UQCRC1 downregulation impairs cognitive function in mice via AMPK inactivation

Animals

C57BL/6 wild-type (WT) mice were obtained from Charles River Laboratories (Chengdu, China), while UQCRC1^+/− heterozygous mice were generated by Professor Zhiyi Zuo (Shan et al., 2019), with all animals housed under specific pathogen-free (SPF) conditions at Army Medical University’s animal facility. Mice aged 8–12 weeks (weighing 20–30 g) were maintained in standard cages (330 × 210 × 170 mm; 5 mice/cage) with ad libitum access to food and water under controlled environmental parameters: 12-h light/dark cycle (08:00–20:00), 20–23 °C ambient temperature, and 50–60% relative humidity.

In addition to 16 female animals (eight UQCRC1^+/− and eight WT) designated for behavioral assessments, this study included 36 male WT mice and 63 male UQCRC1^+/− mice. Following initial behavioral evaluations, eight WT and eight UQCRC1^+/− males were each joined by 10 more genotype-matched males (for a total of n = 18 per genotype) for hippocampus collection at baseline. Six specimens per genotype were assigned to ROS/ATP/caspase assays, six to Western blot analysis, and six to transmission electron microscopy (TEM). For the therapeutic evaluation phase, 45 UQCRC1^+/− males were randomly assigned into three treatment cohorts (solvent vehicle, A-769662, and LH2-051). Eight mice per group received post-treatment behavioral assessment, while seven additional mice per group were included for tissue analysis (six specimens per group were assigned to ROS/ATP/caspase assays, three to Western blot analysis, and six to TEM). A parallel study included 36 WT males following the same experimental timeline and tissue distribution protocols. Animals were euthanized prior to the planned endpoint only if they met predefined humane criteria, including (but were not limited to): severe weight loss (>20% of baseline body weight) or failure to thrive; signs of irreversible distress or pain (e.g., labored breathing, prolonged immobility, inability to access food/water); unexpected complications directly related to the experimental intervention (e.g., neurological deficits). No animals required early euthanasia in this study, as all subjects maintained stable health metrics within predefined thresholds throughout the experimental timeline. No animals were retained beyond the study period due to the terminal nature of the experimental design. Males were euthanized via intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg), while female cohorts were euthanized using gradual CO₂ asphyxiation (30%–99%, 15 min) with confirmation of death. Group assignments were randomized using random number tables. Sample sizes calculated based on power analysis incorporating preliminary data and literature benchmarks (Shan et al., 2019; Lin et al., 2020; Fernandez-Mosquera et al., 2019; Chen et al., 2022; Kim et al., 2021). The experimental protocol was developed prior to study commencement and conducted in compliance with guidelines approved by the Army Medical University’s Laboratory Animal Welfare and Ethics Committee (Approval No.: AMUWEC20245280; Approval date: 10/1/2024).

Behavioral testing

All mice were acclimated to their environment one week prior to the onset of behavioral trials. All behavioral tests commenced at 10 a.m. To minimize environmental stress, mice were transferred to the testing room two hours before each session. Behavioral assessments were spaced one day apart to prevent interference from prior testing. After each test, all equipment was thoroughly cleaned with 75% ethanol to eliminate olfactory cues. During testing, examiners exited the room to reduce external influence. Behavioral data were recorded and analyzed using EthoVision XT 11.5 (Noldus Inc., Wageningen, Netherlands).

Novel object recognition test

The ability of short-term memory in the hippocampus of mice was evaluated using the novel object recognition (NOR) test (Bevins & Besheer, 2006). The experimental setup consisted of an acrylic rectangular enclosure (40 × 40 × 40 cm, Fig. 1A). The test comprised three separate phases. Habituation Phase: mice were allowed to explore the empty apparatus freely for 10 min. Familiarization Phase: conducted 24 h after habituation, two identical objects (Familiar Object, F) were placed symmetrically within the apparatus. Mice were reintroduced into the center of the apparatus and allowed to explore for 10 min. Test Phase: two hours after the second stage, one of the familiar items was replaced with a novel object of the same size but a different shape (Novel Object, N). Mice were again placed in the center and given 10 min to explore. The time spent exploring the familiar object (tF) and the novel object (tN) was recorded. The recognition rate (RR) was calculated as RR = tN / (tF + tN).

