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Mutations in Pre-mRNA Processing Factors 3, 8, and 31 Cause Dysfunction of the Retinal Pigment Epithelium

  • Michael H. Farkas
    Affiliations
    Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
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  • Deborah S. Lew
    Affiliations
    Vision Institute, INSERM, U968, Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, CNRS, UMR_7210, Paris, France
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  • Maria E. Sousa
    Affiliations
    Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
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  • Kinga Bujakowska
    Affiliations
    Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts

    Vision Institute, INSERM, U968, Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, CNRS, UMR_7210, Paris, France
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  • Jonathan Chatagnon
    Affiliations
    Vision Institute, INSERM, U968, Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, CNRS, UMR_7210, Paris, France
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  • Shomi S. Bhattacharya
    Affiliations
    Vision Institute, INSERM, U968, Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, CNRS, UMR_7210, Paris, France

    Institute of Ophthalmology, University College London, London, United Kingdom

    Andalusian Center of Molecular Biology and Regenerative Medicine, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Sevilla, Spain
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  • Eric A. Pierce
    Correspondence
    Eric A. Pierce, M.D., Ph.D., Ocular Genomics Institute, Berman-Gund Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles St., Boston, MA 02114.
    Affiliations
    Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
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  • Emeline F. Nandrot
    Correspondence
    Address correspondence to Emeline F. Nandrot, Ph.D., Institut de la Vision, Therapeutics Department, 17 rue Moreau, 75012 Paris, France.
    Affiliations
    Vision Institute, INSERM, U968, Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, CNRS, UMR_7210, Paris, France
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Open AccessPublished:August 08, 2014DOI:https://doi.org/10.1016/j.ajpath.2014.06.026
      Mutations in the ubiquitously expressed pre-mRNA processing factors 3, 8, and 31 (PRPF3, PRPF8, and PRPF31) cause nonsyndromic dominant retinitis pigmentosa in humans, an inherited retinal degeneration. It is unclear what mechanisms, or which cell types of the retina, are affected. Transgenic mice with the human mutations in these genes display late-onset morphological changes in the retinal pigment epithelium (RPE). To determine whether the observed morphological changes are preceded by abnormal RPE function, we investigated its phagocytic function in Prpf3T494M/T494M, Prpf8H2309P/H2309P, and Prpf31+/− mice. We observe decreased phagocytosis in primary RPE cultures from mutant mice, and this is replicated by shRNA-mediated knockdown of PRPF31 in human ARPE-19 cells. The diurnal rhythmicity of phagocytosis is almost lost, indicated by the marked attenuation of the phagocytic burst 2 hours after light onset. The strength of adhesion between RPE apical microvilli and photoreceptor outer segments also declined during peak adhesion in all mutants. In all models, at least one of the receptors involved in binding and internalization of shed photoreceptor outer segments was subjected to changes in localization. Although the mechanism underlying these changes in RPE function is yet to be elucidated, these data are consistent with the mouse RPE being the primary cell affected by mutations in the RNA splicing factors, and these changes occur at an early age.
      The spliceosome is a ubiquitous, dynamic ribonucleoprotein macromolecule required for removing introns from a nascent RNA.
      • Will C.L.
      • Luhrmann R.
      Spliceosome structure and function.
      Mutations that cause autosomal dominant retinitis pigmentosa (RP) have been identified in five genes that encode proteins (PRPF3, PRPF6, PRPF8, PRPF31, and SNRNP200), which are found in the U4/U6.U5 tri–small nuclear ribonucleoprotein.
      • Liu M.M.
      • Zack D.J.
      Alternative splicing and retinal degeneration.
      In aggregate, mutations in these genes are the second most common cause of dominant RP.
      • Daiger S.P.
      • Bowne S.J.
      • Sullivan L.S.
      Perspective on genes and mutations causing retinitis pigmentosa.
      • Hartong D.T.
      • Berson E.L.
      • Dryja T.P.
      Retinitis pigmentosa.
      • Sullivan L.S.
      • Bowne S.J.
      • Reeves M.J.
      • Blain D.
      • Goetz K.
      • Ndifor V.
      • Vitez S.
      • Wang X.
      • Tumminia S.J.
      • Daiger S.P.
      Prevalence of mutations in eyeGENE probands with a diagnosis of autosomal dominant retinitis pigmentosa.
      Defined by progressive, late-onset vision loss, RP is the most common form of inherited retinal degeneration, affecting approximately 1 in 3500 persons worldwide.
      • Nishiguchi K.M.
      • Rivolta C.
      Genes associated with retinitis pigmentosa and allied diseases are frequently mutated in the general population.
      It is genetically heterogeneous and displays all three modes of Mendelian inheritance.
      • Neveling K.
      • Collin R.W.
      • Gilissen C.
      • van Huet R.A.
      • Visser L.
      • Kwint M.P.
      • Gijsen S.J.
      • Zonneveld M.N.
      • Wieskamp N.
      • de Ligt J.
      • Siemiatkowska A.M.
      • Hoefsloot L.H.
      • Buckley M.F.
      • Kellner U.
      • Branham K.E.
      • den Hollander A.I.
      • Hoischen A.
      • Hoyng C.
      • Klevering B.J.
      • van den Born L.I.
      • Veltman J.A.
      • Cremers F.P.M.
      • Scheffer H.
      Next-generation genetic testing for retinitis pigmentosa.
      Affected tissues include the neural retina, retinal pigment epithelium (RPE), and choroid.
