The Drp1-CoQ10-Coa6-ETC axis represents a therapeutic target for working memory impairment caused by neuronal mitochondrial dysfunction

The Drp1-CoQ10-Coa6-ETC axis represents a therapeutic target for working memory impairment caused by neuronal mitochondrial dysfunction | 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 The Drp1-CoQ10-Coa6-ETC axis represents a therapeutic target for working memory impairment caused by neuronal mitochondrial dysfunction Jingjing Tie, Shujiao Li, Xin Huang, Keke Ren, Ziwei Ni, Xiaodong Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7362769/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Apr, 2026 Read the published version in Translational Neurodegeneration → Version 1 posted 5 You are reading this latest preprint version Abstract Coenzyme Q10 (CoQ10), the third most popular dietary supplement worldwide, shows promise in treating the ten leading non-communicable diseases linked to global mortality. However, its mechanism and potential to address memory deficits caused by cerebellar injury are not fully understood. We explored whether long-term CoQ10 supplementation could help recover working memory loss and examined the underlying mechanisms. Network pharmacology analysis identified DNM1L/Drp1 as a important genetic target of CoQ10 in cerebellar injury-related memory impairment. We generated three lines of mice with Purkinje cell (PC)-specific deficiency in Drp1 (Drp1 −/− mice). Multi-level assessments showed that these mice exhibited: Progressive working memory deficits (assessed via multiple behavioral tests); impaired PC plasticity (evaluated by patch-clamp recordings and morphological analysis); and disrupted mitochondrial membranes (MMs) stability and oxidative phosphorylation (OXPHOS), particularly in complexes III-V (CIII-CV) (assessed through various structural and functional assays). Long-term CoQ10 administration in drinking water for 75 days, beginning at postnatal day 15, effectively ameliorated working memory impairments (5-fold in the percentage of 45° searches), PC plasticity, and mitochondrial dysfunction in Drp1 −/− mice at the animal, cellular, and organelle levels. Furthermore, comprehensive drug-target fishing analyses including thermal proteome profiling (TPP), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), surface plasmon resonance (SPR), and molecular docking demonstrated that CoQ10 directly binds to cytochrome c oxidase assembly factor 6 (Coa6). This CoQ10-Coa6 interaction restored MMs stability and CIII-CV activity, revealing the Drp1-CoQ10-Coa6-electron transport chain (ETC) axis as a promising therapeutic target for memory disorders associated with neurological diseases. Purkinje cells Working memory Coenzyme Q10 Dynamin-related protein 1 Cytochrome c oxidase assembly factor 6. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Neurological diseases have become increasingly prominent in the global disease burden 1 . Working memory refers to the temporary retention and manipulation of relevant information from memory, which guides future actions 2 . Impairment or even loss of working memory is a common symptom of many neurodegenerative diseases, including AD Parkinson's disease (PD), and Huntington's disease (HD) 3 , 4 . Previous studies on working memory mainly focused on the cerebral cortex and hippocampus, while ignoring the role of the cerebellum in it 5 . However, accumulating evidence has begun to redefine the cerebellum as a critical node in cognitive networks 6 . In particular, cerebellar Purkinje cells (PCs) play a key role in regulating working memory, attention, and executive function through their interactions with the prefrontal–cerebellar–thalamic circuitry 7 . Current pharmacological strategies to improve working memory involve medications such as methylphenidate, nimodipine, and lamotrigine 8 – 10 . However, most drugs exhibit complex side effects or prove to be ineffective 11 , 12 . Consequently, it is imperative to identify novel therapeutic agents that can effectively mitigate working memory deficits caused by cerebellar injury. Co-enzyme Q10 (CoQ10) is a critical antioxidant and an essential component of the mitochondrial electron transport chain 13 , 14 . Although CoQ10 is not approved by the US Food and Drug Administration (FDA) as a drug, it is marketed as a dietary supplement and is currently one of the most consumed nutritional supplements 13 , 15 . CoQ10 was considered a promising candidate therapeutic agent for the treatment of various diseases, including cardiovascular disease, neurodegenerative disorders, cancer, and diabetes 15 – 18 . Long-term administration of CoQ10 was shown to effectively alleviate memory function impairment caused by Alzheimer’s disease (AD) 19 , 20 . The CoQ10 headgroup intermediates can restore CoQ10 synthesis in the body, thus alleviating the mitochondrial encephalopathy, including cerebellum damage 21 . However, the molecular targets through which CoQ10 exerts its effects on neurons remain unclear. Therefore, whether CoQ10 can effectively alleviate working memory impairment caused by cerebellar injury and the underlying mechanisms need to be investigated. Here, network pharmacology analysis identified DNM1L/Drp1 as a key genetic target of CoQ10 in cerebellar injury-related memory impairment. We utilized the Cre-LoxP system to generate three distinct PCs-specific Drp1 (Dynamin-related protein 1) knockout mouse models: Drp1 −/− mice (conventional knockout), cKO PC tdTomato mice (enabling specific TdTomato expression in PCs), and cKO PC Mito−GFP mice (enabling specific GFP expression on the outer mitochondrial membrane of PCs). The Drp1 −/− mice exhibited progressive working memory deficits, as demonstrated by eight arm maze and morris water maze tests, along with impaired PC plasticity assessed through Golgi staining and immunofluorescence. Mitochondrial membranes (MMs) instability was confirmed by electron microscopy and mitochondrial membrane potential measurements, while oxidative phosphorylation (OXPHOS) dysfunction - particularly in complexes III-V (CIII-CV) - was evidenced by Western blotting, reactive oxygen species (ROS) levels, and ATP production assays. Following 75-day CoQ10 long-term supplementation in drinking water initiated at postnatal day 15, behavioral tests revealed significant amelioration of working memory impairments in Drp1 −/− mice. Electrophysiological and morphological analyses demonstrated that CoQ10 effectively suppressed aberrant HCN channel activation and restored PC plasticity after injury. Ultrastructural examination by electron microscopy and functional mitochondrial assays showed that CoQ10 treatment reversed Drp1 deficiency-induced MMs instability and OXPHOS dysfunction. Mechanistically, thermal proteome profiling (TPP) identified 12 potential target proteins of CoQ10 in Drp1-deficient mice. Moreover, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and surface plasmon resonance (SPR) confirmed stable binding between CoQ10 and cytochrome c oxidase assembly factor 6 (Coa6). Molecular docking predicted CoQ10 binds within the hydrophobic pocket of Coa6 with strong hydrophobic complementarity. Our findings demonstrate that the CoQ10-Coa6 interaction enhances MMs stability, reduces oxidative stress, and restores CIII-CV activity, establishing the Drp1-CoQ10-Coa6-electron transport chain (ETC) axis as a potential therapeutic mechanism for cerebellar injury-induced cognitive dysfunction. Methods Animals We purchased four types of transgenic mice tool: 1) Pcp2 Cre mice, which were designed by specifically inserting Cre enzyme into all Purkinje cells (PCs), with stock code of 004146 of Jackson Laboratories (US) 22 ; 2) Drp1 flox mice, which were designed by inserting loxP sites flanking the Drp1 sequence, with the serial number of CKOAIS191230RT5 of Cyagen (China) 23 ; 3) B6/JGpt-H11 em1Cin (CAG−LoxP−ZsGreen−Stop−LoxP−tdTomato) /Gpt mice (B6-G/R mice), which were designed by inserting loxP sites flanking the ZsGreen sequence, with the serial number of T006163 of GemPharmatech (China); and 4) RoSA26-CAG-LSL-GFP-MITO-labeled mice (Mito-GFP mice), which were designed by inserting loxP sites flanking the green fluorescent protein (GFP) specifically located on the mitochondrial outer membrane, with a serial number of cas9-ki (Rosa26) of GemPharmatech. All mice were housed (up to five per cage) under a 12-hour light-dark cycle (8 a.m. − 8 p.m.) with ad libitum access to food and water. Generation of Drp1 −/− mice, PC tdTomato mice, cKO PC tdTomato mice, PC Mito−GFP mice, and cKO PC Mito−GFP mice We generated five types conditional knock-out mice (cKO mice), including: 1) Drp1 −/− mice, generated by crossing Pcp2 Cre mice with Drp1 flox mice, in which Drp1 gene was specifically KO in cerebellar PCs; 2) PC tdTomato mice, generated by crossing Pcp2 Cre mice with B6-G/R mic, in which Pcp2-positive PCs carry tdTomato; 3) cKO PC tdTomato mice, generated by crossing Drp1 fl/fl mice with B6-G/R mice, in which the specific Drp1-KO PCs expressed tdTomato; 4) PC Mito−GFP mice, generated by crossing Pcp2 Cre mice with Mito-GFP mice, in which Pcp2-positive PCs carry GFP; and 5) cKO PC Mito−GFP mice, generated by crossing Drp1 fl/fl mice with Mito-GFP mice, in which mitochondrial outer membrane within the specific Drp1-KO PCs expressed GFP. Identification of transgenic mice via PCR analysis The tail DNA was lysed and 200 ul Lysis buffer and 4 ul DNA Release of DNA lysate were mixed for each tail sample. DNA lysate was added to each sample, boiled in a thermostatic water bath for 55℃ for 30 min, then boiled at 98℃ for 5 min, then centrifuged and centrifuged at 12000 rpm / min for 5 min. The obtained supernatant was a DNA sample and was temporarily stored at 4℃ for subsequent experiments. The mouse tail identification kit (TSE014, Tsingke, China) was used for DNA PCR and all these primer sequences were exhibited in Supplementary Table 1 . The PCR protocol included pre-denaturation at 94 ℃ for 3 min, followed by 35 cycles of denaturation at 94℃ for 30 s, annealing at 60 ℃ for 35 s, and finally extended at 72 ℃ for 35 s. DNA products were detected using lysis curves. The quantitative was statistically calculated using the 2 −△△ Ct. Long-term supplementation with Coenzyme Q10 in mice Drp1 −/− mice, cKO PC tdTomato mice, and cKO PC Mito−GFP mice, were administered coenzyme Q10 (CoQ10) supplementation from postnatal day 15 to day 90. CoQ10 (HY-N0111, MCE) was dissolved in a 3% dimethyl sulfoxide (DMSO) solution at a concentration of 500 µM. The CoQ10 solution was added to the drinking water and refreshed daily. Drp1 −/− mice, cKO PC tdTomato mice, and cKO PC Mito−GFP mice in the control group were provided with drinking water containing less than 3% DMSO. Rotarod test For motor coordination assessment, the rotarod test (BZY007, Jiliang, China) started at 4 rpm and increased to 40 rpm within 180 seconds. Each trial lasted 10 minutes, with at least a 15-minute interval. Mice were trained 3 times daily for 3 consecutive days, data were collected on the fourth day. The latency to fall from the rod was recorded. Open field test To evaluate the free and spontaneous movements of transgenic mice, the animals were placed in a 50 × 50 × 50 cm opaque square box (DigBehav, measured), and their movements were recorded using a camera connected to a computer for 15 minutes. The mice's movements were automatically tracked, and the total distance traveled was analyzed using dedicated software. Water maze test We used the Water Maze test (TECHMAN, China) to perform a preliminary exploratory assessment of learning and memory. At least five mice were used for each group: Control and Drp1 −/− . At the start of the training, the water surface was divided into four quadrants based on the position of a marker. A platform was placed in the center of the first quadrant, and the mice were introduced into the pool at random points along the walls of the four quadrants, facing the pool wall. A video recording system was used to capture the time it took for the mice to locate the platform (escape latency) and their swimming paths. Each training session consisted of four trials, with the mice being placed in the water at different starting points (from different quadrants) in each trial. If the mice located the platform or failed to do so within 60 seconds, they were allowed to rest on the platform for 15 seconds before beginning the next trial. The average escape latency across the four trials was recorded as the learning performance for that day. Over five consecutive days of training, the swimming speed (mm/s) and escape latency (s) were measured to evaluate the mice's performance. Eight-arm radial maze test To evaluate the working memory ability of Drp1 −/− mice, we conducted an eight-arm radial maze test (TECHMAN, China). The study included at least five mice in each group: Control, Drp1 −/− , and CoQ10 intervention groups. Animals were acclimated to the experimental environment for one week, during which they were weighed and fasted for 24 hours before the experiment. Following fasting, normal food (2–3 g) was provided daily at the end of training sessions to maintain the body weight of the mice at 80–85% of their normal feeding weight. On days 1 and 2, food pellets were scattered across each arm and the central area of the maze to familiarize the animals with the setup. On the third day, individual training began, where a single food pellet was placed at the end of each arm near the outer food box, allowing the mice to explore and eat freely. From days 4 to 7, the food was placed inside the boxes, and the previous day's training procedure was repeated. Working memory performance was assessed from day 3 to day 7 by recording the latency of mice in locating the food and the number of errors, defined as repeated entries into the same arm. Additionally, the mice's search strategies were manually categorized based on angles of 45°, 90°, 270°, and 360°. Over a period of five consecutive days, we assessed the mice's working memory performance by measuring working memory errors, latency periods, and the percentage of searching strategies categorized at angles of 45°, 90°, 270°, and 360°. Golgi staining Golgi staining was performed using a Golgi staining kit (PK401, FD NeuroTechnologies): After deeply anesthetizing the mice, the brain tissue was quickly extracted, and blood on the surface of the tissue was rinsed off with distilled water. The tissue was immersed in a mixture of solution A and solution B. On the following day, the immersion solution was replaced with fresh solution, and the tissue was kept in the dark at room temperature for two weeks. Subsequently, the tissue was transferred to solution C and kept in the dark at room temperature for at least 72 hours, with the solution changed at least once on the second day.The tissue was then sectioned into 100-µm thick slices at a temperature of -20°C to -22°C using a cryotome and transferred onto slides coated with 3.5% gelatin. The slides were air-dried in the dark at room temperature for three days. The sections were washed twice with double-distilled water for 4 minutes each. Next, the slices were incubated in a mixture of 1 part solution D, 1 part solution E, and 2 parts double-distilled water for 10 minutes, followed by rinsing with distilled water twice for 4 minutes each.The sections were dehydrated through an ethanol series (50%, 75%, and 95% ethanol, each for 4 minutes) and then dehydrated in absolute ethanol four times, for 4 minutes each. Subsequently, the sections were cleared in xylene three times for 4 minutes each. Finally, coverslips were mounted using resin sealant. PCs were observed and photographed under an optical microscope, and Sholl analysis was performed using ImageJ software 24 . The analysis focused on two parameters: the abundance of PCs and the density of their dendritic spines. Immunofluorescence staining PC tdTomato mice were used as the control group, cKO PC tdTomato mice as the experimental group, and the CoQ10 intervention group was also included. All mice were deeply anesthetized with 40 mg/kg pentobarbital sodium (Merck, Germany), and 4% paraformaldehyde was perfused through the heart. The extracted brains were fixed in 4% paraformaldehyde (R20497-10, Source Leaf) for 24 hours and then immersed in 30% sucrose solution for 2 days. Brain sections were cut into 30 µm thick slices using a cryostat (Leica CM1850, Germany) and mounted onto slides.The sections were blocked for 30 minutes with a blocking solution containing 0.3% Triton X-100, 10% calf serum, and PBS, and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Drp1 (1:50, Ab184247, Abcam), mouse anti-PSD95 (1:500, MA1-045, Abcam), and rabbit anti-IBA1 (1:500, 011-27991, Wako). After three washes with PBS, the sections were incubated for 2 hours with secondary antibodies, including DyLight 647 goat anti-mouse IgG (1:5000, A23610, Abbkine) or DyLight 647 goat anti-rabbit IgG (1:5000, A23620, Abbkine). The sections were washed three times with PBS, and cell nuclei were stained with DAPI (1:2500, C1002, Biyuntian) for 5 minutes. After air drying, the slides were sealed with a fluorescent mounting medium and observed using a confocal laser scanning microscope (Leica STELLARIS5). ImageJ software was used for statistical analysis. Mitochondrial network analysis (MiNA) Mitochondrial network analysis (MiNA) uses the ImageJ software. Brain sections of PC Mito−GFP and cKO PC Mito−GFP and CoQ10 intervention mice were photographed under inverted laser scanning confocal microscopy and MiNA analysis was performed. 3 mice were taken from each group, and at least 3 slides were taken from each brain tissue to take at least 27 visual fields for statistics 25 . Transmission electron microscope analysis Three mice from each group (Control, Drp1 −/− , and CoQ10 intervention) were selected for perfusion sampling. The cerebellar tissues were placed in 2.5% glutaraldehyde at 4°C overnight and washed three times with PBS. The tissues were then fixed in 1% osmium tetroxide at 4°C in the dark for 2 hours and washed again three times with PBS. Subsequently, the samples were dehydrated through an ethanol gradient (15 minutes per step) and finally dehydrated in 100% acetone before being embedded in resin.Eleven 70-nm sections were collected onto copper grids and stained with lead nitrate and uranyl acetate for 10 minutes. Images were captured using a transmission electron microscope, and mitochondrial morphology was analyzed with ImageJ software. Mitochondria isolation Mitochondria isolation was performed according to the instruction (SM0020, Solarbio, China). Cerebellum tissues in different groups were rinsed in saline and homogenized in Tris-HCl-based lysis buffer. The homogenization was centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was transferred to a new tube and centrifuged under the same condition. The secondary supernatant was then centrifuged at 12,000 g for 10 min at 4 ℃. The pellet was resuspended in Tris-HCl-based wash buffer and centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was centrifuged at 12,000 g for another 10 min at 4 ℃. Finally, mitochondria were resuspended in phosphate-based store buffer (PH 7.2–7.4). Mitochondrial membrane potential (MMP) detection In accordance with the protocol provided by the Mitochondrial JC-1 Assay Kit (C2005, Bicentennial), the concentration of the isolated mitochondria was adjusted to a standardized level, followed by staining with the JC-1 dye. Subsequently, fluorescence intensity measurements were obtained using a fluorescence microplate reader (Spark TECAN, Switzerland) at excitation wavelengths ranging from 475 to 520 nm. Reactive oxygen species (ROS) detection According to the instructions of Mitochondrial ROS Assay Kit (S0033S, Bicentennial): cerebellar tissue was digested to make cell suspension. Staining was performed according to a 1:1000 dilution of DCFH-DA. This was followed by flow cytometry analysis. The maximum excitation wavelength in flow cytometry was 488 nm and the maximum emission wavelength was 525 nm (B75442, Beckman Coulter, USA). FlowJo 10.8.1 software was used for analysis. Western Blot Cerebellar tissue was homogenized in pre-cooled RIPA lysis buffer (P0013B, Biyuntian), and cells were lysed using a pre-cooled lysis buffer (QIAGEN). The lysates were centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. Protein concentration was measured using a BCA kit (ZJ102L, Yadase). Proteins were separated by 12.5% SDS-PAGE (PG113, Yadase) and transferred onto a 0.45 µm PVDF membrane (Millipore, USA). The membrane was blocked in a rapid protein-free blocking solution (PS108P, Yadase) for 20 minutes.The primary antibodies used included rabbit anti-DRP1 polyclonal antibody (1:1000, Ab184247, Abcam), rabbit anti-SOD1 polyclonal antibody (A0274, ABclonal), rabbit anti-GPX1 polyclonal antibody (A11166, ABclonal), rat anti-Total Oxidative phosphorylation (OXPHOS) polyclonal antibody (1:300, Ab110413, Abcam), COX4 (1:1000, PA5-29992, Invitrogen) and rabbit anti-beta actin polyclonal antibody (1:5000, AC004, ABclonal). The PVDF membrane was incubated with the primary antibodies overnight at 4°C and washed three times with TBST. Secondary antibodies included HRP-conjugated goat anti-mouse IgG (1:5000, A21010, Abbkine) and HRP-conjugated goat anti-rabbit IgG (1:5000, A21020, Abbkine). Protein bands were visualized using Fusion FX EDGE chemiluminescence imaging technology, and the gray values of the bands were quantified using ImageJ software. Network pharmacology analysis We initially performed target gene prediction by searching the keywords of "CoQ10" and "mitochondria" in five datasets, including DrugBank ( https://go.drugbank.com/drugs/ ), PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ), Swiss Target Prediction (STP), Comparative Toxicogenomics (CT) ( https://ctdbase.org/ ), and MitoCarta 3.0 ( https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways ). This process yielded 141 CoQ10-related genes and 1,140 mitochondria-related genes in mice. Subsequently, we intersected the 141 CoQ10-associated genes with the 1,140 mitochondrial genes, resulting in the identification of 30 common target genes. Protein-Protein Interaction (PPI) was performed to analyze the mitochondrial genes related to CoQ10, and arrange them according to the degree of interaction by using STRING Database ( https://cn.string-db.org/ ) and software of Cytoscape 3.9.1. