietary interventions. To investigate the potential mechanisms underlying the cognitive and behavioral impairments observed in LDLr ⁻/⁻ mice, we evaluated hippocampal mitochondrial function. Interestingly, our findings revealed both sex- and age-dependent alterations in hippocampal bioenergetics in LDLr ⁻/⁻ mice. Middle-aged LDLr ⁻/⁻ females exhibited a marked impairment in hippocampal mitochondrial function, characterized by a reduction in oxygen consumption linked to complex I substrates, OXPHOS, ETS, and ATP-linked respiration. In contrast, adult LDLr⁻/⁻ males showed reduced oxygen consumption only when both complex I + II substrates were provided, along with a decline in ETS capacity. This pronounced hippocampal bioenergetic failure in middle-aged females likely reflects a metabolic crisis at the neuronal level, with reduced ATP generation compromising processes essential for synaptic maintenance and plasticity. Given that neuronal ATP is predominantly consumed to sustain synapse formation and activity, key determinants of cognitive performance [ 67 ], these data suggest that impaired mitochondrial energy metabolism may directly contribute to the cognitive and behavioral deficits observed in female LDLr ⁻/⁻ mice at midlife. Collectively, these results highlight significant sex-related differences in hippocampal mitochondrial parameters, reinforcing the concept that mitochondrial bioenergetics are inherently sex-dependent and that aging acts as a critical modifier of the neurobiological consequences of LDLr deficiency on CNS function and behavior. Importantly, these metabolic disturbances are consistent with previous reports describing cognitive and behavioral dysfunctions in LDLr ⁻/⁻ mice, particularly in males. This model has been shown to present impaired synaptic plasticity and hippocampal neurogenesis, resulting in deficits in spatial and working memory, as well as depressive-like behavior [ 24 , 68 ]. However, most previous studies have focused primarily on males, leaving the impact of sex and age largely unexplored. Our findings extend these observations by revealing that female LDLr⁻/⁻ mice are also vulnerable, exhibiting a distinct bioenergetic profile that may underlie their cognitive and affective alterations. One plausible mechanism involves the reduced neuronal uptake of cholesterol caused by LDLr dysfunction, which alters membrane lipid composition, impairs cell signaling, and compromises neuronal survival [ 30 , 69 ]. Additionally, previous studies have demonstrated that LDLr⁻/⁻ mice display increased blood–brain barrier (BBB) permeability, astrogliosis, microgliosis, and hippocampal neuronal death [ 25 ], as well as oxidative stress characterized by elevated ROS production and weakened antioxidant defenses [ 24 , 27 , 31 , 33 ]. These mechanisms likely underlie the early-life hippocampal mitochondrial dysfunction observed in LDLr ⁻/⁻ males, suggesting oxidative stress as a primary driver of bioenergetic impairments. In contrast, LDLr ⁻/⁻ females exhibited a delayed onset of hippocampal mitochondrial dysfunction, which emerged only at middle age and may reflect the progressive loss of estrogen-mediated protection, as estrogens are known to enhance mitochondrial efficiency and reduce oxidative damage. Other studies have also reported sex-specific differences in the metabolic disturbances. A study in Chinese adults over 45 years old found that elevated total and LDL cholesterol were associated with cognitive decline in women, but not in men [ 66 ]. Complementarily, Meng et al. (2023)[ 67 ] reported that ovariectomized LDLr ⁻/⁻ mice, used to model menopause, developed worsened dyslipidemia, increased hippocampal apoptosis, and cognitive deficits, effects linked to reduced estradiol levels and downregulated estrogen receptor expression. Moreover, Pettersson et al. (2012) showed that high-fat diet induced hyperinsulinemia, glucose intolerance, and systemic inflammation in male C57BL/6 mice, whereas females exhibited an anti-inflammatory adipose tissue profile and preserved metabolic function [ 68 ]. Together, these data highlight the central role of estrogen signaling in protecting brain function against metabolic disturbances. A key strength of our study is the focus on middle-aged LDLr ⁻/⁻ mice, a period that closely models the stage at which chronic hypercholesterolemia emerges as a critical risk factor for dementia in humans [ 69 ]. The pronounced hippocampal bioenergetic failure we identified at this stage aligns with, and extends, a growing body of evidence indicating that middle age represents a phase of accelerating neuropathology in this model. Previously, we demonstrated that middle-aged LDLr ⁻/⁻ mice exhibit marked antioxidant imbalance and oxidative damage in the prefrontal cortex, including elevated lipid peroxidation, disrupted glutathione metabolism, and increased acetylcholinesterase activity [ 31 ]. These signs of cellular stress are further compounded by evidence of synaptic decline, as both our group [ 31 ]and Mulder and collaborators [ 70 ] have reported decreased synaptophysin content at this age. Functionally, these molecular and structural alterations are accompanied by impaired spatial memory in the water maze and by pronounced microglial morphological changes indicative of neuroinflammation [ 71 ]. Therefore, the severe mitochondrial dysfunction observed in the present study provides a compelling mechanistic link suggesting that a collapse in energy metabolism may underpin the previously documented cascade of oxidative stress, synaptic decay, and cognitive impairment that characterizes the middle-aged hypercholesterolemic phenotype. Finally, considering behavioral performance alongside hippocampal mitochondrial bioenergetics, we performed a correlation analysis (Supplementary Fig. 2) and identified positive associations between locomotor parameters assessed in the OF and EPM (summarized as locomotor score), hippocampal bioenergetic parameters (hippocampal bioenergetics score), and BAT mitochondrial parameters (BAT thermogenic score). Based on these integrative analyses, we found that increased cholesterol levels positively correlated with hyperlocomotion, but inversely correlated with spatial memory in the OL test. These observations align with reports that hypercholesterolemia impairs cognition and synaptic function, whereas lipid-lowering interventions, such as statins, can improve memory and reduce neuroinflammation [ 72 , 73 ]. Notably, PCSK9 inhibition lowers cholesterol without affecting locomotor or cognitive performance in some models [ 74 ], suggesting that the behavioral effects of cholesterol-lowering strategies depend on experimental context. We also identified a positive correlation between hippocampal and BAT scores, suggesting that broad metabolic disruption, coupled with mitochondrial dysfunction, likely contributes to both central and peripheral alterations in the context of chronic hypercholesterolemia. Interestingly, our findings reveal a mechanistic link between mitochondrial dysfunction in the hippocampus and BAT, highlighting the convergence of systemic and neural metabolism in this model. We also performed an integrative analysis aimed at identifying a “signature” of the LDLr ⁻/⁻ genotype including both sexes (Supplementary Fig. 4). This approach revealed that plasma cholesterol levels, followed by locomotor score, triglyceride levels, time spent in the periphery of the OF, hippocampal bioenergetics score, and location index were the most informative variables for characterizing this model, reinforcing the impact of cholesterol metabolism on both CNS function and behavior. Altogether, our findings emphasize the necessity of including sex as a critical biological variable in studies of neurodegeneration and metabolic disease. The results provide new insight into the age- and sex-dependent hippocampal vulnerability to mitochondrial dysfunction in FH and point to the convergence of systemic and neural metabolism as a key component in the onset of cognitive impairment. It is also important to consider that aging has a profound impact on females, particularly due to hormonal fluctuations and the gradual decline in estrogen levels, which can exacerbate mitochondrial dysfunction and increase neural susceptibility to metabolic and oxidative stress [ 75 ]. Therefore, interventions targeting mitochondrial health and hormonal balance may represent promising strategies for preserving brain function in dyslipidemic conditions. Abbreviations AA: Antimycin A, AChE: Acetylcholinesterase, AD: Alzheimer’s Disease, ADP: Adenosine Diphosphate, ANOVA: Analysis of Variance, Apo B-100: Apolipoprotein B-100, ARRIVE: Reporting of In Vivo Experiments, ASCVD: Atherosclerotic Cardiovascular Disease, ATP: Adenosine Triphosphate, Aβ: Beta-amyloid, BAT: Brown Adipose Tissue, BBB: Blood–Brain Barrier, BSA: Bovine Serum Albumin, Ca²⁺: Calcium, CCCP: Carbonyl Cyanide m-chlorophenylhydrazone, CoQ10: Coenzyme Q10, CVD: Cardiovascular Disease, D: Displaced Object, EPM: Elevated Plus Maze, ETS: Electron Transport System, FFAs: Free Fatty Acids, GDP: Guanosine Diphosphate, HeFH: Heterozygous Familial Hypercholesterolemia, HMG-CoA Reductase: 3-hydroxy-3-methylglutaryl-coenzyme A reductase, HoFH: Homozygous Familial Hypercholesterolemia, HRR: High-Resolution Respirometry, i.p.: Intraperitoneal, IMM: Inner Mitochondrial Membrane, LDL: Low-Density Lipoprotein, LDL-C: Circulating Low-Density Lipoprotein, LDLr: LDL Receptor knockout mice, LI: Location Index, M: Malate, MCI: Mild Cognitive Impairment (for CCL), MWM: Morris Water Maze, NADPH: Nicotinamide Adenine Dinucleotide Phosphate (Reduced), ND: Non-Displaced Object, NIH: National Institutes of Health, O₂: Oxygen, OF: Open Field, OL: Object Location, OMY: Oligomycin, OXPHOS: Oxidative Phosphorylation, P: Pyruvate, PCSK9: Proprotein Convertase Subtilisin/Kexin Type 9, PM: Pyruvate and Malate, RB: Reaction Buffer, ROS: Reactive Oxygen Species, ROT: Rotenone, S: Succinate, SOD: Superoxide Dismutase, TCA: Tricarboxylic Acid Cycle, UCL: University College London, UCP-1: Uncoupling Protein 1, VLDL: Very-Low-Density Lipoprotein, WAT: White Adipose Tissue, WT: Wild-Type. Declarations Ethics approval and consent to participate: All animal procedures complied with the National Institutes of Health (NIH) guidelines for animal care, the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, and were approved by the Animal Ethics Committee of the University of Brasília (SEI Protocol 23106.075489/2023-95). Consent for publication: Not applicable . Availability of data and material: The data that support the findings of this study are available from the first author, N. M. C. P. L. and the corresponding author, A.F.B., upon request. Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have influenced this work. Funding: This work was supported by the following grants: Fundação de Apoio à Pesquisa do Distrito Federal-FAPDF (00193-00002348/2022-07 and 00193-00000884/2021-89); Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (404466/2023-0 and 201648/2024-5); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (88881.465507/2019-01); and Instituto Nacional de Ciência e Tecnologia e Neuro-ImunoModulação - INCT-NIM (485489/2014-1). Author’s contributions: N. P-L.: Conceptualization, Methodology, Validation, Investigation, Formal analysis, Data curation, Writing – original draft, Resources. D. F.: Investigation. L. T.: Investigation. W. S.: Investigation. W. B.: Investigation. H. M.: Data curation. M. M.: Resources. J. G.: Supervision, Data curation. A. A.: Supervision, Data curation. J. O.: Writing – review & editing. P. B.: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing – review & editing, Supervision. A. 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No effects of PCSK9-inhibitor treatment on spatial learning, locomotor activity, and novel object recognition in mice. Behav Brain Res. 2021;396. 10.1016/j.bbr.2020.112875 . Lejri I, Grimm A, Eckert A. Mitochondria, Estrogen and Female Brain Aging. Front Aging Neurosci. 2018;10(APR):124. 