Transmission electron microscope

Mice were initially perfused with ice-cold phosphate-buffered saline (PBS). The hippocampus was carefully dissected and sectioned into small fragments. The tissue samples were subsequently fixed, dehydrated, infiltrated, and embedded according to standard protocols. Ultrathin sections were prepared and stained with 2% uranyl acetate. Quantitative analysis of the images was performed using ImageJ software (NIH, https://imagej.net/ij/, version 1.54).

Western blotting

Hippocampal tissues were harvested and homogenized, and 20 µg of protein was loaded onto the 4–20% gradient gels (CAT# ET15420LGel; ACE Biotechnology) and then transferred to a PVDF membrane using the Bio-Rad system. The membranes were subsequently blocked with rapid blocking buffer (CAT# HY-K1027; MedChemExpress) for 10 min. Primary antibodies were incubated overnight at 4 °C. The primary antibodies used included rabbit polyclonal anti-LC3B (1:1000 dilution, Abcam, CAT# ab48394), rabbit polyclonal anti-AMPKα (1:1000 dilution, CAT# 2532; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-phospho-AMPKα (Thr172) (1:1000 dilution, CAT# 2535; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-GAPDH (1:3000 dilution, CAT# LF211; Epizyme Biotech, Cambridge, MA, USA). After incubation with HRP-conjugated goat anti-rabbit IgG (1:3000 dilution, CAT# ZB-2301; ZSGB-BIO) for one hour at room temperature, membranes were developed. Densitometric analysis was conducted using ImageJ software (version 1.54). Protein expression levels were normalized by dividing the signal intensity of the target protein by that of GAPDH. All wild-type (WT) group values were set to 1.0 to establish baseline expression.

Quantitative PCR (qPCR)

Total RNA was extracted from murine hippocampal tissues using the Magbead RNA Extraction Kit (CAT# W3711S; Cwbiotech), followed by genomic DNA removal using RNase-free DNase I (CAT# EN0521; Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA synthesis and amplification were performed using the PrimeScript RT-PCR Kit (CAT# RR037A; Takara Bio). Quantitative analysis employed the ΔΔCT method with normalization to GAPDH expression. Primer sequences are provided in Table S1.

ATP assay

ATP content was measured following the protocol provided in the ATP content test kit manual (CAT# G4309-48T; Servicebio). Extracted hippocampal tissues were homogenized, lysed, boiled, and cooled to room temperature, followed by centrifugation at 10,000× g for 15 min at 4 °C. Subsequently, 20 µL of the supernatant was mixed with 100 µL of ATP assay reagent. Bioluminescence intensity was subsequently measured using a luminometer (Fig. 2A).

Assessment of reactive oxygen species

ROS levels were assessed using the reactive oxygen species (ROS) Detection Kit (Bestbio, CAT# BB-47051). After hippocampal homogenization, the homogenate was centrifuged at 1,000× g for 3 min at 4 °C. A volume of 2 µL dihydroethidium (DHE) probe was added to 200 µL of the supernatant and incubated in the dark at 37 °C for 30 min. Fluorescence intensity was measured using an excitation wavelength of 510 nm and an emission wavelength of 610 nm. ROS levels were determined by calculating the ratio of fluorescence intensity to protein concentration.

Assessment of caspase 3 and caspase 9

The Caspase 3/Caspase 9 Activity Assay Kit (CAT# APC03/APC09; MultiSciences Biotech Co., Ltd.) was used to evaluate the activation of caspase 3 and caspase 9. Briefly, homogenized hippocampal tissue was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and reaction mixtures were prepared according to the manufacturer’s instructions. Following 4 h of incubation at 37 °C, absorbance was measured at 405 nm.

Intraperitoneal injection

UQCRC1^+/− mice were randomly assigned to three groups: UQCRC1^+/− + A-769662, UQCRC1^+/− + LH2-051, and UQCRC1^+/− + solvent. Mice in the UQCRC1^+/− + A-769662 and UQCRC1^+/− + LH2-051 cohorts received intraperitoneal injections of A-769662 (30 mg/kg, MCE, CAT# HY-50662) or LH2-051 (10 mg/kg, MCE, CAT# HY-161723), administered twice daily for 30 consecutive days (Fig. 3A). Mice in the UQCRC1^+/− + solvent group received an equivalent volume of solvent comprising 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline.