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for key targets were performed utilizing the DAVID database and the Microbiotics platform. CoQ10-related mitochondrial genes were enriched for GO cellular components (GO-CC) analysis, GO biological processes (GO-BP) analysis, and GO molecular function (GO-MF) analysis. KEGG pathway enrichment analysis were visualized by Chiplot software ( https://www.chiplot.online/ ) to screen for possible involvement in the regulation of mitochondrial functions associated with neurodegenerative pathologies. Finally, the target gene prediction was conducted in the Gene Set Enrichment Analysis (GSEA) database ( https://www.gsea-msigdb.org/gsea/index.jsp ). Patch clamp electrophysiologic recording Whole-cell electrophysiological experiments were conducted using control and Drp1 −/− mice. The mice were anesthetized with pentobarbital sodium (40 mg/kg) and perfused with 20 mL of an ice-cold carbonated cutting solution (95% O₂, 5% CO₂), which contained 240 mM sucrose, 2.5 mM KCl, 1.25 mM Na₂HPO₄, 2 mM MgSO₄, 1 mM CaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose. The brain was then quickly removed and placed in the same cold, carbonated cutting solution to prepare for slicing. Sagittal brain slices (250 µm thick) were prepared using a microtome (Leica VT1200S) and incubated in the cutting solution in a holding chamber at 32°C for 30 minutes. The slices were subsequently transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 119 NaCl, 2.3 KCL, 1.0 NaH 2 PO 4 , 26 NaHCO 3 , 11 D-(+)-glucose, 1.3 MgCl 2 ·6H 2 O, and 2.5 CaCl 2 ·2H 2 O (pH 7.4; osmotic pressure 295–300 mOsm/L) and kept at room temperature for at least 1 hour.When Coenzyme Q10 was used to treat Drp1 −/− mice, it was added to the ACSF at a final concentration of 11 µM, and the slices from 3 mice were incubated under these conditions. The sections were placed in a recording chamber maintained at 24–28°C (TC-324B, Warner Instrument) with a perfusion rate of 2.0 mL/min. Whole-cell patch-clamp recordings were performed under infrared differential interference contrast (IR-DIC) visual guidance. Recording pipettes (BF150-86-7.5, Sutter Instrument, Novato, USA) were fabricated using a horizontal pipette puller (P-97, Sutter Instrument, Novato, USA) and had a tip resistance of 3–6 MΩ. The patch pipettes were filled with an intracellular solution containing (in mM): 128 potassium gluconate, 10 HEPES, 10 sodium creatine phosphate, 1.1 EGTA, 5 magnesium ATP, and 0.4 GTP sodium. The pH was adjusted to 7.3 with potassium hydroxide, and the osmotic pressure was adjusted to 300–305 mOsm using sucrose.During recordings, cells with a series resistance greater than 20 MΩ were excluded from further analysis. Neurons with a resting membrane potential more negative than − 60 mV and capable of firing action potentials were selected for subsequent experiments. Liquid junction potentials were not corrected. Currents or membrane potentials were recorded using an Axon 200A amplifier (Molecular Devices, Sunnyvale, USA). The signals were low-pass filtered at 5 kHz and digitized at 20 kHz using a Digidata 1322A system and Clampex 9.0 software (Molecular Devices). The data were stored on a computer for subsequent offline analysis 26 . Thermal proteome profiling (TPP) First, the mouse brain tissue was crushed by a tissue crusher to obtain the protein solution. Detect the concentration of the protein lysis buffer. This experiment consists of 3 groups and 3 replicates. That is, the 3 experimental groups are the DMSO control group and the 10 µM CoQ10 group respectively. 100 µM CoQ10 group. Three repetitions in each group. After high-speed centrifugation of the cell lysate, the supernatant was taken and passed through an ultrafiltration tube with PBS for three times. After determining the protein concentration by BCA, it was equally divided into 9 parts (each part had a concentration of 2mg/ml). DMSO was added to the 10 µM CoQ10 group and the 100 µM CoQ10 group respectively as mentioned above, and incubated at 4 ℃for 2 hours. Heat treatment at 58 ℃ for 7 minutes; Centrifuge at 4 ℃ for 30 minutes at high speed; Take the supernatant and transfer it to a new EP tube, then add pre-cooled acetone and precipitate the protein at -80 ℃. Next, protein profile sample pretreatment and proteomic quantitative analysis of the candidates were conducted on the 9 groups of samples. Proteomic identification and analysis were performed by Obitrap HF-X LC-MS/MS high-resolution mass spectrometry. HFX was used for mass spectrometry signal acquisition (with a 70-minute time gradient for each sample). Data analysis was conducted using maxquant analysis software. The database of mouse proteins annotated with Swiss-prot in uniprot was used for database search. The database search results show that approximately 3,100 proteins have been identified in total. Through differential analysis of the omics data, the data were divided into two priorities. The first-priority cutoff criterion is: the difference between the low-concentration drug and the DMSO group is a presence-versus-absence distinction, and simultaneously, the protein quantification value increases with rising drug concentration. The second-priority cutoff is: the low-concentration drug shows a presence-versus-absence difference compared to the DMSO group, but the protein level does not increase in the high-concentration group. Coa6 carriers Construction and protein purification From the first-priority screening data, the Coa6 protein was selected as the candidate validation target protein. First, the RNA of mouse macrophages was extracted, and cDNA was obtained through reverse transcription. Then, PCR was performed to obtain these two target genes. The gene sequences of the two proteins were constructed into the prokaryotic expression vector PET28A, namely PET28A-Coa6, through homologous recombination. After sequencing and comparison, there were no errors.The plasmids were respectively transformed into competent cells expressing strain BL21(DE3), and then the bacterial solution was transferred to a 1L conical flask. When the OD of the bacterial solution reached about 0.8, 1M of IPTG inducer was added and induced for 12 hours (at 37℃). The process was followed by bacterial collection, ultrasonic disruption and protein lysis buffer collection in sequence. Then the protein supernatant was purified by Ni-beads. The impurity proteins were eluted successively through 20mM imidazole, 50mM imidazole, and 500mM imidazole, and the target proteins were eluted. Finally, the 500mM imidazole elution solution was subjected to SDS-PAGE staining to determine the protein purity. Cell thermal shift assay (CETSA) The Coa6 protein solution was added to separate tubes containing increasing concentrations of CoQ10 (dissolved in DMSO vehicle) respectively. To each 100 µl reaction volume, CoQ10 was added at final concentrations of 0 µM (DMSO control), 0.32 µM, 1.6 µM, 8 µM, 40 µM, and 200 µM. After 1.5-hour incubation at room temperature, samples were heated at 55°C for 7 min in a water bath, followed by high-speed centrifugation (4°C, 20,000 g, 30 min). Subsequently, the supernatant was collected for further SDS-PAGE. As the drug concentration increases, the thermal stability of the target protein bound to the drug improves. The electrophoretic results show that the band intensity of the target protein increases with increasing drug concentrations, which is shown as a rise in target protein content on the electrophoresis gel. Drug affinity responsive target stability (DARTS) The Coa6 protein solution was mixed with varying concentrations of CoQ10. Specifically, 100 µl of the solution was supplemented with CoQ10 at concentrations of 0 µM (DMSO control), 1 µM, 10 µM, 100 µM, and 200 µM, respectively. After incubating for 1.5 hours at room temperature, each sample was treated with pronase E 1:100 mass ratio and incubated in a 37°C water bath for 15 minutes. Following the reaction, the samples were immediately prepared for SDS-loading by boiling, and the supernatants were subjected to SDS-PAGE. As the drug concentration increases, the target protein bound to the drug exhibits enhanced resistance to enzymatic degradation. This is evidenced electrophoretically by the progressive intensification of the target protein band with increasing drug concentrations. Surface plasmon resonance (SPR) The activator is prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection. The CM5 sensor chip is activated for 420 s with the mixture at a flow rate of 10 µl/min. Dilute Coa6 to 20 µg/ml in immobilization buffer, then injected to sample channel Fc2 at a flow rate of 10 µl/min, and typically result in immobilization levels of 12600 RU, the reference channel Fc1 does not need ligand immobilization step. The chip is deactivated by 1 M Ethanolamine hydrochloride at a flow rate of 10 µl/min for 420 s. Dilute CoQ10 with the same analyte buffer to 8 concentrations(0.39-25) µM. CoQ10 is injected to channel Fc1- Fc2 at a flow rate of 20 µl/min for an association phase of 100 s, followed by 180 s dissociation. The association and dissociation process are all handling in the analyte buffer. Repeat 8 cycles of analyte according to analyte concentrations in ascending order. After each cycle of interaction analysis, The chip need to be regenerated. Molecular docking of Coa6 with CoQ10 Relevant methods were based on those described in previous studies 27 . The structure of the Coa6-CoQ10 complex was predicted utilizing the ProtENIX web server under default parameters and subsequently visualized through the academic edition of Maestro. The three-dimensional architectures of the proteins were rendered and explored using ChimeraX. In this three-dimensional schematic diagram, orange-marked regions denote negatively charged binding sites, bluish violet regions denote positively charged binding sites, and purple arrows indicate directional hydrogen bonding with the arrow tail representing the hydrogen bond donor and the arrowhead pointing to the hydrogen bond acceptor. These three types of molecular interactions collectively constitute key binding elements at the CoQ10-Coa6 interaction interface. Statistical methods All statistical analyses were performed using GraphPad Prism 8. For comparisons, a t-test was used for parametric data, while the Mann-Whitney U test was applied for non-parametric data to assess statistical significance. For multiple comparisons, one-way ANOVA followed by Dunnett’s t test was used for parametric data, and the Kruskal-Wallis test was employed for non-parametric data. For repeated measures data, two-way ANOVA followed by Sidak test was used. The degree of linear correlation between dendritic spines and working memory was assessed using Pearson correlation analysis. A correlation coefficient of 1 indicates a perfect positive correlation, while a coefficient of -1 signifies a perfect negative correlation. A P -value of less than 0.05 suggests a statistically significant linear relationship between the variables. Results are presented as the mean ± standard deviation (SD). Western blots, fine immunofluorescence stains, and Golgi stains were analyzed using ImageJ. A p -value of less than 0.05 ( p < 0.05 ) was considered statistically significant. Results Network pharmacology analysis revealed that CoQ10 is linked to the DNM1L/Drp1 gene in cerebellar atrophy-induced cognitive impairment. We performed a Network pharmacology analysis to investigate the potential correlation between Drp1 deficiency and Coenzyme Q10 (CoQ10) supplementation. A total of 30 target genes were predicted through the intersection of a CoQ10-related gene database, which contains 141 genes, and a mitochondria-related gene database, which contains 1140 genes (Fig. 1 A). Protein-protein interact (PPI) analysis of these 30 genes revealed that Sod2, Caspase-3, Sdha, and Dnm1l (also known as Drp1) were the most significant nodes within the network (Fig. 1 B). The GO-CC analysis (Gene Ontology - cellular components) showed that this significantly enriched cellular component included the inner and outer mitochondrial membranes, as well as organelle membranes (Fig. 1 C). The GO-BP analysis (Gene Ontology - biological processes) showed that the enriched biological processes included mitochondrial respiration and apoptotic signaling (Fig. 1 D). The GO-MF analysis (Gene Ontology - molecular function) showed that the enriched molecular function included ubiquitin protein ligase binding, ubiquitin-like protein ligase binding, and BH domain binding (Fig. 1 E). KEGG analysis (Kyoto Encyclopedia of Genes and Genomes) illustrated that the enriched genes were mainly associated with neurodegenerative diseases, such as AD, PD, and HD (Fig. 1 F). A further target gene prediction showed that the DNM1L/Drp1 gene was the most important gene which was linked to all four aspects: CoQ10, cognitive impairment, cerebellar atrophy, and mitochondrial division (Fig. 1 G). The above results suggest that CoQ10 is associated with the DNM1L/Drp1 gene in cognitive impairment caused by cerebellar atrophy. Thus, CoQ10 may have therapeutic potential for DRP1-related disorders. Drp1 Deficiency in PCs causes working memory defects in mice Given the established relationship between CoQ10 and Drp1 (dynamin-related protein 1), we employed the Cre-LoxP system to generate three distinct Purkinje cell (PC)-specific Drp1 knockout mouse models. The first model represents a conventional PC-specific Drp1 knockout (Drp1 −/− mice) (Fig. 2 A). The second model incorporates PC-specific TdTomato reporter expression (cKO PC tdTomato mice), enabling fluorescent visualization of PCs (Fig. 3 G). The third model features mitochondria-targeted GFP expression specifically in PC mitochondria (cKO PCMito-GFP mice), allowing for direct observation of mitochondrial dynamics in these neurons (Fig. 4 A). Gene analysis (Fig. 2 B) indicated that mice numbered 4, 5, 6, 8, 9, 13, and 14 were Drp1 −/− mice. The Western blot results revealed a 40% decrease in Drp1 expression level (Fig. 2 C). The rotarod test results indicated impaired motor coordination in Drp1 −/− mice (Fig. 2 D). Conversely, the open-field test results confirmed the normal locomotor activity of Drp1 −/− mice at 1 month, 2 months, and 3 months of age (1M, 2M, and 3M) (Fig. 2 E, F). These findings suggest that while Drp1 −/− mice exhibit motor incoordination, their ability to walk remains unaffected. Beginning at one month of age, water maze testing revealed age-dependent progressive working memory deficits in Drp1 −/− mice compared to control mice (Fig. 2 G, I). This decline in cognitive function was not attributable to impaired motor function, as evidenced by the normal swimming speeds observed in Drp1 −/− mice across all ages (Fig. 2 H). Subsequently, working memory was further assessed using an eight-arm maze test conducted over seven consecutive days (Fig. 2 J, K). However, no significant differences were observed between control and Drp1 −/− mice at 1M or 2M in working memory errors (Fig. 2 L) or entry latency (Fig. 2 M). Notably, the proportion of 45° searches diminished to 60% and 50% in 1M and 2M Drp1 −/− mice, respectively, and the proportion of 90° searches increased significantly (Fig. 2 N). By 3 months of age, working memory deficits in Drp1 −/− mice exhibited significant exacerbation. This was characterized by: an approximate 200% increase in food search error rate; entry latency exceeding 350 seconds with increased variability; a reduction in 45° searches to only 10% of total searches; and a predominant shift towards a 270° search strategy (Fig. 2 K-N). Based on this pronounced deterioration at 3 months, subsequent investigations focused on characterizing the underlying changes in Drp1 −/− mice at this age. Drp1 deletion impaired synaptic plasticity in PCs of memory-deficient mice We compared the complexity of the PCs structure between Drp1 −/− mice and the control group using Golgi staining (Fig. 3 A). Sholl’s analysis showed that the abundance of PCs in Drp1 −/− mice, as indicated by the number of crossings, was significantly reduced by 70% at both 1M and 2M and by 90% at 3M, compared to the control group (Fig. 3 B). We conducted an in-depth analysis of dendritic spine density in the distal dendrites of PCs (Fig. 3 C). The lengths of dendrites in PCs were significantly reduced in Drp1 −/− mice at all three age points compared to the control group (Fig. 3 D). The dendritic spine densities of PCs were significantly reduced in Drp1 −/− mice at 2M and 3M compared to the control group, although no significant difference was observed between 1M Drp1 −/− mice and the control group (Fig. 3 E). Correlation analysis of dendritic spine density with working memory errors in mice showed a negative correlation (Fig. 3 F). To achieve the accurate counting of cerebellar PCs, we generated cKO PC tdTomato mice by crossing Drp1 −/− mice with B6-G/R mice. In these transgenic mice, the specific Drp1-KO PCs expressed tdTomato (Fig. 3 G). PCR analysis (Fig. 3 H) indicated that mice numbered 3, 5 were the target mice. Confocal microscopy observation of the mid-sagittal brain section demonstrated that tdTomato expression was exclusively observed in PCs (Fig. 3 I). Quantification of tdTomato-positive cells showed a 50% reduction in PCs density at both the 1M and 2M time points, and an approximately 80% reduction at 3M in cKO PC tdTomato mice (Fig. 3 J, K). Immunofluorescence staining further revealed the reduction of Drp1 expression in PCs of cKO PC tdTomato mice (Fig. 3 L, M). Postsynaptic density protein 95 (PSD95) levels in PCs of cKO PC tdTomato mice were significantly reduced at 2M and 3M compared to controls (Fig. 3 N, O), which were consistent with the changes observed in Golgi-stained dendritic spines. The above results suggest that early Drp1 deletion primarily reduces PCs density and dendritic arborization, whereas dendritic spine density is progressively impaired over time. Collectively, our results demonstrate that Drp1 deficiency drives working memory deficits through progressive compromise of PCs dendritic spine structure (Fig. 3 P). Drp1 defect in PCs severely impairs MMs stability and OXPHOS function To visualize the mitochondrial morphological changes caused by Drp1 deletion in PCs, we generated cKO PC Mito−GFP mice by crossing Drp1 −/− mice with Mito-GFP mice (Fig. 4 A). Mice 6 and 9 were identified as the target mice (Fig. 4 B). PC Mito−GFP mice served as the control group. The mitochondria targeted GFP was exclusively expressed in PCs of cKO PC Mito−GFP mice (Fig. 4 C). Mitochondrial network analysis (MiNA) revealed significantly reduced mitochondrial interconnectivity and amount in Purkinje cells (PCs) of cKO PC Mito−GFP mice compared to controls (Fig. 4 D). Specifically, there was a notable decrease in the number of individual mitochondria (Fig. 4 E), the number of mitochondrial networks (Fig. 4 F), the lengths of mitochondrial branches (Fig. 4 G), and the mean branches per mitochondrial network (Fig. 4 H). Moreover, mitochondria in PCs of Drp1 −/− mice exhibited significant swelling and severe cristae damage compared to controls (Fig. 4 I). In detail, the following were observed relative to the control group: reduced mitochondrial perimeter in 3M Drp1 −/− mice (Fig. 4 J); increased mitochondrial area at both 2M and 3M (Fig. 4 K); increased mitochondrial circularity across all three time points (Fig. 4 L); and decreased mitochondrial cristae density at both 2M and 3M (Fig. 4 M). Mitochondrial membrane potential (MMP) in the cerebellum of Drp1 −/− mice was significantly reduced at 3M (Fig. 4 N). These results demonstrate that Drp1 deficiency in PCs induces mitochondrial membranes (MMs) instability. Reactive oxygen species (ROS) levels in the cerebellum of these mice were significantly increased by 1.5-fold at 1M and 2M, and by 3-fold at 3M compared to the control (Fig. 4 O). In addition, WB results from cerebellar tissues showed that superoxide dismutase 1 (SOD1) levels were reduced by 50% at Drp1 −/− 3M (Fig. 4 P), while there was no significant difference in glutathione peroxidase 1 (GPx1) levels (Fig. 4 Q). The content of mitochondrial oxidative phosphorylation complex III-UQCRC2 (CIII) and complex V-ATP5A (CV) was significantly reduced, while no significant differences were observed in complex I-NDUFB8 (CI), complex II-SDHB (CII) and complex IV-MTCO1 (CIV) (Fig. 4 R). These results indicate that Drp1 deficiency in PCs induces severe oxidative stress and OXPHOS dysfunction. Collectively, our results demonstrate that Drp1 deficiency drives PCs damage through severe impairment of mitochondrial membrane stability and OXPHOS function (Fig. 4 S). Long-term administration of CoQ10 effectively ameliorate working memory deficits induced by Drp1 deletion To verify the therapeutic effect of CoQ10 in Drp1-deficient diseases, Drp1 −/− mice, cKO PC tdTomato mice, and cKO PC Mito−GFP mice were long-term administered CoQ10 continuously via drinking water supplementation starting at 15 days and continuing until 90 days (Fig. 5 A). CoQ10, known for its significant mitochondrial protective properties, was dissolved in a 3% DMSO solution at a concentration of 500uM. The mice in the control group were provided with drinking water containing less than 3% DMSO (Vehicle, Veh). Behavioral analyses demonstrated that CoQ10 treatment reduced both working memory errors and latency by approximately 50% (Fig. 5 B-D). Additionally, the percentage of 45° searches increased 5-fold, while the percentages of 135° and 180° searches significantly reduced (Fig. 5 E, F). Immunofluorescence staining revealed an increase in microglia in Drp1-deficient mice at 1M, 2M, and 3M compared to the control group (Fig. 5 G). WB analysis further confirmed the elevated expression of microglia in cerebellar tissue (Fig. 5 H). Following the CoQ10 intervention, the number of microglia was reduced by 60% (Fig. 5 I). These findings indicated that long-term CoQ10 supplementation ameliorates working memory deficits in mice with the specific conditional deletion of the Drp1 (Fig. 5 J). CoQ10 treatment effectively ameliorate PCs dysfunction induced by Drp1 deficiency A significant increase in both the number and complexity of PCs dendritic branches was observed following the CoQ10 intervention (Fig. 6 A, B). Further analysis of Purkinje cell dendritic spines revealed that CoQ10 increased dendritic spine density (Fig. 6 C, D); however, no statistical difference was found in dendritic branch length (Fig. 6 E). Additionally, both PSD95 expression (Fig. 6 F, G) and PCs density (Fig. 6 H, I) enhanced markedly following CoQ10 treatment. We further recorded the electrical activity of PCs