DJ-1 and ATP synthase are required for local protein synthesis in midbrain dopaminergic neurons

DJ-1 and ATP synthase are required for local protein synthesis in midbrain dopaminergic neurons | 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 Article DJ-1 and ATP synthase are required for local protein synthesis in midbrain dopaminergic neurons Elizabeth Jonas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5984062/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Dopaminergic neurons have extensive neuritic arbors therefore local protein synthesis is required. Familial PD risk gene PARK-7 (DJ-1) is a key regulator of ATP synthase and of dopaminergic growth and survival. We now find that inhibition of ATP synthase function completely abrogates protein synthesis in dopaminergic (mesDA) neuronal processes, and we demonstrate that DJ-1 is an mRNA binding protein for F1FO ATP synthase β subunit mRNA. DJ-1 localizes ATP β mRNA into neurites, thus DJ-1 patient fibroblasts and DJ-1-/- mesDA mouse neurons have low ATP β mRNA and protein, reduced growth and low protein synthesis rates. Over-expression of ATP β rescues these defects. We conclude that DJ-1 is required to optimize the stoichiometric structure of the ATP synthase in the neuritic arbors. This supports the local protein synthesis required for normal neuritic growth. Biological sciences/Neuroscience/Cellular neuroscience Biological sciences/Neuroscience Mitochondria Parkinson’s Disease mRNA binding proteins neuronal outgrowth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Tableofthereagents.pdf Table of the Reagents Fig1ExtendedData1242025.pdf Figure 1. Extended data A. Immunoblots with the anti-Mfn1 or 2 antibodies for WT fibroblasts and mutant patients’ fibroblasts show that Mfn levels are low in mutant fibroblasts compared to WT. GAPDH serves as protein control. B. and C. N=3 independent experiments per group (*p<0.02, **p=0.0023; one way ANOVA with multiple comparisons). Fig2Extended1242025.pdf Fig 2 Extended data A. Mut1 fibroblasts grow slowly compared to WT fibroblasts (n=3 different plates and 3 independent experiments). B. ATP5b OE (lane 3) increases the protein level of ATP synthase α subunit protein (ATP α) more than c-subunit whereas ATP5G OE (c-subunit, lane 2) does not increase the protein levels of F1 components. Immunoblots (IB) with antibody targets for ATP α (anti-α), ATP β (anti-β), and ATP c (anti-c) were used. Lane one is the empty vector control. C. Immunoblot of total cell lysate using anti-puro antibody shows amount of puromycin incorporation over 15 min. into WT and Mut1 fibroblasts. D. Immunoblot of total cell lysate using anti-puro antibody shows puromycin incorporation over 15 min. into WT and Mut1 fibroblasts overexpressing DJ-1 or ATP β. E. Quantification of experiments shown in C. Puromycin incorporation (10 ug/ml for 15 minutes) is low in-patient fibroblasts carrying a mutation in DJ-1 (Mut1) (***p=0.0006, t-test; n=3 samples, all samples normalized to the average of the WT). F. Quantification of experiments shown in D. The defect in the rate of protein synthesis in mutant fibroblasts is rescued by ATP β exogenous OE (effects of WT DJ-1 or ATP β OE were first normalized to GAPDH and then normalized to the corresponding empty vector translation rate (*p<0.02; n=3 independent samples, one-way ANOVA followed by multiple comparisons). Cite Share Download PDF Status: Under Review Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5984062","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":416000494,"identity":"4230f108-4bd8-419c-862e-d5ce55c3e4e4","order_by":0,"name":"Elizabeth Jonas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie3RsUoDMRjA8S8E7HLnrSlS20dIuUGkoq9y4aA3FRwdypESyC3qrG9xY8ccgXY56NrR42bhXMQO1p7XFqd4q2D+BMI3/AJJAGy2v1gHcagXUIDgez4HwPUI2EzwnpAj8aE54VcCcCRNjLcRD2NRbeZX8QVAUd5O4yhdZrMXuBsxbiBdgfjwIR+TSw6h/7TQkzRngkIeGQnViAeu1IQqGJ85J2qSKiYJktpIbmqSfcqvA9nGEV0VyQfamgnFaCZcqfbElTigaybrFzATopHAvTzsprq+i/uoh8/rQpBgEfkm4iVJ+fY6v/bo8p6VznvcP12FWVVNRz0T+Qk7QbMPFBx+tb2OarZ+++k2m832z9oB0Z1ebk/UEIcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1624-5999","institution":"Yale