      • Hartong D.T.
      • Berson E.L.
      • Dryja T.P.
      Retinitis pigmentosa.
      Because the components of the spliceosome are ubiquitously expressed in every cell type, it is not clear why mutations in these splicing factors cause only nonsyndromic RP. Further, the specific cell type(s) in the retina affected by these mutations has not yet been identified.
      We have previously reported the characterization of mouse models of RNA splicing factor RP due to mutations in the PRPF3, PRPF8, and PRPF31 genes, including Prpf3, Prpf8, and Prpf31 knockout mice, and Prpf3-T494M and Prpf8-H2309P knockin mice.
      • Graziotto J.J.
      • Farkas M.H.
      • Bujakowska K.
      • Deramaudt B.M.
      • Zhang Q.
      • Nandrot E.F.
      • Inglehearn C.F.
      • Bhattacharya S.S.
      • Pierce E.A.
      Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.
      • Graziotto J.J.
      • Inglehearn C.F.
      • Pack M.A.
      • Pierce E.A.
      Decreased levels of the RNA splicing factor Prpf3 in mice and zebrafish do not cause photoreceptor degeneration.
      On the basis of results from studies of these mouse models and data from human studies, it is believed that mutations in PRPF3 and PRPF8 cause dominant disease via gain-of-function or dominant-negative mechanisms, whereas mutations in PRPF31 cause disease via haploinsufficiency.
      • Graziotto J.J.
      • Inglehearn C.F.
      • Pack M.A.
      • Pierce E.A.
      Decreased levels of the RNA splicing factor Prpf3 in mice and zebrafish do not cause photoreceptor degeneration.
      • Rio Frio T.
      • Wade N.M.
      • Ransijn A.
      • Berson E.L.
      • Beckmann J.S.
      • Rivolta C.
      Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay.
      • Venturini G.
      • Rose A.M.
      • Shah A.Z.
      • Bhattacharya S.S.
      • Rivolta C.
      CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance.
      Morphological changes in the aging RPE, but not the neural retina, of the Prpf3-T494M and Prpf8-H2309P knockin mice and Prpf31+/− mice were of particular interest, in which we observed the loss of basal infoldings, the formation of basal deposits beneath the RPE, and vacuolization in the cytoplasm. These RPE degenerative changes were observed in heterozygous Prpf3T494M/+, Prpf8H2309P/+, and Prpf31+/− mice and were more pronounced in homozygous Prpf3T494M/T494M and Prpf8H2309P/H2309P knockin mice.
      • Graziotto J.J.
      • Farkas M.H.
      • Bujakowska K.
      • Deramaudt B.M.
      • Zhang Q.
      • Nandrot E.F.
      • Inglehearn C.F.
      • Bhattacharya S.S.
      • Pierce E.A.
      Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.
      The RPE is vital for the overall well-being of the retina.
      • Kevany B.M.
      • Palczewski K.
      Phagocytosis of retinal rod and cone photoreceptors.
      The daily elimination of spent photoreceptor outer segment (POS) extremities is a highly coordinated process, and phagocytosis of shed POS extremities occurs on a rhythmic basis.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      Some receptors implicated in POS phagocytosis also participate in overall retinal adhesion and its physiological rhythm.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      Peak phagocytosis and retinal adhesion occur approximately 2 and 3.5 hours after light onset, respectively, and are at their minimum levels roughly 10 hours later.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      The RPE is a professional macrophage in which binding and internalization of a substrate is coordinated by receptors on the RPE cell and ligands in the interphotoreceptor matrix bridging the RPE cell and phosphatidylserines at the POS surface, respectively.
      • Ruggiero L.
      • Connor M.P.
      • Chen J.
      • Langen R.
      • Finnemann S.C.
      Diurnal, localized exposure of phosphatidylserine by rod outer segment tips in wild-type but not Itgb5-/- or Mfge8-/- mouse retina.
      Some receptors are common between phagocytosis and adhesion, but they use different ligands.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      • Nandrot E.F.
      • Finnemann S.C.
      Lack of alphavbeta5 integrin receptor or its ligand MFG-E8: distinct effects on retinal function.
      A loss of regulation of any of these important components of phagocytosis leads to vision loss in human disease and in rodent models.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.
      • Dufour E.M.
      • Provost A.C.
      • Péquignot M.O.
      • Bonnel S.
      • Gogat K.
      • Marchant D.
      • Rouillac C.
      • Sépulchre de Condé B.
      • Bihoreau M.T.
      • Shaver C.
      • Dufier J.L.
      • Marsac C.
      • Lathrop M.
      • Menasche M.
      • Abitbol M.M.
      Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis.
      • Gal A.
      • Li Y.
      • Thompson D.A.
      • Weir J.
      • Orth U.
      • Jacobson S.G.
      • Apfelstedt-Sylla E.
      • Vollrath D.
      Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa.
      • Tschernutter M.
      • Jenkins S.
      • Waseem N.
      • Saihan Z.
      • Holder G.
      • Bird A.
      • Bhattacharya S.
      • Ali R.
      • Webster A.
      Clinical characterisation of a family with retinal dystrophy caused by mutation in the Mertk gene.
      Here, we report results of studies of RPE phagocytosis and adhesion for the Prpf3T494M/T494M, Prpf8H2309P/H2309P, and Prpf31+/− mouse models. Specifically, we measured phagocytosis in primary RPE cultures from 2-week-old mice. Results indicate a deficiency in phagocytosis, which we also found in the human RPE cell line, ARPE-19, after shRNA-mediated knockdown of PRPF31. In addition, a loss of diurnal rhythmicity of phagocytosis and adhesion were detected in vivo. Interestingly, localization of key factors known to be involved in phagocytosis by RPE cells is modified. We conclude that the RPE is likely to be the primary site of pathogenesis in RNA splicing factor RP.