using the membrane clamp technique. No significant differences were observed in access resistance (Ra) (Fig. 6 J, K) or membrane resistance (Rm) (Fig. 6 J, L), indicating that the Drp1 knockout does not disrupt the integrity of the PC membrane. However, Drp1 −/− mice showed a significant decrease in the membrane time constant (tau) and membrane capacitance (Cm), indicative of neuronal atrophy and dendritic spines loss, which was reversed by CoQ10 intervention (Fig. 6 M, N). These findings were consistent with the Golgi staining results. Additionally, in voltage-clamp mode, we observed a significantly increased current response to hyperpolarization activation in the Drp1 −/− mice group, suggesting aberrant activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This abnormality was similarly reversed in the CoQ10-treated group (Fig. 6 O, P). These results indicate that long-term CoQ10 supplementation effectively inhibited aberrant activation of HCN channels and restored the impaired plasticity of PCs caused by Drp1 deletion in PCs (Fig. 6 Q). TPP identifies key target proteins of CoQ10 in Drp1-deficient mice To identify the molecular target of CoQ10 on Drp1-induced mitochondrial disease, we conducted a small molecule drug target fishing experiment. First, we conducted TPP (thermal proteome profiling) analysis and divided the data into two priorities. The first-priority cutoff criterion is: the difference between the low-concentration drug and the DMSO group is a presence-versus-absence distinction, and simultaneously, the protein quantification value increases with rising drug concentration. The second-priority cutoff is: the low-concentration drug shows a presence-versus-absence difference compared to the DMSO group, but the protein level does not increase in the high-concentration group. Twelve candidate targets were screened out in the first priority, and 17 proteins were screened out in the second priority (Fig. 7 A). Secondly, through the analysis of the differences between the low concentration and the control group, 28 significant heat-resistant proteins were screened out. The analysis of the differences between the high concentration and the control group screened out 33 significant heat-resistant proteins (Fig. 7 B). Taking the intersection of the two groups of heat-resistant proteins, 12 common proteins were found (Fig. 7 C), namely Coa6, Mb, Slc25a46. Nde1, Rcc2, Hsdl1, Armc1, Cbx5, Rab3gap2, Rabggta, Ap3m2, and Usp47. And, in the first-priority data, the Coa6 protein was selected as the candidate validation target protein. Therefore, we created the PET28A-Coa6 protein vector and carried out protein purification (Fig. 7 D). CoQ10 rescues memory impairment in DRP1-deficient mice by binding to Coa6 To validate Coa6 as a target protein of CoQ10, we assessed Coa6’s thermal stability and protease resistance using CETSA (Cellular Thermal Shift Assay) and DARTS (Drug Affinity Responsive Target Stability) assays, respectively. The CETSA experiment indicated that the Coa6 protein had a significant anti-thermal stability phenomenon, that is, with the increase of CoQ10 concentration, the Coa6 protein had a significant retention (Fig. 7 E). The DARTS data further revealed a dose-dependent protection of Coa6 against proteolysis, with increasing CoQ10 concentrations correlating with enhanced protein stability (Fig. 7 F). To quantitatively analyze the binding affinity between CoQ10 and Coa6, we further conducted surface plasmon resonance (SPR) experiments. SPR results revealed specific binding between CoQ10 and CM5-immobilized Coa6, yielding a dissociation constant (KD) of 2.40 × 10 − 5 M (Fig. 7 G). These results demonstrate high-affinity binding between Coa6 and CoQ10. We predicted the structure of the Coa6-CoQ10 complex using ProtENIX. Molecular docking prediction indicates that CoQ10 is embedded in the protein-binding pocket of Coa6, forming a strong hydrophobic complementarity. The methoxy group of CoQ10 forms a hydrogen bond with the 18th Lysine residue (K18) of Coa6, and its extended hydrophobic tail establishes hydrophobic interactions with residues including Tryptophan at position 59 (W59), Valine at position 45 (V45), and Proline at position 35 (P35), the 36th Valine (V36), the 94th Tryptophan (W94), the 97th Tyrosine (Y97), the 98th Phenylalanine (F98), the 104th Tyrosine (Y104), and the 107th Phenylalanin Residues (F107). These findings provide strong evidence for the molecular interaction model between Coa6 and CoQ10 (Fig. 7 H). Furthermore, the WB results showed that the expression of Coa6 in Drp1 −/− mice decreased, while it significantly increased in the CoQ10 intervention group (Fig. 7 I). CoQ10 binding stabilizes Coa6, which may facilitate the assembly of mitochondrial Complex IV (CIV) within the mitochondrial inner membrane. According to prior research 28 , 29 , reduced Coa6 expression disrupts complex IV (CIV) assembly, consequently diminishing CIV activity and OXPHOS function. Further studies are needed to determine whether CoQ10 binding to Coa6 rescues mitochondrial oxidative phosphorylation function (Fig. 7 J). CoQ10 improves Drp1 deficiency-induced MMs instability and CIII-CV activity Given that Coa6 is an important subunit of mitochondrial complex IV (CIV), we further examined the effects of CoQ10 on mitochondrial morphology and respiratory function in Drp1 −/− mice. Analysis with MiNA revealed that CoQ10 intervention increased the number of individual mitochondria, mitochondrial networks, branching complexity, and branch length in PCs (Fig. 8 A, B). Electron microscopy analysis of cerebellar tissue showed that CoQ10 decreased the mitochondrial perimeter and area while increasing respiratory cristae occupancy (Fig. 8 C, D). Additionally, mitochondrial membrane potential was elevated in cerebellar tissue following CoQ10 supplement (Fig. 8 E). These results demonstrate that CoQ10 corrects Drp1 deficiency-induced mitochondria membranes (MMs) instability. Furthermore, CoQ10 intervention primarily selectively increased the expression of respiratory chain complexes III (UQCRC2) and V (ATP5A), with no significant changes in complexes I (NDUFB8), II (SDHB), and IV (MTCO1) (Fig. 8 F). We further quantified cytochrome c oxidase subunit IV (COX4) expression and found that CoQ10 significantly rescued the Drp1 deficiency-induced reduction in COX4 levels (Fig. 8 G, H). In cerebellar tissue, CoQ10 supplementation significantly elevated levels of the antioxidant enzymes SOD1 (Fig. 8 I) and GPx1 (Fig. 8 J). Quantitative ROS assays confirmed reduced reactive oxygen species in this region (Fig. 8 K). These results demonstrate that CoQ10 corrects Drp1 deficiency-induced oxidative stress and OXPHOS dysfunction. In conclusion, CoQ10 restores mitochondrial membrane stability and OXPHOS function — specifically complexes III, IV, and V — by binding to Coa6 and elevating its expression (Fig. 8 L). Additionally, the improvement in complex IV function was dependent on the Coa6 assembly factor rather than the structural MTCO1 subunit. Discussion In this study, we used three distinct mouse models with dynamin-related protein 1 (Drp1) - specific knockout in cerebellar Purkinje cells (PCs) and demonstrated the following findings (Fig. 9 ): Left side: 1) Successful generation of Drp1-specific knockout mice in PCs; 2) Reduced expression of cytochrome c oxidase assembly factor 6 (Coa6) in cerebellar mitochondria, decreased levels of mitochondrial respiratory chain complexes III, IV, and V (CIII-CV), and disruption of mitochondrial cristae structure; 3) Impaired synaptic plasticity and deficits in working memory. CoQ10 was administered via drinking water at a concentration of 500 µM for 75 days. The effects observed on the right side include: 1) Direct binding of CoQ10 to mitochondrial Coa6, resulting in increased Coa6 levels; 2) Restoration of mitochondrial respiratory chain CIII, CIV, and CV activity through elevated Coa6 levels, accompanied by recovery of mitochondrial cristae structure; 3) Improved synaptic plasticity in Purkinje cells and enhanced working memory performance. CoQ10 was first isolated and discovered from bovine heart mitochondria by Crane's team in 1957 30 . It is a relatively simple-structured vitamin-like fat-soluble molecule that is widely present in human cell membranes, especially abundant in mitochondria 31 . Its core physiological function is to act as an electron carrier in the electron transport chain, participate in the synthesis of cellular energy, and is an indispensable cofactor for maintaining life activities 32 . Studies have confirmed that CoQ10 can significantly improve the cardiac function and quality of life of patients with heart failure 33 , and is effective for patients with hypertension 34 , migraine 35 , and liver steatosis 36 also has therapeutic effects. In neurological diseases, CoQ10 has a good neuroprotective effect on diseases such as PD and AD 37 . Especially in the recovery of neurological injuries and the intervention of working memory disorders, both animal experiments and clinical studies suggest that it has significant improvement potential 38 . Furthermore, Shi et al. recently reported that CoQ10 headgroup intermediates restore CoQ10 synthesis, alleviating mitochondrial injury-induced cerebellar damage and Purkinje cell degeneration 21 . In previous studies, CoQ10 mainly treated diseases related to cognitive impairment by alleviating neuroinflammation 39 , 40 , inhibiting oxidative stress 41 , 42 and restoring abnormal activity of acetylcholinesterase 43 , 44 . Similarly, our findings demonstrate that CoQ10 can effectively alleviate working memory impairments resulting from cerebellar injury. Mechanistically, CoQ10 reduces microglial activation in the cerebellum, decreases reactive oxygen species (ROS) levels, and enhances the expression of antioxidant enzymes such as SOD1 and GPx1. Our experimental data further reveal that long-term CoQ10 supplementation in Purkinje cell-specific Drp1 knockout mice improves mitochondrial function – including enhanced membrane stability (evidenced by increased mitochondrial cristae connectivity and MMP levels) and improved OXPHOS function (verified by increased CIII, CIV, and CV activity) – delays neuronal structural degeneration, and significantly alleviates working memory deficits. Collectively, these results support the notion that long-term CoQ10 supplementation represents an effective strategy for ameliorating working memory impairments associated with cerebellar PCs injury and mitochondrial dysfunction caused by Drp1 deficiency. Therefore, coenzyme Q10 may have promising therapeutic potential in treating the working memory deficits caused by cerebellar injury. Coenzyme Q10, as a mitochondrial function regulator, demonstrates significant therapeutic potential in addressing cognitive impairments associated with neurological disorders 42 . Previous studies have indicated that CoQ10 can mitigate stress sensitivity induced by elevated Drp1 activity, thereby enhancing mitochondrial ATP production 45 . Li et al. reported that water-soluble CoQ10 has been shown to prevent mitochondrial dynamic imbalance by reducing the expression of Drp1 and Fis1 to pre-rotenone treatment levels and by attenuating rotenone-induced mitochondrial fragmentation 46 . Moreover, CoQ10 primarily alleviates structural and functional mitochondrial damage, suppresses mitochondrial fission, and promotes mitochondrial fusion through the inhibition of phosphorylation at Ser616 and Ser637 residues of Drp1 47 . In this study, we integrated protein-protein interaction (PPI), gene ontology (including cellular component—GO-CC, biological process—GO-BP, and molecular function—GO-MF), and KEGG pathway analyses to perform target gene prediction and network pharmacological analysis. The results revealed that diseases associated with the DNM1L/Drp1 gene are significantly involved in processes such as coenzyme Q10 administration, memory disorders, cerebellar dysfunction, and mitochondrial dysfunction. Moreover, our results demonstrate that Drp1 deficiency reduces UQCRC2 (a core subunit of Complex III), thereby disrupting the equilibrium of the Q cycle. This lead to an accumulation of reduced CoQ (CoQH₂) and depletion of oxidized CoQ in mitochondria. The consequent impairment of the mitochondrial electron transport chain elevates ROS levels and diminishes ATP production in PCs. We observed that CoQ10 supplementation rectified these Drp1 deficiency-induced abnormalities in ROS and ATP homeostasis. Collectively, we propose that CoQ10 alleviates pathological phenotypes in Drp1-deficient mice by restoring electron transport chain functionality compromised by Drp1 ablation. Furthermore, through target fishing experiments, we identified Coa6 as the target molecule of coenzyme Q10. TPP, CETSA, DARTS, and SPR assays qualitatively and quantitatively demonstrated the stable binding between CoQ10 and Coa6. Therefore, we propose that CoQ10 may ameliorate cerebellar injury by binding to the Coa6 protein. Previous studies on CoQ10 have focused on other molecular targets. For example, CoQ10 can attenuate LPS-induced acute lung injury (ALI) by stabilizing its binding to Drp1, thereby regulating mitochondrial dynamics, alleviating oxidative stress, and reducing NLRP3-mediated inflammation 47 . As a key electron carrier in the mitochondrial electron transport chain, CoQ10 supports ATP synthesis and mitigates energy metabolism disorders in neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease 48 . Additionally, CoQ10 has been found to regulate key pathways, including PI3K/Akt, GSK-3β, CREB, and BDNF, thereby influencing cell survival and synaptic plasticity, with potential benefits for cognitive impairment 49 . Some studies also suggest that CoQ10 deficiency may disrupt cholesterol homeostasis, impairing the structure and function of neuronal membranes 50 . In contrast to previous findings, our study revealed—through molecular docking experiments—that CoQ10 exerts its effects in the cerebellum by binding to the mitochondrial cytochrome c oxidase assembly factor Coa6. Coa6 is a mitochondrial intermembrane space protein primarily involved in the assembly of CIV of the respiratory chain 51 . The loss of Coa6 can lead to the combined deficiency of CI and CIV 29 . Coa6 also influences tumor progression in breast and lung cancer by modulating mitochondrial oxidative phosphorylation 52 , 53 . In addition, our study showed that CoQ10 increased the levels of CIII and CV in the cerebellar mitochondrial respiratory chain. Therefore, we hypothesize that CoQ10 may improve Drp1-deficient working memory impairment by enhancing the activity of CIII, CIV, and CV via its interaction with Coa6. This suggests that the CoQ10–Coa6–ETC axis may represent a potential therapeutic target for ameliorating working memory deficits in Drp1 deficiency. In our Drp1-deficient model, molecular docking illustrated that CoQ10 forms a stable complex with Coa6 through single hydrogen bonds and multiple hydrophobic interactions. Previous studies indicate that when CoQ10 binds to proteins, hydrogen bonds are usually involved in conjunction with hydrophobic interactions, enhancing the stability and specificity of the complexes 54 , 55 , which aligns with our results. Hydrogen bonding is known to play a critical role in protein stability, improving ligand binding specificity and affinity 56 . Notably, the methoxy group of CoQ10 formed hydrogen bonds exclusively with the 18th lysine residue (K18) of Coa6, suggesting a highly specific interaction. Moreover, cerebellar Coa6 expression levels significantly increased following CoQ10 supplementation. This may result from CoQ10 promoting Coa6 biogenesis or stabilizing Coa6 by inhibiting its degradation. These effects could enhance mitochondrial respiratory chain CIV activity (evidenced by increased COX4 level), reduce ROS production, and restore neuronal energy metabolism. Although previous studies demonstrated that CoQ10 elevates CI, CII, and CIV levels 39 , recovers decreased CIV activity and CIV-OPA1 binding in aged mice 57 , enhances CI/CII/CIII activity in Parkinson’s disease 58 , and modulates CII/CIII in Huntington’s disease 59 , the precise mechanisms underlying CoQ10’s enhancement of CI, CII, CIII, and CV functions remain incompletely understood. Based on our findings, we propose two potential mechanisms: (1) CoQ10 enhances the stability of Coa6 (a CIV assembly factor), which may subsequently promote the function or stability of CIII and CV through indirect effects on overall respiratory chain integrity or supercomplex formation; or (2) Improved CIV activity enhanced by CoQ10 subsequently promotes increased CIII and CV activities. Crucially, using a cerebellar injury model, our study systematically demonstrates that CoQ10 can simultaneously enhance the functions of complexes III, IV, and V. Considering that the Drp1-deficient mice used in our experiments have ataxia 60 , 61 , which may interfere with the results of the water maze, we used the eight-arm maze test to explore working memory impairment in mice. The eight-arm maze is a classic behavioral tool for evaluating working memory and spatial learning ability, which is widely used in neuroscience and pharmacology research. Its design allows simultaneous measurement of working memory and reference memory, with high sensitivity and repeatability, and is considered one of the reliable methods for studying working memory 62 , 63 . Our findings indicate that Drp1 −/− mice at 1 month of age learn slower, with persistently higher latency and 45° search times reduced to 60% despite gradual improvement, but no significant memory errors were observed. At 2 months, Drp1 −/− mice still showed no significant memory loss, but 45° search frequency further decreased to 50%. After 3 months, Drp1 −/− mice showed marked deterioration in working memory, with error rates increased to 200%, unstable latency exceeding 350 seconds, and 45° search patterns reduced to only 10%. When CoQ10 was administered at postnatal day 15, Drp1 −/− mice showed significant improvement in working memory deficits compared to controls. The above results demonstrated that early alleviation of Drp1-deficient mitochondrial morphological dysfunction could rescue PCs function and working memory impairment. Previous studies reported that Drp1 plays a key role in PC and cerebellar development, as its knockdown results in a 40% reduction in cerebellar volume and mitochondrial abnormalities 23 . Our previous study showed that although mitochondria transplantation in juvenile mice ameliorated motor dysfunction caused by Drp1 deficiency, Drp1 upregulation in PCs at 1 month of age or mitochondria transplantation in adult mice failed to improve ataxia 61 . Consistent with previous studies, our findings highlight an important role for Drp1 in PC development. Another contribution is the identification of HCN channels as a potential target for CoQ10 supplementation therapy. Abnormal activation of HCN channels underlies impairments in higher brain functions, influencing neuronal oscillations, synaptic transmission, and cognition 64 – 66 . HCN hyperactivation is also linked to AD and depression-related cognitive deficits 67 , 68 . HCN inhibitors have been shown to alleviate symptoms of depression 69 and demonstrate potential efficacy in the treatment of neuropathic pain and seizures 70 . Harde E's team has discovered that selective HCN1 inhibitors enhance working memory in rats 71 . Currently, ivabradine is the sole FDA-approved HCN channel inhibitor indicated for the treatment of angina 72 . Ivabradine has been shown to mitigate ROS accumulation and enhance ATP generation in cardiomyocytes 73 , which is crucial for maintaining mitochondrial function 16 , 74 . However, to date, no HCN-targeting drugs have been developed for the treatment of neurological disorders, including cerebellar degeneration diseases resulting from PCs impairment. Our findings for the first time indicate that long-term CoQ10 supplementation exhibits cardiovascular and mitochondrial protective effects comparable to those of ivabradine, modulates HCN channels, and enhances working memory performance. Our research has certain limitations. Firstly, we mainly constructed three transgenic mice, namely D rp1 −/− , cKO PC tdTomato , and cKO PC Mito−GFP , for research. We used molecular docking technology to screen for the direct binding and effect of CoQ10 on Coa6 and verified it from molecular biology techniques. However, the downregulation of Coa6 in transgenic mice for re-verification was not in-depth enough. Secondly, we used the model of Drp1-deficient mice and discovered mitochondrial damage and synaptic plasticity damage in PCs through a large number of morphological studies. We took Drp1-deficient mice as the model for studying working memory damage and found that CoQ10 targeted Coa6 to play a role. However, whether there is a direct relationship between Drp1 and Coa6 has not been explored. Abbreviations 1M/ 2M / 3M one-month-old / two-month-old / three-month-old AD Alzheimer's disease CETSA Cellular thermal shift assay Cm membrane capacitance CoQ10 Coenzyme Q10 Complex I/II/III/IV/V CI-CV COX4 Cytochrome c oxidase 4 CT Comparative Toxicogenomics database DARTS Drug affinity responsive target stability Drp1 / DNM1L dynamin-related protein 1 DMSO dimethyl sulfoxide FDA US Food and Drug Administration GO Gene Ontology GO-BP GO biological processes GO-CC GO cellular components GO-MF GO molecular function GPx1 glutathione peroxidase 1 GSEA Gene Set Enrichment Analysis database HCN hyperpolarization activated cyclic nucleotide gated channels HD Huntington's disease Ih inward hyperpolarization-activated current KEGG Kyoto Encyclopedia of Genes and Genomes KO knock-out MiNA mitochondrial network analysis MMP mitochondrial membrane potential ns no significance OXPHOS oxidative phosphorylation PD Parkinson's disease PKA Protein Kinase A PPI Protein-Protein Interaction Purkinje cells PCs Ra membrane resistance Rm membrane resistance ROS reactive oxygen species SOD1 superoxide dismutase SPR Surface plasmon resonance STP Swiss Target Prediction database tau membrane time constant Declarations Ethics approval and consent to participate All animal testing protocols were approved by Fourth Military Medical University’s Animal Care and Use Committee (IACUC-20190107) and were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals. Consent for publication Not applicable. Availability of data and materials The datasets during and analysed during the current study available from the corresponding author on reasonable request. Acknowledgments We would like to express our sincere thanks to all the personnel at the Teaching Laboratory Center of the Air Force Medical University, and the Neurobiology Laboratory for their support. Funding This work was supported by the National Natural Science Foundation of China (82201627) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2022JQ820) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2024JCZDXM60) by Yanling Yang, the New Clinical Technology of Xi-Jing Hospital (2023XJSY27) by Yanling Yang, Military Medicine Promotion Program of Air Force Military Medical University (2020SWAQ04) by Yayun Wang and Shaanxi Provincial Innovation Capability Support Program (2023CXPT33) by Yayun Wang. Author contributions Conceptualization: YY Wang, YL Yang Methodology: JJ TIE, SJ LI Funding acquisition: FF WU, YY Wang, YL Yang Supervision: YY Wang Writing – original draft: JJ TIE, SJ LI, X HUANG, ZW NI, CL ZHU Writing – review & editing: KK REN, XD LI, H LIU Competing interests Authors declare that they have no competing interests. References Global. regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet Neurol. 