Medical School","correspondingAuthor":true,"prefix":"","firstName":"Elizabeth","middleName":"","lastName":"Jonas","suffix":""}],"badges":[],"createdAt":"2025-02-07 22:30:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5984062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5984062/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76749709,"identity":"265ae5f5-65b9-480f-9ab8-e4bf50e8b634","added_by":"auto","created_at":"2025-02-20 09:46:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":364153,"visible":true,"origin":"","legend":"\u003cp\u003eDJ-1 mutant patients' fibroblasts have abnormal mitochondria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. Immunoblot using anti-DJ-1 antibody showing endogenous DJ-1 levels in fibroblasts. Mut1 has low \u0026nbsp;DJ-1 levels, Mut2 has no detectable DJ-1. GAPDH serves as the protein loading control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. Immunoblot using anti-DJ-1 antibody showing cytosolic and mitochondrial DJ-1 levels in wild type \u0026nbsp;and Mut1 fibroblasts. COXIV and GAPDH serve as protein controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC. Example electron micrographs of WT and DJ-1 mutant patients' fibroblasts showing decreased \u0026nbsp;cristae number and mitochondrial length in the mutants. Images were background-subtracted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. Cristae number per mitochondrion is low in both mutant fibroblast types measured in 10 images for \u0026nbsp;each group. WT n=43 mitochondria; Mut1 n=25 mitochondria; Mut2 n=60 mitochondria \u0026nbsp;(***p=0.0002, **p=0.0089; one-way ANOVA, Tukey's multiple comparisons test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. and F. Mitochondria of Mut2 are small and have an electron dense matrix. WT n=30; Mut1 n=25; \u0026nbsp;Mut2 n=36 (**p=0.0023; ****p\u0026lt;0.0001; one way ANOVA, Tukey's multiple comparisons test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG. Number of mitochondria per cell area per micrograph was elevated in Mut2. WT n=10, Mut1 n=12, \u0026nbsp;Mut2 n=10 micrographs (*p=0.0246; one way ANOVA, Tukey's multiple comparisons).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH. Mitochondrial membrane potential is low in mutant fibroblasts. Shown are flow cytometry readings \u0026nbsp;of intensity of the mitochondrial membrane potential indicator TMRE for each cell type. Blue lines \u0026nbsp;divide the cells into three sub-groups based on the indicator intensity. I. The percentage of total cell number with high or low TMRE signal in each subgroup is plotted from \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig.1H, N = 3 independent experiments (**p \u0026lt; 0.01; one way ANOVA, Tukey's multiple comparisons \u0026nbsp;test) performed separately for high and low intensity subgroups.\u003c/p\u003e\n\u003cp\u003eJ. Endogenous ATP levels are decreased in DJ-1 mutant fibroblasts n=20 wells/ per group \u0026nbsp;(****P\u0026lt;0.0001, ***p=0.0007; one way ANOVA with Tukey's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003eAll data shown are mean ± S.E.\u003c/p\u003e","description":"","filename":"Fig11242025.png","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/11e9b3bf7cae76655485ae71.png"},{"id":76749712,"identity":"3abcf466-c266-4a82-8c11-78c79d485215","added_by":"auto","created_at":"2025-02-20 09:46:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":252415,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic health of fibroblast cultures is dependent on the stoichiometry of ATP synthase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. and B. mRNA expression of ATP synthase c-subunit (ATP5G), is high in human mutant fibroblasts \u0026nbsp;compared to WT while expression of ATP5b is unchanged. The data are quantified by qRT-PCR; \u0026nbsp;at least three independent experiments were performed for each subunit (*p\u0026lt;0.05; one way ANOVA \u0026nbsp;with multiple comparisons). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC. ATP β amount is low in the mutant fibroblasts. Immunoblot shows results for indicated antibodies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. The ratio of mitochondrial ATP β to ATP c in Mut1 fibroblasts vs. WT (at least 3 independent \u0026nbsp;experiments were performed; *p=0.0417, two tailed t test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. Immunoblot of ATP β overexpression (ATP β-OE) in HEK293 cells compared to empty vector \u0026nbsp;control using an anti-ATP β antibody. Mitochondrial protein CoxIV serves as loading control. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eF. Native PAGE using the anti-c subunit antibody shows ATP synthase monomer (720 kDa) and free \u0026nbsp;c-ring (66 kDa) amount.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG. Compared with WT, β-OE improved the ratio of ATP synthase monomer to free c subunit by ~27% \u0026nbsp;(*p=0.049; t-test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH. ATP β-OE rescues protein synthesis rate in DJ-1 mutant fibroblasts. WT and mutant cells were \u0026nbsp;transfected with ATP5b or empty vector. Shown are representative immunocytochemical images \u0026nbsp;with an anti-puromycin antibody.\u003c/p\u003e\n\u003cp\u003eI. Low protein synthesis rate of mutant fibroblasts is rescued by ATP β OE. Cells were exposed to \u0026nbsp;puromycin (10 ug/ml for 15 minutes) and then anti-puro antibody was used to determine puromycin \u0026nbsp;uptake. 7-15 images are analyzed for each group, Vec WT (n=56 cells), Vec-Mut1 (n=52), Vec-Mut2 (n=84), β-sub-WT(n=19), β-sub-Mut1 (n=27), β-sub-Mut2(n=40); ****p\u0026lt;0.0001, two-way \u0026nbsp;ANOVA, multiple comparisons.\u003c/p\u003e","description":"","filename":"Fig21242025.png","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/edb2b1ab7aeeb028caf9b412.png"},{"id":76749708,"identity":"464dcfd1-34ac-45e2-b098-9b7b7378d8d1","added_by":"auto","created_at":"2025-02-20 09:46:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87565,"visible":true,"origin":"","legend":"\u003cp\u003eProtein synthesis rate is attenuated by ATP synthase inhibitor oligomycin.\u003c/p\u003e\n\u003cp\u003eA. Reduced protein synthesis rate after oligomycin. WT exposed to puromycin10 µg/ml with or \u0026nbsp;without oligomycin (2μM) for 15min. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. Group data for A. Oligomycin (2μM) for 15min decreased the rate of puromycin incorporation \u0026nbsp;(n=10 blot pairs; **p=0.0046, t-test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC. Reduced protein synthesis rate in TH+ neurons after oligomycin exposure. Example images of \u0026nbsp;TH+ dopaminergic neurons after puromycin10 µg/ml for 15min, or puromycin10 µg/ml plus 1μM \u0026nbsp;oligomycin for 15min. Reduction in the rate of protein synthesis is apparent in the neuronal \u0026nbsp;processes (white arrows).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. Group data for the experiments in C (somata), puromycin n=4, puromycin/oligomycin n=6 cells \u0026nbsp;from two cultures (***p=0.0002, t-test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. Group data n=15-21 neuronal processes (as in C) for each condition. Puromycin intensity is \u0026nbsp;plotted at increasing distance from the soma. Protein synthesis rate is decreased in the distal \u0026nbsp;compared to proximal neurites; oligomycin attenuates all protein synthesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eF. Colocalization of DJ1 with ATP β increases after neuronal stimulation. Immunolocalized proteins \u0026nbsp;within TH+ neurites shown from 0 to 100 µm from the soma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG. Group data for F. Stimulation increases ATP β and DJ-1. n=18 (Ctrl) and n=21 (+KCl); \u0026nbsp;***p=0.0003, *p=0.0171, t-test). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH. Stimulation increases co-localization (Pearson) of DJ-1 and ATP β (n=18 (Ctrl) and n=21 (+KCl); \u0026nbsp;****p\u0026lt;0.0001, t test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eI. DJ-1 OE increases ATP β in cytosol and mitochondria; anti-GFP antibody shows the exogenous \u0026nbsp;DJ-1-GFP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJ. Interaction between DJ-1 protein and ATP5b mRNA. IP products of flag-tagged WT or mutant \u0026nbsp;DJ-1 from HEK cells were subjected to RT-PCR for ATP5b mRNA. Equal binding of WT or \u0026nbsp;mutant DJ-1 to ATP5b mRNA is shown. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK. Mutant DJ1 sequestered in toxic granules. Left panels show WT fibroblasts, labeled with anti-DJ-1 antibody. Right panels show Mut1 fibroblasts; DJ-1 is sequestered near or in the nucleus in \u0026nbsp;granules; distal cell is relatively devoid of staining.\u003c/p\u003e","description":"","filename":"Fig31242025.png","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/d2ed0bcb5c8c7dd08cb93629.png"},{"id":76751314,"identity":"98084bd2-c852-4a4f-bf2c-db063a9254a0","added_by":"auto","created_at":"2025-02-20 10:02:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":532563,"visible":true,"origin":"","legend":"\u003cp\u003eDJ-1 is required for the normal rate of protein synthesis in neurons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. Protein synthesis rate is increased by stimulation. Images show newly synthesized protein as \u0026nbsp;puncta (15 min puromycin labeled puncta=pink), proximity labeling with anti-puromycin probes in \u0026nbsp;WT TH+ dendrites and DJ-1-/- (knockout (KO)) TH+ dendrites (TH=Green). Increased puro labeling \u0026nbsp;by PLA in response to neuronal stimulation shown in WT compared to DJ-1-/-. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. WT, not DJ-1-/- dendrites synthesize protein in response to neuronal stimulation. Intensity of PLA \u0026nbsp;represents newly synthesized proteins. Intensity for each cell was normalized to average in WT \u0026nbsp;TH+ neurons; 3 independent cultures. (WT n=17 neurons, WT+ KCl n=6, DJ-1-/- n=8, DJ-1-/-+ KCl \u0026nbsp;n=8; **p=0.0022; ***p=0.0003; ****p\u0026lt;0.0001, two-way ANOVA, multiple comparisons).\u003c/p\u003e\n\u003cp\u003eC. ATP β protein synthesis rate enhanced by neuronal stimulation in WT, not DJ1-/-. Representative \u0026nbsp;examples of dendritic processes shown measured from the soma to 50 µm. The largest dendrite of \u0026nbsp;each neuron was measured. Puromycin (30 min) was followed by PLA for newly synthesized ATP \u0026nbsp;β. Oligomycin inhibits new protein synthesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. Group data for experiments as shown in C. 4-5 independent cultures were measured. n=WT 41 \u0026nbsp;dendrites, WT+ KCl 26, WT+ Oligo 7, DJ-1-/- 39, -/-+KCl 27, -/- +Oligo 5. *p=0.0104; ***p=0.003; \u0026nbsp;****p\u0026lt;0.0001; two-way ANOVA, multiple comparisons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. Newly synthesized ATP β is increased by stimulation. ATP β synthesis rate decreases \u0026nbsp;decrementally away from soma. Neuronal stimulation increases newly synthesized ATP β at each \u0026nbsp;point along the neurite. Curves fitted with regression analysis (****p\u0026lt;0.0001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eF. and G. Spatial and temporal fluctuations in ATP β are attenuated in TH+ DJ1-/- neurons. Time \u0026nbsp;course (4 weeks) shows synthesis of ATP β in somata and neurites, comparing WT to DJ-1-/-. 2 \u0026nbsp;independent cultures, 4-7 somata per genotype, 6-15 neurites per genotype measured at each time \u0026nbsp;point, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig41242025.png","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/807382fcc7fc97e01a8fcef2.png"},{"id":76749717,"identity":"09f92034-6a67-495d-a729-9191d7a616ec","added_by":"auto","created_at":"2025-02-20 09:46:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":249289,"visible":true,"origin":"","legend":"\u003cp\u003eDJ-1 is required to localize ATP5b mRNA to dopaminergic processes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. ATPb mRNA is decreased in DJ-1-/- TH+ neurons. Representative images show levels of ATP5b mRNA measured using RNA scope and immunocytochemical localization of TH. ATP5b mRNA in situ is shown as red puncta in WT or DJ-1-/- (KO) somata and neurites.