      Materials and Methods

      Animals

      Animal research was performed under the protocols approved by the Institutional Animal Care and Use Committees at the Massachusetts Eye and Ear Infirmary and the Charles Darwin Animal Experimentation Ethics Committee from the Université Pierre et Marie Curie-Paris. An equal number of male and female mice were used in each of the following experiments.

      Primary RPE Cell Culture

      RPE cells from 9- to 10-day-old animals were isolated as described.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      Briefly, eyecups were digested with 2 mg/mL hyaluronidase (Sigma-Aldrich, St. Louis, MO), and the neural retina was gently peeled from the eyecup. RPE was peeled from the Bruch's membrane after digestion with 1 mg/mL trypsin (Invitrogen, Carlsbad, CA) and seeded onto 5-mm glass coverslips. Cells were grown to confluence for 5 to 10 days in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C, 5% CO2.

      Primary Peritoneal Macrophage Cell Culture

      Resident peritoneal macrophages were isolated as previously described.
      • Davies J.Q.
      • Gordon S.
      Isolation and culture of murine macrophages.
      Euthanized mice were pinned down to a dissection board, and the fur was dampened with 70% ethanol in a horizontal flow hood. The skin was delicately separated from the peritoneal wall by using forceps and scissors. Sterile phosphate-buffered saline (PBS; 5 mL) was injected into the abdominal cavity, and the belly was massaged or the whole body was shaken gently for 20 to 30 seconds. PBS was collected slowly from the cavity, and samples from two to three animals were pooled. Cells were spun for 10 minutes at 300 × g and resuspended in 1 mL of RPMI with 10% FBS. Cells were seeded in 96-well plates at 100,000 to 200,000 cells per well and allowed to adhere for 2 hours. Plates were shaken and wells were rinsed once with sterile PBS. Cells were maintained in medium for 2 to 3 days at 37°C, 5% CO2.

