Senior Dogs have Less Plasma Circulating Exosomes Than Young Dogs, With Altered Sterol and miRNA content

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Las-Casas, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8918755/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Increasing life expectancy has been accompanied by a higher prevalence of age-associated conditions, underscoring the need to better understand biological mechanisms of aging and to identify accessible biomarkers. Extracellular vesicles (EVs) are lipid-bilayer vesicles that mediate intercellular communication by transporting proteins, lipids, and regulatory RNAs, including microRNAs (miRNAs). Here, we investigated whether the abundance, morphology, and molecular cargo of plasma-derived EVs differ between young and senior dogs. Blood samples were obtained from clinically healthy dogs in Gama (Federal District, Brazil), and plasma EVs were isolated by sequential centrifugation, filtration through a 0.22 µm filter, and ultracentrifugation. Vesicle concentration and size distribution were assessed by nanoparticle tracking analysis (NTA), morphology by transmission electron microscopy (TEM), and cargo by quantification of total protein and sterols using commercial assays. In addition, we quantified miR-19b, miR-29c, miR-7, miR-155, and miR-21 by Real Time Quantitative Polymerase Chain Reaction (RT-qPCR). Senior dogs exhibited a lower plasma EV yield and greater size heterogeneity, with a higher proportion of larger vesicles. Total protein and sterol content per starting plasma volume were reduced in the senior group; however, sterol normalized per vesicle was increased, consistent with compositional remodeling of circulating vesicles with age. Finally, EV-associated miRNA levels were reduced in senior dogs, particularly miR-19b and miR-29c. Collectively, these findings indicate that canine aging is associated with marked changes in plasma EV abundance, morphology, and cargo, supporting the use of healthy aged dogs as a translational model for investigating age-related dysregulation and neurodegenerative risk. exosomes aging plasma dog miRNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction As life expectancy increases, population aging has become an increasingly important concern, accompanied by a growing elderly population and a consequent rise in the prevalence of neurodegenerative disorders and other associated diseases and disabilities (1-3). As a result, older adults may experience a decline in quality of life that also affects their families, while increasing strain on healthcare systems and exerting broader economic impacts (4). A similar pattern is observed in veterinary medicine, where increasing pet life expectancy has been accompanied by a broad spectrum of age-associated conditions, including ocular, cardiac, orthopedic, neoplastic, and neurodegenerative disorders (5-7). The biological basis of aging is complex; however, the process can be understood as the cumulative consequence of natural failures in the mechanisms that maintain homeostasis (3, 8). These failures could be studied in humans and animals to better understand the process and identify targets for new therapies that may consequently increase the quality of life of elderly individuals and animals (9). An important hallmark is genomic instability, characterized by DNA replication errors, chromosome segregation defects, oxidative processes and spontaneous hydrolytic reactions that cannot be corrected by the DNA repair machinery, leading to the accumulation of significant mutations (10, 11). Other aging hallmark is the progressive telomeres shortening, a consequence of the incapacity of DNA polymerases to complete the copy of telomeric regions of eukaryotic DNA during replication, which can lead to apoptosis or cellular senescence when telomeres become too short (12). Normally, this process is delayed by telomerase and failure in this enzyme leads to accelerated aging and neurodegenerative disorders such as Alzheimer's disease (13). However, neurodegenerative diseases are frequently more associated with impaired protein homeostasis and proteostasis, leading to the accumulation and aggregation of misfolded or structurally altered proteins that impair the normal functionality of affected brain cells, including those referred to as “co-proteinopathies” (14-17). Given the role of pathological proteins in neurodegenerative processes, it is essential to examine the cellular mechanisms underlying protein transport. The transport of proteins is predominantly carried out by exosomes, small extracellular vesicles (EVs) formed by a membranous lipid bilayer (30 to 100 nm) that are secreted by almost all cells and are responsible for cell-to-cell communication (18). Exosomes carry a plenty of proteins, enzymes and ncRNAs that are released into the cytosol of target cells, regulating gene expression and post-transcriptional modifications, which directly influence important biological processes (19). Recently, exosomes have emerged as promising candidates for the treatment of tissue dysregulation or as disease biomarkers, as they are abundant in biological fluids and provide information about the organ or cell from which they were produced, through their surface or content molecules (18, 20). The role of exosomes in aging has been increasingly explored (21, 22). Studies in humans show that senescent and damaged cells produce more exosomes than younger ones; however, controversially, the plasma exosomes concentration decline with age (19). Furthermore, treating senescent cells with exosomes derived from pluripotent stem cells reduced the reactive oxygen species production and ameliorated the skin aging phenotype (23). Nonetheless, the characteristics of exosomes during canine aging have not been previously described, and dogs represent a reliable translational model for age-related disorders in humans (24, 25). The current study aimed to examine how exosome production changes in relation to the progression of aging in dogs by analyzing exosome yield in the plasma of young and senior dogs, as well as their morphological characteristics and miRNA content. Methodology Sampling population Samples were collected from clinically healthy dogs in Gama, an administrative region of the Federal District (DF), Brazil, through the Veterinary Clinical Neurology Service of the Centro Universitário do Planalto Central Apparecido dos Santos (UNICEPLAC). Owners were invited to authorize the donation of a blood sample for inclusion in the study. The protocol was approved by the Ethics Committee on Animal Use of the University of Brasília (CEUA/UnB: 23106.109701/2024-4) and by the UNICEPLAC Ethics Committee (CEUA/UNICEPLAC: 012/2024). Twelve small-breed dogs (body weight < 10 kg) aged 3–7 years were enrolled and classified as young, and twelve dogs aged up to 13 years were enrolled and classified as old (7, 26). Exosomes Isolation For exosomes isolation, we collected at least 5 mL of blood from dogs using EDTA-containing tubes. The blood samples were centrifuged at 300 g for 5 minutes, and the plasma was collected from the upper liquid phase. Next, 500 µL of plasma was then centrifuged at 10,000 g for 10 minutes to eliminate debris and residual