10.3389/FNAGI.2018.00124 . Additional Declarations No competing interests reported. Supplementary Files APPENDIXA.docx GA.png Sexual dimorphism and aging modulate behavioral and metabolic outcomes in LDLr ⁻/⁻ mice. Female and male mice exhibited distinct age-dependent alterations, including hyperlocomotion, memory impairment, dyslipidemia, and impaired mitochondrial oxygen consumption in the hippocampus and brown adipose tissue. Created in BioRender. De Bem, L. (2025) https:// BioRender.com/7ubt09p. Cite Share Download PDF Status: Published Journal Publication published 10 Apr, 2026 Read the published version in Biology of Sex Differences → Version 1 posted Editorial decision: Revision requested 23 Feb, 2026 Reviews received at journal 23 Feb, 2026 Reviews received at journal 11 Feb, 2026 Reviewers agreed at journal 17 Jan, 2026 Reviewers agreed at journal 16 Jan, 2026 Reviewers invited by journal 14 Jan, 2026 Editor assigned by journal 14 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 09 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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13:49:23","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":216929,"visible":true,"origin":"","legend":"","description":"","filename":"426000706f3d4251bbbced7fd0af2f2c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/af03ddcccf97cd6106a94a80.xml"},{"id":100576448,"identity":"444550ac-9219-4efa-800b-c33e89081d9b","added_by":"auto","created_at":"2026-01-19 10:25:27","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":231498,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/bc78d7c30818da53aec91eee.html"},{"id":100595856,"identity":"5d6befc4-fd67-4c21-a819-abcb91028307","added_by":"auto","created_at":"2026-01-19 13:49:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":288109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of age, sex, and LDLr genotype on circulating lipid levels and brown adipose tissue's thermogenic capacity.\u003c/strong\u003e (A) Eight experimental groups: Male and female, adult and middle-aged, Wild-type (WT) and LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. Adult mice were 6–8 months old, and middle-aged mice were 12–14 months old. These animals were subjected to behavioral testing, including the OF, OL and EPM tasks. Blood was collected post-euthanasia for lipid profiling: (B) Total cholesterol and (C) triglyceride levels. HRR was performed to evaluate BAT mitochondrial respiration for (D) Complex I-linked substrates (PM), (E) UCP-1 activity (GDP), and (F) electron transport system (ETS) uncoupling state. n= 6-10/group. All data were expressed as mean ± SEM. Statistical analysis was performed using a Three-way ANOVA to include sex, age and genotype variables. *Genotype effect (WT vs. LDLr\u003csup\u003e-/-\u003c/sup\u003e), \u003csup\u003e#\u003c/sup\u003eAging effect (WT vs. WT, and LDLr\u003csup\u003e-/-\u003c/sup\u003e vs. LDLr\u003csup\u003e-/-\u003c/sup\u003e), \u003csup\u003e\u0026amp;\u003c/sup\u003eSex effect (Male vs. Female of the same age and genotype), p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/eb3f638c198ea8fe92f5df44.png"},{"id":100576424,"identity":"12ca8942-d0a1-42df-9e20-0151e671a2c2","added_by":"auto","created_at":"2026-01-19 10:25:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":391897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDLr\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice exhibited cognitive impairment and hyperlocomotion. \u003c/strong\u003eLocomotor performance was evaluated in (A) the OF arena for 5 minutes, and (B) the location index was accessed to evaluate cognitive performance. (C) Open arms entries (%) and (D) distance traveled during the EPM test. n= 6-12/group. All data were expressed as mean ± SEM. Statistical analysis was performed using a Three-way ANOVA to include sex, age and genotype variables. *Genotype effect (WT vs. LDLr\u003csup\u003e-/-\u003c/sup\u003e), \u003csup\u003e#\u003c/sup\u003eAge effect (WT vs. WT, and LDLr\u003csup\u003e-/- \u003c/sup\u003evs. LDLr\u003csup\u003e-/-\u003c/sup\u003e), \u003csup\u003e\u0026amp;\u003c/sup\u003eSex effect (Male vs. Female of the same age and genotype), p\u0026lt; 0.05; \u003csup\u003e$\u003c/sup\u003e p\u0026lt; 0.05 vs. 50% chance levels.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/6dc9c3ebea31929fe27287bf.png"},{"id":100595823,"identity":"9620110a-98f9-4298-b77c-5580ed2222c9","added_by":"auto","created_at":"2026-01-19 13:49:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSex-specific hippocampal mitochondrial impairment in LDLr\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003eHRR was performed to evaluate mitochondrial respiratory states: Complex I-linked substrates (PM), Complex I and II-linked substrates (PMS), OXPHOS or state 3 (ADP), state 4 (OMY) and ETS capacity, uncoupled state (CCCP). (A and B) Representative oxygraphs from young male and aged female mice, comparing control and LDLr\u003csup\u003e-/-\u003c/sup\u003e groups. (C) O\u003csub\u003e2 \u003c/sub\u003eflux (pmol/s) per mg of protein in complex I, and (D) Complex I and II, (E) OXPHOS and (F) ETS capacity. (G) ATP-linked oxygen consumption, calculated by the difference between state 3 and state 4. n= 6-10/group. All data were expressed as mean ± SEM. Statistical analysis was performed using a Two-way ANOVA to compare differences between same-sex groups. *Genotype effect (WT vs. LDLr), \u003csup\u003e#\u003c/sup\u003eAge effect (WT vs. WT, and LDLr\u003csup\u003e-/-\u003c/sup\u003e vs. LDLr\u003csup\u003e-/-\u003c/sup\u003e), \u003csup\u003e\u0026amp;\u003c/sup\u003eSex effect (Male vs. Female of the same age and genotype), p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/6e86e03be3e60f7a661ff3f6.png"},{"id":100596015,"identity":"b953c2f8-4b04-4fb9-ae12-b1757acdd217","added_by":"auto","created_at":"2026-01-19 13:50:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations between key metabolic, behavioral, and bioenergetic parameters.\u003c/strong\u003e Scatterplots depicting significant linear relationships between select variables. (A) A strong positive correlation between serum cholesterol (mg/dL) and triglycerides (mg/dL). (B) A positive correlation between serum cholesterol (mg/dL) and the composite locomotor score.\u0026nbsp; (C) A moderate negative correlation between serum cholesterol (mg/dL) and the spatial memory index. (D) The relationship between the BAT Thermogenic Score and the Hippocampal Bioenergetics Score. In all panels, the solid line represents the best-fit line from a linear regression model, and the shaded area indicates the 95% confidence interval. In the correlation data plots, gray dots represent controls, pink dots represent female LDLr\u003csup\u003e-/-\u003c/sup\u003e mice, and blue dots represent male LDLr\u003csup\u003e-/-\u003c/sup\u003e mice. These graphs include animals from both age groups, adult and middle-aged.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/c4c9a65272d0a3ce8508f0a5.png"},{"id":106809871,"identity":"df78dc87-8825-4031-961d-92030eb999b2","added_by":"auto","created_at":"2026-04-13 16:13:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2413298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/186bd764-5759-480e-9eea-b41597e51d60.pdf"},{"id":100576420,"identity":"c7d971aa-9125-40aa-aba6-2dbbc93206a5","added_by":"auto","created_at":"2026-01-19 10:25:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":454055,"visible":true,"origin":"","legend":"","description":"","filename":"APPENDIXA.docx","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/6dc628fa55e399c39e40ad16.docx"},{"id":100595288,"identity":"32b036e7-3cad-44a8-aed9-ddea5a79a014","added_by":"auto","created_at":"2026-01-19 13:48:08","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":216427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSexual dimorphism and aging modulate behavioral and metabolic outcomes in LDLr\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e⁻/⁻ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice. \u003c/strong\u003eFemale and male mice exhibited distinct age-dependent alterations, including hyperlocomotion, memory impairment, dyslipidemia, and impaired mitochondrial oxygen consumption in the hippocampus and brown adipose tissue. Created in BioRender. De Bem, L. (2025) https:// BioRender.com/7ubt09p.\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-8561934/v1/bb53ecc7184c94a8c34d3038.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sex- and Age-Dependent Mitochondrial Dysfunction Links Familial Hypercholesterolemia to Cognitive Impairment","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eLDLr\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003emice exhibited hyperlocomotion; males were affected at both ages and females at middle-age.\u003c/li\u003e\n \u003cli\u003eLDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibited spatial memory impairment, independent of sex and age.\u003c/li\u003e\n \u003cli\u003eAdult male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibited higher susceptibility to hippocampal mitochondrial dysfunction; females were affected at middle-age.\u003c/li\u003e\n \u003cli\u003eCorrelation analyses indicated links between metabolic, mitochondrial, and behavioral outcomes.\u003c/li\u003e\n \u003cli\u003eLDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice showed age- and sex-dependent differences that may elucidate potential determinant factors in HF outcomes.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Plain English Summary","content":"\u003cp\u003eFamilial hypercholesterolemia (FH) is a genetic condition in which cholesterol levels are high from an early age. Because the body cannot remove cholesterol efficiently, it can build up in the blood and in the arteries over time. FH is best known for increasing the risk of heart disease, but long-term high cholesterol may also affect brain function. In this study, we used a mouse model of FH to understand how high cholesterol affects memory, behavior, and mitochondrial metabolism, and whether these effects differ between males and females as they age. To do this, we studied male and female mice during adulthood and middle age. Mice with FH showed difficulties remembering the location of objects, indicating impaired spatial memory. This memory problem was observed in both sexes. However, other changes differed between males and females. Male mice with FH showed increased activity levels at both ages, while female mice showed this change only at middle age. We also examined how mitochondria function in the hippocampus, a brain region important for memory, and in brown fat, a tissue involved in metabolism. Mitochondrial function was altered in FH, with differences between males and females. Overall, our findings show that FH affects brain function and mitochondrial metabolism differently in males and females. Males were susceptible in adulthood, while females showed effects after middle age, emphasizing the role of sex and age in the long-term impact of high cholesterol.