\u003c/p\u003e\n\u003cp\u003eB. Group data of experiments as shown in A. DJ-1-/- dopaminergic neurons have lower amount of \u0026nbsp;ATP5b mRNA in the soma than that in WT dopaminergic neurons. n=9 WT soma, n=12 DJ1-/- soma (*p=0.019, unpaired t-test)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC. Group data of experiments as shown in A. DJ-1-/- dopaminergic neurons have lower amount of \u0026nbsp;ATP5b mRNA localized to the neurites than that of WT dopaminergic neurons. ATP5b mRNA \u0026nbsp;puncta were measured at DIV21 in the main neurite of each cell starting at the soma and extending \u0026nbsp;50 µm. Mask was created to determine mRNA puncta and to exclude non-punctal areas. n=10 WT \u0026nbsp;neurites, n=12 DJ1-/- neurites (****p\u0026lt;0.0001, unpaired t-test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. ATP5b mRNA level varies at different developmental stages in WT dopaminergic neurites. 2nd week \u0026nbsp;n=40 neurites, 3rd week n=45, 4th week n=44, ****p\u0026lt;0.00001, **p=0.0075, one-way ANOVA, \u0026nbsp;multiple comparisons. ATP5b mRNA puncta were counted and shown in graph.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. ATP5b mRNA level varies but the amplitude of the variation is diminished in DJ-1-/- neurons. 2nd week n=43, 3rd week n=34, 4th week n=40, *p=0.02, one-way ANOVA, multiple comparisons. \u0026nbsp;ATP5b mRNA puncta were counted and shown in the graph.\u003c/p\u003e","description":"","filename":"Fig51272025.png","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/6a0109f34888919ebe83d4c5.png"},{"id":76749716,"identity":"1cdca1f0-47b4-4370-9405-5a51a9383426","added_by":"auto","created_at":"2025-02-20 09:46:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":423969,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of ATP β rescues overall protein synthesis rate in DJ1-/- neurons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. Diagram of the AAV-ATP β construct.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. Representative images of AAV-GFP transduced or AAV-ATP β subunit-transduced DJ-1-/- (KO) \u0026nbsp;dopaminergic neurons. AAV virus+ (green), TH+(Red), ATP β antibody (pink).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC. Group data from AAV transduced DJ-1-/- TH+ neurites show a 100% increase in ATP β \u0026nbsp;expression in AAV-ATP β OE neurites compared to AAV-GFP; n=12 AAV-GFP transduced \u0026nbsp;neurons, n=24 AAV-ATP β-GFP transduced neurons, **p=0.0016, t-test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. Representative images of AAV-GFP transduced or AAV-ATP β transduced DJ-1-/- cortical \u0026nbsp;neurons. AAV virus+ (green), ATP β antibody (pink).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. Group data in AAV transduced DJ-1-/- cortical neurons shows a 50% increase in ATP β \u0026nbsp;expression in AAV-ATP β OE neurites compared to AAV-GFP; n=21 AAV-GFP neurons, n=28 \u0026nbsp;AAV-ATP β-GFP neurons, ****p\u0026lt;0.0001, t-test.\u003c/p\u003e\n\u003cp\u003eF. Overexpression of ATP β subunit rescues overall protein synthesis rate in DJ1-/- neurons. \u0026nbsp;Representative images of puro-PLA of cortical DJ-1-/- neurons transduced with AAV-GFP or AAV-ATP β-GFP. AAV virus+ is green, puro-PLA is pink.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG. Overall protein synthesis rate (Puro-PLA) of DJ-1-/- cortical neurons is increased in cells \u0026nbsp;transduced with AAV-ATP β compared to the cells transduced with AAV-GFP. Shown are \u0026nbsp;representative neurite images. AAV virus + is green, puro-PLA is pink.