      Generation of Stable shRNA-PRPF31 Knockdown ARPE-19 and J774.1 Cell Lines and Cell Viability Assay

      Three shRNAs were designed to target human PRPF31 or mouse Prpf31 and cloned into pCAG-mir30 vector that contained a puromycin resistance gene. The sequences for these shRNAs are as follows: human shRNA1, 5′-TGCTGTTGACAGTGAGCGAGCAGATGAGCTCTTAGCTGATTAGTGAAGCCACAGATGTAATCAGCTAAGAGCTCATCTGCCTGCCTACTGCCTCGGA-3′; human shRNA2, 5′-TGCTGTTGACAGTGAGCGAACCCAACCTGTCCATCATTATTAGTGAAGCCACAGATGTAATAATGATGGACAGGTTGGGTGTGCCTACTGCCTCGGA-3′; and human shRNA3, 5′-TGCTGTTGACAGTGAGCGAGCTGAGTTCCTCAAGGTCAAGTAGTGAAGCCACAGATGTACTTGACCTTGAGGAACTCAGCCTGCCTACTGCCTCGGA-3′; mouse shRNA1, 5′-TGCTGTTGACAGTGAGCGCTCAGTCAAGAGCATTGCCAAGTAGTGAAGCCACAGATGTACTTGGCAATGCTCTTGACTGAATGCCTACTGCCTCGGA-3′; mouse shRNA2, 5′-TGCTGTTGACAGTGAGCGACCTGTCTGGCTTCTCTTCTACTAGTGAAGCCACAGATGTAGTAGAAGAGAAGCCAGACAGGGTGCCTACTGCCTCGGA-3′; and mouse shRNA3, 5′-TGCTGTTGACAGTGAGCGAGCCGAGTTCCTCAAGGTCAAGTAGTGAAGCCACAGATGTACTTGACCTTGAGGAACTCGGCCTGCCTACTGCCTCGGA-3′. We also cloned an shRNA to green fluorescence protein into this vector as a nontargeted control (5′-TGCTGTTGACAGTGAGCGCTCTCCGAACGTGTATCACGTTTAGTGAAGCCACAGATGTAAACGTGATACACGTTCGGAGATTGCCTACTGCCTCGGA-3′). The shRNA-containing vectors were linearized with PstI and transfected into separate ARPE-19 (human RPE cell line; ATCC, Manassas, VA) or J774A.1 (mouse macrophage cell line; ATCC) cultures by using the Amaxa electroporation kit V (Amaxa Biosystems, Gaithersburg, MD). Transfected cells were transferred to six-well plates and 2 mL of culture medium (dilution 1:1 DMEM:F-12 with 10% FBS). Transfected cells were grown overnight at 37°C, 5% CO2. Stable cell lines were selected with the addition of 1 (ARPE-19) to 1.25 (J774A.1) μg/mL puromycin (Sigma-Aldrich) 24 hours after transfection. Media and puromycin were refreshed every 2 days for 10 days. After selection, the four ARPE-19 and four J774A.1 knockdown lines were grown to confluence. To determine knockdown efficiency, stable lines were transiently transfected with either V5-tagged PRPF31 in ARPE-19 cells or V5-tagged Prpf31 cloned in a Gateway Destination vector (Invitrogen). Western blot analysis was performed, and V5-tagged PRPF31 was quantified with an Odyssey Infrared Imager (LI-CORE Biosciences, Lincoln, NE). Cell viability assays were performed with the Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) according to the manufacturer's recommendations. Briefly, ARPE-19 cells were seeded at a density of 1000 cells/well of a 96-well cell culture plate (catalog no. 3904; Corning, Corning, NY). Cells were grown for 3 days in DMEM with 10% FBS at 37°C, 5% CO2. After this period, cell viability was measured by luminescence, and statistical significance was determined with the Student's t-test.

      In Vitro Phagocytosis Assays

      POSs were isolated from porcine eyes obtained fresh from the slaughterhouse and covalently labeled with fluorescein isothiocyanate (FITC) dye (Invitrogen) for in vitro phagocytosis assays as previously described.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      Confluent cultured RPE cells were challenged with approximately 10 FITC-POSs per cell for 1.5 hours. Nonspecifically bound POSs were thoroughly removed with three washes in PBS with 1 mmol/L MgCl2 and 0.2 mmol/L CaCl2. To measure internalized POSs, some wells were incubated with trypan blue for 10 minutes to quench fluorescence of surface-bound FITC-labeled POSs as previously described.
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization.
      Cells were fixed with ice-cold methanol, and nuclei were counterstained with Hoechst 33258 (Invitrogen) or DAPI (Euromedex, Strasbourg, France). Cells were imaged with a Nikon Ti2 (Nikon, Tokyo, Japan) or a Leica DM6000 (Leica, Wetzler, Germany) fluorescent microscope at ×20 magnification. For RPE primary cultures, FITC/DAPI ratios were calculated on all picture fields, corresponding to the number of POSs per cell. FITC-POSs were counted on a per cell basis for 100 cells, and the average was determined for three wells for ARPE-19. For peritoneal macrophages, FITC-POS and DAPI-labeled nuclei were quantified by fluorescence plate reading (Infinite M1000; Magellan version 6 software; Tecan, Durham, NC). Binding ratios were calculated by subtracting results obtained in internalization wells (trypan blue-treated) from total phagocytosis (untreated) wells. This was performed for three to six independent assays, and significance was determined with the Student's t-test (P < 0.05).
      Before phagocytosis, confluent cultures of the stable knockdown J774A.1 lines were opsonized with Zymosan A Bioparticles Opsonizing Reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. After opsonization, 1 μg of Zymosan A Bioparticles reconstituted in culture medium were applied to each culture well of a 96-well plate. The cultures were incubated at 37°C, 5% CO2 for 1 hour. Fixation and determination of phagocytosis levels were performed as described above.

      In Vivo Diurnal Rhythm Assays

      Mice were euthanized at 2 hours before light onset (−2), at light onset (0), and 2, 4, and 8 hours (+2, +4, +8, respectively) after light onset, and processed for either electron microscopy or paraffin embedding as previously described.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      For electron microscopy all reagents were purchased from Electron Microscopy Sciences (Hatfield, PA). Mice were perfused with 2% glutaraldehyde + 2% paraformaldehyde, and eyecups were transferred to perfusion buffer by adding 0.2 mol/L sodium cacodylate buffer. Ultrathin sections (60 to 80 nm) were stained with lead citrate/uranyl acetate, and early phagosomes were counted from 200 nm out from the optic nerve. An early phagosome was counted if it met the following criteria: it was contained within the cytoplasm of the RPE and had visible lamellar structure. For light microscopy, eyecups were fixed in formaldehyde/ethanol/acetic acid and embedded in paraffin by using Ottix Plus solvent substitute (DiaPath, Martinengo Berfamo, Italy). Five-micrometer sections were cut, and the paraffin was removed with SafeSolv solvent substitute. The sections were rehydrated and incubated in 5% H2O2 in 1× standard saline citrate for 10 minutes under illumination to bleach pigments. After blocking nonspecific signals by using 10% bovine serum albumin in 1× Tris-buffered saline, sections were stained with an anti-rhodopsin antibody (Millipore, Billerica, MA) and anti-mouse IgG-Alexa Fluor 488 (Invitrogen). Nuclei were stained with DAPI, and slides were mounted with Mowiol (prepared according to standard procedures). Image stacks were acquired on an Olympus FV1000 inverted confocal microscope (Olympus, Tokyo, Japan) with a 60× oil objective, a 4-time zoom, and 0.41-μm step size scans and were processed with the Photoshop CS6 software (Adobe Systems, Mountain View, CA). Areas of at least 100 μm of uninterrupted retina/RPE were counted on 10-scan stacks. In each experiment series, phagosome counts were normalized to length of retina and averaged. Significance was determined with the Student's t-test (P < 0.05) with two to five mice for all experiments.