\u003c/p\u003e"},{"header":"1. INTRODUCTION","content":"\u003cp\u003eFamilial hypercholesterolemia (FH) is one of the most prevalent genetic disorders worldwide, particularly in the heterozygous form [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is mainly caused by mutations in the low-density lipoprotein (LDL) receptor (LDLr) gene and is characterized by chronically elevated LDL-cholesterol levels, resulting in arterial deposition and substantially increased risk of atherosclerotic cardiovascular disease (ASCVD) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Beyond cardiovascular consequences, increasing evidence suggests that FH also compromises brain health. Two aspects are particularly relevant: lifelong exposure to elevated cholesterol levels and LDLr dysfunction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which not only regulate cholesterol uptake but also contribute to synaptic function, neuronal plasticity and amyloid-β (Aβ) clearance [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this context, the Lancet Commission on Dementia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] highlights high LDL cholesterol as one of the main modifiable risk factors for dementia. Supporting this link, familial hypercholesterolemia (FH) has been associated with cognitive decline, mild cognitive impairment (MCI), and dementia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], with memory deficits reported even in young adults aged 18 to 40 years [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Adding to this evidence, recent findings from the ELSA-Brazil cohort showed nonlinear associations between serum lipid levels and cognitive decline, particularly among individuals younger than 60 years and women [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eSexual dimorphism adds further complexity to this scenario. Clinical data indicate a higher prevalence of FH in women, whereas men tend to develop ASCVD earlier and initiate treatment sooner [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. While premenopausal women are relatively protected, menopause attenuates this advantage, raising cardiometabolic and cognitive risks to levels comparable to those observed in men [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In parallel, aging is accompanied by systemic metabolic alterations, including insulin resistance, dyslipidemia and chronic inflammation, which indirectly affect brain function and increase the risk of MCI and dementia across the lifespan [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As a major risk factor for neurodegenerative diseases, aging entails progressive biological changes that disrupt cholesterol homeostasis and compromise both physical and cognitive functions [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, the interplay between sex and aging may critically modulate metabolic and cognitive outcomes in FH.\u003c/p\u003e \u003cp\u003eThe LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, generated by Ishibashi et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], is a well-established model for studying the pathophysiology of FH. Consistent with clinical findings, LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice display elevated LDL-cholesterol levels and a broad spectrum of behavioral alterations, even at young ages, indicating early central nervous system vulnerability. These alterations include deficits in spatial, working and long-term memories [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additionally, young and middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibit increased locomotor activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], as well as emotional dysregulation, characterized by heightened stress sensitivity and depressive-like behavior [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Underlying these cognitive impairments, several cellular and biochemical alterations have been reported in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. These include impaired adult hippocampal neurogenesis, enhanced glial reactivity, blood-brain barrier (BBB) disruption, hippocampal apoptosis, and neuronal and synaptic dysfunctions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Among these pathological processes, mitochondrial dysfunction has emerged as a central mechanism, connecting cognitive and neuronal impairments with systemic metabolic alterations.\u003c/p\u003e \u003cp\u003eIn the brain, LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibit oxidative stress, impaired respiratory chain function, and reduced coenzyme Q10 (CoQ10) levels, worsened by intracellular cholesterol accumulation driven by mevalonate pathway activation [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Peripheral tissues are also affected: white adipose tissue (WAT) shows oxidative stress and inflammation, promoting reactive oxygen species (ROS) accumulation, impairing insulin signaling, and contributing to insulin resistance, glucose intolerance, and metabolic dysfunction [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. WAT from LDLr\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice also exhibits macrophage infiltration, increased proinflammatory cytokines, and altered adipokine secretion, establishing a chronic inflammatory state that disrupts energy homeostasis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In contrast, brown adipose tissue (BAT), a thermogenic and mitochondria-rich depot essential for systemic metabolism, regulating thermogenesis, insulin sensitivity, and lipid metabolism [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], remains underexplored in the context of FH.\u003c/p\u003e \u003cp\u003eNotably, most studies have focused on male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, leaving the influence of sex largely unresolved. However, sex is a critical determinant of both metabolic regulation and brain function. Given that aging also modulates cholesterol metabolism and hormonal status, FH may exert age- and sex-specific effects on cognition and mitochondrial function. Accordingly, the present study investigated the combined influence of genotype, sex, and age on behavioral and metabolic outcomes in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. We assessed cognitive, locomotor and anxiety-like behaviors, as well as mitochondrial function in the hippocampus and BAT, to elucidate mechanisms of vulnerability associated with FH.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eMale and female C57Bl/6 wild-type (WT) and LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e (B6.129S7 LDLrtm1Her/J) mice, aged 6\u0026ndash;8 months (adult group) and 12\u0026ndash;14 months (middle-aged group), were originally purchased from the Jackson Laboratory and were bred in our own breeding colony at the University of Bras\u0026iacute;lia. Mice were housed in acrylic cages under controlled conditions, with a filtered air system (Alesco; 3\u0026ndash;4 mice/cage) at a controlled temperature (23\u0026ndash;25\u0026deg;C), and 12h light/dark cycle (lights on at 6 a.m.). They had \u003cem\u003ead libitum\u003c/em\u003e access to food and water. All animal procedures complied with the National Institutes of Health (NIH) guidelines for animal care, the Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments (ARRIVE) guidelines, and were approved by the Animal Ethics Committee of the University of Bras\u0026iacute;lia (SEI Protocol 23106.075489/2023-95).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental design\u003c/h2\u003e \u003cp\u003eMice were allocated according on sex, age, and genotype into eight groups of 6\u0026ndash;12 animals. Mice weighed between 25\u0026shy;30 g, and were fed a standard rodent diet (SD; 70% carbohydrates, 20% protein, and 10% fat). Behavioral assessments included the open field (OF), object location (OL), and elevated plus maze (EPM) tests. Following this, the animals were anesthetized with ketamine and xylazine via intraperitoneal (i.p.) injection (80:8 mg/kg, respectively) until full analgesia and then were euthanized by cervical dislocation. Blood and tissues, including hippocampi and BAT, were collected immediately after euthanasia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Behavioral tasks\u003c/h2\u003e \u003cp\u003eFor the behavioral tests, the animals were transferred to the testing room at least 1 hour before the beginning of the experiments, to allow acclimation. The testing room was maintained at controlled temperature and humidity, mirroring the conditions of their regular housing. The room was also free from extraneous odors, including those from the experimenter (i.e., perfume, deodorant, or lotion were avoided). All behavioral tests were recorded using AnyMaze\u0026reg; software (RRID:SCR_014289; version 7.1 for Windows) and conducted between 7 a.m. and 4 p.m., during the light phase of the animals\u0026rsquo; light/dark cycle.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Open field (OF)\u003c/h2\u003e \u003cp\u003eOF was used to evaluate the spontaneous locomotor and exploratory activities induced by a novel environment. The OF apparatus was a 30 cm length cubic arena with a white background, containing spatial cues on the sidewalls. Animals were placed individually on the center of the apparatus to freely explore the arena for 5 min [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The arena was cleaned with ethanol 30% between each animal trial. Total distance traveled and time spent in the inner and peripheral quadrants (inner quadrant was drawn 7.50 cm away from the walls) were evaluated using ANY-maze tracking system (Stoelting Co., IL, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Object location test (OL)\u003c/h2\u003e \u003cp\u003eOL test was performed to assess spatial reference memory, following the protocol previously described by Assini et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. During the training session, animals were placed in the arena for 5 minutes with two identical objects positioned parallel to each other, 5 cm away from the walls. After the training phase, the mice were removed from the arena for 90 minutes. Following the inter-trial interval, one object remained in the same location (nondisplaced object [ND]), and the other one was relocated to a new position (displaced object [D]). The animals were then reintroduced into the arena and allowed to explore for another 5 minutes. After each trial, the experimental apparatus was cleaned with 30% ethanol. Exploration time was recorded when the mice sniffed, looked at, touched, or smelled the object at least 1 cm away. A location index (LI) was calculated to evaluate location memory using the formula: LI\u0026thinsp;=\u0026thinsp;TD * 100 / (TD\u0026thinsp;+\u0026thinsp;TND), where TD and TND are the exploration times for the displaced and nondisplaced objects, respectively [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Elevated plus maze (EPM)\u003c/h2\u003e \u003cp\u003eEPM test is currently performed to assess anxiety-like behavior in rodents [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The apparatus is elevated 60 cm from the floor, with four arms (18 cm long, 6 cm wide). Two opposite arms are surrounded by walls (6 cm high, enclosed arms), while the other two arms are devoid of enclosing walls (open arms). The four arms are connected by a central area (6 x 6 cm). The animals were individually placed in the central area and allowed to freely explore for 5 minutes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The apparatus was cleaned with ethanol solution (30% v/v) and dried with paper towels after each trial, to avoid odor impregnation. During the test, the number of entries into each arm and the total distance traveled were recorded using ANY-maze tracking system (Stoelting Co., IL, USA). To normalize the number of entries into the open arms relative to the total number of arm entries, results were expressed as the percentage of entries into the open arms, calculated as follows: 100 * OA/(OA\u0026thinsp;+\u0026thinsp;CA), where OA corresponds to the number of entries into the open arms and CA to the number of entries into the closed arms.