cells, and the supernatant was filtered through 0.22 µm filters and ultracentrifuged at 100,000 g for 1.5 hours. The supernatant was discarded, and the pellet resuspended in Phosphate-Buffered Saline (PBS). The ultracentrifugation was repeated twice, and the final pellet containing exosomes was resuspended in 150 µL PBS. Exosomes Characterization by Yield, Size and Content We characterized the exosomes by assessing their protein content using the Micro BCA Protein Assay Kit (Thermo Fisher) and sterol content with the Amplex Red Cholesterol Assay Kit (Thermo Fisher), following the manufacturers’ recommendations. We measured yield and size through Nanoparticle Tracking Analysis (NTA), and we examined their morphology and size using Transmission Electron Microscopy (TEM), as detailed bellow. Exosomes quantification by NTA The quantification of exosomes was done by NTA (LM10) system coupled to a 488-nm laser, equipped with a camera and flow pump (Malvern Panalytical, Malvern, United Kingdom) and the NTA 3.0 software (Malvern Panalytical). The samples were injected with 1-mL syringes attached to a continuous flow injection pump. Three 60-second videos (camera level at 15, gain at 3) were obtained per sample after the passage of the samples through the light beam. The viscosity of the samples was maintained as that of water. For data analysis, the camera gain was changed to 10 − 15, and the detection limit used was three for all samples. If necessary, the samples were diluted in PBS to achieve the optimal range of 9×10 7 to 2.9×10 9 particles/mL (27). Exosomes Morphological Analysis by Transmission Electron Microscopy (TEM) For optical analysis of the exosomes, the isolated samples were observed using TEM. After homogenization, 50 µL of the exosome suspensions were added to Formvar-coated grids to allow adherence for 60 min at room temperature. Then, the grids were washed with 30 µL of sterile PBS, and the excess buffer was dried with filter paper. The grids were then incubated with 30 µL of Karnovski solution for 10 min, washed three times with cacodylate buffer, and finally dried with filter paper. The samples were counterstained with 5% uranyl acetate for 2 min. The grids were washed once with H 2 O, dried with filter paper, and transferred to a metallizer (Leica EM ACE200), where they were covered with carbon particles for later visualization with a JEOL 1400 Plus microscope with beam acceleration at 90 kV (28). miRNA Quantification For miRNA quantification, we first extracted the miRNA content from the exosome samples using the miRNeasy Mini Kit (Qiagen), following the manufacturer’s recommendations. The resulting miRNA was quantified using the Qubit microRNA Assay Kit (Invitrogen). Then, the volumes were adjusted to ensure the same amount of miRNA for all samples, and they were converted to cDNA using the TaqMan Advanced miRNA cDNA synthesis kit (Thermo Fisher), in accordance with manufacturer’s instructions, and quantified by real time polymerase chain reaction (RT-qPCR) Taqman system (QuantStudio 12K Flex system, Thermo Fisher). The reaction mix contained 2 µl cDNA, 1 µl miRNA specific primers [miR-19b (478264_mir), miR-29c (479229_mir), miR-7 (483061_mir), miR-155 (483064_mir), miR-21(477975_mir), all from Thermo Fisher Scientific], 10 µl TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific) and milli-Q pure water to 20 µl. The qPCR program consisted of two initial cycles (50°C for 2 min, 95°C for 20 s), followed by 40 amplification cycles (95°C for 1 min, 60°C for 1 min). Each run executed in triplicate including negative RT (non-template) controls. Relative expression of microRNAs was expressed by the delta–delta CT method (2 −ΔΔCt ) (29,30). Statistical Analysis The figures in the present study are representative of at least three technical and biological replicates, comprising three independent experiments. The data are presented as the average of the obtained values and their standard error of the mean (SEM). We analyzed if the data followed a normal distribution and statistical analysis was conducted using unpaired T-test, which compares two groups in the software GraphPad Prism, version 9. Significant statistical differences were considered when P < 0,05. P -values represented by * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Results Senior dogs produce fewer exosomes than young dogs After collecting plasma from young and senior dogs and isolating their exosomes, we analyzed exosome concentration and size distribution using NTA. In Figs. 1A and B, we noted great diversity in exosome size and concentration among individuals. However, when considering all samples together, as shown in Fig. 1C, we observed that exosomes from young dogs are homogeneous in size, forming a major peak around 100 nm in diameter, whereas exosomes from elderly dogs are more diverse, whit peaks ranging from 50 nm to 250 nm. We also examined the average exosome diameter and observed that the mean exosomes diameter in senior dogs was significantly higher than that in young dogs, a finding also noted in the groups containing 50% and 90% of exosomes (Fig. 2 A). At the same time, senior dogs presented significantly fewer exosomes in plasma than young dogs (Fig. 2 B). Figure 1 Senior dogs produce less and more diverse exosomes than young dogs. Size distribution and concentration of extracellular vesicles (EV)s measured by Nanoparticle Tracking Analysis (NTA) comparing plasma exosomes from dogs aged 2 to 6 years (young) (a) and dogs over 12 years (senior) (b). In panels a and b, each curve color represents an individual dog donor, showing the prevalence of EVs within each size range. In panel c, the line represents the mean exosomes size distribution comparing exosomes from young and elderly dogs, while the shaded area indicates the standard error of the mean (SEM) Exosomes from elderly dogs have more sterol than those from young dogs Analyzing the total protein and sterol content of isolated exosomes from the same initial plasma volume, we observed a significant reduction in the concentration of these molecules in the exosomes from senior dogs compared to those from young dogs (Fig. 3 A and 3 C). This finding corroborates the NTA data (Figs. 1 and 2 ), and indicates that the total exosome concentration is reduced in elderly dogs. Then, we analyzed the concentration of protein and sterol in relation to each individual sample of exosomes (EV) (Fig. 3 B and D), normalizing the total amount of protein and sterol by dividing each value by the number of exosomes obtained from the NTA data. In this manner, we did not find any differences in the protein ratio (Fig. 3 B); however, we observed a significantly higher concentration of sterol in exosomes from elderly dogs compared to those from young dogs (Fig. 3 D). Additionally, we normalized sterol content relative to protein levels. The resulting sterol/protein ratio per exosome was also higher in senior dogs (Fig. 3 E). This finding may explain the higher prevalence of exosomes with larger diameters in this group (Fig. 2 ), in which the sterol may be aggregating within the phospholipid membrane, prompting us to examine exosomes using microscopy. Age Influences the Morphological Characteristics of Plasma Exosomes Finally, the