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH. and I. Overexpression of ATP β rescues the overall protein synthesis rate in DJ1 -/- neurons. \u0026nbsp;Group data of the puro-PLA intensity in DJ1-/- cortical somata, n=38 AAV-GFP neurons, n=46 \u0026nbsp;AAV-ATP β neurons, ****p\u0026lt;0.0001, t-test. Group data show increased overall protein synthesis \u0026nbsp;rate (puro-PLA intensity) in AAV-β OE dendrites compared to AAV-GFP. n=26 AAV-GFP neurons, \u0026nbsp;n=46 AAV- β subunit neurons, **** p\u0026lt;0.0001, t-test.\u003c/p\u003e","description":"","filename":"Fig61242025.png","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/0cb619af3b3da5e47586b1bc.png"},{"id":76751313,"identity":"5685c477-1794-47b0-bb38-68ff993f9547","added_by":"auto","created_at":"2025-02-20 10:02:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":41397,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of ATP β improves neurite outgrowth of dopaminergic neurons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. AAV-ATP β OE increased the lengths of the neurites in both WT TH+ neurons and DJ-1-/- TH+ \u0026nbsp;neurons. n=26 WT AAV-GFP, n=21 DJ-1-/- AAV-GFP, n=24 WT AAV-ATP β-GFP, n=19 DJ-1-/- AAV-ATP β -GFP. *p=0.0436, ***p=0.0008, ****p\u0026lt;0.0001, two-way ANOVA, multiple \u0026nbsp;comparisons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. AAV-ATP β OE increased the numbers of neurites in WT TH+ neurons, but not in the DJ-1-/- TH+ \u0026nbsp;neurons. n=35 WT AAV-GFP, n=19 DJ-1-/- AAV-GFP, n=40 WT AAV-ATP β-GFP, n=25 DJ-1-/- AAV-ATP β-GFP, *p=0.0289, ***p=0.0005, ****p\u0026lt;0.0001, two-way ANOVA, multiple comparisons. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC. AAV-ATP β OE increased the numbers of branches directly starting from the main neurite in WT \u0026nbsp;TH+ neurons, but not in the DJ-1-/- TH+ neurons. N=34 WT AAV-GFP, n=19 DJ-1-/- AAV-GFP, \u0026nbsp;n=31 WT AAV-ATP β-GFP, n=25 DJ-1-/- AAV-ATP β-GFP, *p=0.0214; *p=0.0116, respectively, \u0026nbsp;two-way ANOVA, multiple comparisons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. Diagram of the working model. DJ-1 (red circle) acts as an RBP and transports ATP5b mRNA \u0026nbsp;(blue squiggle) to distal neurites near mitochondria and ribosomes (purple), increasing ATP β \u0026nbsp;protein (yellow hexagon) synthesis. DJ-1 chaperones ATP β into mitochondria matrix, enhancing \u0026nbsp;ATP production rate by the ATP synthase.\u003c/p\u003e","description":"","filename":"Fig71242025.png","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/2d36fc84d829e111615a08ce.png"},{"id":76752581,"identity":"3dfe3d7d-a251-49d7-a389-70792f9404f8","added_by":"auto","created_at":"2025-02-20 10:10:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1503492,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Chenetal.2025Feb7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1_covered_015ff777-4f66-492c-8e52-0d6ca1cf8c05.pdf"},{"id":76749707,"identity":"8ef96286-3f87-4159-b2d8-a438d16861c5","added_by":"auto","created_at":"2025-02-20 09:46:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":57980,"visible":true,"origin":"","legend":"Table of the Reagents","description":"","filename":"Tableofthereagents.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/91eb01259f77ad2018d6806e.pdf"},{"id":76749711,"identity":"b908c406-5c3b-4ea2-9a1f-ea6710bfe9f5","added_by":"auto","created_at":"2025-02-20 09:46:44","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":552061,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 1. Extended data A. Immunoblots with the anti-Mfn1 or 2 antibodies for WT fibroblasts and mutant patients’ fibroblasts \u0026nbsp;show that Mfn levels are low in mutant fibroblasts compared to WT. GAPDH serves as protein \u0026nbsp;control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. and C. N=3 independent experiments per group (*p\u0026lt;0.02, **p=0.0023; one way ANOVA with \u0026nbsp;multiple comparisons).