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Lipid profile analysis\u003c/h2\u003e \u003cp\u003eAfter euthanasia, approximately 500 \u0026micro;L of whole blood was collected from the animals. The blood was centrifuged at 3000 g for 10 minutes to obtain serum. Subsequently, cholesterol (Labtest, Cat# 76) and triglyceride (Labtest, Cat# 87) levels were measured in the serum using colorimetric assays, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5. High-resolution respirometry\u003c/h2\u003e \u003cp\u003eOxygen (O\u003csub\u003e2\u003c/sub\u003e) consumption was assessed using high-resolution respirometry (HRR) with an Oroboros 2k Oxygraph (Oroboros Instruments, Innsbruck, Austria) at 37\u0026deg;C. The oxygraph system is a closed chamber that measures changes in O\u003csub\u003e2\u003c/sub\u003e concentration. Any variation in O\u003csub\u003e2\u003c/sub\u003e levels is attributed to the samples, which utilize the substrates or drugs added during the experiment, and consume O\u003csub\u003e2\u003c/sub\u003e, enabling the assessment of specific mitochondrial states [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Hippocampal respirometry\u003c/h2\u003e \u003cp\u003eHippocampi were collected and homogenized in 300 \u0026micro;L of reaction buffer [RB: 125 mM sucrose, 65 mM KCl, 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM HEPES, 0.1 mM EGTA, 0.01% bovine serum albumin (BSA)] using a 5 mL glass-teflon homogenizer. The samples (protein \u0026sim;0.200 mg/mL) were added to a 2-mL chamber containing RB. Substrates (all purchased from Sigma-Aldrich) were added sequentially to assess O\u003csub\u003e2\u003c/sub\u003e flux as follows: pyruvate (P; 5mM) and malate (M; 2,5mM) were added to assess complex I activity. For complexes I\u0026thinsp;+\u0026thinsp;II, succinate (S; 10mM) was added. Oxidative phosphorylation (OXPHOS) was evaluated after the addition of 500 \u0026micro;M ADP, and state 4 (leak) was determined following the addition of oligomycin (OMY; 0.1 \u0026micro;g/mL). Uncoupling of the electron transport system (ETS) was induced with titrated carbonyl cyanide 3-chlorophenylhydrazone (CCCP, final concentration 1\u0026ndash;3 \u0026micro;M). Complex II activity was assessed by adding 0.5 \u0026micro;M rotenone, and non-mitochondrial residual respiration was measured after the addition of Antimycin A (AA: 1 \u0026micro;M). ATP-linked respiration was calculated as the difference between oxygen consumption rate in OXPHOS and state 4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. BAT respirometry\u003c/h2\u003e \u003cp\u003eBAT was collected, weighted and cut into 1 mm\u0026sup3; pieces. Around 10 x the volume (\u0026micro;L) of RB corresponding to BAT weight was added to homogenize the tissue into a 5 mL glass-teflon homogenizer. The samples (protein \u0026sim; 0.135 mg/mL) were added to a 2 mL chamber containing RB and 0.1% additional BSA. Initially, PM (5 and 2.5 mM, respectively) were added, followed by guanosine diphosphate (GDP: 1 mM) to evaluate uncoupling protein-1 (UCP-1) activity. Then, CCCP (3\u0026ndash;7 \u0026micro;M final concentration) and rotenone (ROT: 0.5 \u0026micro;M) were sequentially added.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6. STATISTICAL ANALYSIS\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using R (version 4.3), utilizing key packages including tidyverse for data manipulation, car for ANOVA, emmeans for post-hoc comparisons, vegan for multivariate analysis, mediation for causal modeling, and caret for predictive modeling. Prior to analysis, outliers within each of the eight experimental groups were identified for each biological variable using the 1.5x interquartile range (IQR) rule and were excluded from the respective analyses. The assumption of normality of residuals was checked for all parametric tests. The primary statistical method to assess the main effects of sex, age, and phenotype, as well as their interactions, was a three-way analysis of variance (ANOVA). When significant main effects or interactions were detected, simple effects were assessed via pairwise post-hoc comparisons of the estimated marginal means, applying the Holm-Bonferroni method for p-value adjustment. Partial omega-squared (ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e) was calculated as an estimate of effect size for the ANOVA results. Graphs were generated using GraphPad Prism 9.0 (RRID:SCR_002798). All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM unless otherwise stated. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant for all tests.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Behavioral and Metabolic Data Correlation\u003c/h2\u003e \u003cp\u003eTo investigate the linear interrelationships among the phenotypic variables, we calculated Pearson's product-moment correlation coefficients (ρ). The resulting correlation matrix was visualized as a heatmap. To highlight statistically significant associations and facilitate interpretation, only correlation coefficients with an associated p-value of less than 0.05 were numerically displayed within the heatmap cells. Furthermore, we organized the variables a priori into three functional blocks \u0026mdash; 'Metabolic \u0026amp; Behavioral', 'Hippocampal Bioenergetics', and 'BAT Thermogenesis'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Data preprocessing and dimensionality reduction\u003c/h2\u003e \u003cp\u003eOur initial correlation analyses revealed strong collinearity within specific subsets of variables, particularly those related to mitochondrial respiration and locomotor activity. To mitigate the effects of multicollinearity in subsequent statistical models, and to derive robust, composite metrics for these biological domains, we employed Principal Component Analysis (PCA). We conducted three separate PCAs. The first consolidated five hippocampal mitochondrial respiration variables into a single 'Hippocampal Bioenergetics Score'. The second combined three BAT variables to create a 'BAT Thermogenic Score', and the third integrated two locomotor variables into a 'Locomotor Score'. We confirmed the suitability of the data for this approach using Bartlett\u0026rsquo;s test of sphericity and the Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy for each component. For each analysis, we retained the first principal component (PC1), as it explained the majority of the shared variance (84.3% for hippocampal, 52.5% for BAT, and 71.8% for locomotor variables).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. PLS-DA Model\u003c/h2\u003e \u003cp\u003eWe employed a Partial Least Squares Discriminant Analysis (PLS-DA), a supervised machine learning method, to identify the multivariate phenotypic signature capable of discriminating animals by genotype. The predictor set included the metabolic, behavioral, and principal component (PC) scores for bioenergetics; the individual variables comprising the PCs were excluded to prevent multicollinearity. Prior to model training, we autoscored all predictors (mean-centered and scaled to unit variance). We assessed the model's robustness and performance using a 10-fold cross-validation procedure. The optimal number of latent components for the final model was selected based on the maximization of the Area Under the Receiver Operating Characteristic (ROC) curve. We quantified each variable's importance in discriminating between genotypes using the Variable Importance in Projection (VIP) scores, where higher values indicate a greater contribution to group separation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Lipid metabolism and BAT thermogenesis are differently affected in male and female LDLr\u003csup\u003e-/-\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eThe experimental design included three variables \u0026mdash; age (adult vs. middle-aged), genotype (WT vs. LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e), and sex (female vs. male), resulting in eight experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To assess the impact of sex and aging on lipid metabolism in the context of FH, we performed a comprehensive lipid profile analysis. Three-way ANOVA revealed a significant effect of genotype on cholesterol (F(1,51)\u0026thinsp;=\u0026thinsp;51.57, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.68; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and triglycerides (F(1,51)\u0026thinsp;=\u0026thinsp;14.91, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e 0.47; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) levels, as well as a age:genotype interaction on cholesterol levels (F(1,51)\u0026thinsp;=\u0026thinsp;4.38, p\u0026thinsp;=\u0026thinsp;0.0414, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.01). Post-hoc Holm\u0026ndash;Sidak test confirmed that both male (both ages: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and female (adult: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; middle-aged: p\u0026thinsp;=\u0026thinsp;0.0007) LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice displayed significantly elevated serum cholesterol compared with age-matched WT controls. For triglycerides levels, LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females showed a significant increase at the adult stage compared with WT controls (p\u0026thinsp;=\u0026thinsp;0.0032), whereas LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males displayed elevated levels at both adult (p\u0026thinsp;=\u0026thinsp;0.0012) and middle-aged (p\u0026thinsp;=\u0026thinsp;0.0004) stages. Additionally, middle-aged males LDLr\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e exhibited higher triglyceride levels than females LDLr\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e of the same age (p\u0026thinsp;=\u0026thinsp;0.0422).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate mitochondrial thermogenic activity in BAT, we measured oxygen consumption associated with complex I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), the percentage of UCP-linked mitochondrial respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and the maximal uncoupled respiratory capacity (ETS; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). For complex I, three-way ANOVA revealed a significant effect of sex (F(1,51)\u0026thinsp;=\u0026thinsp;9.50, p\u0026thinsp;=\u0026thinsp;0.0033, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.22), and a sex:age interaction (F(1,51)\u0026thinsp;=\u0026thinsp;6.95, p\u0026thinsp;=\u0026thinsp;0.0111, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.08). Post-hoc analysis showed reduced oxygen consumption in middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males compared with controls (p\u0026thinsp;=\u0026thinsp;0.0192), as well as an age effect in WT males, with middle-aged animals exhibited lower oxygen consumption than adult group (p\u0026thinsp;=\u0026thinsp;0.0014). Sex differences were also detected in adult WT (p\u0026thinsp;=\u0026thinsp;0.0298) and adult LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice (p\u0026thinsp;=\u0026thinsp;0.0081), with males exhibiting higher oxygen consumption than females. For UCP1-linked respiration, three-way ANOVA revealed significant effects of sex (F(1,53)\u0026thinsp;=\u0026thinsp;5.14, p\u0026thinsp;=\u0026thinsp;0.0275, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.16), a sex:age:genotype interaction (F(1,53)\u0026thinsp;=\u0026thinsp;7.23, p\u0026thinsp;=\u0026thinsp;0.0096, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.09), and a trend toward a genotype effect (F(1,53)\u0026thinsp;=\u0026thinsp;3.82, p\u0026thinsp;=\u0026thinsp;0.0559). Post-hoc analysis indicated an age effect in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females, as middle-aged animals showed reduced UCP-1 activity compared with the adult group (p\u0026thinsp;=\u0026thinsp;0.0085). Additionally, sex differences were observed in middle-aged WT (p\u0026thinsp;=\u0026thinsp;0.0125) and LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.0097) mice, with males displaying higher UCP-1 activity than females. No significant differences were detected for ETS capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eLdlr\u003c/b\u003e \u003csup\u003e \u003cb\u003e-/-\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice exhibit spatial memory impairment regardless of sex, while hyperactivity is more pronounced in males\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eSpontaneous locomotor activity was evaluated in the OF test by measuring the total distance traveled for 5 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Three-way ANOVA revealed significant effects of sex (F(1,60)\u0026thinsp;=\u0026thinsp;5.54, p\u0026thinsp;=\u0026thinsp;0.0218, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.00) and age (F(1,60)\u0026thinsp;=\u0026thinsp;25.13, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.36), as well as a trend toward a genotype effect (F(1,60)\u0026thinsp;=\u0026thinsp;3.51, p\u0026thinsp;=\u0026thinsp;0.0657, ω\u003csup\u003e2\u003c/sup\u003e\u003csub\u003ep\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.54). Post hoc analysis showed that LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males exhibited increased locomotion at both ages compared to age-matched WT controls (adult: p\u0026thinsp;=\u0026thinsp;0.0005; middle-aged: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females displayed this increase only at middle-age (p\u0026thinsp;=\u0026thinsp;0.0072). An age effect was also detected in WT mice, with middle-aged males (p\u0026thinsp;=\u0026thinsp;0.0247) and females (p\u0026thinsp;=\u0026thinsp;0.0001) traveling shorter distances than their adult counterparts. Similarly, LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females showed reduced locomotion at middle-age compared to adults. In addition, sex differences emerged in middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, with males traveling greater distances than females of the same age. The percentage of time spent on the periphery of the apparatus (Supplementary Fig.