microscopic analysis reinforced the previous results, showing a great diversity of exosomes in terms of size, morphological characteristics, and electron density in the exosomes from senior dogs, while those from younger ones were more homogeneous (Fig. 4 ). We also observed larger exosomes (black arrows in Fig. 4 ) in the elderly samples, with thicker membranes and electron-dense content (ED in Fig. 4 ), which was barely seen in the samples from young dogs (Fig. 4 ). The differences in the particles’ electron density indicate a distinct exosome content in each group. Plasma exosomes from elderly dogs present a reduced content of miR29b and miR29c Considering the differential characteristics of dogs’ exosomes by age, and the importance of these structures in cell-to-cell communication, we analyzed the content of regulatory RNAs, specifically microRNAs, that are typically enriched in exosomes. We selected five miRNAs previously related to neurodegenerative disorders. We observed that exosomes from elderly dogs contained significantly lower levels of miR-19b and miR-29c than those from younger dogs. The fold change values were 0.8 and 0.38 for miR-19b and miR-29c, respectively. In contrast, miR-7, miR-155, and miR-21 were not differentially expressed between the groups (Fig. 5 ). Taking together, our results reinforce the importance of exosomes in transporting diverse molecules throughout the organism, including miRNAs. They also highlight that their morphological characteristics and internal content may influence the aging process. Discussion Comparing the characteristics of exosomes from young and senior dogs is important for highlighting aging biomarkers in this species for identifying potential targets for the development of more effective treatments to enhance the quality of life for elderly pets. Studies on senescence in animals can also contribute to understanding this process in humans, given the population's increased life expectancy. Indeed, exosomes are notably significant cellular components due to their contrasting roles in health and disease. Exosomes exhibit substantial plasticity in their functions, mediating the molecular secretome, intercellular communication, and signal transmission. Conversely, exosomes may play a crucial role in dysregulation of the aging machinery (18, 19). Our study demonstrated that elderly dogs contain fewer serum exosomes than young dogs. This finding is consistent with previous studies involving human plasma, which reported a reduction in exosome concentration with age progression (31). Eitan et al suggested that this reduction may be attributed to an increase in the internalization of circulating exosomes by B cells, rather than a decrease in production by the cells of older individuals (31). In fact, it has been shown that epithelial senescent cells release more exosomes than their young counterparts, accompanied by an increased expression of exosome markers RAB7 and CD63, as well as elevated lysosome content in their cytoplasm (32). The increase in surface markers may explain the elevated exosome internalization by B cells, followed by increased MHCII expression on monocytes. Interestingly, pre-activation with LPS improved the uptake of exosomes by B cells, mimicking an inflammatory condition commonly observed in the elderly (32). The inflammatory microenvironment is initiated by the senescence-associated secretory phenotype (SASP), in which senescent cells secrete a range of pro-inflammatory molecules, including cytokines, chemokines, matrix proteases, and growth factors. These molecules, mainly transported by exosomes, promote the proliferation of neighboring cells and induce tumorigenic effects in pre-malignant recipient cells (33, 34). In contrast, it has been demonstrated that mesenchymal stem cells secrete the rejuvenating factor GDF-11 within exosomes, promoting the polarization switch of macrophages from M1 to M2, thereby enhancing tissue regenerative capacity (35). Our study demonstrated that exosomes from senior dogs exhibit greater polydispersity in size, with exosomes from elderly dogs averaging larger than those from young dogs. The increased size, elevated sterol content, and the prevalence of eletron-dense exosomes in older individuals, along with a thicker lipid membrane observed in microscopy images, indicate a high concentration of LDL (36). A previous study comparing exosomes from endothelial senescent cells and those from young cells did not find differences in size or morphological characteristics (32). Nonetheless, Eitan et al reported a differential protein content in plasma exosomes from older and young donors, as measured by ELISA. In exosomes from older individuals, they observed reduced levels in apoptosis markers, including p53, cleaved PARP, and Caspase-3, along with increased levels of proteins related to tumorigenesis and metastasis, specifically CD151 and MUCIN16 (31). Considering the importance of exosomes for cellular communication, the transport of miRNA is essential for regulating gene expression and maintaining homeostasis, thereby enabling a refined global protein synthesis (37). Multiple groups have demonstrated that miRNAs modulate biological and, unfortunately, pathological aging, including by directly influencing telomere shortening and ROS production (38–45). However, the regulation of these functions through cell-to-cell signaling relies on the transport of microRNAs within exosomes, which promotes their efficient delivery to distant organs and tissues. Previous studies have shown that specific miRNAs, such as miR-21, miR29, and miR-34, are involved in tissue regeneration and homeostasis, potentially impacting life expectancy (19). The miRNAs miR-29c and miR-19b − which were down-regulated in exosomes from senior dogs in the present work, were previously reported to be down-regulated in patients with Parkinson’s disease and other neurodegenerative disorders that exemplify pathological aging (46, 47). Controversially, earlier studies showed that free miR-29 increases in multiple tissues with aging, and its overexpression induces accelerated aging-related phenotypes (48, 49). This discrepancy suggests that more miR-29 is delivered to tissues in the elderly, while it is reduced in blood exosomes. These data indicate that miRNA expression varies across tissues and life phases. For instance, miR-19 increases in plasma from individuals around 70 years old but decreases in plasma from centenarians (50). Additionally, the expression of miR-19 decreases with age in bone samples from mice and in posterior iliac crest bone biopsies from healthy female donors. This miRNA suppresses the expression of p16 Ink4a and p21 Cip1 , thereby influencing the rise of senescence phenotypes (51). Finally, our study demonstrated significant differences in the structural and internal content of plasma exosomes from young or senior dogs. This allow us to conclude that plasma exosomes are promising aging biomarkers, considering the easy of obtaining and processing the samples, as well as the volume of information they provide regarding the homeostasis of different organs and respective patterns of gene expression. This information is crucial for assessing the organism's overall health and identifying