2024;23(4):344–81. Wilhelm M, Sych Y, Fomins A, Alatorre Warren JL, Lewis C, Serratosa Capdevila L, et al. Striatum-projecting prefrontal cortex neurons support working memory maintenance. Nat Commun. 2023;14(1):7016. Goldman JG, Vernaleo BA, Camicioli R, Dahodwala N, Dobkin RD, Ellis T et al. Cognitive impairment in Parkinson's disease: a report from a multidisciplinary symposium on unmet needs and future directions to maintain cognitive health. 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Gutierrez-Mariscal FM, Arenas-de Larriva AP, Limia-Perez L, Romero-Cabrera JL, Yubero-Serrano EM, López-Miranda J. Coenzyme Q(10) Supplementation for the Reduction of Oxidative Stress: Clinical Implications in the Treatment of Chronic Diseases. Int J Mol Sci 2020; 21(21). Supplementary Files Coverletter.docx SupplementaryTable1.xlsx Cite Share Download PDF Status: Published Journal Publication published 27 Apr, 2026 Read the published version in Translational Neurodegeneration → Version 1 posted Reviewers agreed at journal 20 Sep, 2025 Reviewers invited by journal 18 Sep, 2025 Editor invited by journal 17 Aug, 2025 Editor assigned by journal 14 Aug, 2025 First submitted to journal 13 Aug, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":7987236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoQ10 is associated with the DNM1L gene in cognitive impairment caused by cerebellar atrophy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A - G) \u003c/strong\u003eNetwork pharmacology analysis composed of target gene prediction \u003cstrong\u003e(A)\u003c/strong\u003e, PPI \u003cstrong\u003e(B)\u003c/strong\u003e, GO-CC \u003cstrong\u003e(C)\u003c/strong\u003e, GO-BP \u003cstrong\u003e(D)\u003c/strong\u003e, GO-MF \u003cstrong\u003e(E)\u003c/strong\u003e, KEGG \u003cstrong\u003e(F) \u003c/strong\u003eand target gene prediction in GSEA database \u003cstrong\u003e(G)\u003c/strong\u003e. Network pharmacology analysis elucidated the correlation between Drp1 deficiency and CoQ10 supplementation.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/328c9b6ad85b884aeac88b14.png"},{"id":92389236,"identity":"8defb931-f247-4648-b295-f37f8dce9a6d","added_by":"auto","created_at":"2025-09-29 08:20:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20061037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrp1 Deficiency in PCs causes working memory defects in mice.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eConstruction strategy \u003cstrong\u003e(A)\u003c/strong\u003e and gene test \u003cstrong\u003e(B)\u003c/strong\u003e of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Western Blot results and statistical analysis of Drp1 expression level, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Rotarod test results, n = 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Open-field results for one-month-old (1M), two-month-old (2M), and three-month-old (3M) \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, with 2M Pcp2\u003csup\u003eCre\u003c/sup\u003e mice serving as the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Quantitative analysis of total movement distance, n = 5 or 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Traces plot detected by water maze test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Quantitative analysis of swimming speed, n = 5 or 6 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Quantitative analysis of latency period, n = 5 or 6 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eDetection strategy for eight-arm maze test in 1M / 2M / 3M \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, with Pcp2\u003csup\u003eCre\u003c/sup\u003e mice serving as the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K\u003c/strong\u003e) Traces plot detected by eight-arm maze test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eQuantitative analysis of working memory errors, n = 5 or 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M)\u003c/strong\u003e Quantitative analysis of latent period, n = 5 or 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N)\u003c/strong\u003e Quantitative analysis of percentage of different strategy angles, n = 5 or 6.\u0026nbsp; ***\u003cem\u003eP\u0026lt;0.0005\u003c/em\u003e, ****\u003cem\u003eP \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test (C and D), one-way ANOVA (F and H) or two-way ANOVA (I and L-N).\u003c/p\u003e\n\u003cp\u003eNs, no significance.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/17675bf97f9d84d8f976947e.png"},{"id":92389244,"identity":"547014dd-3187-4b63-883a-1328bac04e4e","added_by":"auto","created_at":"2025-09-29 08:20:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54457075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrp1 deletion impaired synaptic plasticity in PCs of memory-deficient mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eGolgi staining images of PCs abundance of 1M / 2M / 3M \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, with 2M Pcp2\u003csup\u003eCre\u003c/sup\u003e mice serving as the control group. Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Quantitative analysis results from \u003cstrong\u003eA\u003c/strong\u003e, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C\u003c/strong\u003e) Golgi staining images of PCs dendrities abundance in different groups. Bar = 1 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eQuantitative analysis of distal branch length of PCs, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Quantitative analysis of dendrite spine density of PCs, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Pearson correlation analysis between dendrite spine density and working memory error on day 7 of eight-arm maze.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G, H) \u003c/strong\u003eConstruction strategy \u003cstrong\u003e(G)\u003c/strong\u003e and gene test \u003cstrong\u003e(H)\u003c/strong\u003e of cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mouse.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eConfocal microscopy image of mid-sagittal brain section of cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice. Bar = 1 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e Confocal microscopy images of tdTomato-positive cells (PCs) in 1M / 2M / 3M cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice, with 2M PC\u003csup\u003etdTomato\u003c/sup\u003e mice serving as the control group. Bar = 1 mm (up) and 300 mm (down).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e Quantitative results of PCs density, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L)\u003c/strong\u003e Immunofluorescence images of Drp1 expression in 2M cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice and PC\u003csup\u003etdTomato\u003c/sup\u003e mice. Bar = 50 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M) \u003c/strong\u003eQuantitative analysis of the proportion of cells co-expressing Drp1 and tdTomato relative to the total population of tdTomato-positive cells, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N) \u003c/strong\u003eImmunofluorescence images of PSD95 expression in 1M / 2M / 3M cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice, with 2M PC\u003csup\u003etdTomato\u003c/sup\u003e mice serving as the control group. Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O)\u003c/strong\u003e Quantitative analysis of the intensity of cells co-expressing PSD95 and tdTomato relative to the total population of tdTomato-positive cells, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(P) \u003c/strong\u003eResult schematic diagram.\u003c/p\u003e\n\u003cp\u003eThe data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by one-way ANOVA (B, D, K, and O), Kruskal-Wallis test (E) or unpaired two-tailed\u003cem\u003e t\u003c/em\u003e-test (M).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/f43fb5acde92795d925e4c6b.png"},{"id":92389016,"identity":"52ce8f30-6b63-495c-8fcb-ae458df61114","added_by":"auto","created_at":"2025-09-29 08:12:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30730099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrp1 defect in PCs severely impairs MMs stability and OXPHOS function.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eConstruction strategy of cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003ePCR results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eConfocal microscopy image of mid-sagittal brain section of cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e mice. Bar = 1 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eConfocal microscopy images of GFP-positive cells (PCs) in 1M / 2M / 3M cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e mice, with 2M PC\u003csup\u003eMito-GFP\u003c/sup\u003e serving as the control group. Additionally, images of these groups following MiNA treatment are provided. Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eQuantitative analysis of individual mitochondrial numbers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eQuantitative analysis of mitochondrial network.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Quantitative analysis of mitochondrial network branch length.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Quantitative analysis of mean branch number of the mitochondrial network. n = 3 (E, F, G, H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I\u003c/strong\u003e) Mitochondrial ultrastructure images under transmission electron microscopy in all groups. Bar = 0.5 mm (up) and 0.2 mm (down).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J - K) \u003c/strong\u003eQuantitative analysis of mitochondrial perimeter and area, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L)\u003c/strong\u003e Quantitative analysis of mitochondrial circularity, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M)\u003c/strong\u003e Quantitative analysis of mitochondrial cristae abundance per mitochondrial area, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N)\u003c/strong\u003e Quantitative analysis of MMP results by JC-1 detection of the purified mitochondria from the cerebellar cortex in 1M / 2M / 3M cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e mice, with 2M PC\u003csup\u003eMito-GFP\u003c/sup\u003e serving as the control group, n = 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O) \u003c/strong\u003eROS results and quantitative analysis by DCFH-DA detection of the proteins isolated from the cerebellar cortex in 1M / 2M / 3M cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e mice, with 2M PC\u003csup\u003eMito-GFP\u003c/sup\u003e serving as the control group, n = 4 or 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(P - R) \u003c/strong\u003eWestern Blot analysis of the expression levels of SOD1 \u003cstrong\u003e(P)\u003c/strong\u003e, GPx1 \u003cstrong\u003e(Q)\u003c/strong\u003e, and mitochondrial OXPHOS complexes I, II, III, IV, and V \u003cstrong\u003e(R)\u003c/strong\u003e, using proteins isolated from the cerebellar cortex in 2M \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice and 3M Pcp2\u003csup\u003eCre\u003c/sup\u003e control mice, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(S) \u003c/strong\u003eResult schematic diagram.\u003c/p\u003e\n\u003cp\u003eThe data were presented as means ± SD. P were calculated by the Kruskal - Wallis test (E-H and J-M), one-way ANOVA (N and O), and unpaired two-tailed t-test (P-R).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/ef1942fdf4bb6eedd16bae40.png"},{"id":92389052,"identity":"58089902-80cd-4a06-bc5b-b7d020a9a8e7","added_by":"auto","created_at":"2025-09-29 08:12:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29941388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term administration of CoQ10 effectively ameliorates working memory deficits and cerebellar inflammation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were administered by drinking water with long - term CoQ10 supplementationand (\u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10) or with vehicle (\u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh) from postnatal day 15 to day 90. At postnatal day 91, the mice were tested for further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Trace plot tested by eight-arm maze tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Quantitative analysis of latent period in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 group and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh groups, n = 5 or 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Quantitative analysis of working memory errors in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 group and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh groups, n = 5 or 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E, F) \u003c/strong\u003eQuantitative analysis of food searching strategies in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 group and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh groups, n = 5 or 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eImmunofluorescence images and analysis of Iba1 (microglia marker) expression in 1M / 2M / 3M cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice, with 2M PC\u003csup\u003etdTomato\u003c/sup\u003e mice serving as the control group. Bar = 100 mm, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eWestern Blot analysis of the expression level of Iba1 in 3M \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice and 3M Pcp2\u003csup\u003eCre\u003c/sup\u003e control mice, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eImmunofluorescence images and quantitative analysis of Iba1 (green) expression in PCs (red) of cKO PC\u003csup\u003etdTomato\u003c/sup\u003e + CoQ10 group and cKO PC\u003csup\u003etdTomato\u003c/sup\u003e + Veh group. Bar = 100 mm, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eResult schematic diagram.\u003c/p\u003e\n\u003cp\u003eThe data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by two-way ANOVA (C-F), Kruskal-Wallis test (G), and unpaired two-tailed t-test (H and I).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/7d8387623b730406bbea766b.png"},{"id":92389032,"identity":"74133c36-0400-491e-a09a-d2dc89acd245","added_by":"auto","created_at":"2025-09-29 08:12:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52353566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRestoration of impaired PCs plasticity induced by Drp1 deletion via CoQ10 treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A\u003c/strong\u003e) Golgi staining images of PCs in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e with long - term CoQ10 supplementation and (\u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003e+ CoQ10) or with vehicle (\u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh). Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Quantitative analysis of the complexity of PCs, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Golgi staining images of PCs dendrites abundance in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 group and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh groups. Bar = 50 mm (up) and 20 mm (down).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D, E)\u003c/strong\u003e Quantitative analysis of density of PCs dendritic spines \u003cstrong\u003e(D) \u003c/strong\u003eand dendritic branch length \u003cstrong\u003e(E)\u003c/strong\u003e, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F\u003c/strong\u003e) Immunofluorescence images and quantitative analysis of PSD95 expression in cKO PC\u003csup\u003etdTomato\u003c/sup\u003e + CoQ10 and cKO PC\u003csup\u003etdTomato\u003c/sup\u003e + Veh groups. Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e \u0026nbsp;of the intensity of cells co-expressing PSD95 and tdTomato relative to the total population of tdTomato-positive cells, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Confocal microscopy images of tdTomato-positive cells (PCs) in cKO PC\u003csup\u003etdTomato\u003c/sup\u003e + CoQ10 group and cKO PC\u003csup\u003etdTomato\u003c/sup\u003e + Veh group, Bar = 50 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e \u0026nbsp;Quantitative results of PCs density, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e Patch clamp electrophysiologic recording of PCs of 2M Pcp2\u003csup\u003eCre\u003c/sup\u003e mice (control), \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh mice, and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K-N)\u003c/strong\u003e Quantitative analysis of Ra \u003cstrong\u003e(K),\u003c/strong\u003e Rm \u003cstrong\u003e(L)\u003c/strong\u003e, tau \u003cstrong\u003e(M)\u003c/strong\u003e, and Cm \u003cstrong\u003e(N)\u003c/strong\u003e, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O\u003c/strong\u003e) Patch clamp electrophysiologic recording at voltage-clamp mode with the holding potential of - 65 mV of PCs of 2M Pcp2\u003csup\u003eCre\u003c/sup\u003e mice (control), \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh mice, and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(P)\u003c/strong\u003e Quantitative analysis of Ih, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Q) \u003c/strong\u003eResult schematic diagram.\u003c/p\u003e\n\u003cp\u003eThe data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed t-test (B and H), Kruskal-Wallis test (D and E), and one-way ANOVA (K-N and P).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/7f2eb3c42372b4a5b68728a5.png"},{"id":92389235,"identity":"e7407144-ff2d-412a-86c4-8c0e67e260b3","added_by":"auto","created_at":"2025-09-29 08:20:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10207928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoQ10 rescues memory impairment in DRP1-deficient mice by binding to Coa6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eProteomic identification and analysis were performed by Obitrap HF-X LC-MS/MS high-resolution mass spectrometry. HFX was used for mass spectrometry signal acquisition (with a 70-minute time gradient for each sample), n=3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Difference analysis between the 10 μM and 100 μM CoQ10 control groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The intersection of the two groups of heat-resistant proteins was analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003ePet28a-coa6 was constructed into the prokaryotic expression vector PET28A by homologous recombination method and was correct after sequencing and comparison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Qualitative analysis of Coa6 content by CETSA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Qualitative analysis of Coa6 content by DARTS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Quantitative analysis of Coa6 content by SPR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e The structure of the Coa6-CoQ10 complex was predicted using ProtENIX. Blue represents Coa6 and purple represents CoQ10. The red doted box showed that the lysine residue at the 18th position forms a hydrogen bond. And its extended hydrophobic tail is associated with Tryptophan at position 59 (W59), Valine at position 45 (V45), and Proline at position 35 (P35), the 36th Valine (V36), the 94th Tryptophan (W94), the 97th Tyrosine (Y97), the 98th Phenylalanine (F98), the 104th Tyrosine (Y104), the 107th Phenylalanin Residues (F107) such as established hydrophobic interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Western Blot analysis of the expression levels of Coa6, using proteins isolated from the cerebellar cortex of Control, \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 mice and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e+ Veh mice, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eResult schematic diagram.\u003c/p\u003e\n\u003cp\u003eThe data were presented as means ± SD. P were calculated by one-way ANOVA (H).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/56546c37e8e884f7ce3c6c79.png"},{"id":92389031,"identity":"5481e0ac-2de4-4333-b3ca-e8b89bcfa658","added_by":"auto","created_at":"2025-09-29 08:12:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":23026792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoQ10 improves Drp1 deficiency-induced MMs instability and ETC dysfunction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A\u003c/strong\u003e) Confocal microscopy images of GFP-positive cells (PCs) in cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e with long-term CoQ10 supplementation and (cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e\u003csup\u003e\u003cem\u003e \u003c/em\u003e\u003c/sup\u003e+ CoQ10) or with vehicle (cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e + Veh). Additionally, images of these groups following MiNA treatment are provided. Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eQuantitative analysis of the individual mitochondrial number, mitochondrial network, mitochondrial network branch length, and mean branch quantity of mitochondrial network in cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e\u003csup\u003e\u003cem\u003e \u003c/em\u003e\u003c/sup\u003e+ CoQ10 group and cKO PC\u003csup\u003eMito-GFP\u003c/sup\u003e + Veh group, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Mitochondrial ultrastructure images under transmission electron microscopy in the \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 group and the \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh group. Bar = 0.5 mm (up) and 0.2 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Quantitative analysis of mitochondrial perimeter, area, circularity, and cristae abundance, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E\u003c/strong\u003e) Quantitative analysis of MMP results by JC-1 detection of the purified mitochondria from the cerebellar cortex of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 mice and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh mice, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Western Blot analysis of the expression levels of mitochondrial OXPHOS complexes I (NDUFB8), II (SDHB), III (UQCRC2), IV (MTCO1), and V (ATP5A), using proteins isolated from the cerebellar cortex of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 mice and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh mice, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G-J)\u003c/strong\u003e Western Blot analysis of the expression levels of COX4\u003cstrong\u003e (G, H)\u003c/strong\u003e, SOD1\u003cstrong\u003e (I)\u003c/strong\u003e and GPx1\u003cstrong\u003e (J)\u003c/strong\u003e in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 mice and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh mice, n = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e ROS results and quantitative analysis by DCFH-DA detection of the proteins isolated from the cerebellar cortex of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + CoQ10 mice and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + Veh mice, n = 4 or 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eResult schematic diagram.\u003c/p\u003e\n\u003cp\u003eThe data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed t-test (B, D-F, and H-K).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/2846d2d07add6a54d3845c57.png"},{"id":92389239,"identity":"0dd08559-126b-4b7d-92a7-ae820e788475","added_by":"auto","created_at":"2025-09-29 08:20:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":16893038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScientific hypothesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeficiency of Drp1 in cerebellar PCs disrupts mitochondrial OXPHOS (including complexes III, IV, and V) and destabilizes the mitochondrial membrane. Reduced Coa6 expression further exacerbates complex IV impairment. These mitochondrial impairments culminate in a reduction of PC numbers, morphological abnormalities, and working memory deficits in mice. CoQ10 directly binds to Coa6 and elevates its expression, restoring CIII/CIV/CV and mitochondrial membrane stability. Consequently, CoQ10 rescues the loss of dendritic spines in PCs and ameliorates working memory deficits in mice.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/144c15a32a4f58366084ba46.png"},{"id":92389010,"identity":"6037d257-151f-4744-b39e-bd934c910559","added_by":"auto","created_at":"2025-09-29 08:12:12","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":13389,"visible":true,"origin":"","legend":"","description":"","filename":"Coverletter.docx","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/44858c43fe916b614a164d39.docx"},{"id":92389233,"identity":"e7d41a00-e26d-4b35-a823-5ff980c21b67","added_by":"auto","created_at":"2025-09-29 08:20:12","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":10443,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7362769/v1/19455b2b9d50def7d47f39df.xlsx"}],"financialInterests":"","formattedTitle":"The Drp1-CoQ10-Coa6-ETC axis represents a therapeutic target for working memory impairment caused by neuronal mitochondrial dysfunction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeurological diseases have become increasingly prominent in the global disease burden \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Working memory refers to the temporary retention and manipulation of relevant information from memory, which guides future actions \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Impairment or even loss of working memory is a common symptom of many neurodegenerative diseases, including AD Parkinson's disease (PD), and Huntington's disease (HD) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Previous studies on working memory mainly focused on the cerebral cortex and hippocampus, while ignoring the role of the cerebellum in it \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, accumulating evidence has begun to redefine the cerebellum as a critical node in cognitive networks \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In particular, cerebellar Purkinje cells (PCs) play a key role in regulating working memory, attention, and executive function through their interactions with the prefrontal\u0026ndash;cerebellar\u0026ndash;thalamic circuitry \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Current pharmacological strategies to improve working memory involve medications such as methylphenidate, nimodipine, and lamotrigine \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, most drugs exhibit complex side effects or prove to be ineffective \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Consequently, it is imperative to identify novel therapeutic agents that can effectively mitigate working memory deficits caused by cerebellar injury.\u003c/p\u003e\u003cp\u003eCo-enzyme Q10 (CoQ10) is a critical antioxidant and an essential component of the mitochondrial electron transport chain \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Although CoQ10 is not approved by the US Food and Drug Administration (FDA) as a drug, it is marketed as a dietary supplement and is currently one of the most consumed nutritional supplements \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. CoQ10 was considered a promising candidate therapeutic agent for the treatment of various diseases, including cardiovascular disease, neurodegenerative disorders, cancer, and diabetes \u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Long-term administration of CoQ10 was shown to effectively alleviate memory function impairment caused by Alzheimer\u0026rsquo;s disease (AD) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The CoQ10 headgroup intermediates can restore CoQ10 synthesis in the body, thus alleviating the mitochondrial encephalopathy, including cerebellum damage \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the molecular targets through which CoQ10 exerts its effects on neurons remain unclear. Therefore, whether CoQ10 can effectively alleviate working memory impairment caused by cerebellar injury and the underlying mechanisms need to be investigated.\u003c/p\u003e\u003cp\u003eHere, network pharmacology analysis identified DNM1L/Drp1 as a key genetic target of CoQ10 in cerebellar injury-related memory impairment. We utilized the Cre-LoxP system to generate three distinct PCs-specific Drp1 (Dynamin-related protein 1) knockout mouse models: \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (conventional knockout), cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice (enabling specific TdTomato expression in PCs), and cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice (enabling specific GFP expression on the outer mitochondrial membrane of PCs). The Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited progressive working memory deficits, as demonstrated by eight arm maze and morris water maze tests, along with impaired PC plasticity assessed through Golgi staining and immunofluorescence. Mitochondrial membranes (MMs) instability was confirmed by electron microscopy and mitochondrial membrane potential measurements, while oxidative phosphorylation (OXPHOS) dysfunction - particularly in complexes III-V (CIII-CV) - was evidenced by Western blotting, reactive oxygen species (ROS) levels, and ATP production assays. Following 75-day CoQ10 long-term supplementation in drinking water initiated at postnatal day 15, behavioral tests revealed significant amelioration of working memory impairments in Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Electrophysiological and morphological analyses demonstrated that CoQ10 effectively suppressed aberrant HCN channel activation and restored PC plasticity after injury. Ultrastructural examination by electron microscopy and functional mitochondrial assays showed that CoQ10 treatment reversed Drp1 deficiency-induced MMs instability and OXPHOS dysfunction. Mechanistically, thermal proteome profiling (TPP) identified 12 potential target proteins of CoQ10 in Drp1-deficient mice. Moreover, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and surface plasmon resonance (SPR) confirmed stable binding between CoQ10 and cytochrome c oxidase assembly factor 6 (Coa6). Molecular docking predicted CoQ10 binds within the hydrophobic pocket of Coa6 with strong hydrophobic complementarity. Our findings demonstrate that the CoQ10-Coa6 interaction enhances MMs stability, reduces oxidative stress, and restores CIII-CV activity, establishing the Drp1-CoQ10-Coa6-electron transport chain (ETC) axis as a potential therapeutic mechanism for cerebellar injury-induced cognitive dysfunction.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eWe purchased four types of transgenic mice tool: 1) Pcp2\u003csup\u003eCre\u003c/sup\u003e mice, which were designed by specifically inserting Cre enzyme into all Purkinje cells (PCs), with stock code of 004146 of Jackson Laboratories (US) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e; 2) Drp1\u003csup\u003eflox\u003c/sup\u003e mice, which were designed by inserting loxP sites flanking the Drp1 sequence, with the serial number of CKOAIS191230RT5 of Cyagen (China) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e; 3) B6/JGpt-H11\u003csup\u003eem1Cin (CAG\u0026minus;LoxP\u0026minus;ZsGreen\u0026minus;Stop\u0026minus;LoxP\u0026minus;tdTomato)\u003c/sup\u003e/Gpt mice (B6-G/R mice), which were designed by inserting loxP sites flanking the ZsGreen sequence, with the serial number of T006163 of GemPharmatech (China); and 4) RoSA26-CAG-LSL-GFP-MITO-labeled mice (Mito-GFP mice), which were designed by inserting loxP sites flanking the green fluorescent protein (GFP) specifically located on the mitochondrial outer membrane, with a serial number of cas9-ki (Rosa26) of GemPharmatech. All mice were housed (up to five per cage) under a 12-hour light-dark cycle (8 a.m. \u0026minus;\u0026thinsp;8 p.m.) with ad libitum access to food and water.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGeneration of\u003c/b\u003e \u003cb\u003eDrp1\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice, PC\u003c/b\u003e\u003csup\u003e\u003cb\u003etdTomato\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice, cKO PC\u003c/b\u003e\u003csup\u003e\u003cb\u003etdTomato\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice, PC\u003c/b\u003e\u003csup\u003e\u003cb\u003eMito\u0026minus;GFP\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice, and cKO PC\u003c/b\u003e\u003csup\u003e\u003cb\u003eMito\u0026minus;GFP\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe generated five types conditional knock-out mice (cKO mice), including: 1) \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, generated by crossing Pcp2\u003csup\u003eCre\u003c/sup\u003e mice with Drp1\u003csup\u003eflox\u003c/sup\u003e mice, in which Drp1 gene was specifically KO in cerebellar PCs; 2) PC\u003csup\u003etdTomato\u003c/sup\u003e mice, generated by crossing Pcp2\u003csup\u003eCre\u003c/sup\u003e mice with B6-G/R mic, in which Pcp2-positive PCs carry tdTomato; 3) cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice, generated by crossing Drp1\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice with B6-G/R mice, in which the specific Drp1-KO PCs expressed tdTomato; 4) PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice, generated by crossing Pcp2\u003csup\u003eCre\u003c/sup\u003e mice with Mito-GFP mice, in which Pcp2-positive PCs carry GFP; and 5) cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice, generated by crossing Drp1\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice with Mito-GFP mice, in which mitochondrial outer membrane within the specific Drp1-KO PCs expressed GFP.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIdentification of transgenic mice via PCR analysis\u003c/h3\u003e\n\u003cp\u003eThe tail DNA was lysed and 200 ul Lysis buffer and 4 ul DNA Release of DNA lysate were mixed for each tail sample. DNA lysate was added to each sample, boiled in a thermostatic water bath for 55℃ for 30 min, then boiled at 98℃ for 5 min, then centrifuged and centrifuged at 12000 rpm / min for 5 min. The obtained supernatant was a DNA sample and was temporarily stored at 4℃ for subsequent experiments. The mouse tail identification kit (TSE014, Tsingke, China) was used for DNA PCR and all these primer sequences were exhibited in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. The PCR protocol included pre-denaturation at 94 ℃ for 3 min, followed by 35 cycles of denaturation at 94℃ for 30 s, annealing at 60 ℃ for 35 s, and finally extended at 72 ℃ for 35 s. DNA products were detected using lysis curves. The quantitative was statistically calculated using the 2\u003csup\u003e\u0026minus;△△\u003c/sup\u003e Ct.\u003c/p\u003e\n\u003ch3\u003eLong-term supplementation with Coenzyme Q10 in mice\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice, and cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice, were administered coenzyme Q10 (CoQ10) supplementation from postnatal day 15 to day 90. CoQ10 (HY-N0111, MCE) was dissolved in a 3% dimethyl sulfoxide (DMSO) solution at a concentration of 500 \u0026micro;M. The CoQ10 solution was added to the drinking water and refreshed daily. \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice, and cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice in the control group were provided with drinking water containing less than 3% DMSO.\u003c/p\u003e\n\u003ch3\u003eRotarod test\u003c/h3\u003e\n\u003cp\u003eFor motor coordination assessment, the rotarod test (BZY007, Jiliang, China) started at 4 rpm and increased to 40 rpm within 180 seconds. Each trial lasted 10 minutes, with at least a 15-minute interval. Mice were trained 3 times daily for 3 consecutive days, data were collected on the fourth day. The latency to fall from the rod was recorded.\u003c/p\u003e\n\u003ch3\u003eOpen field test\u003c/h3\u003e\n\u003cp\u003eTo evaluate the free and spontaneous movements of transgenic mice, the animals were placed in a 50 \u0026times; 50 \u0026times; 50 cm opaque square box (DigBehav, measured), and their movements were recorded using a camera connected to a computer for 15 minutes. The mice's movements were automatically tracked, and the total distance traveled was analyzed using dedicated software.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWater maze test\u003c/h2\u003e\u003cp\u003eWe used the Water Maze test (TECHMAN, China) to perform a preliminary exploratory assessment of learning and memory. At least five mice were used for each group: Control and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e. At the start of the training, the water surface was divided into four quadrants based on the position of a marker. A platform was placed in the center of the first quadrant, and the mice were introduced into the pool at random points along the walls of the four quadrants, facing the pool wall. A video recording system was used to capture the time it took for the mice to locate the platform (escape latency) and their swimming paths. Each training session consisted of four trials, with the mice being placed in the water at different starting points (from different quadrants) in each trial. If the mice located the platform or failed to do so within 60 seconds, they were allowed to rest on the platform for 15 seconds before beginning the next trial. The average escape latency across the four trials was recorded as the learning performance for that day. Over five consecutive days of training, the swimming speed (mm/s) and escape latency (s) were measured to evaluate the mice's performance.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEight-arm radial maze test\u003c/h3\u003e\n\u003cp\u003eTo evaluate the working memory ability of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, we conducted an eight-arm radial maze test (TECHMAN, China). The study included at least five mice in each group: Control, \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, and CoQ10 intervention groups. Animals were acclimated to the experimental environment for one week, during which they were weighed and fasted for 24 hours before the experiment. Following fasting, normal food (2\u0026ndash;3 g) was provided daily at the end of training sessions to maintain the body weight of the mice at 80\u0026ndash;85% of their normal feeding weight. On days 1 and 2, food pellets were scattered across each arm and the central area of the maze to familiarize the animals with the setup. On the third day, individual training began, where a single food pellet was placed at the end of each arm near the outer food box, allowing the mice to explore and eat freely. From days 4 to 7, the food was placed inside the boxes, and the previous day's training procedure was repeated. Working memory performance was assessed from day 3 to day 7 by recording the latency of mice in locating the food and the number of errors, defined as repeated entries into the same arm. Additionally, the mice's search strategies were manually categorized based on angles of 45\u0026deg;, 90\u0026deg;, 270\u0026deg;, and 360\u0026deg;. Over a period of five consecutive days, we assessed the mice's working memory performance by measuring working memory errors, latency periods, and the percentage of searching strategies categorized at angles of 45\u0026deg;, 90\u0026deg;, 270\u0026deg;, and 360\u0026deg;.\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eGolgi staining\u003c/b\u003e\u003c/div\u003e\u003cp\u003eGolgi staining was performed using a Golgi staining kit (PK401, FD NeuroTechnologies): After deeply anesthetizing the mice, the brain tissue was quickly extracted, and blood on the surface of the tissue was rinsed off with distilled water. The tissue was immersed in a mixture of solution A and solution B. On the following day, the immersion solution was replaced with fresh solution, and the tissue was kept in the dark at room temperature for two weeks. Subsequently, the tissue was transferred to solution C and kept in the dark at room temperature for at least 72 hours, with the solution changed at least once on the second day.The tissue was then sectioned into 100-\u0026micro;m thick slices at a temperature of -20\u0026deg;C to -22\u0026deg;C using a cryotome and transferred onto slides coated with 3.5% gelatin. The slides were air-dried in the dark at room temperature for three days. The sections were washed twice with double-distilled water for 4 minutes each. Next, the slices were incubated in a mixture of 1 part solution D, 1 part solution E, and 2 parts double-distilled water for 10 minutes, followed by rinsing with distilled water twice for 4 minutes each.The sections were dehydrated through an ethanol series (50%, 75%, and 95% ethanol, each for 4 minutes) and then dehydrated in absolute ethanol four times, for 4 minutes each. Subsequently, the sections were cleared in xylene three times for 4 minutes each. Finally, coverslips were mounted using resin sealant. PCs were observed and photographed under an optical microscope, and Sholl analysis was performed using ImageJ software \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The analysis focused on two parameters: the abundance of PCs and the density of their dendritic spines.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003ePC\u003csup\u003etdTomato\u003c/sup\u003e mice were used as the control group, cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice as the experimental group, and the CoQ10 intervention group was also included. All mice were deeply anesthetized with 40 mg/kg pentobarbital sodium (Merck, Germany), and 4% paraformaldehyde was perfused through the heart. The extracted brains were fixed in 4% paraformaldehyde (R20497-10, Source Leaf) for 24 hours and then immersed in 30% sucrose solution for 2 days. Brain sections were cut into 30 \u0026micro;m thick slices using a cryostat (Leica CM1850, Germany) and mounted onto slides.The sections were blocked for 30 minutes with a blocking solution containing 0.3% Triton X-100, 10% calf serum, and PBS, and then incubated overnight at 4\u0026deg;C with the following primary antibodies: rabbit anti-Drp1 (1:50, Ab184247, Abcam), mouse anti-PSD95 (1:500, MA1-045, Abcam), and rabbit anti-IBA1 (1:500, 011-27991, Wako). After three washes with PBS, the sections were incubated for 2 hours with secondary antibodies, including DyLight 647 goat anti-mouse IgG (1:5000, A23610, Abbkine) or DyLight 647 goat anti-rabbit IgG (1:5000, A23620, Abbkine). The sections were washed three times with PBS, and cell nuclei were stained with DAPI (1:2500, C1002, Biyuntian) for 5 minutes. After air drying, the slides were sealed with a fluorescent mounting medium and observed using a confocal laser scanning microscope (Leica STELLARIS5). ImageJ software was used for statistical analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial network analysis (MiNA)\u003c/h2\u003e\u003cp\u003eMitochondrial network analysis (MiNA) uses the ImageJ software. Brain sections of PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e and cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e and CoQ10 intervention mice were photographed under inverted laser scanning confocal microscopy and MiNA analysis was performed. 