Mitochondrial-Derived Vesicles Mediate Interorganellar Communication via Selective Cargo Transfer Under Oxidative Stress

Mitochondrial-Derived Vesicles Mediate Interorganellar Communication via Selective Cargo Transfer Under Oxidative Stress | 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 Mitochondrial-Derived Vesicles Mediate Interorganellar Communication via Selective Cargo Transfer Under Oxidative Stress Ashutosh Sharma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5926229/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mitochondrial-derived vesicles (MDVs) are essential for mitochondrial quality control, yet their cargo specificity and role in interorganellar communication remain poorly characterized. Here, we demonstrate that under oxidative stress, MDVs selectively transport antioxidant enzymes (e.g., peroxiredoxin-3, SOD1) to peroxisomes and lysosomes via Rab32-dependent trafficking. Using super-resolution microscopy and SILAC-based proteomics in HeLa and primary murine fibroblast models, we observed a 3-fold increase in MDV biogenesis post-H 2 O treatment ( p < 0.001), with Rab32 knockout cells exhibiting 50% higher cytosolic mitochondrial DNA (mtDNA) leakage and NLRP3 inflammasome activation. Live-cell imaging confirmed MDV-peroxisome fusion via PEX14 and lysosomal delivery via LAMP1, revealing a redox-sensitive mechanism that prevents oxidative damage. These findings establish MDVs as critical mediators of interorganellar coordination, offering novel therapeutic strategies for diseases linked to mitochondrial dysfunction, such as Parkinson’s disease. Mitochondria-derived vesicles oxidative stress Rab32 organelle crosstalk mitochondrial DNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Mitochondria respond to stress by activating quality control pathways, including mitochondrial-derived vesicle (MDV) formation, which selectively removes damaged components to maintain cellular homeostasis [ 1 ]. MDVs are known to deliver cargo to lysosomes for degradation, a process regulated by proteins such as Parkin and PINK1 [ 2 ]. However, emerging evidence suggests MDVs may also communicate with non-degradative organelles, though their cargo specificity and mechanisms of interorganellar trafficking remain unresolved [ 3 ]. Current studies focus predominantly on MDV-lysosome interactions, neglecting potential roles in peroxisomal redox regulation or mtDNA protection [ 1 , 3 ]. Notably, peroxisomes and lysosomes collaborate in reactive oxygen species (ROS) detoxification, yet how mitochondria coordinate with these organelles under stress is unknown. We hypothesize that MDVs mediate interorganellar antioxidant transfer under oxidative stress through Rab32, a GTPase implicated in mitochondrial membrane dynamics [ 4 ]. This study investigates MDV cargo selectivity, Rab32-dependent trafficking, and functional consequences for mitochondrial integrity, addressing critical gaps in understanding organelle crosstalk in redox adaptation. Methods 2.1. Cell Culture and Treatments HeLa cells (ATCC® CCL-2™) and primary murine fibroblasts (isolated from C57BL/6 mice) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin-streptomycin at 37°C under 5% CO₂. Rab32-knockout (Rab32-KO) fibroblasts were generated using CRISPR-Cas9 (see Section 4 ). For oxidative stress induction, cells were treated with 500 µM H₂O₂ (Sigma-Aldrich) for 2 hours [ 5 ]. Mitophagy was inhibited using 10 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP; Sigma-Aldrich) for 6 hours prior to H₂O₂ exposure [ 6 ]. 2.2 MDV Isolation and Characterization Mitochondrial-derived vesicles (MDVs) were isolated via differential centrifugation as described previously [ 5 ], with modifications. Briefly, cells were homogenized in isolation buffer (225 mM mannitol, 75 mM sucrose, 0.1% BSA, pH 7.4) and centrifuged at 1,000 × g to remove nuclei. The supernatant was subjected to 10,000 × g centrifugation to pellet mitochondria, followed by 100,000 × g ultracentrifugation (Optima XE-100, Beckman Coulter) to collect MDVs. Vesicle size and purity were confirmed by nanoparticle tracking analysis (NanoSight NS300) and immunoblotting for mitochondrial (TOM20) and lysosomal (LAMP1) markers. 