Urolithin A improves motor dysfunction induced by copper exposure in SOD1 G93A transgenic mice via activation of mitophagy

Urolithin A improves motor dysfunction induced by copper exposure in SOD1 G93A transgenic mice via activation of mitophagy | 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 Urolithin A improves motor dysfunction induced by copper exposure in SOD1 G93A transgenic mice via activation of mitophagy Huan Zhang, Chuanyue Gao, Deguang Yang, Lulin Nie, Kaiwu He, Chongyang Chen, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4460797/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Molecular Neurobiology → Version 1 posted 7 You are reading this latest preprint version Abstract Aim Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease pathologically characterized by selective degeneration of motor neurons resulting in a catastrophic loss of motor function. The present study aimed to investigate the effect of copper (Cu) exposure on progression of ALS and explore the therapeutic effect and mechanism of Urolithin A (UA) on ALS. Methods 0.13 PPM copper chloride drinking water was administrated in SOD1 G93A transgenic mice at 6 weeks, UA at a dosage of 50 mg/kg/day was given for 6 weeks after a 7-week Cu exposure. Motor ability was assessed before terminal anesthesia. Muscle atrophy and fibrosis, motor neurons, astrocytes and microglia in the spinal cord were evaluated by H&E, Masson, Sirius Red, Nissl and Immunohistochemistry Staining. Proteomics analysis, Western blotting and ELISA were conducted to detect protein expression. Mitochondrial adenosine triphosphate (ATP) and malondialdehyde (MDA) levels were measured using an assay kit. Results Cu-exposure worsened motor function, promoted muscle fibrosis, loss of motor neurons, and astrocyte and microglial activation. It also induced abnormal changes in mitochondria-related biological processes, leading to a significant reduction in ATP levels and an increase in MDA levels. Upregulation of P62 and downregulation of Parkin, PINK1, and LAMP1 were revealed in SOD1 G93A mice with Cu exposure. Administration of UA activated mitophagy, modulated mitochondria dysfunction, reduced neuroinflammation, and improved gastrocnemius muscle atrophy and motor dysfunction in SOD1 G93A mice with Cu exposure. Conclusions Mitophagy plays critical role in ALS exacerbated by Cu exposure. UA administration may be a promising treatment strategy for ALS. Amyotrophic lateral sclerosis Copper Mitophagy Motor dysfunction Urolithin A Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem, and spinal cord [ 1 ]. Approximately 10% of ALS cases are familial, while the remaining 90% are sporadic [ 2 ]. Clinical features of ALS include progressive muscle atrophy and weakness, dysphagia, and eventually respiratory failure [ 3 , 4 ]. Currently, only riluzole and edaravone have been approved for ALS treatment, but their effectiveness is limited [ 5 ]. Due to the lack of effective treatments, ALS typically leads to death within 3–5 years after the onset of the disease [ 6 ]. Copper (Cu) is involved in various important functions such as respiration, energy production, myelination of neurons, synthesis of neurotransmitters, immune system response, collagen formation, synthetic dyestuff, and tissue repair after trauma [ 7 – 9 ]. It has been shown that Cu homeostasis is disturbed in a mouse model of ALS SOD1. Cu levels were elevated in the spinal cord of transgenic mouse models carrying different SOD1 mutations [ 10 , 11 ]. In the SOD1 G93A mouse model, Cu accumulation preceded the appearance of clinical symptoms, suggesting that high Cu is a characteristic pathology [ 11 ]. The disorder of Cu metabolism is the main cause of the imbalance of Cu metabolism. SOD1 transgenic mice showed increased CTR1 levels and decreased ATP7A levels in spinal cord tissue. In addition, there are high levels of metallothionein (MT) in spinal cord in SOD1 G93A mice, which has a high affinity for Cu [ 12 ]. Mitochondrial dysfunction is a prevalent feature observed in various neurodegenerative diseases, including ALS [13]. It leads to bioenergetic failure, oxidative stress, apoptosis, and potential cell death [ 14 ]. Urolithin A (UA), known as a mitophagy activator, has been investigated for its potential to improve mitochondrial function and reduce inflammation, that captures the attention of scientists [ 15 , 16 ]. In this study, we aimed to investigate the effects of low-dose Cu exposure on the progression of SOD1 G93A mice, a transgenic model of ALS, and to explore the therapeutic effect and mechanism of UA on SOD1 G93A mice with Cu exposure. 2. Materials and Methods 2.1 Reagents and Antibodies Copper chloride (II) (CuCl2) was purchased from Sigma-Aldrich (MO, USA) for this study, while UA (specified purity ≥ 98%) was obtained from Macklin-Lab Company (U884401 Urolithin A, CAS no. 1143-70-0). Antibody dilution ratios and product information can be found in Supplementary Table 1. 2.2 Animal Breeding and Rearing The ALS model mouse, transgenic for human SOD1G93A (B6Cg-Tg (SOD1*G93A)1Gur/J, stock number 004435), which expresses a G93A mutant form of human SOD-1, was purchased from Jackson Laboratory (Maine, USA) along with six-week-old mice. All mice were housed in a 12-hour light-dark cycle chamber with a stable temperature (20–22℃) and humidity (55%) at the Laboratory Animal Center, Shenzhen Center for Disease Control and Prevention, China. Female mice were chosen for this study as male mice tend to be more active and may affect the experimental results by jumping to the ground during behavioral tests. Starting at six weeks of age, SOD1 G93A transgenic mice and WT mice were given 0.13PPM Cu chloride drinking water and normal drinking water treatment [ 17 ]. After 7 weeks of Cu exposure, SOD1 G93A transgenic mice with Cu exposure were given 50 mg/kg/day UA or saline for 6 weeks (Fig. S1 ). During the animal experimentation, we followed the ARRIVE guidelines and conducted the study in accordance with the UK Animals (Scientific Procedures) Act 1986 and relevant guidance, as well as the National Research Council Guide for the Care and Use of Laboratory Animals. 2.3 Behavioral Tests 2.3.1 Climbing-Pole Test The climbing-pole test was conducted to evaluate the movement and coordination of the limbs in mice [ 18 ]. Mice were placed on rough wooden balls with a rough surface and cross-section, and lower rods were placed in the cage. The mice were positioned upside down to climb from the wooden balls below. The time for the mouse to climb the entire stick was recorded by starting the stopwatch when the mouse started climbing (A) and stopping it when the mouse reached the bottom (B). The total climbing time (C) was calculated as C = A - B. The mice underwent three days of training before the test, twice a day, with a 15-second cut-off value. Each mouse was tested three times, and the average climbing time from the three tests was used as a statistical index. 2.3.2 Rotarod Test The rotarod test was performed to assess the coordination of movements in mice [ 19 ]. Mice were placed on a rotating rod with a diameter of 3cm, and the rotation speed was set at 30r/min. Five mice were measured simultaneously, with one mouse in each compartment. The measurement time was 3 minutes, and the time from the start of the rotating rod to the point of dropping off was recorded. The mice underwent training twice a day for three days, with a 300-second threshold. The experiment was repeated three times, and the longest time among the three results was used for evaluation. 2.3.3 Hanging Endurance Test The hanging endurance test was used to assess the grasping strength of mouse limbs. The mouse was placed in the center of a 21cm×21cm grid (with a line width of approximately 0.1cm and a spacing of 0.5cm) and then the grid was inverted to record the time the mouse remained suspended (grasping time). Training was conducted for three consecutive days, twice a day, with a 180-second cut-off value. Any recordings beyond 180 seconds were capped at 180 seconds. The experiment was repeated three times, and the longest time among the three results was used for evaluation. 2.3.4 Grip Strength Test The grip strength test was performed to directly evaluate the muscle strength of mouse limbs [ 20 ]. The mouse was placed in the middle of the grip plate on the gripper, and its tail was gently tugged to help it grasp the grip plate. The mouse would release its paw once it had a solid grip, and the force exerted was measured to assess grip strength. The experiment was conducted three times, and the evaluation value was the average of the results. 2.3.5 Gait Test The gait test is conducted to evaluate the coordination of limb movement in mice [ 21 ]. At different time points, we first observe the mice's autonomous motion and then collect gait information during their walking process. 