      In Vivo Retinal Adhesion Assays

      We performed in vivo retinal adhesion assays as described.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      Briefly, lens and cornea were removed from eyecups immediately after death in Hanks’ saline buffer with calcium and magnesium. A radial cut was made to the optic nerve, and the neural retina was gently peeled from the flattened eyecup. Neural retina samples were lyzed in 50 mmol/L Tris-HCl (pH 7.5), 2 mmol/L EDTA, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, and 1% Nonidet P-40, with the addition of a protease inhibitors cocktail (Sigma-Aldrich) and 1 mmol/L phenylmethylsulfonyl fluoride. Proteins from cleared supernatants were quantified with the Bradford assay, and equal concentrations were immunoblotted for RPE65 (Abcam, Cambridge, MA, or Millipore) and β-actin (Abcam or Sigma-Aldrich). Melanin pigments were extracted from the insoluble neural retina pellet with 20% dimethyl sulfoxide, 2N NaOH. Samples and commercial melanin standards (Sigma-Aldrich) were quantified by measuring absorbance at 490 nm. Pigment abundance was normalized to protein concentration in each sample to account for different tissue yields. Bands from immunoblots were quantified with ImageJ version 1.46r (NIH, Bethesda, MD) by using a common sample on all blots as reference; signals were then averaged. Significance was determined with the Student's t-test (P < 0.05) with three to six neural retina samples for all experiments.

      Immunofluorescence Microscopy

      For cryosections, eyecups were fixed in 2% paraformaldehyde and incubated in 30% sucrose overnight at 4°C. Eyecups were embedded in optimum cutting temperature compound (Sakura, Finetek, Inc., Torrance, CA), and 10-μm sections were cut. Sections were individually incubated with primary antibodies against αv integrin (BD Biosciences, Franklin Lakes, NJ), β5 integrin (Santa Cruz Biotechnology, Santa Cruz, CA), MER proto-oncogene tyrosine kinase (MerTK; FabGennix, Frisco, TX), milk fat globule-EGF8 (Mfg-E8) and growth arrest-specific 6 (Gas6; R&D Systems, Minneapolis, MN), focal adhesion kinase (FAK) clone 2A7 (Millipore), and Protein S (Sigma-Aldrich), followed by IgG-Alexa Fluor 488 (Invitrogen). Nuclei were stained with DAPI and mounted with Fluoromount (Electron Microscopy Sciences). Images were taken with a Nikon Eclipse Ti inverted fluorescence microscope with an oil immersion 60× objective. Images were processed with NIS-Elements AR software version 3.22.14 (Nikon).
      For paraffin sections, animals were sacrificed during the phagocytic peak, and eyes were fixed in Davidson fixative for 3 hours at 4°C, then lens and cornea were removed. Eyecups were embedded in paraffin, and 5-μm sections were cut. Sections were treated as described in In Vivo Diurnal Rhythm Assays and individually incubated with primary antibodies against αv integrin (Covance, Princeton, NJ); β5 integrin (Santa Cruz Biotechnology); Mfg-E8, MerTK, and Gas6 (R&D Systems); FAK clone 2A7 (Millipore); and Protein S (Novus Biologicals, Littleton, CO), followed by secondary antibody incubation with IgG-Alexa Fluor 488 (Invitrogen). Nuclei were stained with DAPI, and slides were mounted with Mowiol. Images were taken with a Leica DM6000 B Epifluorescence microscope with a 40× oil immersion objective. Images were processed with ImageJ version 1.46r and Photoshop CS6 (Adobe Systems) software.

      Results

      RPE Phagocytosis Is Decreased in Prpf-Mutant Mice

      In the original characterization of the Prpf-mutant mice, electron microscopy identified morphological changes in the RPE of 1- to 2-year-old mutants.
      • Graziotto J.J.
      • Farkas M.H.
      • Bujakowska K.
      • Deramaudt B.M.
      • Zhang Q.
      • Nandrot E.F.
      • Inglehearn C.F.
      • Bhattacharya S.S.
      • Pierce E.A.
      Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.
      Here, we wanted to determine whether functional changes precede the observed morphological changes. Because the RPE maintains phagocytic activity in culture, we established independent primary RPE cultures from 9- to 10-day-old Prpf3T494M/T494M, Prpf8H2309P/H2309P, Prpf31+/− mice and their corresponding littermate controls. Once the cultures were confluent, we used FITC-labeled porcine POSs and measured the phagocytosis after a 1.5-hour incubation. Images of primary cultures indicate the POS binding/uptake of RPE cells from the Prpf-mutant mice and their littermate controls and the qualitative deficiency in phagocytosis by the mutant mice (Figure 1A). In all three mutant models, 37% to 48% decrease in phagocytosis was observed (n = 3 to 5; P < 0.05) (Figure 1B). To account for nonspecific binding of POSs to the coverslips, we ran a negative control, in which the phagocytosis assay was performed on coverslips that did not contain cells. We observed no nonspecific adhesion of the POSs to the coverslips (data not shown).
      Figure thumbnail gr1
      Figure 1Inhibition of phagocytosis in Prpf-mutant mice. RPE primary cultures were established from 9- to 10-day-old Prpf-mutant mice and their littermate controls, then challenged with FITC-labeled porcine POSs and nuclei labeled with DAPI (blue). A: Qualitative representation of primary RPE cells from WT or Prpf3T494M/T494M, Prpf8H2309P/H2309P, Prpf31+/− MUT mice. A difference in POS uptake (green) was observed between the mutants and controls. B: Quantitative analysis of the phagocytic ratio showed a significant decrease in phagocytosis in the MUT mice compared with WT littermates for the three mutant mouse strains as indicated. C: Comparison of binding and internalization ratios of POSs between Prpf31+/− MUT mice and WT controls showed a significant decrease in Bind, but no significant change was seen in POS Intern in MUT mice. D: A stable line of shRNA-mediated knockdown of PRPF31 in ARPE-19 cells. A difference in POS uptake (green) was observed between the control shRNA-transfected ARPE-19 cells and anti-PRPF31 shRNA-transfected ARPE-19 cells. E: Cell viability assay to determine the effect of PRPF31-knockdown in ARPE-19 cells showed no significant differences in cell growth or viability after shRNA-knockdown of PRPF31 relative to the nontargeted control. F: shRNA-mediated knockdown of PRPF31 in the human ARPE-19 cell line also significantly inhibited phagocytosis as shown by the decreased number of POSs per cell compared with NTCs. Data are expressed as means ± SD. n = 3 to 5 mice (B), n = 2 to 5 mice (C), n = 6 assays (E), n = 3 assays (F). P < 0.05. Bind, binding; Intern, internalization; MUT, mutant; NTC, nontargeting construct.
      We investigated if a specific step of phagocytosis between binding and internalization was preferentially perturbed in Prpf31+/− RPE primary cultures. After performing 1.5-hour phagocytic challenge, we treated the cells to quench the surface (bound POS) fluorescence to quantify solely internalized POSs. POS binding was significantly reduced by 53% ± 11% in mutant cells (n = 2 to 5; P < 0.05), whereas no significant difference was found in POS internalization rates between wild-type (WT) and mutant RPE cultures (Figure 1C).
      Currently, 64 pathogenic mutations are known in PRPF31, of which many result in a frame shift and are degraded via the nonsense-mediated decay pathway.
      • Liu M.M.
      • Zack D.J.
      Alternative splicing and retinal degeneration.
      • Rio Frio T.
      • Wade N.M.
      • Ransijn A.
      • Berson E.L.
      • Beckmann J.S.
      • Rivolta C.
      Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay.
      • Venturini G.
      • Rose A.M.
      • Shah A.Z.
      • Bhattacharya S.S.
      • Rivolta C.
      CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance.
      • Stenson P.D.
      • Mort M.
      • Ball E.V.
      • Shaw K.
      • Phillips A.D.
      • Cooper D.N.
      The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine.
      ARPE-19 is a spontaneously immortalized human RPE cell line that is amenable to transfection and retains the ability to phagocytose.