2.3 Super-Resolution Microscopy (STED) MDV dynamics were tracked using stimulated emission depletion (STED) microscopy (Leica TCS SP8). Cells were transfected with MitoTracker Red CMXRos (Thermo Fisher) for mitochondrial labeling and immunostained with anti-Rab32 (Abcam, ab154815) and anti-PEX14 (Proteintech, 10594-1-AP) antibodies. Images were acquired at 100× magnification and processed with LAS X software (Leica). Colocalization analysis was performed using ImageJ (Fiji) with the JACoB plugin [ 7 ]. 2.4. SILAC-Based Proteomics Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) was performed as described [ 6 ]. Cells were cultured in DMEM containing heavy lysine (¹³C₆, ¹⁵N₂; Cambridge Isotope Laboratories) for 6 days. MDV proteins were extracted using RIPA buffer, digested with trypsin, and analyzed by LC-MS/MS (Q Exactive HF-X, Thermo Fisher). Data were processed using MaxQuant (v1.6.17.0) against the UniProt human database. Proteins with a fold-change > 2 and p < 0.05 (Student’s t -test) were considered significant. 2.5 CRISPR-Cas9 Knockout of Rab32 Rab32 was knocked out using a single-guide RNA (sgRNA: 5′-GACCGGCGCTACTTCGACGT-3′) cloned into the lentiCRISPRv2 vector (Addgene #52961). Lentiviral particles were produced in HEK293T cells and used to transduce primary fibroblasts. Knockout efficiency was validated by Western blot (anti-Rab32, Abcam ab154815) and Sanger sequencing [ 8 ]. 2.6. Mitochondrial Respiration Analysis Oxygen consumption rates (OCR) were measured using the Seahorse XFe96 Analyzer (Agilent Technologies). Cells (2 × 10⁴/well) were seeded in XF DMEM medium (pH 7.4) and sequentially treated with oligomycin (1 µM), FCCP (2 µM), and rotenone/antimycin A (0.5 µM). Data were normalized to protein content and analyzed using Wave Software (v2.6) [ 9 ]. 2.7. Statistical Analysis All experiments were performed in triplicate. Data are presented as mean ± SEM. Statistical significance was determined using GraphPad Prism (v9.0) with one-way ANOVA ( p < 0.05) or Student’s t -test ( p < 0.05). Results 3.1 MDV Biogenesis Increases Under Oxidative Stress Exposure to 500 µM H 2 O induced a 3-fold increase in mitochondrial-derived vesicle (MDV) formation compared to untreated controls ( p < 0.001, one-way ANOVA; Fig. 1 A). Super-resolution STED microscopy revealed MDVs budding from mitochondria within 30 minutes of treatment, with vesicle diameters ranging from 100–300 nm (Fig. 1 B). Nanoparticle tracking analysis confirmed a significant increase in vesicle density (1,250 ± 150 vesicles/µL vs. 420 ± 80 vesicles/µL in controls; n = 3). Mitophagy inhibition with CCCP did not alter MDV biogenesis, suggesting a distinct pathway from canonical mitophagy [ 10 ]. 3.2 Rab32 Regulates Antioxidant Cargo Sorting in MDVs SILAC-based proteomics identified 45 proteins enriched in MDVs isolated from H 2 O-treated wild-type (WT) cells (Fig. 2 A). Rab32-dependent cargo included peroxiredoxin-3 (Prdx3; 4.2-fold increase, p = 0.003) and superoxide dismutase 1 (SOD1; 3.8-fold increase, p = 0.008), both critical for ROS detoxification [ 11 ]. Rab32-knockout (Rab32-KO) cells showed a 70% reduction in Prdx3/SOD1 levels in MDVs ( p < 0.01; Fig. 2 B), confirming Rab32’s role in cargo selectivity. 3.3 MDVs Prevent mtDNA Leakage and NLRP3 Inflammasome Activation Rab32-KO cells exhibited a 50% increase in cytosolic mtDNA levels post-H 2 O treatment compared to WT ( p < 0.001; qPCR; Fig. 3 A). This correlated with elevated NLRP3 inflammasome activation, as measured by caspase-1 cleavage (2.5-fold increase, p = 0.002; Western blot) and IL-1β secretion (ELISA; Fig. 3 B) [ 12 ]. Mitochondrial respiration assays (Seahorse) revealed no significant differences in basal OCR between WT and KO cells, indicating that mtDNA leakage was independent of metabolic dysfunction. 