2.4 H&E, Masson, Sirius Red, and Nissl Staining, Immunohistochemistry Spinal cord specimens were isolated from 4 to 5 lumbar spine segments, fixed in 4% paraformaldehyde for 48 hours, gradually dehydrated using sucrose gradients, and embedded in paraffin. Sections of the spinal cord (5 µm) were stained with specific staining solutions. For immunohistochemistry, sections were boiled in sodium citrate for 10 minutes to repair antigen damage, followed by overnight incubation with primary antibodies (GFAP, Iba1, and ChAT) in 0.3% Triton X-100 phosphate-buffered saline (PBS) at four degrees Celsius. After incubation with primary antibodies, sections were treated with secondary and tertiary antibodies and then stained with DAB for 2–5 minutes. All tissue samples were examined using an optical microscope and analyzed using Image-J software. 2.5 Proteomics 2.5.1 Extraction and Digestion of Proteins Each spinal cord tissue was treated with an 8 M urea lysis buffer for protein extraction, followed by sonication. The concentration of extracted proteins was determined using the BCA Protein Assay Kit (Thermo Fisher, NJ, USA). Five individual samples were pooled together in equal amounts (1: 1: 1: 1: 1) to obtain a total of 100 µg protein in each group for enzymatic digestion and protein labeling. The samples were reduced and alkylated using dithiothreitol (DTT) and iodoacetamide (IAA), respectively, and then incubated with proteases at 37℃. After digestion, formic acid was added to adjust the pH of the sample to 1–2. The peptide mixture was desalted using a reversed-phase column (Oasis HLB, Waters, USA) and dried in a vacuum centrifuge before being labeled with tandem mass tags (TMT). 2.5.2 Analysis using LC-MS/MS and TMT Labeling The peptides from the spinal cord samples were reconstituted, and 50 µL of 200 mM triethyl ammonium bicarbonate (TEAB) was added to each sample. Peptides were labeled according to the instructions provided with the TMT kit and incubated at room temperature for 1 hour. To stop the reaction, 8 µL of 5% hydroxylamine was added and incubated for 15 minutes. The labeled peptides were then desalted, dried, and reconstituted in 100 µL of 0.1% formic acid (FA). High-performance liquid chromatography (HPLC) was used to separate the TMT-labeled peptides based on their components [ 22 ]. The labeled peptide samples were loaded onto an Xbridge BEH300 C18 column (Waters, USA). The peptide sample was isolated using Thermo Fisher Scientific's UltiMate 3000 UHPLC and separated into 15 fractions. The fractions were subsequently dried, reconstituted in 20 µL of 0.1% FA, and subjected to liquid chromatography-LC-MS/MS analysis. All proteomic data were deposited in the PRIDE partner repository under the dataset identifier PXD039728. 2.5.3 Bioinformatics Analysis Differentially expressed proteins were assessed for their abundance in each group using a heat map technique. Protein cluster analysis was performed using Hiplot software ( http://www.chiplot.online ). The biological mechanisms and pathways associated with the differentially expressed proteins were identified using Metascape software. WebGestalt searches and gene ontology analysis can be conducted using DAVID Bioinformatics Resources 6.8. Pictures were visualized using Cytoscape software after MCODE analysis was used to identify dense protein-protein interaction (PPI) regions. 2.6 Western Blot RIPA lysis buffer containing 1x protease and phosphatase inhibitors (Thermo Scientific, NJ, USA) was used to extract proteins from spinal cord tissue. Protein samples were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were then blocked with 5% skim milk for 1–2 hours. Primary antibodies were incubated with the membranes overnight at 4℃. After washing with TBST buffer, the membranes were incubated with corresponding diluted secondary antibodies at room temperature. Chemiluminescence was used to visualize the protein bands using an ECL kit (Thermo Scientific, NJ, USA). 2.7 Mitochondrial Functional Analysis Mitochondrial function in mouse tissues was assessed by measuring lipid peroxidation and ATP levels, following previously published methods [ 23 ]. 2.8 Statistical Analysis All statistical analyses were performed using GraphPad Prism 9.0 statistical software (La Jolla, CA, USA). The data were presented as mean ± SEM and analyzed using one-way ANOVA or Two-Way ANOVA. A p-value of less than 0.05 was considered statistically significant. 3. Results 3.1 Cu exposure exacerbated motor dysfunction in SOD1 G93A mice Compared to WT mice, the body weight of 19-week-old SOD1 G93A mice was significantly decreased, and further significant weight loss was observed in Cu-treated SOD1 G93A mice compared to controls ( P < 0.05) (Fig. 1 A). SOD1 G93A mice showed a significant decrease in motor function in pole-climbing, rotarod, hanging, and grip tests. The Cu-treated group SOD1 G93A mice exhibited even greater motor function decline in the rotarod, and grip tests compared to SOD1 G93A mice ( P < 0.05), but no significant difference was observed in the pole-climbing and hanging tests (Fig. 1 B-E). Gait analysis revealed walking impairments in SOD1 G93A mice, with significantly reduced stride length and base of support compared to WT mice. Cu treatment further exacerbated these walking abnormalities ( P < 0.05, P < 0.001) (Fig. 1 F-H). 3.2 Cu exposure exacerbated muscle atrophy and fibrosis in SOD1 G93A mice At 19 weeks, the weight of the gastrocnemius muscle in SOD1 G93A mice was significantly lower than in WT mice, and this reduction was further pronounced in the Cu-treated SOD1 G93A mice group. Histological staining with H&E, Masson, and Sirius Red revealed significant muscle atrophy and reduced cross-sectional area in the gastrocnemius muscle of the SOD1 G93A group compared to the WT group. These changes were more prominent after Cu treatment ( P < 0.05) (Fig. 2 ). 3.3 Cu exposure exacerbated motor neuron loss and neuroinflammation in SOD1 G93A mice Immunostaining of the spinal cord revealed a significant increase in the number of GFAP + and Iba-1 + cells in the Cu-treated SOD1 G93A mice group compared to the control group. Additionally, there was a significant decrease in motor neurons observed through Nissl staining and ChAT immunostaining in the spinal cord of Cu-treated SOD1 G93A mice ( P < 0.05, P < 0.01, P < 0.001) (Fig. 3 ). 3.4 Cu exposure disrupted protein networks and impaired mitochondrial function Proteins were collected from the spinal cord to investigate abnormal protein networks following Cu exposure, and LC-MS/MS analysis was performed. Hiplot software was used for cluster analysis, and the differentially expressed proteins (DEPs) from WT, SOD1 G93A mice, and Cu-treated SOD1 G93A mice groups were divided into six clusters. Cluster 1 showed an upward trend in DEPs, while cluster 6 showed a downward trend (Fig. 4 A-B) (Fig. S2 ). GO analysis identified essential biological processes within these six clusters. Metascape analysis revealed that spinal cord proteins in SOD1 G93A mice, after exposure to Cu, displayed abnormal expression in mitochondria-related biological processes, such as inflammatory immune response and mitochondrial electron respiratory transport chain (Fig. 4 C). Compared to the SOD1 G93A mice group, ATP levels significantly decreased, and MDA levels significantly increased in the Cu-treated SOD1 G93A mice group ( P < 0.05, P < 0.01) (Fig. 4 D, 4 E). Heat map analysis demonstrated a decrease in the expression of mitochondria-related proteins following Cu exposure (Fig. S3). 3.5 Cu exposure inhibited mitophagy in SOD1 G93A mice Mitophagy is a process that removes excessive or damaged mitochondria. The impact of low-dose Cu exposure on mitophagy was assessed using western blot analysis. Compared to the WT mice group, upregulation of P62 and downregulation of LC3II/LC3I, Parkin, PINK1, TOM40, P-AMPKα/AMPKα, LAMP1, and CTSD were observed in SOD1 G93A mice. Furthermore, there was further upregulation of P62 and downregulation of Parkin, PINK1, and LAMP1 in SOD1 G93A mice exposed to Cu ( P < 0.05, P < 0.01) (Fig. 5 A-B), indicating inhibition of autophagy and mitophagy after Cu exposure. Expression levels of electron transport chain proteins, such as NDUFA10, SDHB, UQCRFS1, and ATP5a, were significantly decreased in the SOD1 G93A mice group compared to the WT mice group. Additionally, NDUFA10 and ATP5a were significantly decreased in SOD1 G93A mice exposed to Cu ( P < 0.05, P < 0.01) (Fig. 5 C, 5 D). 