\u0026nbsp;1A) was also analyzed. Three-way ANOVA revealed significant effects of sex (F(1,56)\u0026thinsp;=\u0026thinsp;19.64, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.10), age (F(1,56)\u0026thinsp;=\u0026thinsp;14.87, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.05), and a sex:age:genotype interaction (F(1,56)\u0026thinsp;=\u0026thinsp;14.52, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.17). Post hoc analysis indicated that LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e adult males (p\u0026thinsp;=\u0026thinsp;0.0003) and LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e middle-aged females (p\u0026thinsp;=\u0026thinsp;0.0003) spent more time in the periphery compared with their respective WT controls. Moreover, WT females at middle-age spent less time in the periphery than adult WT females (p\u0026thinsp;=\u0026thinsp;0.0027). In addition, a sex effect was detected in adult WT mice, with males spending less time in the periphery than females of the same age (p\u0026thinsp;=\u0026thinsp;0.0004).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpatial memory was evaluated using the OL test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Only WT control groups of both sexes exhibited a location index significantly above the chance level of 50%, while LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice failed to discriminate the displaced object. Two-way ANOVA revealed a significant effect of age (F(1,61)\u0026thinsp;=\u0026thinsp;6.50, p\u0026thinsp;=\u0026thinsp;0.0133, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.16), a sex:age:genotype interaction (F(1,61)\u0026thinsp;=\u0026thinsp;6.74, p\u0026thinsp;=\u0026thinsp;0.0118, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.08), and a trend toward a genotype effect (F(1,61)\u0026thinsp;=\u0026thinsp;3.98, p\u0026thinsp;=\u0026thinsp;0.0505, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.32).\u003c/p\u003e \u003cp\u003eAnxiety-like behavior was assessed in the EPM test through the number of entries into open arms (Supplementary Fig.\u0026nbsp;1B), the percentage of open arm entries (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and the total distance traveled (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) over 5 minutes. For the number of entries into open arms, three-way ANOVA revealed significant effects of sex (F(1,58)\u0026thinsp;=\u0026thinsp;7.59, p\u0026thinsp;=\u0026thinsp;0.0078, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.00) and a sex:genotype interaction (F(1,58)\u0026thinsp;=\u0026thinsp;6.67, p\u0026thinsp;=\u0026thinsp;0.0123, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.08), with a trend for an age effect (F(1,58)\u0026thinsp;=\u0026thinsp;3.49, p\u0026thinsp;=\u0026thinsp;0.0669, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.00). Post hoc analysis showed that both adult (p\u0026thinsp;=\u0026thinsp;0.0021) and middle-aged (p\u0026thinsp;=\u0026thinsp;0.0025) LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males displayed increased open arm entries compared to WT controls.\u003c/p\u003e \u003cp\u003eTo account for differences in overall locomotor activity, we also calculated the percentage of entries into the open arms relative to the total number of entries. Three-way ANOVA indicated significant effects of age (F(1,61)\u0026thinsp;=\u0026thinsp;6.44, p\u0026thinsp;=\u0026thinsp;0.0137, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.00) and a sex:age interaction (F(1,61)\u0026thinsp;=\u0026thinsp;6.85, p\u0026thinsp;=\u0026thinsp;0.0112, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.07), as well as a trend toward an age:genotype interaction (F(1,61)\u0026thinsp;=\u0026thinsp;3.45, p\u0026thinsp;=\u0026thinsp;0.0680, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.01). Post hoc analysis revealed that middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females entered the open arms more frequently than WT females of the same age.\u003c/p\u003e \u003cp\u003eFinally, total distance traveled in the EPM was analyzed to assess locomotor activity within the apparatus. Three-way ANOVA showed significant effects of sex (F(1,55)\u0026thinsp;=\u0026thinsp;8.34, p\u0026thinsp;=\u0026thinsp;0.0055, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.00) and a sex:genotype interaction (F(1,55)\u0026thinsp;=\u0026thinsp;18.72, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.26). Post hoc analysis demonstrated that LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males traveled longer distances than their respective WT counterparts at both ages (adult: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; middle-aged: p\u0026thinsp;=\u0026thinsp;0.0012). In addition, sex differences were detected in WT mice, with females traveling longer distances than males at both adulthood (p\u0026thinsp;=\u0026thinsp;0.0442) and middle-age (p\u0026thinsp;=\u0026thinsp;0.0382). A sex effect was also observed in adult LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, with males covering more distance than females.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Sex-specific differences in hippocampal mitochondrial dysfunction in LDLr\u003csup\u003e-/-\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eWe evaluated hippocampal mitochondrial bioenergetics using HRR to measure oxygen consumption under different stimuli. For complex I\u0026ndash;related oxygen consumption (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), three-way ANOVA revealed significant effects of sex (F(1,52)\u0026thinsp;=\u0026thinsp;8.99, p\u0026thinsp;=\u0026thinsp;0.0042, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.00), age (F(1,52)\u0026thinsp;=\u0026thinsp;5.02, p\u0026thinsp;=\u0026thinsp;0.0294, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.25), and a sex:age:genotype interaction (F(1,52)\u0026thinsp;=\u0026thinsp;29.44, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.32), with a trend toward a genotype effect (F(1,52)\u0026thinsp;=\u0026thinsp;3.91, p\u0026thinsp;=\u0026thinsp;0.0533, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.09). Post hoc analysis indicated that middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females exhibited reduced oxygen consumption compared with WT females (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Age effects were observed in WT males (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females (p\u0026thinsp;=\u0026thinsp;0.0001), with middle-aged animals showing lower mitochondrial activity than adults of the same genotype. Sex differences were also detected in WT mice: adult males displayed higher Complex I\u0026ndash;linked respiration than adult females (p\u0026thinsp;=\u0026thinsp;0.0333), whereas middle-aged males exhibited lower activity than females (p\u0026thinsp;=\u0026thinsp;0.0004).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor complex I\u0026thinsp;+\u0026thinsp;II respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), three-way ANOVA revealed significant effects of sex (F(1,53)\u0026thinsp;=\u0026thinsp;10.59, p\u0026thinsp;=\u0026thinsp;0.0020, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.02) and a sex:age:genotype interaction (F(1,53)\u0026thinsp;=\u0026thinsp;10.45, p\u0026thinsp;=\u0026thinsp;0.0021, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.13). Post hoc analysis indicated reduced mitochondrial respiration in adult LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males compared with WT males (p\u0026thinsp;=\u0026thinsp;0.0143). An age effect was observed in WT males, with middle-aged animals displaying lower oxygen consumption than adults (p\u0026thinsp;=\u0026thinsp;0.0143). Additionally, adult WT males exhibited higher respiration than adult females (p\u0026thinsp;=\u0026thinsp;0.0198).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding OXPHOS capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), three-way ANOVA revealed significant effects of sex (F(1,55)\u0026thinsp;=\u0026thinsp;8.74, p\u0026thinsp;=\u0026thinsp;0.0046, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.006) and a sex:age:genotype interaction (F(1,55)\u0026thinsp;=\u0026thinsp;11.84, p\u0026thinsp;=\u0026thinsp;0.0011, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.15). Post hoc analysis showed that middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females exhibited reduced OXPHOS capacity compared with WT females (p\u0026thinsp;=\u0026thinsp;0.0208). Middle-aged WT males also showed a trend toward reduced capacity compared with adults (p\u0026thinsp;=\u0026thinsp;0.0712), and a similar trend was observed for sex differences in adult WT mice, with males showing higher values than females (p\u0026thinsp;=\u0026thinsp;0.0503).\u003c/p\u003e \u003cp\u003eFor ETS capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), three-way ANOVA revealed significant effects of sex (F(1,54)\u0026thinsp;=\u0026thinsp;7.99, p\u0026thinsp;=\u0026thinsp;0.0066, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.00) and a sex:age:genotype interaction (F(1,54)\u0026thinsp;=\u0026thinsp;10.37, p\u0026thinsp;=\u0026thinsp;0.0022, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.13). Post hoc analysis indicated reduced maximal uncoupled respiration in adult LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males (p\u0026thinsp;=\u0026thinsp;0.0401) and middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females (p\u0026thinsp;=\u0026thinsp;0.0019) compared with their WT counterparts. Age effects were also observed in WT males and LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females, as middle-aged animals displayed lower oxygen consumption than adults. Additionally, a trend toward sex differences was observed in adult WT mice (p\u0026thinsp;=\u0026thinsp;0.0527).\u003c/p\u003e \u003cp\u003eFinally, for ATP-linked respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), three-way ANOVA revealed significant effects of sex (F(1,52)\u0026thinsp;=\u0026thinsp;6.21, p\u0026thinsp;=\u0026thinsp;0.0160, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.02) and a sex:age:genotype interaction (F(1,52)\u0026thinsp;=\u0026thinsp;7.58, p\u0026thinsp;=\u0026thinsp;0.0081, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.10), with a trend toward an age effect (F(1,52)\u0026thinsp;=\u0026thinsp;3.67, p\u0026thinsp;=\u0026thinsp;0.0608, ω\u0026sup2;\u003csub\u003ep\u003c/sub\u003e=0.04). Post hoc analysis demonstrated that middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females exhibited reduced ATP-linked respiration compared with WT females (p\u0026thinsp;=\u0026thinsp;0.0119).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. PCA\u0026ndash;based functional scores for locomotor, hippocampal, and BAT domains\u003c/h2\u003e \u003cp\u003eFollowing the correlogram analysis (Supplementary Fig.