      Mao Y, Finnemann S: Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture. Retinal Degeneration. Edited by Weber BH, Langmann T. Humana Press, 2013. pp. 285–295

      To test whether mutations in the splicing factors also affect phagocytosis in a human RPE model, we generated three stable ARPE-19 cell lines with shRNA-mediated knockdown of PRPF31 by using three distinct shRNAs directed against the 5′, 3′, and middle regions of the transcript (Figure 1D). We also generated a fourth stable cell line with an shRNA directed against the green fluorescent protein to use as a control. In each of the three PRPF31 shRNA stable cell lines we achieved approximately 60% to 95% knockdown of PRPF31 (data not shown). Cell viability assays of the shRNA-knockdown and nontargeted control ARPE-19 cells found that no significant decrease occurred in association with the knockdown of PRPF31 (Figure 1E). Phagocytosis was decreased by approximately 40% in each line tested compared with the nontargeted control shRNA line (Figure 1F). As with the phagocytosis assay performed on primary RPE, we also performed a negative control assay and observed no nonspecific adhesion of the POSs to the coverslips (data not shown).
      To determine whether disruption of the phagocytic machinery is an RPE-specific mechanism or can be observed in other phagocytic cells, we knocked down Prpf31 in the mouse macrophage cell line, J774A.1. Similar to the knockdown studies in the ARPE-19 cell line, three distinct shRNAs were directed to the 5′- and 3′-termini and middle of the transcript. We used the same control shRNA as the previous studies. In each of the stable Prpf31 cell lines, we achieved approximately 45% to 70% knockdown of Prpf31 (Supplemental Figure S1A). We observed no phagocytosis deficiency in any of the lines tested (Supplemental Figure S1B). To ensure we did not observe nonspecific POS adhesion, we performed a negative control assay as for the previous experiment series (data not shown). Identical experiments were repeated on mouse primary peritoneal macrophages isolated from Prpf31+/− mice. Interestingly, neither step of phagocytosis, that is, binding or internalization, nor total phagocytosis was affected in Prpf31-mutant macrophages compared with WT macrophages (Supplemental Figure S1C).

      Diurnal Rhythmicity of Phagocytosis Is Disrupted

      Phagocytosis of shed POSs by the RPE followed a strong diurnal, synchronized rhythm, peaking at 2 hours after light onset and remaining relatively inactive for the remainder of the day.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      We measured phagocytosis in vivo at 5 time points throughout the light cycle by using either electron microscopy (Figure 2A) or immunofluorescence (Figure 2B), both recognized techniques to assess the RPE phagocytic rhythm.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      For Prpf3 and Prpf8 control and mutant mice we counted early phagosomes that contained lamellar structures on electron micrographs (Figure 2A). Phagocytosis rhythmicity was determined in Prpf31+/− mice by using paraffin embedding and staining for rhodopsin, and we counted phagosomes present in the RPE cell layer (Figure 2B). We observed a phagocytosis burst at 2 hours after light onset in all control mice, identifying 22 to 26 phagosomes per 100 μm of retinal section (Figure 2C). In contrast, mutant mice only displayed 10 to 14 phagosomes at the same peak time point. During the rest of the light/dark cycle, phagocytosis levels remained relatively low in control mice (off-peak hours, 2 to 12 phagosomes/100 μm retina), and these levels were increased in mutant mice (6 to 14 phagosomes/100 μm retina). These results indicated a decrease in the phagocytic peak intensity in all three types of mutant mice, with a spreading of the time of the peak that lasted longer in Prpf3 and Prpf8 mutants and started earlier in Prpf31 mutants. Further, the Prpf8 mutants have more phagosomes at the off-peak time point (8 hours), relative to the WT controls.
      Figure thumbnail gr2
      Figure 2The diurnal rhythmicity of phagocytosis in Prpf-mutant mice is disrupted. Phagocytosis was assayed in vivo at 2 hours before light onset (−2), at light onset (0), and 2, 4, and 6 (+2, +4, +6) hours after light onset. Representative images at +2 (phagocytic peak) and +8 (outside of the phagocytic peak) hours after light onset (A and B). A: Detection of early phagosomes in Prpf3- and Prpf8-mutant mice was performed with electron microscopy and counting phagosomes that were in the cytoplasm of the RPE and contained visible lamellar structure (black arrowheads are representative of a phagosome; inset represents phagosome). B: The diurnal rhythm of Prpf31+/− mice was determined with immunofluorescent staining for rhodopsin (Ig-Alexa Fluor 488) and detection of phagosomes (white arrowheads) located in the RPE cell layer (DAPI-stained nuclei) across 100 μm of intact retina. C: Phagosome quantification across all time points showed the consistent significant disruption of the phagocytic burst in all Prpf-mutant mice. Data are expressed as means ± SD. n = 2 for Prpf3- and Prpf8-mutant mice, n = 3 to 5 for Prpf31-mutant mice (C). P < 0.05. Original magnification: ×4400 (A, inset). Ch, choroid; OS, photoreceptor outer segment, RPE, retinal pigment epithelium.