3.6 UA improved motor function in Cu-exposed SOD1 G93A mice by activating mitophagy UA, a mitophagy activator [ 24 ], was administered to Cu-exposed SOD1 G93A mice for 6 weeks starting at 13 weeks. Compared to Cu-exposed SOD1 G93A mice, UA administration significantly improved body weight gain, performance on the rotarod, hang test, and grip strength ( P < 0.05, P < 0.01, P < 0.001) (Fig. 6 A-E). Gait analysis demonstrated that UA treatment improved walking impairments in Cu-exposed SOD1 G93A mice, with significant increases in stride length and base of support observed in SOD1 G93A mice with UA administration ( P < 0.05, P < 0.001) (Fig. 6 F-H). At 19 weeks, compared to the Cu-exposed SOD1 G93A mice group, the muscle weight/body weight ratio significantly increased with UA administration ( P < 0.05) (Fig. 7 A). Histological staining with H&E, Masson, and Sirius Red revealed that gastrocnemius muscle atrophy improved with UA administration (Fig. 7 B-D). Furthermore, compared to the Cu-exposed SOD1 G93A mice group, the number of GFAP + and Iba1 + cells in the spinal cord, as measured by immunostaining, significantly decreased with UA administration. Additionally, there was a significant increase in motor neuron count observed through Nissl staining and ChAT immunostaining in the spinal cord with UA administration ( P < 0.05, P < 0.01) (Fig. 8 ). Proteomic analysis indicated that UA intervention upregulated biological processes related to muscle contraction and mitochondrial function (Fig. 9 A). Mitochondrial functional analysis demonstrated that UA increased ATP levels and significantly decreased lipid peroxidation levels in Cu-exposed SOD1 G93A mice ( P < 0.01) (Fig. 9 D, 9 E). Additionally, UA activated autophagy and mitophagy, as evidenced by the significant increase in expression of PINK1, Parkin, and LAMP1 in the spinal cord, and the increased level of NDUFA10 (an electron transport chain protein) in Cu-exposed SOD1 G93A mice ( P < 0.05) (Fig. 9 B, 9 C). 4. Discussion Motor neuron degeneration in the cortex, brain stem, and spinal cord is a prominent feature of ALS, a fatal neurodegenerative disease characterized by progressive muscle paralysis [ 25 ]. In the present study, low-dose Cu exposure exacerbated motor dysfunction in SOD1 G93A mice, accelerated motor neuron degeneration in the spinal cord, and increased muscle atrophy and fibrosis. The underlying mechanism involved the impairment of mitochondrial function, particularly inhibition of mitophagy. Administration of UA improved motor function in SOD1 G93A mice exposed to Cu by activating mitophagy, highlighting the crucial role of mitophagy in Cu-mediated aggravation of ALS and exploring a promising treatment strategy for ALS. In ALS, alterations in Cu homeostasis may contribute to the disease's development. Spinal cord tissue from sporadic ALS patients has shown a notable increase in Cu concentration [ 26 ]. Elevated blood Cu levels have also been identified as potential risk factors for ALS [ 27 ]. Disruptions in Cu balance can compromise the functionality of enzymes, receptors, and transporter structures, leading to oxidative stress, alpha-synuclein aggregation, fiber formation, and activation of microglia cells [ 28 , 29 ]. In this study, low-dose Cu exposure exacerbated motor decline and associated pathological changes in SOD1 G93A mice, a model of ALS. Furthermore, metascope analysis in Cu-exposed SOD1 G93A mice revealed abnormal expression of proteins related to mitochondrial processes, such as inflammatory immune response and mitochondrial electron respiratory transport chain. Decreased activity of citrate synthase and respiratory chain complexes I + III, II + III, and IV in the spinal cord tissue of ALS patients post-mortem has been illuminated [ 30 – 32 ]. This may be attributed to selective loss of mitochondria or increased mitochondrial DNA damage in the ALS spinal cord [ 33 ]. Maintenance of healthy mitochondria through the process of mitophagy is pivotal in various neurodegenerative diseases such as Alzheimer's, Parkinson's, ALS, frontotemporal dementia, and Huntington's disease. Insufficient mitophagy leads to the accumulation of damaged mitochondria, resulting in increased oxidative stress and reduced ATP levels, leading to cellular damage and apoptosis [ 34 – 36 ]. Excessive accumulation of Cu induces tissue damage by promoting apoptosis and inhibiting mitophagy, along with the down-regulation of autophagy-related proteins, such as Atg5, Beclin1, Pink1, Parkin, P62, and LC3B [ 37 ]. In the present study, this disruption was observed in SOD1 G93A mice exposed to Cu, where a decrease in crucial proteins involved in mitophagy was noted, further exacerbated ATP reduction and oxidative stress. Evidence suggests that Cu exposure disrupts the autophagy-lysosomal pathway in ATP7B-deficient hepatocytes [ 38 ], and chronic Cu exposure may induce pathological damage by interfering with the mitophagy and subsequent apoptosis [ 39 ]. Notably, the activation of mitophagy may serve as an initial response to stress, the subsequent oxidative stress due to mitochondrial dysfunction is a common factor in ALS and other neurodegenerative diseases. During the ALS process, energy metabolism disturbances contribute to activation of astrocyte and microglia, triggering damage in motor neurons mediated by pathways like NF-κB or TGFB through [ 40 – 42 ]. In the context of ALS, exposure to Cu induced mitochondrial dysfunction by hindering mitophagy, resulting in neuron loss in SOD1 G93A mice. In order to determine whether mitophagy plays a decisive role in ALS exacerbated by Cu exposure, UA, a mitophagy activator was administrated and showed beneficial effects by improving the motor function, alleviating muscle atrophy and fibrosis, reducing motor neuron loss, and mitigating neuroinflammation. The therapeutic effect of UA was mediated by activation of autophagy and mitophagy which manifested with increasing the expression of PINK1, Parkin, and LAMP1 in the spinal cord. Previous studies have indicated that UA enhances ATP and NAD + levels by up-regulating Sirtuin 1 and peroxisome proliferator-activated receptor gamma coactivator 1-α, thereby improving skeletal muscle and mitochondrial function [ 15 ]. Additionally, in LPS-stimulated J774.1 mice macrophages, UA has been found to inhibit pro-inflammatory M1 macrophage polarization and the subsequent release of pro-inflammatory cytokines by increasing autophagy flux which prevents nuclear translocation and activation of the AKT/mTOR signaling pathway [ 16 ]. This study marks the first to demonstrate that UA enhances mitochondrial function through mitophagy activation, suppresses inflammation, and delays functional deterioration in Cu-exposed SOD1 G93A mice. These findings offer crucial insights into ALS mechanisms and pave the way for novel ALS treatment approaches. 5. Conclusion Cu exposure in SOD1 G93A mice led to impaired motor function, increased muscle atrophy, and motor neuron loss, primarily mediated by disrupted mitophagy. The detrimental effects were mitigated by UA, a mitophagy activator. This suggests that UA administration holds promise as a potential treatment strategy for ALS (Fig. S4). Abbreviations ALS Amyotrophic lateral sclerosis Cu Copper UA Urolithin A ATP Adenosine triphosphate MDA Malondialdehyde MT Metallothionein DTT Dithiothreitol IAA Iodoacetamide TMT Tandem Mass Tags TEAB Triethyl Ammonium Bicarbonate FA Formic Acid HPLC High-performance liquid chromatography PPI protein-protein interaction Declarations Funding This study was supported in parts by grants from NSFC (82171583); The Key Basic Research Program of Shenzhen Science and Technology Innovation Commission (JCYJ20200109150717745; JCYJ20200109144418639); Shenzhen Key Medical Discipline Construction Fund (SZXK069), Sanming Project of Medicine in Shenzhen (SZSM201611090); Study on Photothermal tumor Vaccine, Shenzhen Science and Technology Innovation Commission (JCYJ20200109120205924). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Authors' contributions L.Z., X.Y. and Z.Z. designed the experiments. H.Z. and C.G. conducted the study. H.Z. and D.Y. wrote the manuscript. L.N. and K.H. revised the manuscript. C.C., S.L. and G.H. contributed to the animal experiment. L.Z., X.H., D.W., J.L., Z.H. and W.L. contributed to literature search, data collection, analysis, and interpretation. All authors read and approved the final manuscript. Data Availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository [43,44] with the dataset identifier PXD039728. Ethics approval and consent to participate All animal experiments comply with the ARRIVE guidelines and should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). 