{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:46:33Z","timestamp":1785415593608,"version":"3.56.0"},"posted":{"date-parts":[[2025,7,12]]},"group-title":"Neuroscience","reference-count":32,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,12]],"date-time":"2025-07-12T00:00:00Z","timestamp":1752278400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"funder":[{"award":["AG089761"],"award-info":[{"award-number":["AG089761"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>CRES is the defining member of a reproductive subgroup of family 2 cystatins of cysteine protease inhibitors. We previously showed that CRES and other subgroup members are part of a highly plastic amyloid-containing extracellular matrix (ECM) with host defense functions in the mouse epididymal lumen. Based on parallels between the epididymis and the brain, we hypothesized that CRES and CRES amyloids might also function within the brain including the ECM. Here we show that CRES is produced by hippocampal neurons and astrocytes in the male and female mouse and human brain. Further, approximately 50% of hippocampal astrocytes from aged mice, like the aged human donor samples, had significantly reduced levels of CRES compared to younger mice, suggesting an age-related decline in CRES could contribute to altered brain function. Immunofluorescence experiments showed CRES colocalized with the ECM markers phosphacan and wisteria floribunda agglutinin indicating that CRES is part of the ECM. CRES monomer and high molecular weight SDS-resistant forms were found in insoluble fractions of the hippocampus, cortex, cerebellum, and midbrain and bound to the protein aggregation disease (PAD) ligand, which preferentially binds amyloids but not protein monomers, suggesting a population of CRES exists in the brain as an amyloid structure. Collectively, our studies demonstrate that CRES\/CRES amyloid is present in the mammalian brain and may contribute to ECM structure and function.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>We previously established that the cystatin-related epididymal spermatogenic (CRES) protein is part of an amyloid-containing extracellular matrix (ECM) that protects the male germline in the epididymal lumen. Here we demonstrate that CRES is present within the mouse and human brain. Using cell biological and biochemical approaches, we show that CRES is found in hippocampal astrocytes and specific neuronal populations, including those that possess perineuronal nets, and colocalized with ECM markers suggesting it is part of the ECM.<\/jats:p>\n                  <jats:p>Biochemical analyses suggested a population of CRES is present as an ordered amyloid structure. Our studies reveal CRES is present in the male and female mammalian brain and may contribute to brain structure and function as a biological amyloid.<\/jats:p>\n                  <jats:p>Keyword: hippocampus, mouse, human, plasticity<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.07.08.663757","type":"posted-content","created":{"date-parts":[[2025,7,12]],"date-time":"2025-07-12T15:55:14Z","timestamp":1752335714000},"source":"Crossref","is-referenced-by-count":0,"title":["The Functional Epididymal Amyloid Cystatin-Related Epididymal Spermatogenic (CRES) is a Component of the Mammalian Brain Extracellular Matrix"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-8694-3083","authenticated-orcid":false,"given":"Alejandra","family":"Gomez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2788-382X","authenticated-orcid":false,"given":"Petar N.","family":"Grozdanov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2903-1690","authenticated-orcid":false,"given":"Gail A.","family":"Cornwall","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025071514403097000_2025.07.08.663757v1.1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1215369"},{"key":"2025071514403097000_2025.07.08.663757v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/S0303-7207(02)00408-2"},{"key":"2025071514403097000_2025.07.08.663757v1.3","doi-asserted-by":"crossref","unstructured":"Corsi, A. , Bombieri, C. , Valenti, M. T. & Romanelli, M. G . 2022. Tau Isoforms: Gaining Insight into MAPT Alternative Splicing. International Journal of Molecular Sciences, 23.","DOI":"10.3390\/ijms232315383"},{"key":"2025071514403097000_2025.07.08.663757v1.4","doi-asserted-by":"crossref","unstructured":"Dankovich, T. M. & Rizzoli, S. O . 2022. Extracellular Matrix Recycling as a Novel Plasticity Mechanism With a Potential Role in Disease. Frontiers in Cellular Neuroscience, 16.","DOI":"10.3389\/fncel.2022.854897"},{"key":"2025071514403097000_2025.07.08.663757v1.5","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA120.013012"},{"key":"2025071514403097000_2025.07.08.663757v1.6","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M600544200"},{"key":"2025071514403097000_2025.07.08.663757v1.7","doi-asserted-by":"crossref","unstructured":"Do, H. Q. , Hewetson, A. , Borcik, C. G. , Hastert, M. C. , Whelly, S. , Wylie, B. J. , Sutton, R. B. & Cornwall, G. A . 2021. Cross-seeding between the functional amyloidogenic CRES and CRES3 family members and their regulation of A\u03b2 assembly. Journal of Biological Chemistry, 296.","DOI":"10.1074\/jbc.RA120.015307"},{"key":"2025071514403097000_2025.07.08.663757v1.8","doi-asserted-by":"crossref","unstructured":"Dong, C. , Madar, A. D. & Sheffield, M. E. J . 