{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T07:47:18Z","timestamp":1782978438554,"version":"3.54.5"},"reference-count":54,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2022,3,31]],"date-time":"2022-03-31T00:00:00Z","timestamp":1648684800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100004807","name":"DFG","doi-asserted-by":"publisher","award":["PA1751\/10-1"],"award-info":[{"award-number":["PA1751\/10-1"]}],"id":[{"id":"10.13039\/100004807","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000780","name":"European Union","doi-asserted-by":"publisher","award":["H2020-MSCA-765441"],"award-info":[{"award-number":["H2020-MSCA-765441"]}],"id":[{"id":"10.13039\/501100000780","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Calpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the calpain-specific substrate t-BOC-Leu-Met-CMAC to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and RhoP23H\/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell-death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD suitable for combination with non-invasive imaging techniques.<\/jats:p>","DOI":"10.3390\/ijms23073892","type":"journal-article","created":{"date-parts":[[2022,3,31]],"date-time":"2022-03-31T21:34:29Z","timestamp":1648762469000},"page":"3892","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Visualizing Cell Death in Live Retina: Using Calpain Activity Detection as a Biomarker for Retinal Degeneration"],"prefix":"10.3390","volume":"23","author":[{"given":"Soumaya","family":"Belhadj","sequence":"first","affiliation":[{"name":"Cell Death Mechanisms Group, Institute for Ophthalmic Research, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"},{"name":"Graduate Training Center of Neuroscience, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nina Sofia","family":"Hermann","sequence":"additional","affiliation":[{"name":"Graduate Training Center of Neuroscience, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Zhu","sequence":"additional","affiliation":[{"name":"Cell Death Mechanisms Group, Institute for Ophthalmic Research, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"},{"name":"Graduate Training Center of Neuroscience, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9786-7588","authenticated-orcid":false,"given":"Gustav","family":"Christensen","sequence":"additional","affiliation":[{"name":"Cell Death Mechanisms Group, Institute for Ophthalmic Research, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"},{"name":"Graduate Training Center of Neuroscience, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7725-7961","authenticated-orcid":false,"given":"Torsten","family":"Strasser","sequence":"additional","affiliation":[{"name":"Applied Vision Research Group, Institute for Ophthalmic Research, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"},{"name":"University Eye Hospital T\u00fcbingen, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7355-5742","authenticated-orcid":false,"given":"Fran\u00e7ois","family":"Paquet-Durand","sequence":"additional","affiliation":[{"name":"Cell Death Mechanisms Group, Institute for Ophthalmic Research, Eberhard-Karls-Universit\u00e4t T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"217","DOI":"10.3109\/09286586.2014.929710","article-title":"Prevalence of generalized retinal dystrophy in Denmark","volume":"21","author":"Bertelsen","year":"2014","journal-title":"Ophthalmic Epidemiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/j.preteyeres.2010.03.004","article-title":"The molecular basis of human retinal and vitreoretinal diseases","volume":"29","author":"Berger","year":"2010","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1637","DOI":"10.1016\/j.drudis.2019.03.004","article-title":"Drug delivery to retinal photoreceptors","volume":"24","author":"Himawan","year":"2019","journal-title":"Drug Discov. Today"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1038\/s41591-021-01382-x","article-title":"Biomarkers for neurodegenerative diseases","volume":"27","author":"Hansson","year":"2021","journal-title":"Nat. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1126\/science.186.4162.449","article-title":"Cyclic guanosine monophosphate: Elevation in degenerating photoreceptor cells of the C3H mouse retina","volume":"186","author":"Farber","year":"1974","journal-title":"Science"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"100772","DOI":"10.1016\/j.preteyeres.2019.07.005","article-title":"Cellular mechanisms of hereditary photoreceptor degeneration-Focus on cGMP","volume":"74","author":"Power","year":"2020","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1411","DOI":"10.1007\/s00424-021-02556-9","article-title":"The role of cGMP-signalling and calcium-signalling in photoreceptor cell death: Perspectives for therapy development","volume":"473","author":"Das","year":"2021","journal-title":"Pflug. Arch."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1152\/physrev.00029.2002","article-title":"The calpain system","volume":"83","author":"Goll","year":"2003","journal-title":"Physiol. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1038\/nsb0402-239","article-title":"How calpain is activated by calcium","volume":"9","author":"Khorchid","year":"2002","journal-title":"Nat. Struct. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"823","DOI":"10.2174\/1570159X17666190228112451","article-title":"Calpain-1 and Calpain-2 in the Brain: Dr. Jekill and Mr Hyde?","volume":"17","author":"Baudry","year":"2019","journal-title":"Curr. Neuropharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.tins.2016.01.007","article-title":"Calpain-1 and Calpain-2: The Yin and Yang of Synaptic Plasticity and Neurodegeneration","volume":"39","author":"Baudry","year":"2016","journal-title":"Trends Neurosci."