biomarkers indicative of disease and pathological aging before symptom intensification. Declarations Ethics approval: This study was performed in line with the principles of the Ethics Committee on Animal Use of the University of Brasília and of UNICEPLAC and was approved by both Ethics Committees (CEUA/UnB: 23106.109701/2024-4) and (CEUA/UNICEPLAC: 012/2024). Funding: This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (305584/2023–5) and Fundação de Apoio à Pesquisa do Distrito Federal (00193.00000227/2023-01) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES. Statements: The authors declare no conflict of interest. Author Contribution Study conception and design was performed by Clara Luna Marina, Anamélia Lorenzetti Bocca, Simoneide Souza Titze-de-Almeida, Fernando Francisco Borges Resende, Fabiano José de Sant’Anna and Ricardo Titze-de-Almeida. Material preparation, data collection and analysis were performed by Clara Luna Marina, Alexandra Mazer Greuel, Lucas Las-Casas, Evilly Lopes, Sabrina Simplício de Araújo Romero Ferrari, Marcio Lourenço Rodrigues, Flavia C. G. dos Reis and Fernando Francisco Borges Resende. The first draft of the manuscript was written by Clara Luna Marina and Fernando Francisco Borges Resende and Ricardo Titze-de-Almeida commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and analysed during the current study are not publicly available due to ongoing analyses and related unpublished work, but are available from the corresponding author on reasonable request. References Martinez R, Morsch P, Soliz P, Hommes C, Ordunez P, Vega E (2021) Life expectancy, healthy life expectancy, and burden of disease in older people in the Americas, 1990–2019: a population-based study. Rev Panam Salud Publica 45:e114. https://doi.org/10.26633/RPSP.2021.114 Guo J, Huang X, Dou L et al (2022) Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. 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Commun Biol 7:1055. https://doi.org/10.1038/s42003-024-06735-z Takeda T, Tanabe H (2016) Lifespan and reproduction in brain-specific miR-29 knockdown mice. Biochem Biophys Res Commun 471:454–458. https://doi.org/10.1016/j.bbrc.2016.02.063 Morsiani C, Terlecki-Zaniewicz L, Skalicky S et al (2021) Circulating miR-19a-3p and miR-19b-3p characterize the human aging process. Aging Cell 20:e13409. https://doi.org/10.1111/acel.13409 Kaur J, Saul D, Doolittle ML, Farr JN, Khosla S, Monroe DG (2023) MicroRNA-19a-3p decreases with age and inhibits osteoblast senescence. JBMR Plus 7:e10745. https://doi.org/10.1002/jbm4.10745 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Feb, 2026 Editor assigned by journal 25 Feb, 2026 Submission checks completed at journal 25 Feb, 2026 First submitted to journal 19 Feb, 2026 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-8918755","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598155490,"identity":"30bb43dd-9016-4d68-aca0-8b9a31608da3","order_by":0,"name":"Clara Luna Marina","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDACdhiDGYg/MDAkMEgQ0sKMxGCcQZoWEJuHGC38zczPPvxg2CZv3s578LNt2508BuneB3i1SBxmM57Zw3DbcM5hvmTp3LZnxQwyxw3wajFgZjBm4GG4zTiDmccAqOVwYoNEGn6HGTCzf2b8w3DbHqjF+LclcVp4jIG+vp0I1GImzUiMFonDPMXMMga3k0FaLHvOHS5mkzmGXwt/e/tmxjcVt21n8J8xvvGj7HAev3Qbfi1Q5yGx2YjRMApGwSgYBaMAPwAAQeo6YZplWf8AAAAASUVORK5CYII=","orcid":"","institution":"University of Brasília","correspondingAuthor":true,"prefix":"","firstName":"Clara","middleName":"Luna","lastName":"Marina","suffix":""},{"id":598155492,"identity":"41d60f58-b364-4b9b-bfe5-631890767f6c","order_by":1,"name":"Alexandra Mazer Greuel","email":"","orcid":"","institution":"University of Brasília","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"Mazer","lastName":"Greuel","suffix":""},{"id":598155494,"identity":"4b706f58-2c6c-4f1c-a232-37a2122b0a33","order_by":2,"name":"Lucas de O. Las-Casas","email":"","orcid":"","institution":"Carlos Chagas Institute, Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"de O.","lastName":"Las-Casas","suffix":""},{"id":598155502,"identity":"9daf443c-0906-48dd-8dc2-ff4fc71daf97","order_by":3,"name":"Evilly Lopes","email":"","orcid":"","institution":"University of Brasília","correspondingAuthor":false,"prefix":"","firstName":"Evilly","middleName":"","lastName":"Lopes","suffix":""},{"id":598155514,"identity":"989ad441-5de8-4cc7-a296-d0ed89836012","order_by":4,"name":"Sabrina Simplício de Araújo Romero Ferrari","email":"","orcid":"","institution":"University of Brasília","correspondingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"Simplício de Araújo Romero","lastName":"Ferrari","suffix":""},{"id":598155517,"identity":"9ffd2a44-8152-44c6-9089-b0b70287b6ca","order_by":5,"name":"Marcio Lourenço Rodrigues","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Marcio","middleName":"Lourenço","lastName":"Rodrigues","suffix":""},{"id":598155519,"identity":"4a55f867-acf2-40ec-9087-a2c7f41bc81f","order_by":6,"name":"Flavia C. G. dos Reis","email":"","orcid":"","institution":"Carlos Chagas Institute, Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Flavia","middleName":"C. G. dos","lastName":"Reis","suffix":""},{"id":598155520,"identity":"881fbf4c-783a-4c8e-afaa-d75ddc454755","order_by":7,"name":"Anamélia Lorenzetti Bocca","email":"","orcid":"","institution":"Bi-Institutional Platform for Translational Medicine, Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Anamélia","middleName":"Lorenzetti","lastName":"Bocca","suffix":""},{"id":598155521,"identity":"79f0f097-0603-4846-a548-d6cf038bbd29","order_by":8,"name":"Simoneide Souza Titze-de-Almeida","email":"","orcid":"","institution":"University of Brasília","correspondingAuthor":false,"prefix":"","firstName":"Simoneide","middleName":"Souza","lastName":"Titze-de-Almeida","suffix":""},{"id":598155522,"identity":"bff1cfc3-c318-4d29-87e8-641432e4c4df","order_by":9,"name":"Fernando Francisco Borges Resende","email":"","orcid":"","institution":"University Center of the Central Highlands Apparecido dos Santos (UNICEPLAC)","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"Francisco Borges","lastName":"Resende","suffix":""},{"id":598155523,"identity":"646f868e-002d-4305-93ff-7f226a9e618c","order_by":10,"name":"Fabiano José de Sant’Anna","email":"","orcid":"","institution":"University of Brasília","correspondingAuthor":false,"prefix":"","firstName":"Fabiano","middleName":"José","lastName":"de Sant’Anna","suffix":""},{"id":598155524,"identity":"0d779255-56d8-4c46-813e-81205abfaa22","order_by":11,"name":"Ricardo Titze-de-Almeida","email":"","orcid":"","institution":"University of Brasília","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Titze-de-Almeida","suffix":""}],"badges":[],"createdAt":"2026-02-19 15:40:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8918755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8918755/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104601938,"identity":"e7e4d73f-2296-47f6-afe6-95fa5b63479b","added_by":"auto","created_at":"2026-03-13 21:00:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSenior dogs produce less and more diverse exosomes than young dogs. \u003c/strong\u003eSize distribution and concentration of extracellular vesicles (EV)s measured by Nanoparticle Tracking Analysis (NTA) comparing plasma exosomes