3 mice were taken from each group, and at least 3 slides were taken from each brain tissue to take at least 27 visual fields for statistics \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTransmission electron microscope analysis\u003c/h2\u003e\u003cp\u003eThree mice from each group (Control, \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, and CoQ10 intervention) were selected for perfusion sampling. The cerebellar tissues were placed in 2.5% glutaraldehyde at 4\u0026deg;C overnight and washed three times with PBS. The tissues were then fixed in 1% osmium tetroxide at 4\u0026deg;C in the dark for 2 hours and washed again three times with PBS. Subsequently, the samples were dehydrated through an ethanol gradient (15 minutes per step) and finally dehydrated in 100% acetone before being embedded in resin.Eleven 70-nm sections were collected onto copper grids and stained with lead nitrate and uranyl acetate for 10 minutes. Images were captured using a transmission electron microscope, and mitochondrial morphology was analyzed with ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMitochondria isolation\u003c/h2\u003e\u003cp\u003eMitochondria isolation was performed according to the instruction (SM0020, Solarbio, China). Cerebellum tissues in different groups were rinsed in saline and homogenized in Tris-HCl-based lysis buffer. The homogenization was centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was transferred to a new tube and centrifuged under the same condition. The secondary supernatant was then centrifuged at 12,000 g for 10 min at 4 ℃. The pellet was resuspended in Tris-HCl-based wash buffer and centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was centrifuged at 12,000 g for another 10 min at 4 ℃. Finally, mitochondria were resuspended in phosphate-based store buffer (PH 7.2\u0026ndash;7.4).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial membrane potential (MMP) detection\u003c/h2\u003e\u003cp\u003eIn accordance with the protocol provided by the Mitochondrial JC-1 Assay Kit (C2005, Bicentennial), the concentration of the isolated mitochondria was adjusted to a standardized level, followed by staining with the JC-1 dye. Subsequently, fluorescence intensity measurements were obtained using a fluorescence microplate reader (Spark TECAN, Switzerland) at excitation wavelengths ranging from 475 to 520 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eReactive oxygen species (ROS) detection\u003c/h2\u003e\u003cp\u003eAccording to the instructions of Mitochondrial ROS Assay Kit (S0033S, Bicentennial): cerebellar tissue was digested to make cell suspension. Staining was performed according to a 1:1000 dilution of DCFH-DA. This was followed by flow cytometry analysis. The maximum excitation wavelength in flow cytometry was 488 nm and the maximum emission wavelength was 525 nm (B75442, Beckman Coulter, USA). FlowJo 10.8.1 software was used for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot\u003c/h2\u003e\u003cp\u003eCerebellar tissue was homogenized in pre-cooled RIPA lysis buffer (P0013B, Biyuntian), and cells were lysed using a pre-cooled lysis buffer (QIAGEN). The lysates were centrifuged at 12,000 rpm for 10 minutes at 4\u0026deg;C, and the supernatant was collected. Protein concentration was measured using a BCA kit (ZJ102L, Yadase). Proteins were separated by 12.5% SDS-PAGE (PG113, Yadase) and transferred onto a 0.45 \u0026micro;m PVDF membrane (Millipore, USA). The membrane was blocked in a rapid protein-free blocking solution (PS108P, Yadase) for 20 minutes.The primary antibodies used included rabbit anti-DRP1 polyclonal antibody (1:1000, Ab184247, Abcam), rabbit anti-SOD1 polyclonal antibody (A0274, ABclonal), rabbit anti-GPX1 polyclonal antibody (A11166, ABclonal), rat anti-Total Oxidative phosphorylation (OXPHOS) polyclonal antibody (1:300, Ab110413, Abcam), COX4 (1:1000, PA5-29992, Invitrogen) and rabbit anti-beta actin polyclonal antibody (1:5000, AC004, ABclonal). The PVDF membrane was incubated with the primary antibodies overnight at 4\u0026deg;C and washed three times with TBST. Secondary antibodies included HRP-conjugated goat anti-mouse IgG (1:5000, A21010, Abbkine) and HRP-conjugated goat anti-rabbit IgG (1:5000, A21020, Abbkine). Protein bands were visualized using Fusion FX EDGE chemiluminescence imaging technology, and the gray values of the bands were quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eNetwork pharmacology analysis\u003c/h2\u003e\u003cp\u003eWe initially performed target gene prediction by searching the keywords of \"CoQ10\" and \"mitochondria\" in five datasets, including DrugBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/drugs/\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/drugs/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Swiss Target Prediction (STP), Comparative Toxicogenomics (CT) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ctdbase.org/\u003c/span\u003e\u003cspan address=\"https://ctdbase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and MitoCarta 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways\u003c/span\u003e\u003cspan address=\"https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This process yielded 141 CoQ10-related genes and 1,140 mitochondria-related genes in mice. Subsequently, we intersected the 141 CoQ10-associated genes with the 1,140 mitochondrial genes, resulting in the identification of 30 common target genes. Protein-Protein Interaction (PPI) was performed to analyze the mitochondrial genes related to CoQ10, and arrange them according to the degree of interaction by using STRING Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and software of Cytoscape 3.9.1. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for key targets were performed utilizing the DAVID database and the Microbiotics platform. CoQ10-related mitochondrial genes were enriched for GO cellular components (GO-CC) analysis, GO biological processes (GO-BP) analysis, and GO molecular function (GO-MF) analysis. KEGG pathway enrichment analysis were visualized by Chiplot software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to screen for possible involvement in the regulation of mitochondrial functions associated with neurodegenerative pathologies. Finally, the target gene prediction was conducted in the Gene Set Enrichment Analysis (GSEA) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsea-msigdb.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"https://www.gsea-msigdb.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003ePatch clamp electrophysiologic recording\u003c/h2\u003e\u003cp\u003eWhole-cell electrophysiological experiments were conducted using control and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. The mice were anesthetized with pentobarbital sodium (40 mg/kg) and perfused with 20 mL of an ice-cold carbonated cutting solution (95% O₂, 5% CO₂), which contained 240 mM sucrose, 2.5 mM KCl, 1.25 mM Na₂HPO₄, 2 mM MgSO₄, 1 mM CaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose. The brain was then quickly removed and placed in the same cold, carbonated cutting solution to prepare for slicing. Sagittal brain slices (250 \u0026micro;m thick) were prepared using a microtome (Leica VT1200S) and incubated in the cutting solution in a holding chamber at 32\u0026deg;C for 30 minutes. The slices were subsequently transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 119 NaCl, 2.3 KCL, 1.0 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 26 NaHCO\u003csub\u003e3\u003c/sub\u003e, 11 D-(+)-glucose, 1.3 MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, and 2.5 CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO (pH 7.4; osmotic pressure 295\u0026ndash;300 mOsm/L) and kept at room temperature for at least 1 hour.When Coenzyme Q10 was used to treat \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, it was added to the ACSF at a final concentration of 11 \u0026micro;M, and the slices from 3 mice were incubated under these conditions. The sections were placed in a recording chamber maintained at 24\u0026ndash;28\u0026deg;C (TC-324B, Warner Instrument) with a perfusion rate of 2.0 mL/min. Whole-cell patch-clamp recordings were performed under infrared differential interference contrast (IR-DIC) visual guidance. Recording pipettes (BF150-86-7.5, Sutter Instrument, Novato, USA) were fabricated using a horizontal pipette puller (P-97, Sutter Instrument, Novato, USA) and had a tip resistance of 3\u0026ndash;6 MΩ. The patch pipettes were filled with an intracellular solution containing (in mM): 128 potassium gluconate, 10 HEPES, 10 sodium creatine phosphate, 1.1 EGTA, 5 magnesium ATP, and 0.4 GTP sodium. The pH was adjusted to 7.3 with potassium hydroxide, and the osmotic pressure was adjusted to 300\u0026ndash;305 mOsm using sucrose.During recordings, cells with a series resistance greater than 20 MΩ were excluded from further analysis. Neurons with a resting membrane potential more negative than \u0026minus;\u0026thinsp;60 mV and capable of firing action potentials were selected for subsequent experiments. Liquid junction potentials were not corrected. Currents or membrane potentials were recorded using an Axon 200A amplifier (Molecular Devices, Sunnyvale, USA). The signals were low-pass filtered at 5 kHz and digitized at 20 kHz using a Digidata 1322A system and Clampex 9.0 software (Molecular Devices). The data were stored on a computer for subsequent offline analysis \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eThermal proteome profiling (TPP)\u003c/h2\u003e\u003cp\u003eFirst, the mouse brain tissue was crushed by a tissue crusher to obtain the protein solution. Detect the concentration of the protein lysis buffer. This experiment consists of 3 groups and 3 replicates. That is, the 3 experimental groups are the DMSO control group and the 10 \u0026micro;M CoQ10 group respectively. 100 \u0026micro;M CoQ10 group. Three repetitions in each group. After high-speed centrifugation of the cell lysate, the supernatant was taken and passed through an ultrafiltration tube with PBS for three times. After determining the protein concentration by BCA, it was equally divided into 9 parts (each part had a concentration of 2mg/ml). DMSO was added to the 10 \u0026micro;M CoQ10 group and the 100 \u0026micro;M CoQ10 group respectively as mentioned above, and incubated at 4 ℃for 2 hours. Heat treatment at 58 ℃ for 7 minutes; Centrifuge at 4 ℃ for 30 minutes at high speed; Take the supernatant and transfer it to a new EP tube, then add pre-cooled acetone and precipitate the protein at -80 ℃. Next, protein profile sample pretreatment and proteomic quantitative analysis of the candidates were conducted on the 9 groups of samples. Proteomic identification and analysis were performed by Obitrap HF-X LC-MS/MS high-resolution mass spectrometry. HFX was used for mass spectrometry signal acquisition (with a 70-minute time gradient for each sample). Data analysis was conducted using maxquant analysis software. The database of mouse proteins annotated with Swiss-prot in uniprot was used for database search. The database search results show that approximately 3,100 proteins have been identified in total. Through differential analysis of the omics data, the data were divided into two priorities. The first-priority cutoff criterion is: the difference between the low-concentration drug and the DMSO group is a presence-versus-absence distinction, and simultaneously, the protein quantification value increases with rising drug concentration. The second-priority cutoff is: the low-concentration drug shows a presence-versus-absence difference compared to the DMSO group, but the protein level does not increase in the high-concentration group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eCoa6 carriers Construction and protein purification\u003c/h2\u003e\u003cp\u003eFrom the first-priority screening data, the Coa6 protein was selected as the candidate validation target protein. First, the RNA of mouse macrophages was extracted, and cDNA was obtained through reverse transcription. Then, PCR was performed to obtain these two target genes. The gene sequences of the two proteins were constructed into the prokaryotic expression vector PET28A, namely PET28A-Coa6, through homologous recombination. After sequencing and comparison, there were no errors.The plasmids were respectively transformed into competent cells expressing strain BL21(DE3), and then the bacterial solution was transferred to a 1L conical flask. When the OD of the bacterial solution reached about 0.8, 1M of IPTG inducer was added and induced for 12 hours (at 37℃). The process was followed by bacterial collection, ultrasonic disruption and protein lysis buffer collection in sequence. Then the protein supernatant was purified by Ni-beads. The impurity proteins were eluted successively through 20mM imidazole, 50mM imidazole, and 500mM imidazole, and the target proteins were eluted. Finally, the 500mM imidazole elution solution was subjected to SDS-PAGE staining to determine the protein purity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eCell thermal shift assay (CETSA)\u003c/h2\u003e\u003cp\u003eThe Coa6 protein solution was added to separate tubes containing increasing concentrations of CoQ10 (dissolved in DMSO vehicle) respectively. To each 100 \u0026micro;l reaction volume, CoQ10 was added at final concentrations of 0 \u0026micro;M (DMSO control), 0.32 \u0026micro;M, 1.6 \u0026micro;M, 8 \u0026micro;M, 40 \u0026micro;M, and 200 \u0026micro;M. After 1.5-hour incubation at room temperature, samples were heated at 55\u0026deg;C for 7 min in a water bath, followed by high-speed centrifugation (4\u0026deg;C, 20,000 g, 30 min). Subsequently, the supernatant was collected for further SDS-PAGE. As the drug concentration increases, the thermal stability of the target protein bound to the drug improves. The electrophoretic results show that the band intensity of the target protein increases with increasing drug concentrations, which is shown as a rise in target protein content on the electrophoresis gel.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eDrug affinity responsive target stability (DARTS)\u003c/h2\u003e\u003cp\u003eThe Coa6 protein solution was mixed with varying concentrations of CoQ10. Specifically, 100 \u0026micro;l of the solution was supplemented with CoQ10 at concentrations of 0 \u0026micro;M (DMSO control), 1 \u0026micro;M, 10 \u0026micro;M, 100 \u0026micro;M, and 200 \u0026micro;M, respectively. After incubating for 1.5 hours at room temperature, each sample was treated with pronase E 1:100 mass ratio and incubated in a 37\u0026deg;C water bath for 15 minutes. Following the reaction, the samples were immediately prepared for SDS-loading by boiling, and the supernatants were subjected to SDS-PAGE. As the drug concentration increases, the target protein bound to the drug exhibits enhanced resistance to enzymatic degradation. This is evidenced electrophoretically by the progressive intensification of the target protein band with increasing drug concentrations.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eSurface plasmon resonance (SPR)\u003c/h2\u003e\u003cp\u003eThe activator is prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection. The CM5 sensor chip is activated for 420 s with the mixture at a flow rate of 10 \u0026micro;l/min. Dilute Coa6 to 20 \u0026micro;g/ml in immobilization buffer, then injected to sample channel Fc2 at a flow rate of 10 \u0026micro;l/min, and typically result in immobilization levels of 12600 RU, the reference channel Fc1 does not need ligand immobilization step. The chip is deactivated by 1 M Ethanolamine hydrochloride at a flow rate of 10 \u0026micro;l/min for 420 s. Dilute CoQ10 with the same analyte buffer to 8 concentrations(0.39-25) \u0026micro;M. CoQ10 is injected to channel Fc1- Fc2 at a flow rate of 20 \u0026micro;l/min for an association phase of 100 s, followed by 180 s dissociation. The association and dissociation process are all handling in the analyte buffer. Repeat 8 cycles of analyte according to analyte concentrations in ascending order. After each cycle of interaction analysis, The chip need to be regenerated.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eMolecular docking of Coa6 with CoQ10\u003c/h2\u003e\u003cp\u003eRelevant methods were based on those described in previous studies \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The structure of the Coa6-CoQ10 complex was predicted utilizing the ProtENIX web server under default parameters and subsequently visualized through the academic edition of Maestro. The three-dimensional architectures of the proteins were rendered and explored using ChimeraX. In this three-dimensional schematic diagram, orange-marked regions denote negatively charged binding sites, bluish violet regions denote positively charged binding sites, and purple arrows indicate directional hydrogen bonding with the arrow tail representing the hydrogen bond donor and the arrowhead pointing to the hydrogen bond acceptor. These three types of molecular interactions collectively constitute key binding elements at the CoQ10-Coa6 interaction interface.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eStatistical methods\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism 8. For comparisons, a t-test was used for parametric data, while the Mann-Whitney \u003cem\u003eU\u003c/em\u003e test was applied for non-parametric data to assess statistical significance. For multiple comparisons, one-way ANOVA followed by Dunnett\u0026rsquo;s t test was used for parametric data, and the Kruskal-Wallis test was employed for non-parametric data. For repeated measures data, two-way ANOVA followed by Sidak test was used. The degree of linear correlation between dendritic spines and working memory was assessed using Pearson correlation analysis. A correlation coefficient of 1 indicates a perfect positive correlation, while a coefficient of -1 signifies a perfect negative correlation. A \u003cem\u003eP\u003c/em\u003e-value of less than 0.05 suggests a statistically significant linear relationship between the variables. Results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Western blots, fine immunofluorescence stains, and Golgi stains were analyzed using ImageJ. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eNetwork pharmacology analysis revealed that CoQ10 is linked to the DNM1L/Drp1 gene in cerebellar atrophy-induced cognitive impairment.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe performed a Network pharmacology analysis to investigate the potential correlation between Drp1 deficiency and Coenzyme Q10 (CoQ10) supplementation. A total of 30 target genes were predicted through the intersection of a CoQ10-related gene database, which contains 141 genes, and a mitochondria-related gene database, which contains 1140 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Protein-protein interact (PPI) analysis of these 30 genes revealed that Sod2, Caspase-3, Sdha, and Dnm1l (also known as Drp1) were the most significant nodes within the network (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The GO-CC analysis (Gene Ontology - cellular components) showed that this significantly enriched cellular component included the inner and outer mitochondrial membranes, as well as organelle membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The GO-BP analysis (Gene Ontology - biological processes) showed that the enriched biological processes included mitochondrial respiration and apoptotic signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The GO-MF analysis (Gene Ontology - molecular function) showed that the enriched molecular function included ubiquitin protein ligase binding, ubiquitin-like protein ligase binding, and BH domain binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). KEGG analysis (Kyoto Encyclopedia of Genes and Genomes) illustrated that the enriched genes were mainly associated with neurodegenerative diseases, such as AD, PD, and HD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA further target gene prediction showed that the DNM1L/Drp1 gene was the most important gene which was linked to all four aspects: CoQ10, cognitive impairment, cerebellar atrophy, and mitochondrial division (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The above results suggest that CoQ10 is associated with the DNM1L/Drp1 gene in cognitive impairment caused by cerebellar atrophy. Thus, CoQ10 may have therapeutic potential for DRP1-related disorders.\u003c/p\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eDrp1 Deficiency in PCs causes working memory defects in mice\u003c/h2\u003e\u003cp\u003eGiven the established relationship between CoQ10 and Drp1 (dynamin-related protein 1), we employed the Cre-LoxP system to generate three distinct Purkinje cell (PC)-specific Drp1 knockout mouse models. The first model represents a conventional PC-specific Drp1 knockout (Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The second model incorporates PC-specific TdTomato reporter expression (cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice), enabling fluorescent visualization of PCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The third model features mitochondria-targeted GFP expression specifically in PC mitochondria (cKO PCMito-GFP mice), allowing for direct observation of mitochondrial dynamics in these neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGene analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) indicated that mice numbered 4, 5, 6, 8, 9, 13, and 14 were \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. The Western blot results revealed a 40% decrease in Drp1 expression level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The rotarod test results indicated impaired motor coordination in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Conversely, the open-field test results confirmed the normal locomotor activity of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice at 1 month, 2 months, and 3 months of age (1M, 2M, and 3M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). These findings suggest that while \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibit motor incoordination, their ability to walk remains unaffected.\u003c/p\u003e\u003cp\u003eBeginning at one month of age, water maze testing revealed age-dependent progressive working memory deficits in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, I). This decline in cognitive function was not attributable to impaired motor function, as evidenced by the normal swimming speeds observed in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice across all ages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Subsequently, working memory was further assessed using an eight-arm maze test conducted over seven consecutive days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, K). However, no significant differences were observed between control and \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice at 1M or 2M in working memory errors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL) or entry latency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Notably, the proportion of 45\u0026deg; searches diminished to 60% and 50% in 1M and 2M \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, respectively, and the proportion of 90\u0026deg; searches increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN). By 3 months of age, working memory deficits in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited significant exacerbation. This was characterized by: an approximate 200% increase in food search error rate; entry latency exceeding 350 seconds with increased variability; a reduction in 45\u0026deg; searches to only 10% of total searches; and a predominant shift towards a 270\u0026deg; search strategy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-N). Based on this pronounced deterioration at 3 months, subsequent investigations focused on characterizing the underlying changes in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice at this age.