University of Kentucky UKnowledge Spinal Cord and Brain Injury Research Center Spinal Cord and Brain Injury Research Faculty Publications 5-2016 Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses Kellie A. Schaefer University of Iowa Marcus A. Toral University of Iowa Gabriel Velez University of Iowa Allison J. Cox University of Iowa Sheila A. Baker University of Iowa See next page for additional authors Right click to open a feedback form in a new tab to let us know how this document benefits oy u. Follow this and additional works at: https://uknowledge.uky.edu/scobirc_facpub Part of the Neurology Commons Repository Citation Schaefer, Kellie A.; Toral, Marcus A.; Velez, Gabriel; Cox, Allison J.; Baker, Sheila A.; Borcherding, Nicholas C.; Colgan, Diana F.; Bondada, Vimala; Mashburn, Charles B.; Yu, Chen Guang; Geddes, James W.; Tsang, Stephen H.; Bassuk, Alexander G.; and Mahajan, Vinit B., "Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses" (2016). Spinal Cord and Brain Injury Research Center Faculty Publications. 12. https://uknowledge.uky.edu/scobirc_facpub/12 This Article is brought to you for free and open access by the Spinal Cord and Brain Injury Research at UKnowledge. It has been accepted for inclusion in Spinal Cord and Brain Injury Research Center Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Authors Kellie A. Schaefer, Marcus A. Toral, Gabriel Velez, Allison J. Cox, Sheila A. Baker, Nicholas C. Borcherding, Diana F. Colgan, Vimala Bondada, Charles B. Mashburn, Chen Guang Yu, James W. Geddes, Stephen H. Tsang, Alexander G. Bassuk, and Vinit B. Mahajan Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses Notes/Citation Information Published in Investigative Ophthalmology & Visual Science, v. 57, no. 6, p. 2509-2521. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Digital Object Identifier (DOI) https://doi.org/10.1167/iovs.15-18680 This article is available at UKnowledge: https://uknowledge.uky.edu/scobirc_facpub/12 Retinal Cell Biology Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses Kellie A. Schaefer,1,2 Marcus A. Toral,1–3 Gabriel Velez,1–3 Allison J. Cox,4 Sheila A. Baker,2,5 Nicholas C. Borcherding,1,3 Diana F. Colgan,1,2 Vimala Bondada,6 Charles B. Mashburn,6 Chen-Guang Yu,6 James W. Geddes,6 Stephen H. Tsang,7,8 Alexander G. Bassuk,4,9 and Vinit B. Mahajan1,2 1Omics Laboratory, University of Iowa, Iowa City, Iowa, United States 2Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States 3Medical Scientist Training Program, University of Iowa, Iowa City, Iowa, United States 4Department of Pediatrics, University of Iowa, Iowa City, Iowa, United States 5Department of Biochemistry, University of Iowa, Iowa City, Iowa, United States 6Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky, United States 7Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, New York, New York, United States 8Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, New York, New York, United States 9Neurology, University of Iowa, Iowa City, Iowa, United States Correspondence: Vinit B. Mahajan, PURPOSE. We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular Department of Ophthalmology and compartments. Visual Sciences, The University of Iowa,200HawkinsDrive,IowaCity, METHODS. CAPN5 gene variants were classified using the exome variant server, and RNA- IA 52242, USA; sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina [email protected]. and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and Submitted: November 19, 2015 mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines Accepted: February 28, 2016 and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. Citation: Schaefer KA, Toral MA, Velez G, et al. Calpain-5 expression in the RESULTS. Most CAPN5 genetic variation occurs outside its protease core; and searches of retina localizes to photoreceptor syn- cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating apses. Invest Ophthalmol Vis Sci. retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of 2016;57:2509–2521. DOI:10.1167/ nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of iovs.15-18680 CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS. CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles. Keywords: CAPN5, calpain, autosomal dominant neovascular inflammatory vitreoretinopathy, ADNIV utation of the calcium-activated protease calpain-5 disease allele reduces the calcium level required for protease M (CAPN5) can cause a severe blinding disease, autosomal activity.7 Thus, the eye-restricted phenotype likely reflects the dominant neovascular inflammatory vitreoretinopathy (ADNIV, extraordinarily high calcium concentrations in the retina, OMIM #193235).1–5 In their twenties, ADNIV patients begin to where such a hyperactive calcium-dependent protease could display a synaptic signaling defect and intraocular inflammation be particularly damaging.3,5 (uveitis). Over the ensuing five decades, they experience retinal Increased calpain activity is a feature of many eye-related degeneration, retinal neovascularization, and intraocular fibro- pathologies, including retinal degeneration,8,9 retinal hypox- sis, culminating in phthisis and blindness.1–3 Although CAPN5 ia,10–13 retinitis pigmentosa,14–16 retinal detachment,17 and is expressed in many tissues, ADNIV patients only manifest glaucoma.18,19 Retinal damage from these pathologies can be disease in the eye.6 Autosomal dominant neovascular inflam- lessened by administering the calpain inhibitor SJA6017.8,20–22 matory vitreoretinopathy CAPN5 is hyperactive, since the However, since the human retina expresses several calpains, it iovs.arvojournals.org j ISSN: 1552-5783 2509 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/935270/ on 07/10/2017 Calpain-5 Expression in the Retina IOVS j May 2016 j Vol. 57 j No. 6 j 2510 is not known which isoform(s) SJA6017 inhibits. Both CAPN1 Collaboration (phs000298). Only those consented for autism and CAPN2 are expressed in the retina and show increased research only (AO) were downloaded. The data set(s) were activity in other neurodegenerative conditions and hypoxic deposited by the ARRA Autism Sequencing Collaborative, an cell death.8,20 CAPN10 and calpastatin also are expressed in ARRA funded research initiative. Support for the Autism the retina23,24 and CAPN3 expresses a retina-specific splice Sequencing Collaborative was provided by Grants R01- variant in rats.8,25 Although CAPN3 is linked to limb-girdle MH089208 awarded to Mark Daly, R01-MH089175 awarded muscular dystrophy type 2A,26 it is not associated with any to Richard Gibbs, R01-MH089025 awarded to Joseph Bux- known retinal disease. CAPN5, the most distant calpain family baum, R01-MH089004 awarded to Gerard Schellenberg, and ortholog,7 is the only retinal calpain known directly to trigger R01-MH089482 awarded to James Sutcliffe. retinal disease in humans. Inhibition of CAPN5 might be Exome vcf files from the Epi4k Epilepsy Phenome/Genome therapeutic, but a specific inhibitor has never been isolated; Project (EPGP) were requested and downloaded from dbGAP and sequence analysis shows CAPN5 does not bind calpastatin, (dbGAP Study Accession, phs000653.v2.p1). Our appreciation the endogenous calpain inhibitor.7,27 goes to the Epilepsy Epi4k consortium: Discovery in Epilepsy To increase our understanding of CAPN5 in the healthy study (NINDS U01-NS077303) and the Epilepsy Genome/ retina and during ADNIV, we characterized CAPN5 mRNA and Phenome Project (EPGP-NINDS U01-NS053998).31,32 protein expression in the normal retina. We also drew from rich compilations of genetic-variance expression databases and Cell Culture, Reagents, and Antibodies performed antibody epitope-structure analysis, immunohisto- chemistry, and subcellular fractionation. Cell lines HEK293T, SH-SY5Y, and Y79 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Antibodies used were anti-GAPDH antibody (sc-32233), goat anti- METHODS rabbit, and goat anti-mouse secondary antibodies (Santa