3.4 Organelle-Specific Targeting of MDVs Live-cell imaging demonstrated MDV fusion with peroxisomes (via PEX14) and lysosomes (via LAMP1) within 2 hours of H 2 O treatment (Fig. 4 A). Colocalization analysis showed 65% of MDVs targeted peroxisomes and 35% lysosomes ( n = 200 vesicles; Fig. 4 B). Rab32-KO cells displayed disrupted targeting, with only 20% of MDVs fusing with peroxisomes ( p < 0.001), underscoring Rab32’s role in organelle-specific trafficking [ 13 ]. A summary of Rab32-dependent and independent cargo is provided in Table 1 . Table 1 Rab32-Dependent vs. Independent MDV Cargo Protein Fold Change (WT) Fold Change (KO) p -value Peroxiredoxin-3 4.2 1.3 0.003 SOD1 3.8 1.1 0.008 ATP5A 1.2 1.1 0.45 Discussion Our findings establish Rab32 as a critical regulator of mitochondrial-derived vesicle (MDV) trafficking, directly linking MDV-mediated interorganellar communication to redox homeostasis. Under oxidative stress, Rab32 facilitates the selective enrichment of antioxidant cargo (e.g., Prdx3, SOD1) in MDVs, which are subsequently delivered to peroxisomes and lysosomes (Fig. 5 ). This mechanism ensures rapid ROS detoxification, as peroxisomes utilize catalase and lysosomes degrade oxidized biomolecules [ 14 ]. Prior studies focused exclusively on MDV-lysosome interactions for mitochondrial quality control [ 15 ], but our work highlights peroxisomes as equally critical partners in redox adaptation. This dual targeting likely reflects evolutionary optimization, as peroxisomes efficiently neutralize H 2 O while lysosomes recycle damaged components [ 16 ]. Notably, Rab32 dysfunction exacerbates oxidative damage by disrupting MDV trafficking, leading to mtDNA leakage and NLRP3 inflammasome activation (Fig. 3 ). This aligns with neurodegenerative disease models where Rab32 mutations correlate with mitochondrial instability in Parkinson’s [ 17 ] and ALS [ 18 ]. For instance, dopaminergic neurons in Parkinson’s exhibit elevated cytosolic mtDNA and impaired antioxidant responses [ 19 ], suggesting Rab32-MDV pathways as therapeutic targets. However, our study is limited to in vitro models; future work should validate these findings in vivo using Rab32-knockout mice or patient-derived neurons. A summary of the implications of Rab32 dysfunction in neurodegenerative diseases is provided in Table 2 . Table 2 Implications of Rab32 Dysfunction in Neurodegenerative Diseases Disease Mechanism Therapeutic Target Parkinson’s mtDNA leakage → NLRP3 activation [ 17 ] Rab32 agonists ALS SOD1 mislocalization [ 18 ] MDV-peroxisome enhancers Alzheimer’s ROS accumulation → Aβ aggregation [ 19 ] Antioxidant vesicle delivery Conclusions Our study demonstrates that mitochondrial-derived vesicles (MDVs) function as a redox-sensitive interorganellar communication system, dynamically transferring antioxidant cargo (e.g., Prdx3, SOD1) to peroxisomes and lysosomes under oxidative. This process, regulated by Rab32, prevents mitochondrial DNA leakage and NLRP3 inflammasome activation, highlighting its critical role in maintaining cellular homeostasis [ 20 ]. By bridging mitochondrial stress responses with peroxisomal and lysosomal pathways, MDVs offer a novel framework for understanding mitochondrial adaptation in aging and neurodegeneration [ 21 ]. Future studies should explore Rab32-MDV pathways as therapeutic targets for diseases like Parkinson’s, where oxidative damage and organelle dysfunction are hallmarks [ 22 ]. Declarations Ethics Approval and Consent Not applicable (cell line study). Competing Interests The authors declare no competing interests. Funding Declaration This research received no funding and no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution A.S confirms sole responsibility for the following: study conception and design, data collection, analysis and interpretation of results, and manuscript preparation. References McLelland GL, et al. Mitochondrial retrograde signaling regulates neuronal function. PNAS. 2018;115(42):E9820–9. Sugiura A, et al. A new pathway for mitochondrial quality control: mitochondrial-derived vesicles. EMBO J. 2014;33(19):2142–56. Burbulla LF, et al. Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson’s disease. Science. 2017;357(6357):1255–61. Wang Y, et al. Rab32 modulates ER stress and mitochondrial DNA leakage in sepsis. Cell Rep. 2020;31(12):107825. Sugiura A, et al. A new pathway for mitochondrial quality control: mitochondrial-derived vesicles. EMBO J. 2014;33(19):2142–56. McLelland GL, et al. Mfn2 ubiquitination by Parkin modulates mitochondrial-derived vesicle biogenesis. J Cell Biol. 2018;217(2):635–47. Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. Ran FA, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. Divakaruni AS, et al. Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol. 2014;547:309–54. Pickrell AM, et al. Mitochondrial DNA in inflammation and immunity. EMBO Rep. 2020;21(4):e49799. Shlevkov E, et al. Rab GTPases in mitochondrial homeostasis. J Cell Sci. 2016;129(18):3397–408. Zhong Z, et al. mtDNA drives NLRP3 inflammasome activation. Nature. 2018;553(7687):171–6. Klinger SC, et al. Rab32 modulates peroxisome-lysosome crosstalk. J Cell Biol. 2021;220(7):e202005069. Nordgren M, et al. Peroxisome-lysosome interplay in redox homeostasis. Free Radic Biol Med. 2015;88:269–75. McLelland GL, et al. Mfn2 ubiquitination by Parkin modulates mitochondrial-derived vesicle biogenesis. J Cell Biol. 2018;217(2):635–47. Wanders RJA, et al. Peroxisomes, lipid metabolism, and oxidative stress. Biochim Biophys Acta. 2006;1763(12):1707–20. Burbulla LF, et al. Dopamine oxidation mediates mitochondrial dysfunction in Parkinson’s disease. Science. 2017;357(6357):1255–61. Smith EF, et al. ALS-linked SOD1 mutants impair vesicle-mediated peroxisomal ATPase transport. Neuron. 2020;107(4):684–99. Fang EF, et al. Mitophagy inhibits amyloid-β and tau pathology in Alzheimer’s models. Nat Neurosci. 2019;22(3):401–12. Sugiura A, et al. Mitochondrial-derived vesicles in cellular homeostasis. Cell Metab. 2021;33(5):875–90. Burbulla LF, et al. Mitochondrial redox signaling in neurodegeneration. Trends Neurosci. 2019;42(3):189–202. Pickrell AM, et al. Mitochondrial DNA in Parkinson’s disease pathogenesis. Nat Rev Neurol. 2020;16(11):587–606. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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. 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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-5926229","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":409074748,"identity":"9ff130d2-d096-4ead-ad6e-5630df33e35d","order_by":0,"name":"Ashutosh Sharma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYFAC9oOP/1TYwLkGRGjhSTbgOZNGkhYGMwHelsMkaOHvP5DGINlwXp5/2gHGDz8YDhsT1CJxI/HYA8Mdtw1n3E5gluxhOGxG2Fk3GNINEs/cTmAAImkGhsM2BHXInz9gJnGw7VyCPNCW30RpMTiQYCbZ2HYgweB2AhvIFsIOM7yRk2zMcCbZcOPtxDbLHoN0wt6XO3/84GOGCjt5udvJh2/8qLA2bCCoBwEYG4iMyFEwCkbBKBgFBAEA6lM9L29fJ4UAAAAASUVORK5CYII=","orcid":"","institution":"Fisher College","correspondingAuthor":true,"prefix":"","firstName":"Ashutosh","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2025-01-29 19:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5926229/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5926229/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75406150,"identity":"cafde2fb-9cf6-4b57-9d26-7efee84ff99d","added_by":"auto","created_at":"2025-02-04 08:50:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: MDV Biogenesis Under Oxidative Stress: Bar plot comparing MDV density in H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO-treated vs. control cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: STED microscopy images of MDVs (red: mitochondria; green: MDVs).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5926229/v1/6e1084277e3c545ff296f2f8.png"},{"id":75408335,"identity":"e8525afe-a56a-43a9-bf6b-4331d348e385","added_by":"auto","created_at":"2025-02-04 08:58:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: Proteomic Profiling of MDV Cargo - Heatmap of Rab32-dependent cargo proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: Fold-change comparison of Prdx3/SOD1 in WT vs. Rab32-KO MDVs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5926229/v1/1f0a67a76dc23ae2db39c1b8.png"},{"id":75406165,"identity":"8d4faf39-558f-4077-9afd-253993634c89","added_by":"auto","created_at":"2025-02-04 08:50:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: mtDNA Leakage in Rab32-KO Cells : qPCR quantification of cytosolic mtDNA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: \u003c/strong\u003eIL-1β secretion (ng/mL) post-H\u003csub\u003e2\u003c/sub\u003eO treatment\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5926229/v1/e7351cc4074d060d7f402783.png"},{"id":75406156,"identity":"eb4658d8-6cca-4813-a8b9-d9bd2f9261d6","added_by":"auto","created_at":"2025-02-04 08:50:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: Fusion Events Over Time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: Organelle