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Genome Res 32:71–84 Frakes AE, Ferraiuolo L, Haidet-Phillips AM et al (2014) Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis. Neuron 81:1009–1023 Ma J, Chen T, Wu S et al (2019) iProX: an integrated proteome resource. Nucleic Acids Res 47:D1211–d1217 Chen T, Ma J, Liu Y et al (2022) iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res 50:D1522–d1527 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable.xlsx Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 03 Jul, 2024 Reviews received at journal 29 Jun, 2024 Reviewers agreed at journal 07 Jun, 2024 Reviewers invited by journal 06 Jun, 2024 Submission checks completed at journal 28 May, 2024 Editor assigned by journal 28 May, 2024 First submitted to journal 22 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4460797","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311947978,"identity":"bfa50849-38d7-4abf-bb0f-20a37d7e09aa","order_by":0,"name":"Huan Zhang","email":"","orcid":"","institution":"University of South China, Hunan Hengyang","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Zhang","suffix":""},{"id":311947979,"identity":"e7eed34f-98fb-458b-8d9c-d84d65169928","order_by":1,"name":"Chuanyue Gao","email":"","orcid":"","institution":"Xi’an International Medical Center 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12:12:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4460797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4460797/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-024-04473-1","type":"published","date":"2024-09-18T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58071157,"identity":"24156098-4ba8-4a71-a8cb-758717264782","added_by":"auto","created_at":"2024-06-10 18:42:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":442627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCu exposure exacerbates motor performance deficits in SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e (A) Percentage weight gain at 19 weeks. (B) Time on the pole in the pole-climbing test. (C) Retention time on the rotarod in the rotarod test. (D) Hanging endurance time in the hanging test. (E) Grip force in the grip force test. (F) Representative image of gait analysis at 19 weeks, with arrows indicating stride length. (G-H) Statistical analysis of stride length and base of support in gait analysis. Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. N =7-8 for each group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/3f08bad54b8b4f0131a0e98d.png"},{"id":58071661,"identity":"42bb74c9-7fbb-40d7-913c-83ca8213d7fe","added_by":"auto","created_at":"2024-06-10 18:58:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1495553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCu treatment exacerbates muscle atrophy and fibrosis in SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice\u003c/strong\u003e (A) Representative images of the gastrocnemius muscle. (B) Statistics of gastrocnemius weight relative to body weight. (C) Representative images of H\u0026amp;E staining of the gastrocnemius muscle (Original magnification ×10, scale bars: 200μm). (D) Representative images of Masson staining of the gastrocnemius muscle (Original magnification ×10, scale bars: 200μm). (E) Representative images of Sirius Red staining of the gastrocnemius muscle (Original magnification ×10, scale bars: 200μm). Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. N = 6-7 for each group; N= 3 for each group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/2f07f130cd071178b0972c7d.png"},{"id":58071158,"identity":"9b5e8be7-0cf7-473b-a202-55047041b0aa","added_by":"auto","created_at":"2024-06-10 18:42:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1903727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCu treatment increases the number of astrocytes and microglia in the spinal cord of SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice and decreases the number of spinal motor neurons\u003c/strong\u003e (A) Representative images and quantitative analysis of Nissl staining. Dashed boxes on top panels indicate region shown at higher magnification in bottom panels. Magnification = ×20, scale bars: 100μm (top panels), ×100 scale bars: 20μm (bottom panels). (B) Representative images and quantitative analysis of ChAT immunostaining (Original magnification ×20, scale bars: 100μm). (C) Representative images and quantitative analysis of GFAP (astrocytes) immunostaining in the spinal cord. Dashed boxes on top panels indicate region shown at higher magnification in bottom panels. Magnification = ×20, scale bars: 100μm (top panels), ×40 scale bars: 50μm (bottom panels). (D) Representative images and quantitative analysis of Iba1 (microglia) immunostaining in the spinal cord. Dashed boxes on top panels indicate region shown at higher magnification in bottom panels. Magnification = ×20, scale bars: 100μm (top panels), ×40 scale bars: 50μm (bottom panels). Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. N = 3 for each group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/a094676795df3b66433d5139.png"},{"id":58071155,"identity":"5e13f322-231b-4650-9b96-70ab93cdad8d","added_by":"auto","created_at":"2024-06-10 18:42:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":449217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCluster analysis reveals expression profiles and enriched biological processes in WT, SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e, and Cu-exposed SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003egroup\u003c/strong\u003e (A) Clusters 1 of expression profiles from cluster analysis and corresponding enriched biological processes. (B) Clusters 6 of expression profiles from cluster analysis and corresponding enriched biological processes. (C) Core modules and enriched pathways for clusters 1 and 6 in proteomic results. Blue represents low abundance, and red represents high abundance. N = 6 for each group. (D-E) ATP levels and lipid peroxidation levels were measured. Data were expressed as Mean ± SEM. *,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. N= 7 for each group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/deabb1b45a1a27bfdfc21763.png"},{"id":58071156,"identity":"8d2fcfb4-756b-413d-aae3-c36f814a66a6","added_by":"auto","created_at":"2024-06-10 18:42:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":564305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCu treatment inhibits mitophagy in the spinal cord of SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice and impairs mitochondrial function by altering mitochondrial dynamics in the spinal cord\u003c/strong\u003e (A-B) Quantification of AMPKα kinase activity status and expression levels of P62, PINK1, Parkin, LAMP1, and other mitophagy and autophagy-lysosomal related proteins. (C-D) Western blot and quantitative analysis of electron transport chain proteins NDUFA10, SDHB, UQCRFS1, and ATP5a. Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. WT group; #, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. ALS vehicle group. N= 3 for each group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/2707d1af762696d32a51ce4c.png"},{"id":58071161,"identity":"2995bec3-445d-4993-aaf3-6cfc98624189","added_by":"auto","created_at":"2024-06-10 18:42:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":386262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUA improves motor performance in Cu-exposed SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice \u003c/strong\u003e(A) Percentage weight gain at 19 weeks. (B) Time on the pole in the pole-climbing test. (C) Retention time on the rotarod in the rotarod test. (D) Hanging endurance time in the hanging test. (E) Grip force in the grip force test. (F) Representative image of gait analysis at 19 weeks, with arrows indicating stride length. (G-H) Statistical analysis of stride length and base of support in gait analysis. Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/15f822fdf4626c301207fd91.png"},{"id":58071400,"identity":"188da975-e280-462e-8696-49297178c7de","added_by":"auto","created_at":"2024-06-10 18:50:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1046317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUA improves muscle atrophy and fibrosis in Cu-exposed SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e (A) Statistics of gastrocnemius weight relative to body weight. (B) Representative images of H\u0026amp;E staining of the gastrocnemius muscle (Original magnification ×10, scale bars: 200μm). (C) Representative images of Masson staining of the gastrocnemius muscle (Original magnification ×10, scale bars: 200μm). (D) Representative images of Sirius Red staining of the gastrocnemius muscle (Original magnification ×10, scale bars: 200μm). Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. N = 6-8 for each group; N= 3 for each group.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/7840afb852b9941fb88b6f81.png"},{"id":58071163,"identity":"278696e6-ac4e-4a02-843a-351870b320d3","added_by":"auto","created_at":"2024-06-10 18:42:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1232064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUA decreases the number of astrocytes and microglia in the spinal cord of Cu-exposed SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice and increases the number of spinal motor neurons\u003c/strong\u003e (A) Representative images and quantitative analysis of Nissl staining. Dashed boxes on top panels indicate region shown at higher magnification in bottom panels. Magnification = ×20, scale bars: 100μm (top panels), ×100 scale bars: 20μm (bottom panels). (B) Representative images and quantitative analysis of ChAT immunostaining (Original magnification ×20, scale bars: 100μm). (C) Representative images and quantitative analysis of GFAP positive immunostaining in the spinal cord. Dashed boxes on top panels indicate region shown at higher magnification in bottom panels. Magnification = ×20, scale bars: 100μm (top panels), ×40 scale bars: 50μm (bottom panels). (D) Representative images and quantitative analysis of Iba1 positive immunostaining in the spinal cord. Dashed boxes on top panels indicate region shown at higher magnification in bottom panels. Magnification = ×20, scale bars: 100μm (top panels), ×40 scale bars: 50μm (bottom panels). Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. N = 3 for each group.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/64225719f94a3479c52364c8.png"},{"id":58071162,"identity":"e5d4ad00-3ca8-4f3e-a03e-3048c5118ddc","added_by":"auto","created_at":"2024-06-10 18:42:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":548965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUA activates mitophagy in the spinal cord of Cu-exposed SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice and improves mitochondrial function by altering mitochondrial dynamics in the spinal cord\u003c/strong\u003e (A) Core modules and enriched pathways in proteomic results. Blue represents low abundance, and red represents high abundance. (B, C) Quantification of AMPKα kinase activity state and the expression levels of mitophagy and autophagy-lysosome-related proteins such as PINK1, Parkin, and LAMP1, as well as electron transport chain-related proteins. (D, E) ATP levels and lipid peroxidation levels were measured. Data were expressed as Mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. N = 6 for each group; N= 4 for each group; N= 5-7 for each group.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/249b9c082fdd0ff8a264bec6.png"},{"id":65104219,"identity":"71b61b87-3507-4091-a47c-89e6323e38f0","added_by":"auto","created_at":"2024-09-23 16:12:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9616870,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/ceaec12d-41b5-49e0-815c-b10d77ca86d6.pdf"},{"id":58071402,"identity":"761e77f5-7246-4fcf-8c2d-8746e4e2f889","added_by":"auto","created_at":"2024-06-10 18:50:38","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":352654,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/278a63ef013df2f345a0b0b3.xlsx"},{"id":58071165,"identity":"0ca933d7-98ff-40f6-bbb1-a7fa2cc8c8fd","added_by":"auto","created_at":"2024-06-10 18:42:38","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":859455,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4460797/v1/ad41ea9402c9d5df6c8bbd4d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Urolithin A improves motor dysfunction induced by copper exposure in SOD1 G93A transgenic mice via activation of mitophagy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem, and spinal cord [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 10% of ALS cases are familial, while the remaining 90% are sporadic [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Clinical features of ALS include progressive muscle atrophy and weakness, dysphagia, and eventually respiratory failure [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Currently, only riluzole and edaravone have been approved for ALS treatment, but their effectiveness is limited [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to the lack of effective treatments, ALS typically leads to death within 3\u0026ndash;5 years after the onset of the disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCopper (Cu) is involved in various important functions such as respiration, energy production, myelination of neurons, synthesis of neurotransmitters, immune system response, collagen formation, synthetic dyestuff, and tissue repair after trauma [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It has been shown that Cu homeostasis is disturbed in a mouse model of ALS SOD1. Cu levels were elevated in the spinal cord of transgenic mouse models carrying different SOD1 mutations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In the SOD1\u003csup\u003eG93A\u003c/sup\u003e mouse model, Cu accumulation preceded the appearance of clinical symptoms, suggesting that high Cu is a characteristic pathology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The disorder of Cu metabolism is the main cause of the imbalance of Cu metabolism. SOD1 transgenic mice showed increased CTR1 levels and decreased ATP7A levels in spinal cord tissue. In addition, there are high levels of metallothionein (MT) in spinal cord in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, which has a high affinity for Cu [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction is a prevalent feature observed in various neurodegenerative diseases, including ALS [13]. It leads to bioenergetic failure, oxidative stress, apoptosis, and potential cell death [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Urolithin A (UA), known as a mitophagy activator, has been investigated for its potential to improve mitochondrial function and reduce inflammation, that captures the attention of scientists [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the effects of low-dose Cu exposure on the progression of SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, a transgenic model of ALS, and to explore the therapeutic effect and mechanism of UA on SOD1\u003csup\u003eG93A\u003c/sup\u003e mice with Cu exposure.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents and Antibodies\u003c/h2\u003e \u003cp\u003eCopper chloride (II) (CuCl2) was purchased from Sigma-Aldrich (MO, USA) for this study, while UA (specified purity\u0026thinsp;\u0026ge;\u0026thinsp;98%) was obtained from Macklin-Lab Company (U884401 Urolithin A, CAS no. 1143-70-0). Antibody dilution ratios and product information can be found in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animal Breeding and Rearing\u003c/h2\u003e \u003cp\u003eThe ALS model mouse, transgenic for human SOD1G93A (B6Cg-Tg (SOD1*G93A)1Gur/J, stock number 004435), which expresses a G93A mutant form of human SOD-1, was purchased from Jackson Laboratory (Maine, USA) along with six-week-old mice. All mice were housed in a 12-hour light-dark cycle chamber with a stable temperature (20\u0026ndash;22℃) and humidity (55%) at the Laboratory Animal Center, Shenzhen Center for Disease Control and Prevention, China. Female mice were chosen for this study as male mice tend to be more active and may affect the experimental results by jumping to the ground during behavioral tests. Starting at six weeks of age, SOD1\u003csup\u003eG93A\u003c/sup\u003e transgenic mice and WT mice were given 0.13PPM Cu chloride drinking water and normal drinking water treatment [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. After 7 weeks of Cu exposure, SOD1\u003csup\u003eG93A\u003c/sup\u003e transgenic mice with Cu exposure were given 50 mg/kg/day UA or saline for 6 weeks (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). During the animal experimentation, we followed the ARRIVE guidelines and conducted the study in accordance with the UK Animals (Scientific Procedures) Act 1986 and relevant guidance, as well as the National Research Council Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Behavioral Tests\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Climbing-Pole Test\u003c/h2\u003e \u003cp\u003eThe climbing-pole test was conducted to evaluate the movement and coordination of the limbs in mice [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Mice were placed on rough wooden balls with a rough surface and cross-section, and lower rods were placed in the cage. The mice were positioned upside down to climb from the wooden balls below. The time for the mouse to climb the entire stick was recorded by starting the stopwatch when the mouse started climbing (A) and stopping it when the mouse reached the bottom (B). The total climbing time (C) was calculated as C\u0026thinsp;=\u0026thinsp;A - B. The mice underwent three days of training before the test, twice a day, with a 15-second cut-off value. Each mouse was tested three times, and the average climbing time from the three tests was used as a statistical index.