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 iovs.arvojournals.org on 02/20/2020 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 Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-CAPN5 Human ADNIV Electroretinogram (ERG) polyclonal antibodies (GTX103264; GeneTex, Irvine, CA, USA; The collection of data used in this study was approved by the 12373-1-AP; Proteintech Group, Inc., Chicago, IL, USA; sc-50500 Institutional Review Board for Human Subjects Research at the and sc-271271; Santa Cruz Biotechnology, Inc.) rabbit anti-myc University of Iowa, was compliant with the Health Insurance tag antibody (ab9106) and other anti-CAPN5 antibodies Portability and Accountability Act, and adhered to the tenets of (ab28280, ab38943, and ab97534; Abcam, Cambridge, MA, USA). the Declaration of Helsinki. A full-field ERG was performed HEK293T adherent cells were grown in complete Dulbec- according to international standards. Briefly, the eyes were co’s modified Eagle’s medium (DMEM) with 10% fetal bovine dilated and dark adapted for 30 minutes. Electroretinograms system (FBS). SH-SY5Y adherent cells were cultured in a 1:1 were recorded simultaneously from both eyes using Burian- mix of RPMI 1640 and F-12 media containing 10% FBS. Human Allen bipolar contact lens electrodes as described previously.28 retinoblastoma Y79 cells were grown in RPMI-1640 medium Evoked waveforms, a 100 lV calibration pulse, and a stimulus containing 20% FBS. All cultures contained penicillin (100 U/ artifact were recorded on Polaroid film. mL) and streptomycin (100 lg/mL). RNA Preparation and Next-Generation Sequencing Western Blot The Institutional Animal Care and Use Committee (IACUC) Levels of CAPN5 were detected by immunoblot. Cells were approved all experiments. Rodents were used in accordance lysed 24 hours after transfection, and total cellular protein with the ARVO Statement for the Use of Animals in Ophthalmic measured using NanoDrop 2000c (Thermo Fisher Scientific, and Vision Research, as well as the Policy for the Use of Inc., Rockford, IL, USA). Samples were electrophoretically Animals in Neuroscience Research of the Society for Neuro- resolved by 4% to 12% Bis-Tris PAGE (Life Technologies; Grand science. Total RNA was extracted from mouse retinas and cell Island, NY, USA) and transferred to nitrocellulose (iBlot; Life lines using