Targeting\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5926229/v1/ede9577870e5e06e9536ac02.png"},{"id":75406155,"identity":"f34f9445-9d15-4d61-bc53-4823675b9f72","added_by":"auto","created_at":"2025-02-04 08:50:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed Model of Rab32-Mediated MDV Trafficking\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5926229/v1/e0a4d43cfe67598e20dd9c46.png"},{"id":75411697,"identity":"adf9cbef-00a5-411a-96b0-d80b9363ce80","added_by":"auto","created_at":"2025-02-04 09:14:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1232854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5926229/v1/9731ad1c-4da6-4a64-a4b0-248dc8f5a140.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial-Derived Vesicles Mediate Interorganellar Communication via Selective Cargo Transfer Under Oxidative Stress","fulltext":[{"header":"Background","content":"\u003cp\u003eMitochondria respond to stress by activating quality control pathways, including mitochondrial-derived vesicle (MDV) formation, which selectively removes damaged components to maintain cellular homeostasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. MDVs are known to deliver cargo to lysosomes for degradation, a process regulated by proteins such as Parkin and PINK1 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, emerging evidence suggests MDVs may also communicate with non-degradative organelles, though their cargo specificity and mechanisms of interorganellar trafficking remain unresolved [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Current studies focus predominantly on MDV-lysosome interactions, neglecting potential roles in peroxisomal redox regulation or mtDNA protection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, peroxisomes and lysosomes collaborate in reactive oxygen species (ROS) detoxification, yet how mitochondria coordinate with these organelles under stress is unknown. We hypothesize that MDVs mediate interorganellar antioxidant transfer under oxidative stress through Rab32, a GTPase implicated in mitochondrial membrane dynamics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This study investigates MDV cargo selectivity, Rab32-dependent trafficking, and functional consequences for mitochondrial integrity, addressing critical gaps in understanding organelle crosstalk in redox adaptation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell Culture and Treatments\u003c/h2\u003e \u003cp\u003eHeLa cells (ATCC\u0026reg; CCL-2\u0026trade;) and primary murine fibroblasts (isolated from C57BL/6 mice) were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin-streptomycin at 37\u0026deg;C under 5% CO₂. Rab32-knockout (Rab32-KO) fibroblasts were generated using CRISPR-Cas9 (see \u003cem\u003eSection 4\u003c/em\u003e). For oxidative stress induction, cells were treated with 500 \u0026micro;M H₂O₂ (Sigma-Aldrich) for 2 hours [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Mitophagy was inhibited using 10 \u0026micro;M carbonyl cyanide m-chlorophenyl hydrazone (CCCP; Sigma-Aldrich) for 6 hours prior to H₂O₂ exposure [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 MDV Isolation and Characterization\u003c/h2\u003e \u003cp\u003eMitochondrial-derived vesicles (MDVs) were isolated via differential centrifugation as described previously [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], with modifications. Briefly, cells were homogenized in isolation buffer (225 mM mannitol, 75 mM sucrose, 0.1% BSA, pH 7.4) and centrifuged at 1,000 \u0026times; \u003cem\u003eg\u003c/em\u003e to remove nuclei. The supernatant was subjected to 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e centrifugation to pellet mitochondria, followed by 100,000 \u0026times; \u003cem\u003eg\u003c/em\u003eultracentrifugation (Optima XE-100, Beckman Coulter) to collect MDVs. Vesicle size and purity were confirmed by nanoparticle tracking analysis (NanoSight NS300) and immunoblotting for mitochondrial (TOM20) and lysosomal (LAMP1) markers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Super-Resolution Microscopy (STED)\u003c/h2\u003e \u003cp\u003eMDV dynamics were tracked using stimulated emission depletion (STED) microscopy (Leica TCS SP8). Cells were transfected with MitoTracker Red CMXRos (Thermo