\u0026nbsp;2), to condense correlated variables into meaningful biological indices, we performed three separated principal component analyses (PCAs). In all analyses, the first principal component (PC1) accounted for the majority of the variance, a finding visually confirmed by the scree plot analysis, where PC1 was the only component across all three domains with an eigenvalue greater than 1.0 (Kaiser\u0026rsquo;s criterion) (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLocomotor Score: the two locomotor variables (OF and EPM) were also condensed into a single component (Bartlett's χ2 (1)\u0026thinsp;=\u0026thinsp;12.31, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; KMO\u0026thinsp;=\u0026thinsp;0.50). PC1 explained 71.8% of the combined variance, with both distance variables contributing equally (loadings\u0026thinsp;=\u0026thinsp;0.707). The \"Locomotor Score\" thus effectively represents an overall measure of locomotor activity.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eHippocampal Bioenergetics Score: the five variables related to hippocampal mitochondrial respiration were highly suitable for PCA, as indicated by a significant Bartlett's test of sphericity (χ2 (10)\u0026thinsp;=\u0026thinsp;373.57, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a good KMO value of 0.74. PC1, designated the \"Hippocampal Bioenergetics Score,\" explained 84.3% of the total variance. All five input variables demonstrated strong, negative loadings on this component (loadings from \u0026minus;\u0026thinsp;0.41 to -0.47), indicating that PC1 represents a robust, integrated measure of overall mitochondrial respiratory function.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eBAT Thermogenic Score: similarly, the three variables measuring BAT mitochondrial function were appropriate for PCA (Bartlett's χ2 (3)\u0026thinsp;=\u0026thinsp;12.2, p\u0026thinsp;=\u0026thinsp;0.007; KMO\u0026thinsp;=\u0026thinsp;0.62). The resulting PC1, termed the \"BAT Thermogenic Score\", accounted for 52.5% of the shared variance. The variables for complex I-linked, maximal, and uncoupled respirations all contributed positively and with similar weights (loadings from 0.56 to 0.59), reflecting a composite index of thermogenic potential.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Correlational Analysis Reveals Links Between Metabolic, Mitochondrial, and Behavioral Genotypes\u003c/h2\u003e \u003cp\u003eTo elucidate the interrelationships between the observed alterations, we performed a comprehensive Spearman's correlational analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Critically, metabolic dysregulation was tightly linked to the behavioral genotype: the composite locomotion score was strongly and positively correlated with cholesterol levels (ρ\u0026thinsp;=\u0026thinsp;0.620, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, cognitive performance was inversely related to metabolic health, with the spatial memory index showing significant negative correlations with serum cholesterol levels (ρ = \u0026minus;0.421, p\u0026thinsp;=\u0026thinsp;0.001). Furthermore, a novel positive correlation was found between overall hippocampal bioenergetic and BAT thermogenic scores (ρ\u0026thinsp;=\u0026thinsp;0.392, p\u0026thinsp;=\u0026thinsp;0.006), which may reflect an association between central and peripheral bioenergetic impairment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Predictive Modeling Identifies Dyslipidemia and Hyperactivity as the Core Genotypic Signature\u003c/h2\u003e \u003cp\u003eTo move beyond identifying group differences and instead determine the core \"genotypic signature\" of the LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e model, we employed a supervised machine learning approach, Partial Least Squares Discriminant Analysis (PLS-DA), to classify animals based on their complete biological profile. Remarkably, the PLS-DA model distinguished LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e from control mice with perfect accuracy in cross-validation (ROC-AUC\u0026thinsp;=\u0026thinsp;1.00), indicating that the measured variables created a highly consistent and separable biological fingerprint for the genotype. An analysis of Variable Importance in Projection (VIP) scores provided a crucial insight into this signature: the most powerful and reliable predictors were serum cholesterol (VIP\u0026thinsp;=\u0026thinsp;100.00), the composite locomotor score (VIP\u0026thinsp;=\u0026thinsp;93.55), and serum triglycerides (VIP\u0026thinsp;=\u0026thinsp;91.33) (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eEpidemiological studies over the past decades have consistently shown that high cholesterol is linked to a greater risk of dementia. This connection is even stronger in familial hypercholesterolemia (FH), a condition long known for its cardiovascular complications and now increasingly associated with cognitive problems. In this regard, LDLr⁻/⁻ mice are widely used to explore how chronically elevated cholesterol affects tissues that are sensitive to metabolic stress, including the brain, providing an important model for interpreting our findings [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our study, both adult and middle-aged male and female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, fed a standard diet, exhibited impaired cholesterol clearance and significantly elevated plasma cholesterol levels compared to age- and sex-matched WT controls. The increase was approximately two-fold, consistent with the well-characterized hypercholesterolemic phenotype of the model. However, we did not detect sex differences in cholesterol levels at either age. Previous studies have reported varying results: Ishibashi et al. (1993,1994) reported higher cholesterol in female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice at 56 days and 6 months [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], while Marsh et al. (1999) observed higher levels in male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice at 4 months [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and Ghosh et al. (2020) described no sex differences at 6 months in LDLr\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on a standard diet [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Similarly, Rinninger et al. (2014) reported that both male and female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice showed markedly elevated and triglycerides compared to WT [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The discrepancies between studies may stem from methodological and physiological variables, including fasting status, diet composition, age, and hormonal influences, all of which can markedly affect lipid metabolism and mask potential sex-related differences.\u003c/p\u003e \u003cp\u003eConversely, sex- and age-dependent differences in triglyceride (TG) levels were observed in our study. Adult female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice displayed elevated TG compared to WT, while male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibited increased TG at both ages. While some studies did not observe differences between LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e and WT [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], others described pronounced TG elevations in male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, even under standard diet conditions [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The Jackson Laboratory also notes that TG increases can be detected in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice on chow diet, though they become more evident under high-fat feeding. A similar profile has been reported in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e rats, with increased plasma cholesterol and TG under standard diet [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This aligns with the classification of genetic dyslipidemias, in which this form of hypercholesterolemia may include higher TG levels, as LDL particles\u0026mdash;while mainly cholesterol-rich\u0026mdash;also carry a smaller TG fraction. Given that elevated serum TG reflects atherogenic lipoproteins and adds cardiovascular risk [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], these observations further underscore the systemic metabolic burden caused by LDLr dysfunction, paralleling features seen in FH patients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThermogenic adipocytes substantially contribute to systemic lipid clearance by accelerating the uptake, oxidation and re-esterification of circulating fatty acids, thereby lowering plasma triglyceride and cholesterol levels [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. White adipocytes are unilocular with a single large lipid droplet and relatively few elongated mitochondria, whereas brown adipocytes are multilocular and densely packed with mitochondria that are rich in iron and cytochromes, features that underlie BAT\u0026rsquo;s characteristic coloration and high oxidative capacity [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. BAT is also highly vascularized and extensively sympathetically innervated, structural attributes that support rapid fuel delivery and heat generation [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Mechanistically, BAT thermogenesis is mediated by uncoupling protein 1 (UCP-1), which dissipates the proton motive force across the inner mitochondrial membrane and uncouples electron transport from ATP synthesis, converting chemical energy into heat [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In our oximetry analyses, middle-aged male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibited reduced oxygen consumption linked to complex I substrates, while middle-aged female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice showed a selective reduction in UCP-1\u0026ndash;related oxygen consumption, indicating age-dependent declines in thermogenic function in both sexes. We also identified sex-related differences in BAT bioenergetics: adult WT males displayed higher respiration rates linked to complex I substrates compared with adult females, and middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males showed higher respiration rates than LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females of the same age. Functionally, such BAT dysfunction is expected to impair thermogenic capacity and lipid clearance, thereby contributing to the exacerbated dyslipidemia observed in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eThese experimental observations are consistent with interventional studies in the LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e model: pharmacological activation of thermogenic adipocytes with the selective β3-adrenergic agonist CL316,243 reduced plasma lipids and induced regression of atherosclerotic plaques in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Conversely, surgical removal of adipose depots, including BAT, aggravated metabolic disturbances in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice under high-fat feeding [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], demonstrating the protective, lipid-clearing role of brown and beige fat. More recently, β3-adrenoceptor agonists used clinically, such as mirabegron, were reported to lower plasma triglycerides in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, further supporting the translational relevance of BAT activation for lipid control [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Notably, however, most of these studies do not disaggregate outcomes by sex, and evidence specifically addressing UCP-1 activity in female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice remains limited.