Statistical analysis

All experiments and analyses were conducted under blinded conditions, with investigators unaware of group allocations during both experimental procedures and data interpretation. All acquired data were retained for statistical analysis without exclusion. The Shapiro–Wilk test was employed to assess data normality. Parametric tests were applied to normally distributed data, while non-parametric tests were applied when normality assumptions were not met. For two-way ANOVA, no outliers were detected based on studentized residuals exceeding ±3. The Kruskal–Wallis H test, unpaired two-tailed t-tests, Mann–Whitney U test, one-way ANOVA, and two-way ANOVA were used to assess differences between groups. Statistical significance was defined as a p-value of less than 0.05. Data analysis was performed using GraphPad Prism (version 9.5; GraphPad Software, Boston, MA, USA) and SPSS (version 27.0; IBM, Armonk, NY, USA). Statistical significance was indicated as ^∗p < 0.05, ^∗∗p <  0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p <  0.0001.

AI tool usage statement

The DeepSeek-R1 model (developed by DeepSeek) was employed exclusively for grammatical error detection, spelling correction, and sentence structure refinement. The tool did not contribute to data generation, conceptual development, or conclusion formulation. All modifications suggested by the tool underwent rigorous manual verification by the authors, who assume full responsibility for the perspectives, data analysis, and scholarly argumentation presented herein.

The downregulation of UQCRC1 impairs cognitive function

In the NOR test, both male (Fig. 1B; p < 0.0001) and female (Fig. 1B; p < 0.05) UQCRC1^+/− mice demonstrated a markedly reduced recognition rate compared to WT controls. In the NBT, male UQCRC1^+/− mice exhibited impaired performance (Fig. 1D; p < 0.01). Starting on day 3 of the Barnes maze training phase, male UQCRC1^+/− mice exhibited substantially longer escape latencies (Fig. 1F; p < 0.05 on day 3; p < 0.01 on day 4). During the testing phase, these mice also demonstrated significantly longer escape latencies (Fig. 1G; p < 0.05) and a notably higher number of attempts to find the target box (Fig. 1H; p < 0.05). Female UQCRC1^+/− mice also required more attempts to locate the target box (Fig. 1H; p < 0.05).

These results suggested that mitochondrial dysfunction caused by UQCRC1 downregulation is strongly correlated with cognitive deficits in male mice, whereas its impact on female mice appeared to be more variable. Consequently, subsequent investigations focused primarily on male mice.

The downregulation of UQCRC1 leads to autophagy impairment

Our findings showed that the downregulation of UQCRC1 significantly reduced hippocampus-dependent cognitive functions. To further investigate this phenomenon, we assessed cellular energy status and oxidative stress levels. A significant decrease in ATP levels was observed in UQCRC1^+/− mice (Fig. 2B; p < 0.01), accompanied by an increase in ROS levels (Fig. 2C; p < 0.05). Lipofuscin, a yellowish-brown granular pigment that accumulates in ageing cells, is formed through the cross-linking of incompletely degraded lipids and proteins. Its accumulation serves as an indicator of impaired lysosomal degradation capacity. In our study, increased lipofuscin accumulation was detected (Figs. 2D, 2E; p < 0.05), along with elevated expression of LC3B-II (Figs. 2F, 2G; p < 0.01). These results implied that UQCRC1 downregulation results in defective autophagic flux. Consistent with previous reports (Fernandez-Mosquera et al., 2019), our data showed a significant reduction in pAMPK levels (Figs. 2F, 2H; p < 0.0001) and in pAMPK/AMPK ratio (Figs. 2F, 2J; p < 0.0001), whereas total AMPK levels remained unchanged (Figs. 2F, 2I). To date, folliculin (FLCN), a known tumor suppressor, is the only identified direct negative regulator of AMPK activity. FLCN forms a functional heterotrimeric complex with its interacting proteins FNIP1 and FNIP2, which together inhibit AMPK signaling (Xiao et al., 2021). Notably, previous studies have demonstrated upregulation of FLCN, FNIP1, and FNIP2 in UQCRC1 knockdown cells (Fernandez-Mosquera et al., 2019). In alignment with these findings, our study confirmed the transcriptional upregulation of FLCN (Fig. 2K; p < 0.05), FNIP1 (Fig. 2K; p < 0.05), and FNIP2 (Fig. 2K; p < 0.05). Together, these findings indicate that UQCRC1 downregulation reduced AMPK activation in the hippocampus and impaired autolysosomal degradation. Moreover, we observed increased activation of caspase 3 (Fig. 2I; p < 0.0001) and caspase 9 (Fig. 2I; p < 0.001), suggesting enhanced apoptotic activity as a consequence of UQCRC1 downregulation. Notably, no differences were observed in ATP levels, ROS levels, or the expression of pAMPK, AMPK, and LC3B between female UQCRC1+/− mice and female WT mice (Fig. S1).