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eDrp1 deletion impaired synaptic plasticity in PCs of memory-deficient mice\u003c/h2\u003e\u003cp\u003eWe compared the complexity of the PCs structure between \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and the control group using Golgi staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Sholl\u0026rsquo;s analysis showed that the abundance of PCs in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, as indicated by the number of crossings, was significantly reduced by 70% at both 1M and 2M and by 90% at 3M, compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We conducted an in-depth analysis of dendritic spine density in the distal dendrites of PCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The lengths of dendrites in PCs were significantly reduced in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice at all three age points compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The dendritic spine densities of PCs were significantly reduced in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice at 2M and 3M compared to the control group, although no significant difference was observed between 1M \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Correlation analysis of dendritic spine density with working memory errors in mice showed a negative correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eTo achieve the accurate counting of cerebellar PCs, we generated cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice by crossing \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with B6-G/R mice. In these transgenic mice, the specific Drp1-KO PCs expressed tdTomato (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). PCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) indicated that mice numbered 3, 5 were the target mice.\u003c/p\u003e\u003cp\u003eConfocal microscopy observation of the mid-sagittal brain section demonstrated that tdTomato expression was exclusively observed in PCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Quantification of tdTomato-positive cells showed a 50% reduction in PCs density at both the 1M and 2M time points, and an approximately 80% reduction at 3M in cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, K). Immunofluorescence staining further revealed the reduction of Drp1 expression in PCs of cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL, M). Postsynaptic density protein 95 (PSD95) levels in PCs of cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice were significantly reduced at 2M and 3M compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN, O), which were consistent with the changes observed in Golgi-stained dendritic spines. The above results suggest that early Drp1 deletion primarily reduces PCs density and dendritic arborization, whereas dendritic spine density is progressively impaired over time. Collectively, our results demonstrate that Drp1 deficiency drives working memory deficits through progressive compromise of PCs dendritic spine structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDrp1 defect in PCs severely impairs MMs stability and OXPHOS function\u003c/h3\u003e\n\u003cp\u003eTo visualize the mitochondrial morphological changes caused by Drp1 deletion in PCs, we generated cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice by crossing \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with Mito-GFP mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Mice 6 and 9 were identified as the target mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice served as the control group. The mitochondria targeted GFP was exclusively expressed in PCs of cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Mitochondrial network analysis (MiNA) revealed significantly reduced mitochondrial interconnectivity and amount in Purkinje cells (PCs) of cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Specifically, there was a notable decrease in the number of individual mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), the number of mitochondrial networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), the lengths of mitochondrial branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), and the mean branches per mitochondrial network (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Moreover, mitochondria in PCs of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited significant swelling and severe cristae damage compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). In detail, the following were observed relative to the control group: reduced mitochondrial perimeter in 3M \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ); increased mitochondrial area at both 2M and 3M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK); increased mitochondrial circularity across all three time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL); and decreased mitochondrial cristae density at both 2M and 3M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). Mitochondrial membrane potential (MMP) in the cerebellum of \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice was significantly reduced at 3M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). These results demonstrate that Drp1 deficiency in PCs induces mitochondrial membranes (MMs) instability.\u003c/p\u003e\u003cp\u003eReactive oxygen species (ROS) levels in the cerebellum of these mice were significantly increased by 1.5-fold at 1M and 2M, and by 3-fold at 3M compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO). In addition, WB results from cerebellar tissues showed that superoxide dismutase 1 (SOD1) levels were reduced by 50% at \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e 3M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eP), while there was no significant difference in glutathione peroxidase 1 (GPx1) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ). The content of mitochondrial oxidative phosphorylation complex III-UQCRC2 (CIII) and complex V-ATP5A (CV) was significantly reduced, while no significant differences were observed in complex I-NDUFB8 (CI), complex II-SDHB (CII) and complex IV-MTCO1 (CIV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eR). These results indicate that Drp1 deficiency in PCs induces severe oxidative stress and OXPHOS dysfunction. Collectively, our results demonstrate that Drp1 deficiency drives PCs damage through severe impairment of mitochondrial membrane stability and OXPHOS function (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eS).\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eLong-term administration of CoQ10 effectively ameliorate working memory deficits induced by Drp1 deletion\u003c/h2\u003e\u003cp\u003eTo verify the therapeutic effect of CoQ10 in Drp1-deficient diseases, \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, cKO PC\u003csup\u003etdTomato\u003c/sup\u003e mice, and cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e mice were long-term administered CoQ10 continuously via drinking water supplementation starting at 15 days and continuing until 90 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). CoQ10, known for its significant mitochondrial protective properties, was dissolved in a 3% DMSO solution at a concentration of 500uM. The mice in the control group were provided with drinking water containing less than 3% DMSO (Vehicle, Veh).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBehavioral analyses demonstrated that CoQ10 treatment reduced both working memory errors and latency by approximately 50% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). Additionally, the percentage of 45\u0026deg; searches increased 5-fold, while the percentages of 135\u0026deg; and 180\u0026deg; searches significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Immunofluorescence staining revealed an increase in microglia in Drp1-deficient mice at 1M, 2M, and 3M compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). WB analysis further confirmed the elevated expression of microglia in cerebellar tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Following the CoQ10 intervention, the number of microglia was reduced by 60% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eThese findings indicated that long-term CoQ10 supplementation ameliorates working memory deficits in mice with the specific conditional deletion of the Drp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eCoQ10 treatment effectively ameliorate PCs dysfunction induced by Drp1 deficiency\u003c/h2\u003e\u003cp\u003eA significant increase in both the number and complexity of PCs dendritic branches was observed following the CoQ10 intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Further analysis of Purkinje cell dendritic spines revealed that CoQ10 increased dendritic spine density (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D); however, no statistical difference was found in dendritic branch length (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Additionally, both PSD95 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G) and PCs density (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, I) enhanced markedly following CoQ10 treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe further recorded the electrical activity of PCs using the membrane clamp technique. No significant differences were observed in access resistance (Ra) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, K) or membrane resistance (Rm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, L), indicating that the Drp1 knockout does not disrupt the integrity of the PC membrane. However, \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed a significant decrease in the membrane time constant (tau) and membrane capacitance (Cm), indicative of neuronal atrophy and dendritic spines loss, which was reversed by CoQ10 intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM, N). These findings were consistent with the Golgi staining results. Additionally, in voltage-clamp mode, we observed a significantly increased current response to hyperpolarization activation in the \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice group, suggesting aberrant activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This abnormality was similarly reversed in the CoQ10-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO, P). These results indicate that long-term CoQ10 supplementation effectively inhibited aberrant activation of HCN channels and restored the impaired plasticity of PCs caused by Drp1 deletion in PCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eQ).\u003c/p\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003eTPP identifies key target proteins of CoQ10 in Drp1-deficient mice\u003c/h2\u003e\u003cp\u003eTo identify the molecular target of CoQ10 on Drp1-induced mitochondrial disease, we conducted a small molecule drug target fishing experiment. First, we conducted TPP (thermal proteome profiling) analysis and divided the data into two priorities. The first-priority cutoff criterion is: the difference between the low-concentration drug and the DMSO group is a presence-versus-absence distinction, and simultaneously, the protein quantification value increases with rising drug concentration. The second-priority cutoff is: the low-concentration drug shows a presence-versus-absence difference compared to the DMSO group, but the protein level does not increase in the high-concentration group. Twelve candidate targets were screened out in the first priority, and 17 proteins were screened out in the second priority (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSecondly, through the analysis of the differences between the low concentration and the control group, 28 significant heat-resistant proteins were screened out. The analysis of the differences between the high concentration and the control group screened out 33 significant heat-resistant proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Taking the intersection of the two groups of heat-resistant proteins, 12 common proteins were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), namely Coa6, Mb, Slc25a46. Nde1, Rcc2, Hsdl1, Armc1, Cbx5, Rab3gap2, Rabggta, Ap3m2, and Usp47. And, in the first-priority data, the Coa6 protein was selected as the candidate validation target protein. Therefore, we created the PET28A-Coa6 protein vector and carried out protein purification (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003eCoQ10 rescues memory impairment in DRP1-deficient mice by binding to Coa6\u003c/h2\u003e\u003cp\u003eTo validate Coa6 as a target protein of CoQ10, we assessed Coa6\u0026rsquo;s thermal stability and protease resistance using CETSA (Cellular Thermal Shift Assay) and DARTS (Drug Affinity Responsive Target Stability) assays, respectively. The CETSA experiment indicated that the Coa6 protein had a significant anti-thermal stability phenomenon, that is, with the increase of CoQ10 concentration, the Coa6 protein had a significant retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The DARTS data further revealed a dose-dependent protection of Coa6 against proteolysis, with increasing CoQ10 concentrations correlating with enhanced protein stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). To quantitatively analyze the binding affinity between CoQ10 and Coa6, we further conducted surface plasmon resonance (SPR) experiments. SPR results revealed specific binding between CoQ10 and CM5-immobilized Coa6, yielding a dissociation constant (KD) of 2.40 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). These results demonstrate high-affinity binding between Coa6 and CoQ10.\u003c/p\u003e\u003cp\u003eWe predicted the structure of the Coa6-CoQ10 complex using ProtENIX. Molecular docking prediction indicates that CoQ10 is embedded in the protein-binding pocket of Coa6, forming a strong hydrophobic complementarity. The methoxy group of CoQ10 forms a hydrogen bond with the 18th Lysine residue (K18) of Coa6, and its extended hydrophobic tail establishes hydrophobic interactions with residues including Tryptophan at position 59 (W59), Valine at position 45 (V45), and Proline at position 35 (P35), the 36th Valine (V36), the 94th Tryptophan (W94), the 97th Tyrosine (Y97), the 98th Phenylalanine (F98), the 104th Tyrosine (Y104), and the 107th Phenylalanin Residues (F107). These findings provide strong evidence for the molecular interaction model between Coa6 and CoQ10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eFurthermore, the WB results showed that the expression of Coa6 in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice decreased, while it significantly increased in the CoQ10 intervention group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). CoQ10 binding stabilizes Coa6, which may facilitate the assembly of mitochondrial Complex IV (CIV) within the mitochondrial inner membrane. According to prior research \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, reduced Coa6 expression disrupts complex IV (CIV) assembly, consequently diminishing CIV activity and OXPHOS function. Further studies are needed to determine whether CoQ10 binding to Coa6 rescues mitochondrial oxidative phosphorylation function (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eCoQ10 improves Drp1 deficiency-induced MMs instability and CIII-CV activity\u003c/h3\u003e\n\u003cp\u003eGiven that Coa6 is an important subunit of mitochondrial complex IV (CIV), we further examined the effects of CoQ10 on mitochondrial morphology and respiratory function in \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Analysis with MiNA revealed that CoQ10 intervention increased the number of individual mitochondria, mitochondrial networks, branching complexity, and branch length in PCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). Electron microscopy analysis of cerebellar tissue showed that CoQ10 decreased the mitochondrial perimeter and area while increasing respiratory cristae occupancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, D). Additionally, mitochondrial membrane potential was elevated in cerebellar tissue following CoQ10 supplement (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). These results demonstrate that CoQ10 corrects Drp1 deficiency-induced mitochondria membranes (MMs) instability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, CoQ10 intervention primarily selectively increased the expression of respiratory chain complexes III (UQCRC2) and V (ATP5A), with no significant changes in complexes I (NDUFB8), II (SDHB), and IV (MTCO1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). We further quantified cytochrome c oxidase subunit IV (COX4) expression and found that CoQ10 significantly rescued the Drp1 deficiency-induced reduction in COX4 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG, H). In cerebellar tissue, CoQ10 supplementation significantly elevated levels of the antioxidant enzymes SOD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI) and GPx1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). Quantitative ROS assays confirmed reduced reactive oxygen species in this region (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). These results demonstrate that CoQ10 corrects Drp1 deficiency-induced oxidative stress and OXPHOS dysfunction.\u003c/p\u003e\u003cp\u003eIn conclusion, CoQ10 restores mitochondrial membrane stability and OXPHOS function \u0026mdash; specifically complexes III, IV, and V \u0026mdash; by binding to Coa6 and elevating its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL). Additionally, the improvement in complex IV function was dependent on the Coa6 assembly factor rather than the structural MTCO1 subunit.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used three distinct mouse models with dynamin-related protein 1 (Drp1) - specific knockout in cerebellar Purkinje cells (PCs) and demonstrated the following findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e): Left side: 1) Successful generation of Drp1-specific knockout mice in PCs; 2) Reduced expression of cytochrome c oxidase assembly factor 6 (Coa6) in cerebellar mitochondria, decreased levels of mitochondrial respiratory chain complexes III, IV, and V (CIII-CV), and disruption of mitochondrial cristae structure; 3) Impaired synaptic plasticity and deficits in working memory. CoQ10 was administered via drinking water at a concentration of 500 \u0026micro;M for 75 days. The effects observed on the right side include: 1) Direct binding of CoQ10 to mitochondrial Coa6, resulting in increased Coa6 levels; 2) Restoration of mitochondrial respiratory chain CIII, CIV, and CV activity through elevated Coa6 levels, accompanied by recovery of mitochondrial cristae structure; 3) Improved synaptic plasticity in Purkinje cells and enhanced working memory performance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCoQ10 was first isolated and discovered from bovine heart mitochondria by Crane's team in 1957 \u003csup\u003e30\u003c/sup\u003e. It is a relatively simple-structured vitamin-like fat-soluble molecule that is widely present in human cell membranes, especially abundant in mitochondria \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Its core physiological function is to act as an electron carrier in the electron transport chain, participate in the synthesis of cellular energy, and is an indispensable cofactor for maintaining life activities \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Studies have confirmed that CoQ10 can significantly improve the cardiac function and quality of life of patients with heart failure \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and is effective for patients with hypertension \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, migraine \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and liver steatosis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e also has therapeutic effects. In neurological diseases, CoQ10 has a good neuroprotective effect on diseases such as PD and AD \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Especially in the recovery of neurological injuries and the intervention of working memory disorders, both animal experiments and clinical studies suggest that it has significant improvement potential \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Furthermore, Shi et al. recently reported that CoQ10 headgroup intermediates restore CoQ10 synthesis, alleviating mitochondrial injury-induced cerebellar damage and Purkinje cell degeneration \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In previous studies, CoQ10 mainly treated diseases related to cognitive impairment by alleviating neuroinflammation \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, inhibiting oxidative stress \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and restoring abnormal activity of acetylcholinesterase \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Similarly, our findings demonstrate that CoQ10 can effectively alleviate working memory impairments resulting from cerebellar injury. Mechanistically, CoQ10 reduces microglial activation in the cerebellum, decreases reactive oxygen species (ROS) levels, and enhances the expression of antioxidant enzymes such as SOD1 and GPx1. Our experimental data further reveal that long-term CoQ10 supplementation in Purkinje cell-specific Drp1 knockout mice improves mitochondrial function \u0026ndash; including enhanced membrane stability (evidenced by increased mitochondrial cristae connectivity and MMP levels) and improved OXPHOS function (verified by increased CIII, CIV, and CV activity) \u0026ndash; delays neuronal structural degeneration, and significantly alleviates working memory deficits. Collectively, these results support the notion that long-term CoQ10 supplementation represents an effective strategy for ameliorating working memory impairments associated with cerebellar PCs injury and mitochondrial dysfunction caused by Drp1 deficiency. Therefore, coenzyme Q10 may have promising therapeutic potential in treating the working memory deficits caused by cerebellar injury.