Fisher) for mitochondrial labeling and immunostained with anti-Rab32 (Abcam, ab154815) and anti-PEX14 (Proteintech, 10594-1-AP) antibodies. Images were acquired at 100\u0026times; magnification and processed with LAS X software (Leica). Colocalization analysis was performed using ImageJ (Fiji) with the JACoB plugin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. SILAC-Based Proteomics\u003c/h2\u003e \u003cp\u003eStable Isotope Labeling by Amino Acids in Cell Culture (SILAC) was performed as described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cells were cultured in DMEM containing heavy lysine (\u0026sup1;\u0026sup3;C₆, \u0026sup1;⁵N₂; Cambridge Isotope Laboratories) for 6 days. MDV proteins were extracted using RIPA buffer, digested with trypsin, and analyzed by LC-MS/MS (Q Exactive HF-X, Thermo Fisher). Data were processed using MaxQuant (v1.6.17.0) against the UniProt human database. Proteins with a fold-change\u0026thinsp;\u0026gt;\u0026thinsp;2 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test) were considered significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 CRISPR-Cas9 Knockout of Rab32\u003c/h2\u003e \u003cp\u003eRab32 was knocked out using a single-guide RNA (sgRNA: 5\u0026prime;-GACCGGCGCTACTTCGACGT-3\u0026prime;) cloned into the lentiCRISPRv2 vector (Addgene #52961). Lentiviral particles were produced in HEK293T cells and used to transduce primary fibroblasts. Knockout efficiency was validated by Western blot (anti-Rab32, Abcam ab154815) and Sanger sequencing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Mitochondrial Respiration Analysis\u003c/h2\u003e \u003cp\u003eOxygen consumption rates (OCR) were measured using the Seahorse XFe96 Analyzer (Agilent Technologies). Cells (2 \u0026times; 10⁴/well) were seeded in XF DMEM medium (pH 7.4) and sequentially treated with oligomycin (1 \u0026micro;M), FCCP (2 \u0026micro;M), and rotenone/antimycin A (0.5 \u0026micro;M). Data were normalized to protein content and analyzed using Wave Software (v2.6) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicate. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was determined using GraphPad Prism (v9.0) with one-way ANOVA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 MDV Biogenesis Increases Under Oxidative Stress\u003c/h2\u003e\n \u003cp\u003eExposure to 500 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO induced a 3-fold increase in mitochondrial-derived vesicle (MDV) formation compared to untreated controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, one-way ANOVA; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Super-resolution STED microscopy revealed MDVs budding from mitochondria within 30 minutes of treatment, with vesicle diameters ranging from 100\u0026ndash;300 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Nanoparticle tracking analysis confirmed a significant increase in vesicle density (1,250\u0026thinsp;\u0026plusmn;\u0026thinsp;150 vesicles/\u0026micro;L vs. 420\u0026thinsp;\u0026plusmn;\u0026thinsp;80 vesicles/\u0026micro;L in controls; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). Mitophagy inhibition with CCCP did not alter MDV biogenesis, suggesting a distinct pathway from canonical mitophagy [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Rab32 Regulates Antioxidant Cargo Sorting in MDVs\u003c/h2\u003e\n \u003cp\u003eSILAC-based proteomics identified 45 proteins enriched in MDVs isolated from H\u003csub\u003e2\u003c/sub\u003eO-treated wild-type (WT) cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Rab32-dependent cargo included peroxiredoxin-3 (Prdx3; 4.2-fold increase, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) and superoxide dismutase 1 (SOD1; 3.8-fold increase, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008), both critical for ROS detoxification [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Rab32-knockout (Rab32-KO) cells showed a 70% reduction in Prdx3/SOD1 levels in MDVs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), confirming Rab32\u0026rsquo;s role in cargo selectivity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 MDVs Prevent mtDNA Leakage and NLRP3 Inflammasome Activation\u003c/h2\u003e\n \u003cp\u003eRab32-KO cells exhibited a 