Enhancing AMPK activity or improving lysosomal function ameliorates autophagy in UQCRC1^+/− mice

A-769662 is a direct, allosteric activator of AMPK that increases its activity through two complementary mechanisms: by inhibiting Thr-172 dephosphorylation and directly inducing allosteric activation of AMPK (Cool et al., 2006; Göransson et al., 2007). LH2−051 improves lysosomal function through the DAT-CDK9-TFEB signaling pathway, thereby promoting the degradation of toxic protein aggregates such as β-amyloid (Yin et al., 2023a; Yin et al., 2023b). In this study, we investigated the effects of these compounds in UQCRC1^+/− mice. Following intraperitoneal administration of A-769662, we observed a significant restoration in ATP content (Fig. 3B; p < 0.001), a reduction in ROS levels (Fig. 3C; p < 0.001), and decreased lipofuscin accumulation (Figs. 3D, 3E; p < 0.05). Furthermore, AMPK activity (Figs. 3F, 3H–3J; p < 0.01) was notably increased, alongside a marked reduction in the LC3B-II expression (Figs. 3F, 3G; p < 0.0001), activation of caspase 3 (Fig. 3K; p < 0.0001) and caspase 9 (Fig. 3K; p < 0.001). In parallel, LH2-051 administration led to significant improvements in ATP content (Fig. 3B; p < 0.05), reductions in ROS levels (Fig. 3C; p < 0.05), and decreased lipofuscin deposition (Figs. 3D, 3E; p < 0.05). LC3B-II expression (Figs. 3F, 3G; p < 0.0001) and caspase 9 activation (Fig. 3K; p < 0.01) were also restored. Although caspase 3 levels showed partial recovery (Fig. 3K; p < 0.0001), they did not return to the levels observed in the control group. Notably, no significant changes were detected in AMPK expression, pAMPK expression, or the pAMPK/AMPK ratio (Figs. 3F, 3H–3J).

Collectively, these findings confirm that lysosomal dysfunction is closely associated with mitochondrial impairment in the hippocampal tissue of UQCRC1-deficient mice. Both AMPK activation (via A-769662) and lysosomal enhancement (via LH2-051) alleviate these deficits, highlighting their therapeutic potential. However, the incomplete functional rescue suggests the involvement of additional pathological mechanisms beyond AMPK and lysosomal pathways.

Increasing AMPK activity or enhancing lysosomal function can both rescue cognitive deficits in UQCRC1^+/− mice

To investigate the potential causal relationship between autophagy dysfunction and the cognitive abnormalities observed in UQCRC1^+/− mice, we performed a series of behavioral evaluations following rescue therapies. The results demonstrated that augmenting AMPK activity significantly improved the performance of UQCRC1^+/− mice in the novel object recognition test (Figs. 4A, 4B; p < 0.001), the nest-building test (Figs. 4C, 4D; p < 0.01), and the Barnes maze (Figs. 4E–4H).

Enhancing lysosomal function partially ameliorated the cognitive deficits in UQCRC1^+/− mice. Specifically, treated mice exhibited a higher recognition rate in the novel object recognition test (Figs. 4A, 4B; p < 0.01), as well as reduced latency during both the training phase (Fig. 4F; p < 0.01) and testing phase (Fig. 4G; p < 0.05) of the Barnes maze. However, no significant improvement was observed in the nesting score during the nest-building test (Figs. 4C, 4D), nor in the number of attempts required to locate the target box in the Barnes maze (Fig. 4H).

These findings indicate that AMPK plays a pivotal role in the cognitive impairments associated with mitochondrial dysfunction, and that lysosomal dysfunction is also implicated in this pathological process.

Limitation

This study has several limitations that warrant consideration. First, our findings suggest sex-dependent differences in the effects of UQCRC1 downregulation on murine cognitive function, yet the underlying mechanisms remain unclear. Second, although behavioral assessments preliminarily identified the hippocampus as the primary region affected, the extensive neuronal networks involved in cognition prevent the complete exclusion of contributions from extra-hippocampal areas. Third, while we demonstrated that UQCRC1-mediated mitochondrial dysfunction impairs cognition through AMPK-dependent autophagic dysregulation, two critical mechanistic gaps persist: (1) the precise signaling cascades linking AMPK activation to autophagic modulation, and (2) the exact neurobiological mechanisms through which autophagy perturbations mediate cognitive deficits.