from dogs aged 2 to 6 years (young) (a) and dogs over 12 years (senior) (b). In panels a and b, each curve color represents an individual dog donor, showing the prevalence of EVs within each size range. In panel c, the line represents the mean exosomes size distribution comparing exosomes from young and elderly dogs, while the shaded area indicates the standard error of the mean (SEM)\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918755/v1/6279a8aadc1f96cc9bbdd70b.jpg"},{"id":104601934,"identity":"442d515d-7ce9-4f6e-be83-2de6eec17104","added_by":"auto","created_at":"2026-03-13 21:00:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":318647,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918755/v1/f8def36b4c12e30c91ffecdb.jpg"},{"id":104601937,"identity":"f85b0516-d100-406d-bb76-7a7d5817a96a","added_by":"auto","created_at":"2026-03-13 21:00:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":504294,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918755/v1/577e52a673f0ccb16a0078b4.jpg"},{"id":104782271,"identity":"b1375837-668f-48f7-bde1-fe58f7bf6faf","added_by":"auto","created_at":"2026-03-17 07:57:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":660515,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918755/v1/6ace516aef0572776736c599.jpg"},{"id":104601936,"identity":"4f65d065-ecbe-4153-9cf0-2fb1e592e4fb","added_by":"auto","created_at":"2026-03-13 21:00:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":337151,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918755/v1/40131f0cbbd7629d6a0b0ae1.jpg"},{"id":104784836,"identity":"d5a231c0-4cfe-41bf-bac8-abcbc69297b0","added_by":"auto","created_at":"2026-03-17 08:09:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2648668,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8918755/v1/bd066bdd-ceaa-4761-b203-b19649931bd9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Senior Dogs have Less Plasma Circulating Exosomes Than Young Dogs, With Altered Sterol and miRNA content","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs life expectancy increases, population aging has become an increasingly important concern, accompanied by a growing elderly population and a consequent rise in the prevalence of neurodegenerative disorders and other associated diseases and disabilities (1-3). As a result, older adults may experience a decline in quality of life that also affects their families, while increasing strain on healthcare systems and exerting broader economic impacts (4). A similar pattern is observed in veterinary medicine, where increasing pet life expectancy has been accompanied by a broad spectrum of age-associated conditions, including ocular, cardiac, orthopedic, neoplastic, and neurodegenerative disorders (5-7).\u003c/p\u003e\n\u003cp\u003eThe biological basis of aging is complex; however, the process can be understood as the cumulative consequence of natural failures in the mechanisms that maintain homeostasis (3, 8). These failures could be studied in humans and animals to better understand the process and identify targets for new therapies that may consequently increase the quality of life of elderly individuals and animals (9). An important hallmark is genomic instability, characterized by DNA replication errors, chromosome segregation defects, oxidative processes and spontaneous hydrolytic reactions that cannot be corrected by the DNA repair machinery, leading to the accumulation of significant mutations (10, 11). Other aging hallmark is the progressive telomeres shortening, a consequence of the incapacity of DNA polymerases to complete the copy of telomeric regions of eukaryotic DNA during replication, which can lead to apoptosis or cellular senescence when telomeres become too short (12). Normally, this process is delayed by telomerase and failure in this enzyme leads to accelerated aging and neurodegenerative disorders such as Alzheimer's disease (13). However, neurodegenerative diseases are frequently more associated with impaired protein homeostasis and proteostasis, leading to the accumulation and aggregation of misfolded or structurally altered proteins that impair the normal functionality of affected brain cells, including those referred to as “co-proteinopathies” (14-17).\u003c/p\u003e\n\u003cp\u003eGiven the role of pathological proteins in neurodegenerative processes, it is essential to examine the cellular mechanisms underlying protein transport. The transport of proteins is predominantly carried out by exosomes, small extracellular vesicles (EVs) formed by a membranous lipid bilayer (30 to 100 nm) that are secreted by almost all cells and are responsible for cell-to-cell communication (18). Exosomes carry a plenty of proteins, enzymes and ncRNAs that are released into the cytosol of target cells, regulating gene expression and post-transcriptional modifications, which directly influence important biological processes (19). Recently, exosomes have emerged as promising candidates for the treatment of tissue dysregulation or as disease biomarkers, as they are abundant in biological fluids and provide information about the organ or cell from which they were produced, through their surface or content molecules (18, 20).\u003c/p\u003e\n\u003cp\u003eThe role of exosomes in aging has been increasingly explored (21, 22). Studies in humans show that senescent and damaged cells produce more exosomes than younger ones; however, controversially, the plasma exosomes concentration decline with age (19). Furthermore, treating senescent cells with exosomes derived from pluripotent stem cells reduced the reactive oxygen species production and ameliorated the skin aging phenotype (23). Nonetheless, the characteristics of exosomes during canine aging have not been previously described, and dogs represent a reliable translational model for age-related disorders in humans (24, 25). The current study aimed to examine how exosome production changes in relation to the progression of aging in dogs by analyzing exosome yield in the plasma of young and senior dogs, as well as their morphological characteristics and miRNA content.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cp\u003eSampling population\u003c/p\u003e \u003cp\u003eSamples were collected from clinically healthy dogs in Gama, an administrative region of the Federal District (DF), Brazil, through the Veterinary Clinical Neurology Service of the Centro Universit\u0026aacute;rio do Planalto Central Apparecido dos Santos (UNICEPLAC). Owners were invited to authorize the donation of a blood sample for inclusion in the study. The protocol was approved by the Ethics Committee on Animal Use of the University of Bras\u0026iacute;lia (CEUA/UnB: 23106.109701/2024-4) and by the UNICEPLAC Ethics Committee (CEUA/UNICEPLAC: 012/2024). Twelve small-breed dogs (body weight\u0026thinsp;\u0026lt;\u0026thinsp;10 kg) aged 3\u0026ndash;7 years were enrolled and classified as young, and twelve dogs aged up to 13 years were enrolled and classified as old (7, 26).