\u003c/p\u003e \u003cp\u003eBeyond systemic metabolism, the LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice has also been used to study neurocognitive function, including behavioral outcomes and brain cellular homeostasis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the OF test, we found that adult male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibited hyperlocomotion at both ages, traveling significantly greater distances than WT males (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whereas female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice displayed hyperlocomotion only at the middle-age. Aging also influenced female behavior, with middle-aged females showing reduced locomotion compared to adults of the same genotype. These findings extend prior studies that reported hyperlocomotion in male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice even under standard diet [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], but which did not include females or assess the role of age.\u003c/p\u003e \u003cp\u003eIn the object location (OL) task, both male and female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, independent of age, failed to discriminate the displaced object, unlike WT controls, that explored the relocated object more than 50% of the time. Spatial memory impairment in OL and cognitive decline has been previously reported in male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, even when fed a standard diet [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In the present study we demonstrated that female exhibited this memory impairment like males. Furthermore, LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice have been shown to be particularly susceptible to cognitive disturbances triggered by various stimuli that induce memory deficits, such as intracerebroventricular Aβ₁\u0026ndash;₄₀ injection, a peptide implicated in Alzheimer\u0026rsquo;s disease pathophysiology. These mice display impairments in both spatial and working memory even in the absence of exogenous Aβ, as reflected by poor performance in the object location and spontaneous alternation tasks. In addition, only LDLr⁻/⁻ mice injected with Aβ₁\u0026ndash;₄₀ exhibited impaired experience-dependent avoidance behavior in the EPM retest, a form of learning in which prior exposure to the apparatus normally promotes increased open-arm avoidance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings align with recent human studies showing that FH contributes to cellular dysfunction in the brain and cognitive decline. For instance, a clinical study comparing Heterozygous FH (HeFH) patients with age-matched controls with no history of cognitive-affecting disorders over 50 years old reported that 21.3% of HeFH patients exhibited MCI, compared with only 2.9% of controls, indicating a significant difference. The authors proposed that early exposure to high cholesterol levels or LDLr dysfunction may constitute a risk factor for MCI [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, another study found that HeFH patients aged 18\u0026ndash;40 performed worse on tasks assessing verbal memory and executive function compared with controls, and impairments in executive function correlated with higher serum LDL-C levels in the HeFH group [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering other behavioral assessments, we employed the EPM to investigate the impact of FH on anxiety-like behavior, particularly given the lack of literature on male and female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice in the context of aging and sexual dimorphism. In this study, LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males exhibited a higher number of open-arm entries in the EPM at both ages compared with WT controls (Supplementary Fig.\u0026nbsp;1B). However, when the percentage of open-arm entries was analyzed, no differences were observed in males, whereas middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females entered the open arms more frequently than WT females of the same age. To clarify whether this pattern reflected changes on behavior or locomotor activity, we further examined distance traveled in the EPM. This analysis confirmed hyperlocomotion in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males, consistent with findings from the OF test. Therefore, the increased number of open-arm entries in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males may reflect disinhibited behavior, but it should not be interpreted as altered anxiety-like behavior, since this effect is likely driven by hyperlocomotion in the absence of pharmacological or dietary interventions. Moreover, we identified sex-related differences, in which WT females displayed higher locomotor activity than WT males, whereas adult LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males traveled greater distances than LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females. To date, no studies have specifically examined EPM performance in the LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e model. However, previous work has reported depressive-like behavior in these animals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In line with this, a study using C57BL/6 mice showed that a high-cholesterol diet promotes both depressive- and anxiety-like behaviors [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], further supporting a potential link between cholesterol dysregulation and emotional alterations. Nevertheless, further studies are required to better elucidate the nature and mechanisms of these behavioral changes in the context of LDLr dysfunction.\u003c/p\u003e \u003cp\u003eThe hippocampus is essential for learning, memory, and mood regulation [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Previous studies indicate that FH can impact brain regions involved in cognitive processes, such as the hippocampus[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and the prefrontal cortex [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], independent of dietary interventions. To investigate the potential mechanisms underlying the cognitive and behavioral impairments observed in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, we evaluated hippocampal mitochondrial function. Interestingly, our findings revealed both sex- and age-dependent alterations in hippocampal bioenergetics in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. Middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females exhibited a marked impairment in hippocampal mitochondrial function, characterized by a reduction in oxygen consumption linked to complex I substrates, OXPHOS, ETS, and ATP-linked respiration. In contrast, adult LDLr⁻/⁻ males showed reduced oxygen consumption only when both complex I\u0026thinsp;+\u0026thinsp;II substrates were provided, along with a decline in ETS capacity. This pronounced hippocampal bioenergetic failure in middle-aged females likely reflects a metabolic crisis at the neuronal level, with reduced ATP generation compromising processes essential for synaptic maintenance and plasticity. Given that neuronal ATP is predominantly consumed to sustain synapse formation and activity, key determinants of cognitive performance [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], these data suggest that impaired mitochondrial energy metabolism may directly contribute to the cognitive and behavioral deficits observed in female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice at midlife. Collectively, these results highlight significant sex-related differences in hippocampal mitochondrial parameters, reinforcing the concept that mitochondrial bioenergetics are inherently sex-dependent and that aging acts as a critical modifier of the neurobiological consequences of LDLr deficiency on CNS function and behavior. Importantly, these metabolic disturbances are consistent with previous reports describing cognitive and behavioral dysfunctions in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, particularly in males. This model has been shown to present impaired synaptic plasticity and hippocampal neurogenesis, resulting in deficits in spatial and working memory, as well as depressive-like behavior [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. However, most previous studies have focused primarily on males, leaving the impact of sex and age largely unexplored. Our findings extend these observations by revealing that female LDLr⁻/⁻ mice are also vulnerable, exhibiting a distinct bioenergetic profile that may underlie their cognitive and affective alterations. One plausible mechanism involves the reduced neuronal uptake of cholesterol caused by LDLr dysfunction, which alters membrane lipid composition, impairs cell signaling, and compromises neuronal survival [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Additionally, previous studies have demonstrated that LDLr⁻/⁻ mice display increased blood\u0026ndash;brain barrier (BBB) permeability, astrogliosis, microgliosis, and hippocampal neuronal death [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], as well as oxidative stress characterized by elevated ROS production and weakened antioxidant defenses [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These mechanisms likely underlie the early-life hippocampal mitochondrial dysfunction observed in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e males, suggesting oxidative stress as a primary driver of bioenergetic impairments. In contrast, LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e females exhibited a delayed onset of hippocampal mitochondrial dysfunction, which emerged only at middle age and may reflect the progressive loss of estrogen-mediated protection, as estrogens are known to enhance mitochondrial efficiency and reduce oxidative damage. Other studies have also reported sex-specific differences in the metabolic disturbances. A study in Chinese adults over 45 years old found that elevated total and LDL cholesterol were associated with cognitive decline in women, but not in men [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Complementarily, Meng et al. (2023)[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] reported that ovariectomized LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, used to model menopause, developed worsened dyslipidemia, increased hippocampal apoptosis, and cognitive deficits, effects linked to reduced estradiol levels and downregulated estrogen receptor expression. Moreover, Pettersson et al. (2012) showed that high-fat diet induced hyperinsulinemia, glucose intolerance, and systemic inflammation in male C57BL/6 mice, whereas females exhibited an anti-inflammatory adipose tissue profile and preserved metabolic function [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Together, these data highlight the central role of estrogen signaling in protecting brain function against metabolic disturbances.\u003c/p\u003e \u003cp\u003eA key strength of our study is the focus on middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, a period that closely models the stage at which chronic hypercholesterolemia emerges as a critical risk factor for dementia in humans [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The pronounced hippocampal bioenergetic failure we identified at this stage aligns with, and extends, a growing body of evidence indicating that middle age represents a phase of accelerating neuropathology in this model. Previously, we demonstrated that middle-aged LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibit marked antioxidant imbalance and oxidative damage in the prefrontal cortex, including elevated lipid peroxidation, disrupted glutathione metabolism, and increased acetylcholinesterase activity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These signs of cellular stress are further compounded by evidence of synaptic decline, as both our group [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]and Mulder and collaborators [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] have reported decreased synaptophysin content at this age. Functionally, these molecular and structural alterations are accompanied by impaired spatial memory in the water maze and by pronounced microglial morphological changes indicative of neuroinflammation [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Therefore, the severe mitochondrial dysfunction observed in the present study provides a compelling mechanistic link suggesting that a collapse in energy metabolism may underpin the previously documented cascade of oxidative stress, synaptic decay, and cognitive impairment that characterizes the middle-aged hypercholesterolemic phenotype.