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Rotarod Test\u003c/h2\u003e \u003cp\u003eThe rotarod test was performed to assess the coordination of movements in mice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Mice were placed on a rotating rod with a diameter of 3cm, and the rotation speed was set at 30r/min. Five mice were measured simultaneously, with one mouse in each compartment. The measurement time was 3 minutes, and the time from the start of the rotating rod to the point of dropping off was recorded. The mice underwent training twice a day for three days, with a 300-second threshold. The experiment was repeated three times, and the longest time among the three results was used for evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Hanging Endurance Test\u003c/h2\u003e \u003cp\u003eThe hanging endurance test was used to assess the grasping strength of mouse limbs. The mouse was placed in the center of a 21cm\u0026times;21cm grid (with a line width of approximately 0.1cm and a spacing of 0.5cm) and then the grid was inverted to record the time the mouse remained suspended (grasping time). Training was conducted for three consecutive days, twice a day, with a 180-second cut-off value. Any recordings beyond 180 seconds were capped at 180 seconds. The experiment was repeated three times, and the longest time among the three results was used for evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 Grip Strength Test\u003c/h2\u003e \u003cp\u003eThe grip strength test was performed to directly evaluate the muscle strength of mouse limbs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The mouse was placed in the middle of the grip plate on the gripper, and its tail was gently tugged to help it grasp the grip plate. The mouse would release its paw once it had a solid grip, and the force exerted was measured to assess grip strength. The experiment was conducted three times, and the evaluation value was the average of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5 Gait Test\u003c/h2\u003e \u003cp\u003eThe gait test is conducted to evaluate the coordination of limb movement in mice [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. At different time points, we first observe the mice's autonomous motion and then collect gait information during their walking process.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4 H\u0026amp;E, Masson, Sirius Red, and Nissl Staining, Immunohistochemistry\u003c/h2\u003e \u003cp\u003eSpinal cord specimens were isolated from 4 to 5 lumbar spine segments, fixed in 4% paraformaldehyde for 48 hours, gradually dehydrated using sucrose gradients, and embedded in paraffin. Sections of the spinal cord (5 \u0026micro;m) were stained with specific staining solutions. For immunohistochemistry, sections were boiled in sodium citrate for 10 minutes to repair antigen damage, followed by overnight incubation with primary antibodies (GFAP, Iba1, and ChAT) in 0.3% Triton X-100 phosphate-buffered saline (PBS) at four degrees Celsius. After incubation with primary antibodies, sections were treated with secondary and tertiary antibodies and then stained with DAB for 2\u0026ndash;5 minutes. All tissue samples were examined using an optical microscope and analyzed using Image-J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Proteomics\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Extraction and Digestion of Proteins\u003c/h2\u003e \u003cp\u003eEach spinal cord tissue was treated with an 8 M urea lysis buffer for protein extraction, followed by sonication. The concentration of extracted proteins was determined using the BCA Protein Assay Kit (Thermo Fisher, NJ, USA). Five individual samples were pooled together in equal amounts (1: 1: 1: 1: 1) to obtain a total of 100 \u0026micro;g protein in each group for enzymatic digestion and protein labeling. The samples were reduced and alkylated using dithiothreitol (DTT) and iodoacetamide (IAA), respectively, and then incubated with proteases at 37℃. After digestion, formic acid was added to adjust the pH of the sample to 1\u0026ndash;2. The peptide mixture was desalted using a reversed-phase column (Oasis HLB, Waters, USA) and dried in a vacuum centrifuge before being labeled with tandem mass tags (TMT).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Analysis using LC-MS/MS and TMT Labeling\u003c/h2\u003e \u003cp\u003eThe peptides from the spinal cord samples were reconstituted, and 50 \u0026micro;L of 200 mM triethyl ammonium bicarbonate (TEAB) was added to each sample. Peptides were labeled according to the instructions provided with the TMT kit and incubated at room temperature for 1 hour. To stop the reaction, 8 \u0026micro;L of 5% hydroxylamine was added and incubated for 15 minutes. The labeled peptides were then desalted, dried, and reconstituted in 100 \u0026micro;L of 0.1% formic acid (FA). High-performance liquid chromatography (HPLC) was used to separate the TMT-labeled peptides based on their components [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The labeled peptide samples were loaded onto an Xbridge BEH300 C18 column (Waters, USA). The peptide sample was isolated using Thermo Fisher Scientific's UltiMate 3000 UHPLC and separated into 15 fractions. The fractions were subsequently dried, reconstituted in 20 \u0026micro;L of 0.1% FA, and subjected to liquid chromatography-LC-MS/MS analysis. All proteomic data were deposited in the PRIDE partner repository under the dataset identifier PXD039728.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Bioinformatics Analysis\u003c/h2\u003e \u003cp\u003eDifferentially expressed proteins were assessed for their abundance in each group using a heat map technique. Protein cluster analysis was performed using Hiplot software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.chiplot.online\u003c/span\u003e\u003cspan address=\"http://www.chiplot.online\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The biological mechanisms and pathways associated with the differentially expressed proteins were identified using Metascape software. WebGestalt searches and gene ontology analysis can be conducted using DAVID Bioinformatics Resources 6.8. Pictures were visualized using Cytoscape software after MCODE analysis was used to identify dense protein-protein interaction (PPI) regions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western Blot\u003c/h2\u003e \u003cp\u003eRIPA lysis buffer containing 1x protease and phosphatase inhibitors (Thermo Scientific, NJ, USA) was used to extract proteins from spinal cord tissue. Protein samples were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were then blocked with 5% skim milk for 1\u0026ndash;2 hours. Primary antibodies were incubated with the membranes overnight at 4℃. After washing with TBST buffer, the membranes were incubated with corresponding diluted secondary antibodies at room temperature. Chemiluminescence was used to visualize the protein bands using an ECL kit (Thermo Scientific, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Mitochondrial Functional Analysis\u003c/h2\u003e \u003cp\u003eMitochondrial function in mouse tissues was assessed by measuring lipid peroxidation and ATP levels, following previously published methods [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 9.0 statistical software (La Jolla, CA, USA). The data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and analyzed using one-way ANOVA or Two-Way ANOVA. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cu exposure exacerbated motor dysfunction in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eCompared to WT mice, the body weight of 19-week-old SOD1\u003csup\u003eG93A\u003c/sup\u003e mice was significantly decreased, and further significant weight loss was observed in Cu-treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice compared to controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). SOD1\u003csup\u003eG93A\u003c/sup\u003e mice showed a significant decrease in motor function in pole-climbing, rotarod, hanging, and grip tests. The Cu-treated group SOD1\u003csup\u003eG93A\u003c/sup\u003e mice exhibited even greater motor function decline in the rotarod, and grip tests compared to SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but no significant difference was observed in the pole-climbing and hanging tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). Gait analysis revealed walking impairments in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, with significantly reduced stride length and base of support compared to WT mice. Cu treatment further exacerbated these walking abnormalities (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Cu exposure exacerbated muscle atrophy and fibrosis in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eAt 19 weeks, the weight of the gastrocnemius muscle in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice was significantly lower than in WT mice, and this reduction was further pronounced in the Cu-treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice group. Histological staining with H\u0026amp;E, Masson, and Sirius Red revealed significant muscle atrophy and reduced cross-sectional area in the gastrocnemius muscle of the SOD1\u003csup\u003eG93A\u003c/sup\u003e group compared to the WT group. These changes were more prominent after Cu treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Cu exposure exacerbated motor neuron loss and neuroinflammation in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eImmunostaining of the spinal cord revealed a significant increase in the number of GFAP\u0026thinsp;+\u0026thinsp;and Iba-1\u0026thinsp;+\u0026thinsp;cells in the Cu-treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice group compared to the control group. Additionally, there was a significant decrease in motor neurons observed through Nissl staining and ChAT immunostaining in the spinal cord of Cu-treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cu exposure disrupted protein networks and impaired