\u003c/p\u003e \u003cp\u003eExosomes Isolation\u003c/p\u003e \u003cp\u003eFor exosomes isolation, we collected at least 5 mL of blood from dogs using EDTA-containing tubes. The blood samples were centrifuged at 300 g for 5 minutes, and the plasma was collected from the upper liquid phase. Next, 500 \u0026micro;L of plasma was then centrifuged at 10,000 g for 10 minutes to eliminate debris and residual cells, and the supernatant was filtered through 0.22 \u0026micro;m filters and ultracentrifuged at 100,000 g for 1.5 hours. The supernatant was discarded, and the pellet resuspended in Phosphate-Buffered Saline (PBS). The ultracentrifugation was repeated twice, and the final pellet containing exosomes was resuspended in 150 \u0026micro;L PBS.\u003c/p\u003e \u003cp\u003eExosomes Characterization by Yield, Size and Content\u003c/p\u003e \u003cp\u003eWe characterized the exosomes by assessing their protein content using the Micro BCA Protein Assay Kit (Thermo Fisher) and sterol content with the Amplex Red Cholesterol Assay Kit (Thermo Fisher), following the manufacturers\u0026rsquo; recommendations. We measured yield and size through Nanoparticle Tracking Analysis (NTA), and we examined their morphology and size using Transmission Electron Microscopy (TEM), as detailed bellow.\u003c/p\u003e \u003cp\u003eExosomes quantification by NTA\u003c/p\u003e \u003cp\u003eThe quantification of exosomes was done by NTA (LM10) system coupled to a 488-nm laser, equipped with a camera and flow pump (Malvern Panalytical, Malvern, United Kingdom) and the NTA 3.0 software (Malvern Panalytical). The samples were injected with 1-mL syringes attached to a continuous flow injection pump. Three 60-second videos (camera level at 15, gain at 3) were obtained per sample after the passage of the samples through the light beam. The viscosity of the samples was maintained as that of water. For data analysis, the camera gain was changed to 10\u0026thinsp;\u0026minus;\u0026thinsp;15, and the detection limit used was three for all samples. If necessary, the samples were diluted in PBS to achieve the optimal range of 9\u0026times;10\u003csup\u003e7\u003c/sup\u003e to 2.9\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL (27).\u003c/p\u003e \u003cp\u003eExosomes Morphological Analysis by Transmission Electron Microscopy (TEM)\u003c/p\u003e \u003cp\u003eFor optical analysis of the exosomes, the isolated samples were observed using TEM. After homogenization, 50 \u0026micro;L of the exosome suspensions were added to Formvar-coated grids to allow adherence for 60 min at room temperature. Then, the grids were washed with 30 \u0026micro;L of sterile PBS, and the excess buffer was dried with filter paper. The grids were then incubated with 30 \u0026micro;L of Karnovski solution for 10 min, washed three times with cacodylate buffer, and finally dried with filter paper. The samples were counterstained with 5% uranyl acetate for 2 min. The grids were washed once with H\u003csub\u003e2\u003c/sub\u003eO, dried with filter paper, and transferred to a metallizer (Leica EM ACE200), where they were covered with carbon particles for later visualization with a JEOL 1400 Plus microscope with beam acceleration at 90 kV (28).\u003c/p\u003e \u003cp\u003emiRNA Quantification\u003c/p\u003e \u003cp\u003eFor miRNA quantification, we first extracted the miRNA content from the exosome samples using the miRNeasy Mini Kit (Qiagen), following the manufacturer\u0026rsquo;s recommendations. The resulting miRNA was quantified using the Qubit microRNA Assay Kit (Invitrogen). Then, the volumes were adjusted to ensure the same amount of miRNA for all samples, and they were converted to cDNA using the TaqMan Advanced miRNA cDNA synthesis kit (Thermo Fisher), in accordance with manufacturer\u0026rsquo;s instructions, and quantified by real time polymerase chain reaction (RT-qPCR) Taqman system (QuantStudio 12K Flex system, Thermo Fisher). The reaction mix contained 2 \u0026micro;l cDNA, 1 \u0026micro;l miRNA specific primers [miR-19b (478264_mir), miR-29c (479229_mir), miR-7 (483061_mir), miR-155 (483064_mir), miR-21(477975_mir), all from Thermo Fisher Scientific], 10 \u0026micro;l TaqMan\u0026reg; Fast Advanced Master Mix (Thermo Fisher Scientific) and milli-Q pure water to 20 \u0026micro;l. The qPCR program consisted of two initial cycles (50\u0026deg;C for 2 min, 95\u0026deg;C for 20 s), followed by 40 amplification cycles (95\u0026deg;C for 1 min, 60\u0026deg;C for 1 min). Each run executed in triplicate including negative RT (non-template) controls. Relative expression of microRNAs was expressed by the delta\u0026ndash;delta CT method (2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e) (29,30).\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe figures in the present study are representative of at least three technical and biological replicates, comprising three independent experiments. The data are presented as the average of the obtained values and their standard error of the mean (SEM). We analyzed if the data followed a normal distribution and statistical analysis was conducted using unpaired T-test, which compares two groups in the software GraphPad Prism, version 9. Significant statistical differences were considered when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05. \u003cem\u003eP\u003c/em\u003e-values represented by *\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, and ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSenior dogs produce fewer exosomes than young dogs\u003c/p\u003e \u003cp\u003eAfter collecting plasma from young and senior dogs and isolating their exosomes, we analyzed exosome concentration and size distribution using NTA. In Figs.\u0026nbsp;1A and B, we noted great diversity in exosome size and concentration among individuals. However, when considering all samples together, as shown in Fig.\u0026nbsp;1C, we observed that exosomes from young dogs are homogeneous in size, forming a major peak around 100 nm in diameter, whereas exosomes from elderly dogs are more diverse, whit peaks ranging from 50 nm to 250 nm.\u003c/p\u003e \u003cp\u003eWe also examined the average exosome diameter and observed that the mean exosomes diameter in senior dogs was significantly higher than that in young dogs, a finding also noted in the groups containing 50% and 90% of exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At the same time, senior dogs presented significantly fewer exosomes in plasma than young dogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1 Senior dogs produce less and more diverse exosomes than young dogs.\u003c/b\u003e Size distribution and concentration of extracellular vesicles (EV)s measured by Nanoparticle Tracking Analysis (NTA) comparing plasma exosomes from dogs aged 2 to 6 years (young) (a) and dogs over 12 years (senior) (b). In panels a and b, each curve color represents an individual dog donor, showing the prevalence of EVs within each size range. In panel c, the line represents the mean exosomes size distribution comparing exosomes from young and elderly dogs, while the shaded area indicates the standard error of the mean (SEM)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExosomes from elderly dogs have more sterol than those from young dogs\u003c/p\u003e \u003cp\u003eAnalyzing the total protein and sterol content of isolated exosomes from the same initial plasma volume, we observed a significant reduction in the concentration of these molecules in the exosomes from senior dogs compared to those from young dogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This finding corroborates the NTA data (Figs.