2021. Distinct place cell dynamics in CA1 and CA3 encode experience in new environments. Nature Communications, 12.","DOI":"10.1038\/s41467-021-23260-3"},{"key":"2025071514403097000_2025.07.08.663757v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41380-022-01634-3"},{"key":"2025071514403097000_2025.07.08.663757v1.10","doi-asserted-by":"crossref","unstructured":"Fornasiero, E. F. , Mandad, S. , Wildhagen, H. , Alevra, M. , Rammner, B. , Keihani, S. , Opazo, F. , Urban, I. , Ischebeck, T. , Sakib, M. S. , Fard, M. K. , Kirli, K. , Centeno, T. P. , Vidal, R. O. , Rahman, R. U. , Benito, E. , Fischer, A. , Dennerlein, S. , Rehling, P. , Feussner, I. , Bonn, S. , Simons, M. , Urlaub, H. & Rizzoli, S. O . 2018. Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions. Nature Communications, 9.","DOI":"10.1038\/s41467-018-06519-0"},{"key":"2025071514403097000_2025.07.08.663757v1.11","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M211721200"},{"key":"2025071514403097000_2025.07.08.663757v1.12","doi-asserted-by":"publisher","DOI":"10.32607\/20758251-2015-7-2-42-47"},{"key":"2025071514403097000_2025.07.08.663757v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2020.01.002"},{"key":"2025071514403097000_2025.07.08.663757v1.14","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-019-03182-6"},{"key":"2025071514403097000_2025.07.08.663757v1.15","doi-asserted-by":"crossref","unstructured":"Laham, B. J. & Gould, E . 2022. How Stress Influences the Dynamic Plasticity of the Brain\u2019s Extracellular Matrix. Frontiers in Cellular Neuroscience, 15.","DOI":"10.3389\/fncel.2021.814287"},{"key":"2025071514403097000_2025.07.08.663757v1.16","doi-asserted-by":"crossref","unstructured":"Lensjo, K. K. , Christensen, A. C. , Tennoe, S. , Fyhn, M. & Hafting, T . 2017. Differential Expression and Cell-Type Specificity of Perineuronal Nets in Hippocampus, Medial Entorhinal Cortex, and Visual Cortex Examined in the Rat and Mouse. Eneuro, 4.","DOI":"10.1523\/ENEURO.0379-16.2017"},{"key":"2025071514403097000_2025.07.08.663757v1.17","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod.102.014100"},{"key":"2025071514403097000_2025.07.08.663757v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/nrm3902"},{"key":"2025071514403097000_2025.07.08.663757v1.19","doi-asserted-by":"crossref","unstructured":"Myers, C. , Atkins, G. , Villarreal, J. , Sutton, R. B. & Cornwall, G. A. 2024. The mouse epididymal amyloid matrix is a mammalian counterpart of a bacterial biofilm. iScience, 27.","DOI":"10.1016\/j.isci.2024.110152"},{"key":"2025071514403097000_2025.07.08.663757v1.20","doi-asserted-by":"crossref","unstructured":"Myers, C. , Hastert, M. C. & Cornwall, G. A . 2022. Host defense functions of the epididymal amyloid matrix. Molecular Human Reproduction, 28.","DOI":"10.1093\/molehr\/gaac038"},{"key":"2025071514403097000_2025.07.08.663757v1.21","doi-asserted-by":"crossref","unstructured":"P\u00e9rez-Gonz\u00e1lez, R. , Sahoo, S. , Gauthier, S. A. , Kim, Y. , Li, M. H. , Kumar, A. , Pawlik, M. , Benussi, L. , Ghidoni, R. & Levy, E. 2019. Neuroprotection mediated by cystatin C-loaded extracellular vesicles. Scientific Reports, 9.","DOI":"10.1038\/s41598-019-47524-7"},{"key":"2025071514403097000_2025.07.08.663757v1.22","doi-asserted-by":"publisher","DOI":"10.1177\/0271678X211004307"},{"key":"2025071514403097000_2025.07.08.663757v1.23","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.matbio.2022.05.003","article-title":"The alternative matrisome: Alternative splicing of ECM proteins in development, homeostasis and tumor progression","volume":"111","year":"2022","journal-title":"Matrix Biology"},{"key":"2025071514403097000_2025.07.08.663757v1.24","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/6.5.489"},{"key":"2025071514403097000_2025.07.08.663757v1.25","doi-asserted-by":"publisher","DOI":"10.1177\/002215549904700601"},{"key":"2025071514403097000_2025.07.08.663757v1.26","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.2351-16.2016"},{"key":"2025071514403097000_2025.07.08.663757v1.27","doi-asserted-by":"crossref","unstructured":"Su, C. H. , Dhananjaya, D. & Tarn, W. Y . 2018. Alternative Splicing in Neurogenesis and Brain Development. Frontiers in Molecular Biosciences, 5.","DOI":"10.3389\/fmolb.2018.00012"},{"key":"2025071514403097000_2025.07.08.663757v1.28","doi-asserted-by":"publisher","DOI":"10.2164\/jandrol.106.000331"},{"key":"2025071514403097000_2025.07.08.663757v1.29","doi-asserted-by":"crossref","unstructured":"Whelly, S. , Johnson, S. , Powell, J. , Borchardt, C. , Hastert, M. C. & Cornwall, G. A . 2012. Nonpathological extracellular amyloid is present during normal epididymal sperm maturation. Plos One, 7.","DOI":"10.1371\/journal.pone.0036394"},{"key":"2025071514403097000_2025.07.08.663757v1.30","doi-asserted-by":"publisher","DOI":"10.1093\/molehr\/gaw049"},{"key":"2025071514403097000_2025.07.08.663757v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/0006-8993(93)90941-F"},{"key":"2025071514403097000_2025.07.08.663757v1.32","doi-asserted-by":"crossref","unstructured":"Zhang, Z. L. , Ma, Z. , Zou, W. Y. , Guo, H. , Liu, M. , Ma, Y. L. & Zhang, L. X . 2019. The Appropriate Marker for Astrocytes: Comparing the Distribution and Expression of Three Astrocytic Markers in Different Mouse Cerebral Regions. Biomed Research International, 2019.","DOI":"10.1155\/2019\/9605265"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.07.08.663757","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:04:29Z","timestamp":1782277469000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.07.08.663757"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,12]]},"references-count":32,"URL":"https:\/\/doi.org\/10.1101\/2025.07.08.663757","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1002\/jnr.70113","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,7,12]]},"subtype":"preprint"}}