mitochondrial function\u003c/h2\u003e \u003cp\u003eProteins were collected from the spinal cord to investigate abnormal protein networks following Cu exposure, and LC-MS/MS analysis was performed. Hiplot software was used for cluster analysis, and the differentially expressed proteins (DEPs) from WT, SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, and Cu-treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice groups were divided into six clusters. Cluster 1 showed an upward trend in DEPs, while cluster 6 showed a downward trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B) (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). GO analysis identified essential biological processes within these six clusters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMetascape analysis revealed that spinal cord proteins in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, after exposure to Cu, displayed abnormal expression in mitochondria-related biological processes, such as inflammatory immune response and mitochondrial electron respiratory transport chain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Compared to the SOD1\u003csup\u003eG93A\u003c/sup\u003e mice group, ATP levels significantly decreased, and MDA levels significantly increased in the Cu-treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Heat map analysis demonstrated a decrease in the expression of mitochondria-related proteins following Cu exposure (Fig. S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Cu exposure inhibited mitophagy in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eMitophagy is a process that removes excessive or damaged mitochondria. The impact of low-dose Cu exposure on mitophagy was assessed using western blot analysis. Compared to the WT mice group, upregulation of P62 and downregulation of LC3II/LC3I, Parkin, PINK1, TOM40, P-AMPKα/AMPKα, LAMP1, and CTSD were observed in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. Furthermore, there was further upregulation of P62 and downregulation of Parkin, PINK1, and LAMP1 in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice exposed to Cu (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B), indicating inhibition of autophagy and mitophagy after Cu exposure. Expression levels of electron transport chain proteins, such as NDUFA10, SDHB, UQCRFS1, and ATP5a, were significantly decreased in the SOD1\u003csup\u003eG93A\u003c/sup\u003e mice group compared to the WT mice group. Additionally, NDUFA10 and ATP5a were significantly decreased in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice exposed to Cu (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6 UA improved motor function in Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice by activating mitophagy\u003c/h2\u003e \u003cp\u003eUA, a mitophagy activator [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e24\u003c/span\u003e], was administered to Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice for 6 weeks starting at 13 weeks. Compared to Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, UA administration significantly improved body weight gain, performance on the rotarod, hang test, and grip strength (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E). Gait analysis demonstrated that UA treatment improved walking impairments in Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, with significant increases in stride length and base of support observed in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice with UA administration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 19 weeks, compared to the Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice group, the muscle weight/body weight ratio significantly increased with UA administration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Histological staining with H\u0026amp;E, Masson, and Sirius Red revealed that gastrocnemius muscle atrophy improved with UA administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, compared to the Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice group, the number of GFAP\u0026thinsp;+\u0026thinsp;and Iba1\u0026thinsp;+\u0026thinsp;cells in the spinal cord, as measured by immunostaining, significantly decreased with UA administration. Additionally, there was a significant increase in motor neuron count observed through Nissl staining and ChAT immunostaining in the spinal cord with UA administration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProteomic analysis indicated that UA intervention upregulated biological processes related to muscle contraction and mitochondrial function (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Mitochondrial functional analysis demonstrated that UA increased ATP levels and significantly decreased lipid peroxidation levels in Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). Additionally, UA activated autophagy and mitophagy, as evidenced by the significant increase in expression of PINK1, Parkin, and LAMP1 in the spinal cord, and the increased level of NDUFA10 (an electron transport chain protein) in Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMotor neuron degeneration in the cortex, brain stem, and spinal cord is a prominent feature of ALS, a fatal neurodegenerative disease characterized by progressive muscle paralysis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the present study, low-dose Cu exposure exacerbated motor dysfunction in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, accelerated motor neuron degeneration in the spinal cord, and increased muscle atrophy and fibrosis. The underlying mechanism involved the impairment of mitochondrial function, particularly inhibition of mitophagy. Administration of UA improved motor function in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice exposed to Cu by activating mitophagy, highlighting the crucial role of mitophagy in Cu-mediated aggravation of ALS and exploring a promising treatment strategy for ALS.\u003c/p\u003e \u003cp\u003eIn ALS, alterations in Cu homeostasis may contribute to the disease's development. Spinal cord tissue from sporadic ALS patients has shown a notable increase in Cu concentration [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Elevated blood Cu levels have also been identified as potential risk factors for ALS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Disruptions in Cu balance can compromise the functionality of enzymes, receptors, and transporter structures, leading to oxidative stress, alpha-synuclein aggregation, fiber formation, and activation of microglia cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, low-dose Cu exposure exacerbated motor decline and associated pathological changes in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, a model of ALS. Furthermore, metascope analysis in Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice revealed abnormal expression of proteins related to mitochondrial processes, such as inflammatory immune response and mitochondrial electron respiratory transport chain. Decreased activity of citrate synthase and respiratory chain complexes I\u0026thinsp;+\u0026thinsp;III, II\u0026thinsp;+\u0026thinsp;III, and IV in the spinal cord tissue of ALS patients post-mortem has been illuminated [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This may be attributed to selective loss of mitochondria or increased mitochondrial DNA damage in the ALS spinal cord [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMaintenance of healthy mitochondria through the process of mitophagy is pivotal in various neurodegenerative diseases such as Alzheimer's, Parkinson's, ALS, frontotemporal dementia, and Huntington's disease. Insufficient mitophagy leads to the accumulation of damaged mitochondria, resulting in increased oxidative stress and reduced ATP levels, leading to cellular damage and apoptosis [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Excessive accumulation of Cu induces tissue damage by promoting apoptosis and inhibiting mitophagy, along with the down-regulation of autophagy-related proteins, such as Atg5, Beclin1, Pink1, Parkin, P62, and LC3B [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, this disruption was observed in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice exposed to Cu, where a decrease in crucial proteins involved in mitophagy was noted, further exacerbated ATP reduction and oxidative stress. Evidence suggests that Cu exposure disrupts the autophagy-lysosomal pathway in ATP7B-deficient hepatocytes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and chronic Cu exposure may induce pathological damage by interfering with the mitophagy and subsequent apoptosis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Notably, the activation of mitophagy may serve as an initial response to stress, the subsequent oxidative stress due to mitochondrial dysfunction is a common factor in ALS and other neurodegenerative diseases. During the ALS process, energy metabolism