\u0026nbsp;1 and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and indicates that the total exosome concentration is reduced in elderly dogs.\u003c/p\u003e \u003cp\u003eThen, we analyzed the concentration of protein and sterol in relation to each individual sample of exosomes (EV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and D), normalizing the total amount of protein and sterol by dividing each value by the number of exosomes obtained from the NTA data. In this manner, we did not find any differences in the protein ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); however, we observed a significantly higher concentration of sterol in exosomes from elderly dogs compared to those from young dogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e Additionally, we normalized sterol content relative to protein levels. The resulting sterol/protein ratio per exosome was also higher in senior dogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This finding may explain the higher prevalence of exosomes with larger diameters in this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e), in which the sterol may be aggregating within the phospholipid membrane, prompting us to examine exosomes using microscopy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAge Influences the Morphological Characteristics of Plasma Exosomes\u003c/p\u003e \u003cp\u003eFinally, the microscopic analysis reinforced the previous results, showing a great diversity of exosomes in terms of size, morphological characteristics, and electron density in the exosomes from senior dogs, while those from younger ones were more homogeneous (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We also observed larger exosomes (black arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) in the elderly samples, with thicker membranes and electron-dense content (ED in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which was barely seen in the samples from young dogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The differences in the particles\u0026rsquo; electron density indicate a distinct exosome content in each group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlasma exosomes from elderly dogs present a reduced content of miR29b and miR29c\u003c/p\u003e \u003cp\u003eConsidering the differential characteristics of dogs\u0026rsquo; exosomes by age, and the importance of these structures in cell-to-cell communication, we analyzed the content of regulatory RNAs, specifically microRNAs, that are typically enriched in exosomes. We selected five miRNAs previously related to neurodegenerative disorders. We observed that exosomes from elderly dogs contained significantly lower levels of miR-19b and miR-29c than those from younger dogs. The fold change values were 0.8 and 0.38 for miR-19b and miR-29c, respectively. In contrast, miR-7, miR-155, and miR-21 were not differentially expressed between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaking together, our results reinforce the importance of exosomes in transporting diverse molecules throughout the organism, including miRNAs. They also highlight that their morphological characteristics and internal content may influence the aging process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eComparing the characteristics of exosomes from young and senior dogs is important for highlighting aging biomarkers in this species for identifying potential targets for the development of more effective treatments to enhance the quality of life for elderly pets. Studies on senescence in animals can also contribute to understanding this process in humans, given the population's increased life expectancy. Indeed, exosomes are notably significant cellular components due to their contrasting roles in health and disease. Exosomes exhibit substantial plasticity in their functions, mediating the molecular secretome, intercellular communication, and signal transmission. Conversely, exosomes may play a crucial role in dysregulation of the aging machinery (18, 19).\u003c/p\u003e \u003cp\u003eOur study demonstrated that elderly dogs contain fewer serum exosomes than young dogs. This finding is consistent with previous studies involving human plasma, which reported a reduction in exosome concentration with age progression (31). Eitan \u003cem\u003eet al\u003c/em\u003e suggested that this reduction may be attributed to an increase in the internalization of circulating exosomes by B cells, rather than a decrease in production by the cells of older individuals (31). In fact, it has been shown that epithelial senescent cells release more exosomes than their young counterparts, accompanied by an increased expression of exosome markers RAB7 and CD63, as well as elevated lysosome content in their cytoplasm (32). The increase in surface markers may explain the elevated exosome internalization by B cells, followed by increased MHCII expression on monocytes. Interestingly, pre-activation with LPS improved the uptake of exosomes by B cells, mimicking an inflammatory condition commonly observed in the elderly (32).\u003c/p\u003e \u003cp\u003eThe inflammatory microenvironment is initiated by the senescence-associated secretory phenotype (SASP), in which senescent cells secrete a range of pro-inflammatory molecules, including cytokines, chemokines, matrix proteases, and growth factors. These molecules, mainly transported by exosomes, promote the proliferation of neighboring cells and induce tumorigenic effects in pre-malignant recipient cells (33, 34). In contrast, it has been demonstrated that mesenchymal stem cells secrete the rejuvenating factor GDF-11 within exosomes, promoting the polarization switch of macrophages from M1 to M2, thereby enhancing tissue regenerative capacity (35).\u003c/p\u003e \u003cp\u003eOur study demonstrated that exosomes from senior dogs exhibit greater polydispersity in size, with exosomes from elderly dogs averaging larger than those from young dogs. The increased size, elevated sterol content, and the prevalence of eletron-dense exosomes in older individuals, along with a thicker lipid membrane observed in microscopy images, indicate a high concentration of LDL (36). A previous study comparing exosomes from endothelial senescent cells and those from young cells did not find differences in size or morphological characteristics (32). Nonetheless, Eitan \u003cem\u003eet al\u003c/em\u003e reported a differential protein content in plasma exosomes from older and young donors, as measured by ELISA. In exosomes from older individuals, they observed reduced levels in apoptosis markers, including p53, cleaved PARP, and Caspase-3, along with increased levels of proteins related to tumorigenesis and metastasis, specifically CD151 and MUCIN16 (31).\u003c/p\u003e \u003cp\u003eConsidering the importance of exosomes for cellular communication, the transport of miRNA is essential for regulating gene expression and maintaining homeostasis, thereby enabling a refined global protein synthesis (37). Multiple groups have demonstrated that miRNAs modulate biological and, unfortunately, pathological aging, including by directly influencing telomere shortening and ROS production (38\u0026ndash;45). However, the regulation of these functions through cell-to-cell signaling relies on the transport of microRNAs within exosomes, which promotes their efficient delivery to distant organs and tissues.