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Arango-Gonzalez, B., Trifunovic, D., Sahaboglu, A., Kranz, K., Michalakis, S., Farinelli, P., Koch, S., Koch, F., Cottet, S., and Janssen-Bienhold, U. (2014). Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0112142"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"802","DOI":"10.1111\/j.1471-4159.2005.03628.x","article-title":"Calpain is activated in degenerating photoreceptors in the rd1 mouse","volume":"96","author":"Azadi","year":"2006","journal-title":"J. Neurochem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5431","DOI":"10.1167\/iovs.14-14325","article-title":"Characterization of a mouse model with complete RPE loss and its use for RPE cell transplantation","volume":"55","author":"Carido","year":"2014","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"18612","DOI":"10.1038\/srep18612","article-title":"The temporal topography of the N-Methyl- N-nitrosourea induced photoreceptor degeneration in mouse retina","volume":"5","author":"Tao","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1002\/jnr.21151","article-title":"Calpain activity in retinal degeneration","volume":"85","author":"Johnson","year":"2007","journal-title":"J. Neurosci. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1002\/cne.24807","article-title":"Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration","volume":"528","author":"Power","year":"2020","journal-title":"J. Comp. Neurol."},{"key":"ref_18","first-page":"533","article-title":"Involvement of calpain isoforms in retinal degeneration in WBN\/Kob rats","volume":"54","author":"Azuma","year":"2004","journal-title":"Comp. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3478","DOI":"10.2174\/0929867321666140601201337","article-title":"Novel in situ activity assays for the quantitative molecular analysis of neurodegenerative processes in the retina","volume":"21","author":"Ekstrom","year":"2014","journal-title":"Curr. Med. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"7130","DOI":"10.1016\/S0021-9258(19)42083-8","article-title":"Glutathione S-transferases. The first enzymatic step in mercapturic acid formation","volume":"249","author":"Habig","year":"1974","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"23593","DOI":"10.1016\/S0021-9258(19)49504-5","article-title":"Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach","volume":"268","author":"Rosser","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1167\/iovs.04-1051","article-title":"Permeability of retinal pigment epithelium: Effects of permeant molecular weight and lipophilicity","volume":"46","author":"Pitkanen","year":"2005","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1007\/s12035-019-01723-5","article-title":"Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation","volume":"57","author":"Comitato","year":"2020","journal-title":"Mol. Neurobiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e30577","DOI":"10.7554\/eLife.30577","article-title":"Small molecule Photoregulin3 prevents retinal degeneration in the Rho(P23H) mouse model of retinitis pigmentosa","volume":"6","author":"Nakamura","year":"2017","journal-title":"eLife"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1083\/jcb.119.3.493","article-title":"Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation","volume":"119","author":"Gavrieli","year":"1992","journal-title":"J. Cell Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"39537","DOI":"10.1038\/srep39537","article-title":"Olaparib significantly delays photoreceptor loss in a model for hereditary retinal degeneration","volume":"6","author":"Sahaboglu","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e488","DOI":"10.1038\/cddis.2013.12","article-title":"Retinitis pigmentosa: Rapid neurodegeneration is governed by slow cell death mechanisms","volume":"4","author":"Sahaboglu","year":"2013","journal-title":"Cell Death Dis."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"S12","DOI":"10.2337\/diabetes.53.2007.S12","article-title":"Structure, activation, and biology of calpain","volume":"53","author":"Suzuki","year":"2004","journal-title":"Diabetes"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1038\/35018098","article-title":"A one-hit model of cell death in inherited neuronal degenerations","volume":"406","author":"Clarke","year":"2000","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Bille, J.F. (2019). Scanning Laser Ophthalmoscopy (SLO). High Resolution Imaging in Microscopy and Ophthalmology: New Frontiers in Biomedical Optics, Springer.","DOI":"10.1007\/978-3-030-16638-0"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1111\/j.1365-2818.2012.03619.x","article-title":"Optical coherence tomography","volume":"247","author":"Podoleanu","year":"2012","journal-title":"J. Microsc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e142740","DOI":"10.1172\/jci.insight.142740","article-title":"Inhibiting calpain 1 and 2 in cyclin G associated kinase-knockout mice mitigates podocyte injury","volume":"5","author":"Tian","year":"2020","journal-title":"JCI Insight"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1177\/1535370218779780","article-title":"Bicalutamide enhances fodrin-mediated apoptosis through calpain in LNCaP","volume":"243","author":"Lee","year":"2018","journal-title":"Exp. Biol. Med."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1097\/01.WCB.0000035040.10031.B0","article-title":"Traumatic axonal injury results in biphasic calpain activation and retrograde transport impairment in mice","volume":"23","author":"Saatman","year":"2003","journal-title":"J. Cereb. Blood Flow Metab."