\u003c/p\u003e","description":"","filename":"Fig1ExtendedData1242025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/5cea1c594e024ea89b55ea1a.pdf"},{"id":76750760,"identity":"3a58cb14-3539-4838-9927-0051bf7ffabb","added_by":"auto","created_at":"2025-02-20 09:54:44","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1054603,"visible":true,"origin":"","legend":"\u003cp\u003eFig 2 Extended data \u0026nbsp;A. Mut1 fibroblasts grow slowly compared to WT fibroblasts (n=3 different plates and 3 independent experiments).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. ATP5b OE (lane 3) increases the protein level of ATP synthase α subunit protein (ATP α) more than c-subunit whereas ATP5G OE (c-subunit, lane 2) does not increase the protein levels of F1 \u0026nbsp;components. Immunoblots (IB) with antibody targets for ATP α (anti-α), ATP β (anti-β), and ATP c (anti-c) were used. Lane one is the empty vector control. C. Immunoblot of total cell lysate using anti-puro antibody shows amount of puromycin incorporation over 15 min. into WT and Mut1 fibroblasts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. Immunoblot of total cell lysate using anti-puro antibody shows puromycin incorporation over 15 min. into WT and Mut1 fibroblasts overexpressing DJ-1 or ATP β.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. Quantification of experiments shown in C. Puromycin incorporation (10 ug/ml for 15 minutes) is low in-patient fibroblasts carrying a mutation in DJ-1 (Mut1) (***p=0.0006, t-test; n=3 samples, all \u0026nbsp;samples normalized to the average of the WT). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eF. Quantification of experiments shown in D. The defect in the rate of protein synthesis in mutant \u0026nbsp;fibroblasts is rescued by ATP β exogenous OE (effects of WT DJ-1 or ATP β OE were first normalized to GAPDH and then normalized to the corresponding empty vector translation rate (*p\u0026lt;0.02; n=3 independent samples, one-way ANOVA followed by multiple comparisons).\u003c/p\u003e","description":"","filename":"Fig2Extended1242025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5984062/v1/020a311ff5161d46366efa1b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"DJ-1 and ATP synthase are required for local protein synthesis in midbrain dopaminergic neurons","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mitochondria, Parkinson’s Disease, mRNA binding proteins, neuronal outgrowth","lastPublishedDoi":"10.21203/rs.3.rs-5984062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5984062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Dopaminergic neurons have extensive neuritic arbors therefore local protein synthesis is required. Familial PD risk gene PARK-7 (DJ-1) is a key regulator of ATP synthase and of dopaminergic growth and survival. We now find that inhibition of ATP synthase function completely abrogates protein synthesis in dopaminergic (mesDA) neuronal processes, and we demonstrate that DJ-1 is an mRNA binding protein for F1FO ATP synthase β subunit mRNA. DJ-1 localizes ATP β mRNA into neurites, thus DJ-1 patient fibroblasts and DJ-1-/- mesDA mouse neurons have low ATP β mRNA and protein, reduced growth and low protein synthesis rates. Over-expression of ATP β rescues these defects. We conclude that DJ-1 is required to optimize the stoichiometric structure of the ATP synthase in the neuritic arbors. This supports the local protein synthesis required for normal neuritic growth.","manuscriptTitle":"DJ-1 and ATP synthase are required for local protein synthesis in midbrain dopaminergic neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-20 09:46:39","doi":"10.21203/rs.3.rs-5984062/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"30830ddf-dfb9-4d94-99a0-bb13953a84a4","owner":[],"postedDate":"February 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44364802,"name":"Biological sciences/Neuroscience/Cellular neuroscience"},{"id":44364803,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-03-09T13:01:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-20 09:46:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5984062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5984062","identity":"rs-5984062","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}