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that specific miRNAs, such as miR-21, miR29, and miR-34, are involved in tissue regeneration and homeostasis, potentially impacting life expectancy (19). The miRNAs miR-29c and miR-19b\u0026thinsp;\u0026minus;\u0026thinsp;which were down-regulated in exosomes from senior dogs in the present work, were previously reported to be down-regulated in patients with Parkinson\u0026rsquo;s disease and other neurodegenerative disorders that exemplify pathological aging (46, 47). Controversially, earlier studies showed that free miR-29 increases in multiple tissues with aging, and its overexpression induces accelerated aging-related phenotypes (48, 49). This discrepancy suggests that more miR-29 is delivered to tissues in the elderly, while it is reduced in blood exosomes. These data indicate that miRNA expression varies across tissues and life phases. For instance, miR-19 increases in plasma from individuals around 70 years old but decreases in plasma from centenarians (50). Additionally, the expression of miR-19 decreases with age in bone samples from mice and in posterior iliac crest bone biopsies from healthy female donors. This miRNA suppresses the expression of p16\u003csup\u003eInk4a\u003c/sup\u003e and p21\u003csup\u003eCip1\u003c/sup\u003e, thereby influencing the rise of senescence phenotypes (51).\u003c/p\u003e \u003cp\u003eFinally, our study demonstrated significant differences in the structural and internal content of plasma exosomes from young or senior dogs. This allow us to conclude that plasma exosomes are promising aging biomarkers, considering the easy of obtaining and processing the samples, as well as the volume of information they provide regarding the homeostasis of different organs and respective patterns of gene expression. This information is crucial for assessing the organism's overall health and identifying biomarkers indicative of disease and pathological aging before symptom intensification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval:\u003c/h2\u003e \u003cp\u003e This study was performed in line with the principles of the Ethics Committee on Animal Use of the University of Bras\u0026iacute;lia and of UNICEPLAC and was approved by both Ethics Committees (CEUA/UnB: 23106.109701/2024-4) and (CEUA/UNICEPLAC: 012/2024).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico - CNPq (305584/2023\u0026ndash;5) and Funda\u0026ccedil;\u0026atilde;o de Apoio \u0026agrave; Pesquisa do Distrito Federal (00193.00000227/2023-01) and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - CAPES.\u003c/p\u003e \u003cp\u003eStatements: The authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eStudy conception and design was performed by Clara Luna Marina, Anam\u0026eacute;lia Lorenzetti Bocca, Simoneide Souza Titze-de-Almeida, Fernando Francisco Borges Resende, Fabiano Jos\u0026eacute; de Sant\u0026rsquo;Anna and Ricardo Titze-de-Almeida. Material preparation, data collection and analysis were performed by Clara Luna Marina, Alexandra Mazer Greuel, Lucas Las-Casas, Evilly Lopes, Sabrina Simpl\u0026iacute;cio de Ara\u0026uacute;jo Romero Ferrari, Marcio Louren\u0026ccedil;o Rodrigues, Flavia C. G. dos Reis and Fernando Francisco Borges Resende. The first draft of the manuscript was written by Clara Luna Marina and Fernando Francisco Borges Resende and Ricardo Titze-de-Almeida commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and analysed during the current study are not publicly available due to ongoing analyses and related unpublished work, but are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMartinez R, Morsch P, Soliz P, Hommes C, Ordunez P, Vega E (2021) Life expectancy, healthy life expectancy, and burden of disease in older people in the Americas, 1990\u0026ndash;2019: a population-based study. 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JBMR Plus 7:e10745. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jbm4.10745\u003c/span\u003e\u003cspan address=\"10.1002/jbm4.10745\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"exosomes, aging, plasma, dog, miRNA","lastPublishedDoi":"10.21203/rs.3.rs-8918755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8918755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIncreasing life expectancy has been accompanied by a higher prevalence of age-associated conditions, underscoring the need to better understand biological mechanisms of aging and to identify accessible biomarkers. Extracellular vesicles (EVs) are lipid-bilayer vesicles that mediate intercellular communication by transporting proteins, lipids, and regulatory RNAs, including microRNAs (miRNAs). Here, we investigated whether the abundance, morphology, and molecular cargo of plasma-derived EVs differ between young and senior dogs. Blood samples were obtained from clinically healthy dogs in Gama (Federal District, Brazil), and plasma EVs were isolated by sequential centrifugation, filtration through a 0.22 µm filter, and ultracentrifugation. Vesicle concentration and size distribution were assessed by nanoparticle tracking analysis (NTA), morphology by transmission electron microscopy (TEM), and cargo by quantification of total protein and sterols using commercial assays. In addition, we quantified miR-19b, miR-29c, miR-7, miR-155, and miR-21 by Real Time Quantitative Polymerase Chain Reaction (RT-qPCR). Senior dogs exhibited a lower plasma EV yield and greater size heterogeneity, with a higher proportion of larger vesicles. Total protein and sterol content per starting plasma volume were reduced in the senior group; however, sterol normalized per vesicle was increased, consistent with compositional remodeling of circulating vesicles with age. Finally, EV-associated miRNA levels were reduced in senior dogs, particularly miR-19b and miR-29c. Collectively, these findings indicate that canine aging is associated with marked changes in plasma EV abundance, morphology, and cargo, supporting the use of healthy aged dogs as a translational model for investigating age-related dysregulation and neurodegenerative risk.\u003c/p\u003e","manuscriptTitle":"Senior Dogs have Less Plasma Circulating Exosomes Than Young Dogs, With Altered Sterol and miRNA content","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 21:00:21","doi":"10.21203/rs.3.rs-8918755/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-27T12:38:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T14:32:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T14:26:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2026-02-19T14:48:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d07b7a27-1f43-4cae-8d5d-4f16ba8a9a14","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T23:08:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 21:00:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8918755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8918755","identity":"rs-8918755","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}