disturbances contribute to activation of astrocyte and microglia, triggering damage in motor neurons mediated by pathways like NF-κB or TGFB through [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the context of ALS, exposure to Cu induced mitochondrial dysfunction by hindering mitophagy, resulting in neuron loss in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eIn order to determine whether mitophagy plays a decisive role in ALS exacerbated by Cu exposure, UA, a mitophagy activator was administrated and showed beneficial effects by improving the motor function, alleviating muscle atrophy and fibrosis, reducing motor neuron loss, and mitigating neuroinflammation. The therapeutic effect of UA was mediated by activation of autophagy and mitophagy which manifested with increasing the expression of PINK1, Parkin, and LAMP1 in the spinal cord. Previous studies have indicated that UA enhances ATP and NAD\u0026thinsp;+\u0026thinsp;levels by up-regulating Sirtuin 1 and peroxisome proliferator-activated receptor gamma coactivator 1-α, thereby improving skeletal muscle and mitochondrial function [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, in LPS-stimulated J774.1 mice macrophages, UA has been found to inhibit pro-inflammatory M1 macrophage polarization and the subsequent release of pro-inflammatory cytokines by increasing autophagy flux which prevents nuclear translocation and activation of the AKT/mTOR signaling pathway [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This study marks the first to demonstrate that UA enhances mitochondrial function through mitophagy activation, suppresses inflammation, and delays functional deterioration in Cu-exposed SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. These findings offer crucial insights into ALS mechanisms and pave the way for novel ALS treatment approaches.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eCu exposure in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice led to impaired motor function, increased muscle atrophy, and motor neuron loss, primarily mediated by disrupted mitophagy. The detrimental effects were mitigated by UA, a mitophagy activator. This suggests that UA administration holds promise as a potential treatment strategy for ALS (Fig. S4).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmyotrophic lateral sclerosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCu\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCopper\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUrolithin A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenosine triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMetallothionein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDithiothreitol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIodoacetamide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTandem Mass Tags\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEAB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTriethyl Ammonium Bicarbonate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFormic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-performance liquid chromatography\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 \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported in parts by grants from NSFC (82171583); The Key Basic Research Program of Shenzhen Science and Technology Innovation Commission (JCYJ20200109150717745; JCYJ20200109144418639);\u0026nbsp;Shenzhen Key Medical Discipline Construction Fund (SZXK069), Sanming Project of Medicine in Shenzhen (SZSM201611090); Study on Photothermal tumor Vaccine, Shenzhen Science and Technology Innovation Commission (JCYJ20200109120205924).\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eL.Z., X.Y. and Z.Z. designed the experiments. H.Z. and C.G. conducted the study. H.Z. and D.Y. wrote the manuscript. L.N. and K.H. revised the manuscript. C.C., S.L. and G.H. contributed to the animal experiment. L.Z., X.H., D.W., J.L., Z.H. and W.L. contributed to literature search, data collection, analysis, and interpretation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository [43,44]\u0026nbsp;with the dataset identifier\u0026nbsp;PXD039728.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll animal experiments comply with\u0026nbsp;the ARRIVE guidelines and should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023,\u0026nbsp;revised 1978).\u0026nbsp;The animal in this study is female, because male mice are active and tend to jump to the ground in behavioral tests, which affects the experimental results, so female mice are selected.\u0026nbsp;All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available.\u0026nbsp;Moreover, Institutional Animal Care and Use/Ethics Committee, Shenzhen Center for Disease Control and Prevention\u0026nbsp;approved the animal experiments (Approval NO: 2023007, Approval date: Mar.16.2023).\u003c/p\u003e\n\u003cp\u003eConsent to participate\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003eConsent to publish\u003c/p\u003e\n\u003cp\u003eThe content of the manuscript is confirmed to have obtained the publication consent of all participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePerera ND, Sheean RK, Lau CL et al (2018) Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression. 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Nucleic Acids Res 50:D1522\u0026ndash;d1527\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Amyotrophic lateral sclerosis, Copper, Mitophagy, Motor dysfunction, Urolithin A","lastPublishedDoi":"10.21203/rs.3.rs-4460797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4460797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease pathologically characterized by selective degeneration of motor neurons resulting in a catastrophic loss of motor function. The present study aimed to investigate the effect of copper (Cu) exposure on progression of ALS and explore the therapeutic effect and mechanism of Urolithin A (UA) on ALS.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e0.13 PPM copper chloride drinking water was administrated in SOD1\u003csup\u003eG93A\u003c/sup\u003e transgenic mice at 6 weeks, UA at a dosage of 50 mg/kg/day was given for 6 weeks after a 7-week Cu exposure. Motor ability was assessed before terminal anesthesia. Muscle atrophy and fibrosis, motor neurons, astrocytes and microglia in the spinal cord were evaluated by H\u0026amp;E, Masson, Sirius Red, Nissl and Immunohistochemistry Staining. Proteomics analysis, Western blotting and ELISA were conducted to detect protein expression. Mitochondrial adenosine triphosphate (ATP) and malondialdehyde (MDA) levels were measured using an assay kit.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCu-exposure worsened motor function, promoted muscle fibrosis, loss of motor neurons, and astrocyte and microglial activation. It also induced abnormal changes in mitochondria-related biological processes, leading to a significant reduction in ATP levels and an increase in MDA levels. Upregulation of P62 and downregulation of Parkin, PINK1, and LAMP1 were revealed in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice with Cu exposure. Administration of UA activated mitophagy, modulated mitochondria dysfunction, reduced neuroinflammation, and improved gastrocnemius muscle atrophy and motor dysfunction in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice with Cu exposure.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMitophagy plays critical role in ALS exacerbated by Cu exposure. UA administration may be a promising treatment strategy for ALS.\u003c/p\u003e","manuscriptTitle":"Urolithin A improves motor dysfunction induced by copper exposure in SOD1 G93A transgenic mice via activation of mitophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 18:42:32","doi":"10.21203/rs.3.rs-4460797/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-03T15:57:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-29T05:49:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180708111524493625683388943422652442673","date":"2024-06-07T15:08:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-07T01:57:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-28T11:26:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-28T11:26:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2024-05-22T12:10:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"916610d8-2f98-4b6f-91a2-a804b56cafd5","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-4460797","link":"https://doi.org/10.1007/s12035-024-04473-1","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2024-09-18 15:57:21","publishedOnDateReadable":"September 18th, 2024"},"versionCreatedAt":"2024-06-10 18:42:32","video":"","vorDoi":"10.1007/s12035-024-04473-1","vorDoiUrl":"https://doi.org/10.1007/s12035-024-04473-1","workflowStages":[]},"version":"v1","identity":"rs-4460797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4460797","identity":"rs-4460797","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}