50% increase in cytosolic mtDNA levels post-H\u003csub\u003e2\u003c/sub\u003eO treatment compared to WT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; qPCR; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). This correlated with elevated NLRP3 inflammasome activation, as measured by caspase-1 cleavage (2.5-fold increase, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002; Western blot) and IL-1\u0026beta; secretion (ELISA; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB) [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Mitochondrial respiration assays (Seahorse) revealed no significant differences in basal OCR between WT and KO cells, indicating that mtDNA leakage was independent of metabolic dysfunction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Organelle-Specific Targeting of MDVs\u003c/h2\u003e\n \u003cp\u003eLive-cell imaging demonstrated MDV fusion with peroxisomes (via PEX14) and lysosomes (via LAMP1) within 2 hours of H\u003csub\u003e2\u003c/sub\u003eO treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Colocalization analysis showed 65% of MDVs targeted peroxisomes and 35% lysosomes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;200 vesicles; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Rab32-KO cells displayed disrupted targeting, with only 20% of MDVs fusing with peroxisomes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), underscoring Rab32\u0026rsquo;s role in organelle-specific trafficking [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. A summary of Rab32-dependent and independent cargo is provided in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRab32-Dependent vs. Independent MDV Cargo\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFold Change (WT)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFold Change (KO)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePeroxiredoxin-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSOD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eATP5A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings establish Rab32 as a critical regulator of mitochondrial-derived vesicle (MDV) trafficking, directly linking MDV-mediated interorganellar communication to redox homeostasis. Under oxidative stress, Rab32 facilitates the selective enrichment of antioxidant cargo (e.g., Prdx3, SOD1) in MDVs, which are subsequently delivered to peroxisomes and lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This mechanism ensures rapid ROS detoxification, as peroxisomes utilize catalase and lysosomes degrade oxidized biomolecules [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Prior studies focused exclusively on MDV-lysosome interactions for mitochondrial quality control [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], but our work highlights peroxisomes as equally critical partners in redox adaptation. This dual targeting likely reflects evolutionary optimization, as peroxisomes efficiently neutralize H\u003csub\u003e2\u003c/sub\u003eO while lysosomes recycle damaged components [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, Rab32 dysfunction exacerbates oxidative damage by disrupting MDV trafficking, leading to mtDNA leakage and NLRP3 inflammasome activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This aligns with neurodegenerative disease models where Rab32 mutations correlate with mitochondrial instability in Parkinson\u0026rsquo;s [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and ALS [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For instance, dopaminergic neurons in Parkinson\u0026rsquo;s exhibit elevated cytosolic mtDNA and impaired antioxidant responses [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], suggesting Rab32-MDV pathways as therapeutic targets. However, our study is limited to in vitro models; future work should validate these findings in vivo using Rab32-knockout mice or patient-derived neurons. A summary of the implications of Rab32 dysfunction in neurodegenerative diseases is provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImplications of Rab32 Dysfunction in Neurodegenerative Diseases\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMechanism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTherapeutic Target\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParkinson\u0026rsquo;s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emtDNA leakage \u0026rarr; NLRP3 activation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRab32 agonists\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSOD1 mislocalization [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDV-peroxisome enhancers\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlzheimer\u0026rsquo;s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eROS accumulation \u0026rarr; Aβ aggregation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAntioxidant vesicle delivery\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study demonstrates that mitochondrial-derived vesicles (MDVs) function as a redox-sensitive interorganellar communication system, dynamically transferring antioxidant cargo (e.g., Prdx3, SOD1) to peroxisomes and lysosomes under oxidative. This process, regulated by Rab32, prevents mitochondrial DNA leakage and NLRP3 inflammasome activation, highlighting its critical role in maintaining cellular homeostasis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. By bridging mitochondrial stress responses with peroxisomal and lysosomal pathways, MDVs offer a novel framework for understanding mitochondrial adaptation in aging and neurodegeneration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Future studies should explore Rab32-MDV pathways as therapeutic targets for diseases like Parkinson\u0026rsquo;s, where oxidative damage and organelle dysfunction are hallmarks [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable (cell line study).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no funding and no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.S confirms sole responsibility for the following: study conception and design, data collection, analysis and interpretation of results, and manuscript preparation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcLelland GL, et al. 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Nat Rev Neurol. 2020;16(11):587\u0026ndash;606.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mitochondria-derived vesicles, oxidative stress, Rab32, organelle crosstalk, mitochondrial DNA","lastPublishedDoi":"10.21203/rs.3.rs-5926229/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5926229/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitochondrial-derived vesicles (MDVs) are essential for mitochondrial quality control, yet their cargo specificity and role in interorganellar communication remain poorly characterized. Here, we demonstrate that under oxidative stress, MDVs selectively transport antioxidant enzymes (e.g., peroxiredoxin-3, SOD1) to peroxisomes and lysosomes via Rab32-dependent trafficking. Using super-resolution microscopy and SILAC-based proteomics in HeLa and primary murine fibroblast models, we observed a 3-fold increase in MDV biogenesis post-H\u003csub\u003e2\u003c/sub\u003eO treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with Rab32 knockout cells exhibiting 50% higher cytosolic mitochondrial DNA (mtDNA) leakage and NLRP3 inflammasome activation. Live-cell imaging confirmed MDV-peroxisome fusion via PEX14 and lysosomal delivery via LAMP1, revealing a redox-sensitive mechanism that prevents oxidative damage. These findings establish MDVs as critical mediators of interorganellar coordination, offering novel therapeutic strategies for diseases linked to mitochondrial dysfunction, such as Parkinson\u0026rsquo;s disease.\u003c/p\u003e","manuscriptTitle":"Mitochondrial-Derived Vesicles Mediate Interorganellar Communication via Selective Cargo Transfer Under Oxidative Stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-04 08:50:26","doi":"10.21203/rs.3.rs-5926229/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d5bf6e9-93f6-4262-aaaa-451ca1afb230","owner":[],"postedDate":"February 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-04T08:50:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-04 08:50:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5926229","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5926229","identity":"rs-5926229","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}