      Decreased Retinal Adhesion Is Observed at the Peak Time Point

      Adhesion between the RPE apical microvilli and distal tips of the POSs followed a synchronized rhythm with maximum strength occurring 3.5 hours after light onset, slightly after the phagocytic peak.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      Adhesion could be determined by peeling the retina from a flattened eyecup immediately after euthanasia, then quantifying both the RPE melanin content and apical RPE protein markers, such as RPE65, transferred to the retina. With the use of this method, we assessed adhesion in Prpf-mutant mice and littermate controls at 3.5 and 8.5 hours after light onset (peak and off-peak adhesion, respectively). RPE adhesion was quantified first by using a standard melanin quantification procedure,
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      then Western blot analysis for the presence of RPE65 to confirm the melanin results. We noted a decrease of 56% ± 16% (n = 6; P < 0.05; variation is equal to the SD) of the melanin content in the Prpf3T494M/T494M mice at peak time and no significant change in adhesion at the off-peak time point (Figure 3A). Western blot analysis confirmed this observation with a 30% ± 2% decrease in peak adhesion (Figure 3B). Melanin quantification in Prpf8H2309P/H2309P mice found that adhesion was significantly decreased by 61% ± 28% at the peak time point and 51% ± 16% at the off-peak time point (n = 6; P < 0.05 for both time points) (Figure 3A). Western blot analysis, however, confirmed a significant 36% ± 11% decrease only at the peak time point (Figure 3B). In the Prpf31+/− mice, a 15% ± 1% decrease was observed at the peak time point (Figure 3A) and was confirmed by immunoblot analysis (14% ± 1%; n = 3 to 7; P < 0.05 for both panels) (Figure 3B).
      Figure thumbnail gr3
      Figure 3Alterations in retinal adhesion in Prpf-mutant mice at the peak time point. Adhesive strength between RPE apical microvilli and POSs was determined by quantifying the amount of RPE pigments or proteins that adheres to the neural retina, relative to the WT control. A: Melanin quantification showed that adhesion was decreased in all three mutant mice strains at the peak time point 3.5 hours after light onset and in Prpf8H2309P/H2309P mice at the off-peak time point. B: Quantitative measurements of RPE65 proteins on immunoblots confirm the melanin findings in all three mutant mice strains at the peak time point; however, only a trend was observed for decrease in adhesion at the off-peak time point in Prpf8-mutant mice. Data are expressed as means ± SD. n = 3 to 7 mice (A), n = 4 to 7 mice (B). P < 0.05.

      Localization of Phagocytosis and Adhesion Markers

      RPE cells are highly polarized, and their function depends on this polarity.
      • Marmorstein A.D.
      The polarity of the retinal pigment epithelium.
      The specific localization of many proteins expressed in the RPE is important, and irregularities in localization may cause retinal dystrophies such as RP or Best disease.
      • Davidson A.E.
      • Millar I.D.
      • Urquhart J.E.
      • Burgess-Mullan R.
      • Shweikh Y.
      • Parry N.
      • O'Sullivan J.
      • Maher G.J.
      • McKibbin M.
      • Downes S.M.
      • Lotery A.J.
      • Jacobson S.G.
      • Brown P.D.
      • Black G.C.
      • Manson F.D.
      Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa.
      • Lopes V.S.
      • Gibbs D.
      • Libby R.T.
      • Aleman T.S.
      • Welch D.L.
      • Lillo C.
      • Jacobson S.G.
      • Radu R.A.
      • Steel K.P.
      • Williams D.S.
      The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65.
      Given the disruption of the diurnal rhythm of both phagocytosis and adhesion in all three Prpf-mutant mouse models, we wanted to characterize the localization of the proteins that are known to be important for these processes. Protein localization was assayed on cryosections for Prpf3- and Prpf8-mutant mice (Figure 4) and on paraffin sections for Prpf31-mutant mice (Figure 5).
      Figure thumbnail gr4
      Figure 4Localization and expression of some adhesion and phagocytosis markers are perturbed in Prpf3- and Prpf8-mutant mice. Representative images of the expression and localization of αv and β5 integrin receptor subunits and associated Mfg-E8 ligand (A), FAK intracellular signaling protein (B), and MerTK receptors and associated Gas6 and Protein S ligands (C) on WT control as well as Prpf3- and Prpf8-mutant retinal cryosections as indicated. Images from sections probed with nonimmune IgG (IgG) are included for each antigen. Localization of β5 integrin to the basal side of the RPE was observed in both Prpf3- and Prpf8-mutant mice. In addition, FAK was mislocalized in Prpf8-mutant mice to the basal side of the RPE. Each protein of interest was stained with Ig-Alexa Fluor 488, and nuclei were stained with DAPI. ONL, outer nuclear layer; OS, photoreceptor outer segments, RPE, retinal pigment epithelium.
      Figure thumbnail gr5
      Figure 5Localization and expression of some adhesion and phagocytosis markers are perturbed in Prpf31-mutant mice. Representative images of the expression and localization of αv and β5 integrin receptor subunits and associated Mfg-E8 ligand (A), FAK intracellular signaling protein (B), and MerTK receptors and associated Gas6 and Protein S ligands (C) or nonimmune IgG (IgG) on WT control and Prpf31-mutant retinal paraffin sections as indicated. The most notable change in Prpf31-mutant mice was the mislocalization of β5 integrin to the basal side of the RPE, whereas localization of MerTK was also perturbed. Each protein of interest was stained with IgG-Alexa Fluor 488, and nuclei were stained with DAPI. ONL, outer nuclear layer; OS, photoreceptor outer segments, RPE, retinal pigment epithelium.
      As reported previously, the main phagocytic receptors (αvβ5 integrin and MerTK) localized at the RPE apical surface,
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization.
      whereas their ligands were expressed throughout the POS and RPE.
      • Prasad D.
      • Rothlin C.V.
      • Burrola P.
      • Burstyn-Cohen T.
      • Lu Q.
      • Garcia de Frutos P.
      • Lemke G.
      TAM receptor function in the retinal pigment epithelium.
      Interestingly, extracellular ligands expressed in the interphotoreceptor matrix can be synthesized by both RPE and photoreceptor cells.
      It was reported that αvβ5-integrin with its associated ligand Mfg-E8 are important for phagocytosis and are responsible for the diurnal rhythmicity of this function.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      In addition, αvβ5-integrin participates in retinal adhesion and its rhythm, but with a ligand different from Mfg-E8.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      • Nandrot E.F.
      • Finnemann S.C.
      Lack of alphavbeta5 integrin receptor or its ligand MFG-E8: distinct effects on retinal function.
      αv Integrin subunits associate in complexes with several β integrin subunits in RPE cells
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      ; therefore, it was more relevant to analyze the expression of β5 integrin subunits. Thus, we probed for the αv and β5 subunits of the αvβ5 integrin receptor separately. In WT tissues each integrin localized primarily to the apical side of the RPE, with some expression throughout the RPE cells. In all three Prpf-mutant tissues, no change was observed in αv-integrin localization (Figures 4A and 5A). In contrast, β5 integrin localized primarily to the basal side of the RPE in the Prpf3- and Prpf31-mutant tissues, whereas it displayed expression equally throughout the RPE in Prpf8-mutant RPE cells. We observed no change in the localization of Mfg-E8 in either the RPE or POS.
      The downstream signaling protein FAK provided a sequential activation link between αvβ5 integrin and MerTK receptors both in vitro and in vivo.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Finnemann S.C.
      Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors.
      • Qin S.
      • Rodrigues G.A.
      Roles of alphavbeta5, FAK and MerTK in oxidative stress inhibition of RPE cell phagocytosis.
      FAK was found throughout the RPE, and no change to this pattern was observed in the Prpf3- or the Prpf31-mutant mice (Figures 4B and 5B). Prpf8-mutant mice, however, showed FAK localization to the basal side of the RPE.
      Phagocytosis is driven by the timely activation of MerTK via phosphorylation at the time of the activity peak.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      • Nandrot E.F.
      • Silva K.E.
      • Scelfo C.
      • Finnemann S.C.
      Retinal pigment epithelial cells use a MerTK-dependent mechanism to limit the phagocytic particle binding activity of alphavbeta5 integrin.
      Gas6 and Protein S are ligands of MerTK that can stimulate uptake of shed outer segments in vitro.
      • Hall M.O.
      • Obin M.S.
      • Heeb M.J.
      • Burgess B.L.
      • Abrams T.A.
      Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells.
      Both ligands are necessary to the internalization of POS because double knockout animals recapitulate the rapid retinal degeneration that occurs in rats in whose MerTK receptors are absent.
      • Burstyn-Cohen T.
      • Lew E.D.
      • Través P.G.
      • Burrola P.G.
      • Hash J.C.
      • Lemke G.
      Genetic dissection of TAM receptor-ligand interaction in retinal pigment epithelial cell phagocytosis.
      MerTK expression in WT tissues was localized to both the apical and basal membranes of the RPE, whereas MerTK was localized solely to the apical side of Prpf31-mutant RPE cells (Figures 4C and 5C). The first MerTK ligand Gas6 localized to the POS and apical layer of the RPE in WT tissues. Diffuse expression was seen throughout the RPE of Prpf3-mutant mice. Prpf8-mutant mice maintained Gas6 expression in the POS but appeared to lose apical localization in the RPE, also indicating a diffuse expression throughout the RPE. No localization changes could be observed in Prpf31-mutant mice. The expression of the second MerTK ligand Protein S was localized specifically to the POS in WT and all Prpf-mutant mice (Figures 4C and 5C).