\u003c/p\u003e \u003cp\u003eFinally, considering behavioral performance alongside hippocampal mitochondrial bioenergetics, we performed a correlation analysis (Supplementary Fig.\u0026nbsp;2) and identified positive associations between locomotor parameters assessed in the OF and EPM (summarized as locomotor score), hippocampal bioenergetic parameters (hippocampal bioenergetics score), and BAT mitochondrial parameters (BAT thermogenic score). Based on these integrative analyses, we found that increased cholesterol levels positively correlated with hyperlocomotion, but inversely correlated with spatial memory in the OL test. These observations align with reports that hypercholesterolemia impairs cognition and synaptic function, whereas lipid-lowering interventions, such as statins, can improve memory and reduce neuroinflammation [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Notably, PCSK9 inhibition lowers cholesterol without affecting locomotor or cognitive performance in some models [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], suggesting that the behavioral effects of cholesterol-lowering strategies depend on experimental context. We also identified a positive correlation between hippocampal and BAT scores, suggesting that broad metabolic disruption, coupled with mitochondrial dysfunction, likely contributes to both central and peripheral alterations in the context of chronic hypercholesterolemia. Interestingly, our findings reveal a mechanistic link between mitochondrial dysfunction in the hippocampus and BAT, highlighting the convergence of systemic and neural metabolism in this model. We also performed an integrative analysis aimed at identifying a \u0026ldquo;signature\u0026rdquo; of the LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e genotype including both sexes (Supplementary Fig.\u0026nbsp;4). This approach revealed that plasma cholesterol levels, followed by locomotor score, triglyceride levels, time spent in the periphery of the OF, hippocampal bioenergetics score, and location index were the most informative variables for characterizing this model, reinforcing the impact of cholesterol metabolism on both CNS function and behavior.\u003c/p\u003e \u003cp\u003eAltogether, our findings emphasize the necessity of including sex as a critical biological variable in studies of neurodegeneration and metabolic disease. The results provide new insight into the age- and sex-dependent hippocampal vulnerability to mitochondrial dysfunction in FH and point to the convergence of systemic and neural metabolism as a key component in the onset of cognitive impairment. It is also important to consider that aging has a profound impact on females, particularly due to hormonal fluctuations and the gradual decline in estrogen levels, which can exacerbate mitochondrial dysfunction and increase neural susceptibility to metabolic and oxidative stress [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Therefore, interventions targeting mitochondrial health and hormonal balance may represent promising strategies for preserving brain function in dyslipidemic conditions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAA: Antimycin A, AChE: Acetylcholinesterase, AD: Alzheimer\u0026rsquo;s Disease, ADP: Adenosine Diphosphate, ANOVA: Analysis of Variance, Apo B-100: Apolipoprotein B-100, ARRIVE: Reporting of In Vivo Experiments, ASCVD: Atherosclerotic Cardiovascular Disease, ATP: Adenosine Triphosphate, A\u0026beta;: Beta-amyloid, BAT: Brown Adipose Tissue, BBB: Blood\u0026ndash;Brain Barrier, BSA: Bovine Serum Albumin, Ca\u0026sup2;⁺: Calcium, CCCP: Carbonyl Cyanide m-chlorophenylhydrazone, CoQ10: Coenzyme Q10, CVD: Cardiovascular Disease, D: Displaced Object, EPM: Elevated Plus Maze, ETS: Electron Transport System, FFAs: Free Fatty Acids, GDP: Guanosine Diphosphate, HeFH: Heterozygous Familial Hypercholesterolemia, HMG-CoA Reductase: 3-hydroxy-3-methylglutaryl-coenzyme A reductase, HoFH: Homozygous Familial Hypercholesterolemia, HRR: High-Resolution Respirometry, i.p.: Intraperitoneal, IMM: Inner Mitochondrial Membrane, LDL: Low-Density Lipoprotein, LDL-C: Circulating Low-Density Lipoprotein, LDLr: LDL Receptor knockout mice, LI: Location Index, M: Malate, MCI: Mild Cognitive Impairment (for CCL), MWM: Morris Water Maze, NADPH: Nicotinamide Adenine Dinucleotide Phosphate (Reduced), ND: Non-Displaced Object, NIH: National Institutes of Health, O₂: Oxygen, OF: Open Field, OL: Object Location, OMY: Oligomycin, OXPHOS: Oxidative Phosphorylation, P: Pyruvate, PCSK9: Proprotein Convertase Subtilisin/Kexin Type 9, PM: Pyruvate and Malate, RB: Reaction Buffer, ROS: Reactive Oxygen Species, ROT: Rotenone, S: Succinate, SOD: Superoxide Dismutase, TCA: Tricarboxylic Acid Cycle, UCL: University College London, UCP-1: Uncoupling Protein 1, VLDL: Very-Low-Density Lipoprotein, WAT: White Adipose Tissue, WT: Wild-Type.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eAll animal procedures complied with the National Institutes of Health (NIH) guidelines for animal care, the Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments (ARRIVE) guidelines, and were approved by the Animal Ethics Committee of the University of Brasília (SEI Protocol 23106.075489/2023-95).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the first author, N. M. C. P. L. and the corresponding author, A.F.B., upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the following grants:\u0026nbsp;Fundação de Apoio à Pesquisa do Distrito Federal-FAPDF (00193-00002348/2022-07 and 00193-00000884/2021-89); Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (404466/2023-0 and 201648/2024-5); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (88881.465507/2019-01); and Instituto Nacional de Ciência e Tecnologia e Neuro-ImunoModulação - INCT-NIM (485489/2014-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions:\u0026nbsp;\u003c/strong\u003eN. P-L.: Conceptualization, Methodology, Validation, Investigation, Formal analysis, Data curation, Writing – original draft, Resources. D. F.: Investigation. L. T.: Investigation. W. S.: Investigation. W. B.: Investigation. H. M.: Data curation. M. M.: Resources. J. G.: Supervision, Data curation. A. A.: Supervision, Data curation. J. O.: Writing – review \u0026amp; editing. P. B.: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing – review \u0026amp; editing, Supervision. A. B.: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing – review \u0026amp; editing, Validation, Supervision, Resources, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ information (optional):\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlves RJ, Takao Suehiro Junior A, Brailowsky Pellegrino L. Hipercolesterolemia Familiar Homozig\u0026oacute;tica E Heterozig\u0026oacute;tica Grave: Epidemiologia, Diagn\u0026oacute;stico E Tratamento. 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Front Aging Neurosci. 2018;10(APR):124. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/FNAGI.2018.00124\u003c/span\u003e\u003cspan address=\"10.3389/FNAGI.2018.00124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biology-of-sex-differences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bosd","sideBox":"Learn more about [Biology of Sex Differences](http://bsd.biomedcentral.com)","snPcode":"13293","submissionUrl":"https://submission.nature.com/new-submission/13293/3","title":"Biology of Sex Differences","twitterHandle":"@BiologySexDiff","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"aging, sexual dimorphism, behavior, hypercholesterolemia, mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-8561934/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8561934/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFamilial hypercholesterolemia (FH) is a genetic disorder of cholesterol metabolism caused by loss-of-function variants in the low-density lipoprotein receptor (LDLR), resulting in persistently elevated LDL-cholesterol levels in plasma. Although hypercholesterolemia, especially the high levels of LDL, has been linked to an increased risk of dementia, the underlying mechanisms remain unclear. Here, we investigated the effects of sexual dimorphism and aging on metabolic and cognitive functions in a murine model of FH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult and middle-aged, male and female, C57BL/6 and LDLr\u003csup\u003e−/−\u003c/sup\u003e mice were used in this study. Behavioral assessments included locomotor activity, spatial memory, and anxiety-like behavior. Plasma lipid profiles were measured, and mitochondrial function in the hippocampus and brown adipose tissue (BAT) was assessed using high-resolution respirometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice of both sexes exhibited increased cholesterol and triglycerides levels. Male LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice displayed hyperlocomotion in the Open Field (OF) and Elevated Plus Maze (EPM) at both ages, whereas this phenotype emerged in middle-aged female LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice only in OF. Spatial memory impairments were observed in LDLr\u003csup\u003e⁻/⁻\u003c/sup\u003e mice regardless of sex or age. Hippocampal oxygen consumption was reduced in adult males and middle-aged female mice, whereas BAT respiration was impaired in both sexes at middle-aged animals, affecting distinct respiratory parameters. Correlation analyses revealed that elevated cholesterol levels were associated with memory deficits and hyperlocomotion, along with positive correlations between hippocampal and BAT mitochondrial function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that FH induces sex- and age-dependent alterations in behavior and mitochondrial metabolism, providing mechanistic insights into the link between FH and neurodegenerative disease risk.\u003c/p\u003e","manuscriptTitle":"Sex- and Age-Dependent Mitochondrial Dysfunction Links Familial Hypercholesterolemia to Cognitive Impairment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 10:25:17","doi":"10.21203/rs.3.rs-8561934/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-23T15:50:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T15:44:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T14:11:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63567232950810970894232007248159994432","date":"2026-01-17T15:29:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315274509992762783004751104503342153534","date":"2026-01-16T12:05:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-14T21:56:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-14T05:43:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T05:41:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biology of Sex Differences","date":"2026-01-09T14:12:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biology-of-sex-differences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bosd","sideBox":"Learn more about [Biology of Sex Differences](http://bsd.biomedcentral.com)","snPcode":"13293","submissionUrl":"https://submission.nature.com/new-submission/13293/3","title":"Biology of Sex Differences","twitterHandle":"@BiologySexDiff","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c3cd9fc-fbfa-4c13-ba16-e9debbe3ef90","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:09:36+00:00","versionOfRecord":{"articleIdentity":"rs-8561934","link":"https://doi.org/10.1186/s13293-026-00893-x","journal":{"identity":"biology-of-sex-differences","isVorOnly":false,"title":"Biology of Sex Differences"},"publishedOn":"2026-04-10 15:58:35","publishedOnDateReadable":"April 10th, 2026"},"versionCreatedAt":"2026-01-19 10:25:17","video":"","vorDoi":"10.1186/s13293-026-00893-x","vorDoiUrl":"https://doi.org/10.1186/s13293-026-00893-x","workflowStages":[]},"version":"v1","identity":"rs-8561934","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8561934","identity":"rs-8561934","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}