\u003c/p\u003e\u003cp\u003eCoenzyme Q10, as a mitochondrial function regulator, demonstrates significant therapeutic potential in addressing cognitive impairments associated with neurological disorders \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Previous studies have indicated that CoQ10 can mitigate stress sensitivity induced by elevated Drp1 activity, thereby enhancing mitochondrial ATP production \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Li et al. reported that water-soluble CoQ10 has been shown to prevent mitochondrial dynamic imbalance by reducing the expression of Drp1 and Fis1 to pre-rotenone treatment levels and by attenuating rotenone-induced mitochondrial fragmentation \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Moreover, CoQ10 primarily alleviates structural and functional mitochondrial damage, suppresses mitochondrial fission, and promotes mitochondrial fusion through the inhibition of phosphorylation at Ser616 and Ser637 residues of Drp1 \u003csup\u003e47\u003c/sup\u003e. In this study, we integrated protein-protein interaction (PPI), gene ontology (including cellular component\u0026mdash;GO-CC, biological process\u0026mdash;GO-BP, and molecular function\u0026mdash;GO-MF), and KEGG pathway analyses to perform target gene prediction and network pharmacological analysis. The results revealed that diseases associated with the DNM1L/Drp1 gene are significantly involved in processes such as coenzyme Q10 administration, memory disorders, cerebellar dysfunction, and mitochondrial dysfunction. Moreover, our results demonstrate that Drp1 deficiency reduces UQCRC2 (a core subunit of Complex III), thereby disrupting the equilibrium of the Q cycle. This lead to an accumulation of reduced CoQ (CoQH₂) and depletion of oxidized CoQ in mitochondria. The consequent impairment of the mitochondrial electron transport chain elevates ROS levels and diminishes ATP production in PCs. We observed that CoQ10 supplementation rectified these Drp1 deficiency-induced abnormalities in ROS and ATP homeostasis. Collectively, we propose that CoQ10 alleviates pathological phenotypes in Drp1-deficient mice by restoring electron transport chain functionality compromised by Drp1 ablation.\u003c/p\u003e\u003cp\u003eFurthermore, through target fishing experiments, we identified Coa6 as the target molecule of coenzyme Q10. TPP, CETSA, DARTS, and SPR assays qualitatively and quantitatively demonstrated the stable binding between CoQ10 and Coa6. Therefore, we propose that CoQ10 may ameliorate cerebellar injury by binding to the Coa6 protein. Previous studies on CoQ10 have focused on other molecular targets. For example, CoQ10 can attenuate LPS-induced acute lung injury (ALI) by stabilizing its binding to Drp1, thereby regulating mitochondrial dynamics, alleviating oxidative stress, and reducing NLRP3-mediated inflammation \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. As a key electron carrier in the mitochondrial electron transport chain, CoQ10 supports ATP synthesis and mitigates energy metabolism disorders in neurodegenerative diseases such as Parkinson\u0026rsquo;s disease and Alzheimer\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Additionally, CoQ10 has been found to regulate key pathways, including PI3K/Akt, GSK-3β, CREB, and BDNF, thereby influencing cell survival and synaptic plasticity, with potential benefits for cognitive impairment \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Some studies also suggest that CoQ10 deficiency may disrupt cholesterol homeostasis, impairing the structure and function of neuronal membranes \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In contrast to previous findings, our study revealed\u0026mdash;through molecular docking experiments\u0026mdash;that CoQ10 exerts its effects in the cerebellum by binding to the mitochondrial cytochrome c oxidase assembly factor Coa6. Coa6 is a mitochondrial intermembrane space protein primarily involved in the assembly of CIV of the respiratory chain \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The loss of Coa6 can lead to the combined deficiency of CI and CIV \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Coa6 also influences tumor progression in breast and lung cancer by modulating mitochondrial oxidative phosphorylation \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In addition, our study showed that CoQ10 increased the levels of CIII and CV in the cerebellar mitochondrial respiratory chain. Therefore, we hypothesize that CoQ10 may improve Drp1-deficient working memory impairment by enhancing the activity of CIII, CIV, and CV via its interaction with Coa6. This suggests that the CoQ10\u0026ndash;Coa6\u0026ndash;ETC axis may represent a potential therapeutic target for ameliorating working memory deficits in Drp1 deficiency.\u003c/p\u003e\u003cp\u003eIn our Drp1-deficient model, molecular docking illustrated that CoQ10 forms a stable complex with Coa6 through single hydrogen bonds and multiple hydrophobic interactions. Previous studies indicate that when CoQ10 binds to proteins, hydrogen bonds are usually involved in conjunction with hydrophobic interactions, enhancing the stability and specificity of the complexes \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, which aligns with our results. Hydrogen bonding is known to play a critical role in protein stability, improving ligand binding specificity and affinity \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Notably, the methoxy group of CoQ10 formed hydrogen bonds exclusively with the 18th lysine residue (K18) of Coa6, suggesting a highly specific interaction. Moreover, cerebellar Coa6 expression levels significantly increased following CoQ10 supplementation. This may result from CoQ10 promoting Coa6 biogenesis or stabilizing Coa6 by inhibiting its degradation. These effects could enhance mitochondrial respiratory chain CIV activity (evidenced by increased COX4 level), reduce ROS production, and restore neuronal energy metabolism. Although previous studies demonstrated that CoQ10 elevates CI, CII, and CIV levels \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, recovers decreased CIV activity and CIV-OPA1 binding in aged mice \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, enhances CI/CII/CIII activity in Parkinson\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, and modulates CII/CIII in Huntington\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, the precise mechanisms underlying CoQ10\u0026rsquo;s enhancement of CI, CII, CIII, and CV functions remain incompletely understood. Based on our findings, we propose two potential mechanisms: (1) CoQ10 enhances the stability of Coa6 (a CIV assembly factor), which may subsequently promote the function or stability of CIII and CV through indirect effects on overall respiratory chain integrity or supercomplex formation; or (2) Improved CIV activity enhanced by CoQ10 subsequently promotes increased CIII and CV activities. Crucially, using a cerebellar injury model, our study systematically demonstrates that CoQ10 can simultaneously enhance the functions of complexes III, IV, and V.\u003c/p\u003e\u003cp\u003eConsidering that the Drp1-deficient mice used in our experiments have ataxia \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, which may interfere with the results of the water maze, we used the eight-arm maze test to explore working memory impairment in mice. The eight-arm maze is a classic behavioral tool for evaluating working memory and spatial learning ability, which is widely used in neuroscience and pharmacology research. Its design allows simultaneous measurement of working memory and reference memory, with high sensitivity and repeatability, and is considered one of the reliable methods for studying working memory \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice at 1 month of age learn slower, with persistently higher latency and 45\u0026deg; search times reduced to 60% despite gradual improvement, but no significant memory errors were observed. At 2 months, Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice still showed no significant memory loss, but 45\u0026deg; search frequency further decreased to 50%. After 3 months, Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed marked deterioration in working memory, with error rates increased to 200%, unstable latency exceeding 350 seconds, and 45\u0026deg; search patterns reduced to only 10%. When CoQ10 was administered at postnatal day 15, Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed significant improvement in working memory deficits compared to controls. The above results demonstrated that early alleviation of Drp1-deficient mitochondrial morphological dysfunction could rescue PCs function and working memory impairment. Previous studies reported that Drp1 plays a key role in PC and cerebellar development, as its knockdown results in a 40% reduction in cerebellar volume and mitochondrial abnormalities \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Our previous study showed that although mitochondria transplantation in juvenile mice ameliorated motor dysfunction caused by Drp1 deficiency, Drp1 upregulation in PCs at 1 month of age or mitochondria transplantation in adult mice failed to improve ataxia \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Consistent with previous studies, our findings highlight an important role for Drp1 in PC development.\u003c/p\u003e\u003cp\u003eAnother contribution is the identification of HCN channels as a potential target for CoQ10 supplementation therapy. Abnormal activation of HCN channels underlies impairments in higher brain functions, influencing neuronal oscillations, synaptic transmission, and cognition \u003csup\u003e\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. HCN hyperactivation is also linked to AD and depression-related cognitive deficits \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. HCN inhibitors have been shown to alleviate symptoms of depression \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e and demonstrate potential efficacy in the treatment of neuropathic pain and seizures \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Harde E's team has discovered that selective HCN1 inhibitors enhance working memory in rats \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Currently, ivabradine is the sole FDA-approved HCN channel inhibitor indicated for the treatment of angina \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Ivabradine has been shown to mitigate ROS accumulation and enhance ATP generation in cardiomyocytes \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, which is crucial for maintaining mitochondrial function \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. However, to date, no HCN-targeting drugs have been developed for the treatment of neurological disorders, including cerebellar degeneration diseases resulting from PCs impairment. Our findings for the first time indicate that long-term CoQ10 supplementation exhibits cardiovascular and mitochondrial protective effects comparable to those of ivabradine, modulates HCN channels, and enhances working memory performance.\u003c/p\u003e\u003cp\u003eOur research has certain limitations. Firstly, we mainly constructed three transgenic mice, namely D\u003cem\u003erp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, cKO PC\u003csup\u003etdTomato\u003c/sup\u003e, and cKO PC\u003csup\u003eMito\u0026minus;GFP\u003c/sup\u003e, for research. We used molecular docking technology to screen for the direct binding and effect of CoQ10 on Coa6 and verified it from molecular biology techniques. However, the downregulation of Coa6 in transgenic mice for re-verification was not in-depth enough. Secondly, we used the model of Drp1-deficient mice and discovered mitochondrial damage and synaptic plasticity damage in PCs through a large number of morphological studies. We took Drp1-deficient mice as the model for studying working memory damage and found that CoQ10 targeted Coa6 to play a role. However, whether there is a direct relationship between Drp1 and Coa6 has not been explored.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e1M/ 2M / 3M\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eone-month-old / two-month-old / three-month-old\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAlzheimer's disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCETSA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCellular thermal shift assay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCm\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emembrane capacitance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCoQ10\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoenzyme Q10\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eComplex I/II/III/IV/V\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCI-CV\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCOX4\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytochrome c oxidase 4\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eComparative Toxicogenomics database\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDARTS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDrug affinity responsive target stability\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDrp1 / DNM1L\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edynamin-related protein 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edimethyl sulfoxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUS Food and Drug Administration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO-BP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGO biological processes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO-CC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGO cellular components\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO-MF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGO molecular function\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGPx1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eglutathione peroxidase 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Set Enrichment Analysis database\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHCN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehyperpolarization activated cyclic nucleotide gated channels\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHuntington's disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIh\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einward hyperpolarization-activated current\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eknock-out\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMiNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emitochondrial network analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMMP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emitochondrial membrane potential\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ens\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eno significance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOXPHOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eoxidative phosphorylation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eParkinson's disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePKA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProtein Kinase A\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePPI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProtein-Protein Interaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePurkinje cells\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePCs\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRa\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emembrane resistance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRm\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emembrane resistance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereactive oxygen species\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSOD1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esuperoxide dismutase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSPR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSurface plasmon resonance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSwiss Target Prediction database\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003etau\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emembrane time constant\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal testing protocols were approved by Fourth Military Medical University\u0026rsquo;s Animal Care and Use Committee (IACUC-20190107) and were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets during and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere thanks to all the personnel at the Teaching Laboratory Center of the Air Force Medical University, and the Neurobiology Laboratory for their support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82201627) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2022JQ820) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2024JCZDXM60) by Yanling Yang, the New Clinical Technology of Xi-Jing Hospital (2023XJSY27) by Yanling Yang, Military Medicine Promotion Program of Air Force Military Medical University (2020SWAQ04) by Yayun Wang and Shaanxi Provincial Innovation Capability Support Program (2023CXPT33) by Yayun Wang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: YY Wang, YL Yang\u003c/p\u003e\n\u003cp\u003eMethodology: JJ TIE, SJ LI\u003c/p\u003e\n\u003cp\u003eFunding acquisition: FF WU, YY Wang, YL Yang\u003c/p\u003e\n\u003cp\u003eSupervision: YY Wang\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: JJ TIE, SJ LI, X HUANG, ZW NI, CL ZHU\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: KK REN, XD LI, H LIU\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003cstrong\u003e\u003cbr /\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlobal. regional, and national burden of disorders affecting the nervous system, 1990\u0026ndash;2021: a systematic analysis for the Global Burden of Disease Study 2021. 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Int J Mol Sci 2020; 21(21).\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":true,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Purkinje cells, Working memory, Coenzyme Q10, Dynamin-related protein 1, Cytochrome c oxidase assembly factor 6.","lastPublishedDoi":"10.21203/rs.3.rs-7362769/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7362769/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCoenzyme Q10 (CoQ10), the third most popular dietary supplement worldwide, shows promise in treating the ten leading non-communicable diseases linked to global mortality. However, its mechanism and potential to address memory deficits caused by cerebellar injury are not fully understood. We explored whether long-term CoQ10 supplementation could help recover working memory loss and examined the underlying mechanisms. Network pharmacology analysis identified DNM1L/Drp1 as a important genetic target of CoQ10 in cerebellar injury-related memory impairment. We generated three lines of mice with Purkinje cell (PC)-specific deficiency in Drp1 (Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice). Multi-level assessments showed that these mice exhibited: Progressive working memory deficits (assessed via multiple behavioral tests); impaired PC plasticity (evaluated by patch-clamp recordings and morphological analysis); and disrupted mitochondrial membranes (MMs) stability and oxidative phosphorylation (OXPHOS), particularly in complexes III-V (CIII-CV) (assessed through various structural and functional assays). Long-term CoQ10 administration in drinking water for 75 days, beginning at postnatal day 15, effectively ameliorated working memory impairments (5-fold in the percentage of 45\u0026deg; searches), PC plasticity, and mitochondrial dysfunction in Drp1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice at the animal, cellular, and organelle levels. Furthermore, comprehensive drug-target fishing analyses including thermal proteome profiling (TPP), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), surface plasmon resonance (SPR), and molecular docking demonstrated that CoQ10 directly binds to cytochrome c oxidase assembly factor 6 (Coa6). This CoQ10-Coa6 interaction restored MMs stability and CIII-CV activity, revealing the Drp1-CoQ10-Coa6-electron transport chain (ETC) axis as a promising therapeutic target for memory disorders associated with neurological diseases.\u003c/p\u003e","manuscriptTitle":"The Drp1-CoQ10-Coa6-ETC axis represents a therapeutic target for working memory impairment caused by neuronal mitochondrial dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 08:12:07","doi":"10.21203/rs.3.rs-7362769/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-20T09:18:12+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-18T09:03:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Translational Neurodegeneration","date":"2025-08-18T02:57:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T09:57:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Neurodegeneration","date":"2025-08-13T04:41:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43472571-3931-4cdf-a35a-270803914191","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:02:43+00:00","versionOfRecord":{"articleIdentity":"rs-7362769","link":"https://doi.org/10.1186/s40035-026-00552-6","journal":{"identity":"translational-neurodegeneration","isVorOnly":false,"title":"Translational Neurodegeneration"},"publishedOn":"2026-04-27 15:58:26","publishedOnDateReadable":"April 27th, 2026"},"versionCreatedAt":"2025-09-29 08:12:07","video":"","vorDoi":"10.1186/s40035-026-00552-6","vorDoiUrl":"https://doi.org/10.1186/s40035-026-00552-6","workflowStages":[]},"version":"v1","identity":"rs-7362769","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7362769","identity":"rs-7362769","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}