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"39672","DOI":"10.1074\/jbc.M608803200","article-title":"A mouse model for monitoring calpain activity under physiological and pathological conditions","volume":"281","author":"Bartoli","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/j.jmb.2004.11.039","article-title":"Imaging calpain protease activity by multiphoton FRET in living mice","volume":"346","author":"Stockholm","year":"2005","journal-title":"J. Mol. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1002\/1098-1004(2001)17:1<42::AID-HUMU5>3.0.CO;2-K","article-title":"Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies","volume":"17","author":"Sohocki","year":"2001","journal-title":"Hum. Mutat."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"9745","DOI":"10.1074\/jbc.M513516200","article-title":"Calpain is required for normal osteoclast function and is down-regulated by calcitonin","volume":"281","author":"Marzia","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"858","DOI":"10.1016\/j.jmb.2010.01.014","article-title":"TRPM7 activates m-calpain by stress-dependent stimulation of p38 MAPK and c-Jun N-terminal kinase","volume":"396","author":"Su","year":"2010","journal-title":"J. Mol. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1002\/(SICI)1098-1136(200006)30:4<329::AID-GLIA20>3.0.CO;2-R","article-title":"Evaluation of glutathione-sensitive fluorescent dyes in cortical culture","volume":"30","author":"Tauskela","year":"2000","journal-title":"Glia"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/s002390010209","article-title":"Both the conserved and the unique gene structure of stomach-specific calpains reveal processes of calpain gene evolution","volume":"53","author":"Hata","year":"2001","journal-title":"J. Mol. Evol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1038\/nsb917","article-title":"Calpain silencing by a reversible intrinsic mechanism","volume":"10","author":"Moldoveanu","year":"2003","journal-title":"Nat. Struct. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Dutt, P., Croall, D.E., Arthur, J.S., Veyra, T.D., Williams, K., Elce, J.S., and Greer, P.A. (2006). m-Calpain is required for preimplantation embryonic development in mice. BMC Dev. Biol., 6.","DOI":"10.1186\/1471-213X-6-3"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4097","DOI":"10.1128\/MCB.05189-11","article-title":"Vital role of the calpain-calpastatin system for placental-integrity-dependent embryonic survival","volume":"31","author":"Takano","year":"2011","journal-title":"Mol. Cell. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.yexcr.2004.05.021","article-title":"Isoform specific function of calpain 2 in regulating membrane protrusion","volume":"299","author":"Franco","year":"2004","journal-title":"Exp. Cell Res."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Santos, D.M., Xavier, J.M., Morgado, A.L., Sol\u00e1, S., and Rodrigues, C.M. (2012). Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0033468"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"BSR20200552","DOI":"10.1042\/BSR20200552","article-title":"Calpain-2 participates in the process of calpain-1 inactivation","volume":"40","author":"Shindo","year":"2020","journal-title":"Biosci. Rep."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"8883","DOI":"10.1016\/S0021-9258(18)32139-2","article-title":"Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney","volume":"258","author":"Yoshimura","year":"1983","journal-title":"J. Biol. Chem."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"930","DOI":"10.1111\/j.1471-4159.2010.06983.x","article-title":"Photoreceptor rescue and toxicity induced by different calpain inhibitors","volume":"115","author":"Sanges","year":"2010","journal-title":"J. Neurochem."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Das, S., Popp, V., Power, M., Groeneveld, K., Melle, C., Rogerson, L., Achury, M., Schwede, F., Strasser, T., and Euler, T. (2021). Redefining the role of Ca2+-permeable channels in photoreceptor degeneration using diltiazem. bioRxiv.","DOI":"10.1101\/2020.12.04.411827"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1007\/BF00519723","article-title":"Comparative light and electron microscopic study of retinal histogenesis in normal and rd mutant mice","volume":"142","author":"Sanyal","year":"1973","journal-title":"Z. Anat. Entwicklungsgesch."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"10551","DOI":"10.1074\/jbc.M110.209759","article-title":"Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations","volume":"286","author":"Sakami","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_53","first-page":"e61868","article-title":"Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium","volume":"165","author":"Belhadj","year":"2020","journal-title":"J. Vis. Exp."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1038\/s41433-020-01286-z","article-title":"Adaptive optics: Principles and applications in ophthalmology","volume":"35","author":"Akyol","year":"2021","journal-title":"Eye"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/7\/3892\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:47:52Z","timestamp":1760136472000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/7\/3892"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,31]]},"references-count":54,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2022,4]]}},"alternative-id":["ijms23073892"],"URL":"https:\/\/doi.org\/10.3390\/ijms23073892","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.12.06.471350","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,31]]}}}