      Discussion

      The molecular mechanism by which mutations in the five RNA splicing factor genes associated with RP cause disease has yet to be determined. A first step toward understanding the pathogenesis of RNA splicing factor RP is the identification of which of the >60 cell types in the retina is primarily affected by mutations in the RNA splicing factors.
      • Yin J.
      • Brocher J.
      • Fischer U.
      • Winkler C.
      Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for retinitis pigmentosa.
      • Masland R.H.
      Cell populations of the retina: the Proctor lecture.
      Although these findings must be confirmed in patients with RNA splicing factor RP, generation of the Prpf3-T494M, Prpf8-H2309P, and Prpf31+/− mouse models provided, for the first time, insight into the specific cell type affected by mutations in these genes.
      • Graziotto J.J.
      • Farkas M.H.
      • Bujakowska K.
      • Deramaudt B.M.
      • Zhang Q.
      • Nandrot E.F.
      • Inglehearn C.F.
      • Bhattacharya S.S.
      • Pierce E.A.
      Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.
      Morphological changes in the RPE suggested that the RPE, and not the neural retina, was the primary site of pathogenesis. In this study, we investigated the RPE phenotype in more detail and found that phagocytosis and retinal adhesion, two critical RPE functions for the retina, are impaired from an early age. We found that adhesion between the apical RPE microvilli and POSs and phagocytosis of normal spent POSs are both decreased. In addition, the diurnal rhythmicity of these two functions is disturbed. We observed changes to the localization of some of the major receptors and ligands that facilitate these processes. These findings provide strong evidence that the RPE is likely to be the primary cell affected by mutations in the RNA splicing factors in the mouse.
      One of the main functions that the RPE performs is the rhythmic binding and internalization of shed POSs daily.
      • Kevany B.M.
      • Palczewski K.
      Phagocytosis of retinal rod and cone photoreceptors.
      To complete phagocytosis, the apical microvilli of the RPE specifically recognize the POS via αvβ5 integrin receptor binding to its ligand Mfg-E8.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      Timely POS recognition by αvβ5 integrin receptors initiates a signaling cascade, leading to phosphorylation of MerTK, which is responsible for internalization of the bound particles.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Finnemann S.C.
      • Nandrot E.F.
      MerTK activation during RPE phagocytosis in vivo requires alphaVbeta5 integrin.
      In the primary RPE cultures of the Prpf mutant mice, we found a significant decrease in phagocytosis. Because these studies were performed with isolated RPE cells and normal POSs pooled from several porcine eyes, the results indicate that the decreased phagocytosis observed in the mutant cells is due to a cell autonomous process. In Prpf31-mutant mice the binding of POS, rather than internalization, was decreased. Binding and internalization are functionally distinct, suggesting that binding of POS is the primary function disrupted in this model.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      Because these assays were performed with cells from young animals, these results suggest that mutations in the RNA splicing factors cause alterations in RPE function at an early age and are intrinsically present in RPE cells. Therefore, the phenotype appearing in aging animals also suggests that degeneration is possibly the result of a cumulative process.
      Haploinsufficiency is likely the cause of dominant disease with mutations in PRPF31, and this provides an opportunity to develop a knockdown model of disease in the human ARPE-19 cell line.
      • Liu M.M.
      • Zack D.J.
      Alternative splicing and retinal degeneration.
      • Rio Frio T.
      • Wade N.M.
      • Ransijn A.
      • Berson E.L.
      • Beckmann J.S.
      • Rivolta C.
      Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay.
      With the use of shRNAs directed against PRPF31, we generated three stable knockdown lines in ARPE-19 cells. The phagocytosis assays were performed in the same manner as in the primary mouse culture system, and a similar deficiency in phagocytosis was observed. This not only adds to the evidence that haploinsufficiency is the mechanism of disease in cases of PRPF31 mutations but also suggests that the functional mechanism observed in mice is likely to apply to affected humans.
      RNA splicing RP is characterized as a nonsyndromic disease, and this allows us to study whether defects in phagocytosis of the RPE are caused by RPE-specific mechanisms or generalized defects in common phagocytic processes. We knocked down Prpf31 in the mouse macrophage cell line, J774A.1, tested peritoneal macrophages from the Prpf31-mutant mice, and observed no loss of phagocytic function. This finding suggests that general macrophage function is not affected, and RPE-specific phagocytic mechanisms appear to be the cause of the deficiency. Further, patients with RNA splicing factor RP do not present with any macrophage-associated disease, adding additional evidence in support of an RPE-specific deficiency.
      Knockout mouse models of β5 integrin have found the importance of αvβ5 integrin receptors for both RPE phagocytosis and retinal adhesion.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      Peak phagocytosis occurs before peak adhesion 2 hours after light onset, and a loss of this synchronicity leads to retinal dysfunction with age.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      In the Prpf-mutant models, we used two independent techniques, electron microscopy in Prpf3 and Prpf8 models and immunofluorescence in the Prpf31 model, to study the phagocytic diurnal rhythm, both techniques previously validated as giving comparable results.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      We found that peak phagocytosis, in all cases, was reduced at 2 hours after light onset, with a peak activity appearing attenuated and more widespread in time. Peak adhesion occurs at 3.5 hours after light onset, after a similar synchronous rhythm occurring just after peak phagocytosis.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      In each of the Prpf-mutant mouse models studied, we found that adhesion was significantly decreased at the peak time point, down to levels detected at the off-peak time. These data suggest that functional deficiencies in both phagocytosis and adhesion in the RPE are a result of these mutations and may contribute to the pathogenesis of disease. Interestingly, the peak activity time of both functions is perturbed in vivo, whereas phagocytosis and adhesion seem relatively normal at other times. These results suggest that cyclic activities may be affected and need to be studied in more detail.
      The coordination of the phagocytic diurnal rhythm is reliant on many factors, including the expression of the proper receptors, the polarity of the RPE, the expressed ligands, and signaling pathways.
      • Kevany B.M.
      • Palczewski K.
      Phagocytosis of retinal rod and cone photoreceptors.
      Mfg-E8 has been identified as an extracellular ligand that is required for maintaining the phagocytic but not the adhesive synchronicity of the RPE.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis.
      The RPE in mice lacking either β5 integrin receptors or Mfg-E8 ligands do not experience a phagocytic peak; however, the neural retina does not degenerate in any of these two models even if vision loss occurs in β5 knockout mice.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      The Prpf-mutant mice display a similar phenomenon, suggesting that the retina is tolerant of a dysfunctional RPE, at least for a period of time. But unlike β5 integrin- and Mfg-E8–deficient mice, it is unknown which factors are responsible for the phagocytosis and adhesion deficiencies in the Prpf-mutant mice; therefore, we decided to interrogate the known phagocytosis-related receptors and ligands.
      Immunofluorescence analyses of the localization of the primary RPE phagocytosis ligands and receptors in the RPE and POSs found mislocalization of β5-integrin in all three mutant mouse models and FAK in Prpf8-mutant mice. Aberrant splicing resulting in direct protein mislocalization has been implicated in diseases such as Fukuyama muscular dystrophy.
      • Taniguchi-Ikeda M.
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      • Yu C.C.
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      • Yokota T.
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      • Toda T.
      Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy.
      Alternatively, mislocalization could be the result of aberrant splicing of secondary factors such as components required for protein trafficking.
      • Dell’Angelica E.C.
      AP-3-dependent trafficking and disease: the first decade.
      It is unknown if the extent of dysfunction observed in our study can be the result of the mislocalized receptors and ligands. Various methods are available to examine if aberrantly spliced transcripts are produced in RPE cells, such as reverse transcription PCR of full-length transcripts, or of specific gene portions, and Northern blot analysis. However, they all present major flaws because aberrant transcripts might not be detected because of primer or probe design or because of the lack of information on which exons would be targeted by the missplicing. Currently, the most effective method for detecting aberrant transcripts is the use of RNA-Seq analyses. Indeed, RNA-Seq allowed us to discover thousands of alternative terminal exons in the human retinal transcriptome.
      • Farkas M.H.
      • Grant G.R.
      • White J.A.
      • Sousa M.E.
      • Consugar M.B.
      • Pierce E.A.
      Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes.
      We have established models that permit us to perform these much broader studies of RNA splicing in these forms of RP, although those experiments are beyond the scope of the present study.
      Here, we report the first functional characterization of the RPE in mice with mutations in the RNA splicing factors Prpf3, Prpf8, and Prpf31. As we have previously reported, the mutant mice do not experience photoreceptor degeneration but rather morphological changes in the RPE.
      • Graziotto J.J.
      • Farkas M.H.
      • Bujakowska K.
      • Deramaudt B.M.
      • Zhang Q.
      • Nandrot E.F.
      • Inglehearn C.F.
      • Bhattacharya S.S.
      • Pierce E.A.
      Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.
      Because RNA splicing factor RP is a late-onset disease, these results are not surprising, and the models afford us the ability to study the mechanisms leading to the onset of disease. Our results indicate that the RPE is likely to be the primary cell type affected by mutations in these three RNA splicing factors in the mouse and in humans, given the similar phagocytic deficiency observed in PRPF31-knockdown human ARPE-19 cells. Although the exact mechanism of disease pathogenesis remains to be identified, these data will allow for future research to be focused on the RPE. For example, the identification of the RPE as the primary cell type affected in these disorders will make it possible to extend these studies to human cells, because it is now possible to generate RPE cells from human induced pluripotent stem cells of patients with inherited retinal diseases.
      • Buchholz D.E.
      • Pennington B.O.
      • Croze R.H.
      • Hinman C.R.
      • Coffey P.J.
      • Clegg D.O.
      Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium.
      • Meyer J.S.
      • Howden S.E.
      • Wallace K.A.
      • Verhoeven A.D.
      • Wright L.S.
      • Capowski E.E.
      • Pinilla I.
      • Martin J.M.
      • Tian S.
      • Stewart R.
      • Pattnaik B.
      • Thomson J.A.
      • Gamm D.M.
      Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.
      • Singh R.
      • Phillips M.J.
      • Kuai D.
      • Meyer J.
      • Martin J.M.
      • Smith M.A.
      • Perez E.T.
      • Shen W.
      • Wallace K.A.
      • Capowski E.E.
      • Wright L.S.
      • Gamm D.M.
      Functional analysis of serially expanded human iPS cell-derived RPE cultures.
      One of the most vital questions that remains to be answered is whether mutations in these splicing factors specifically alter the proteins that are required for proper adhesion and phagocytosis of shed POSs. Because it appears that splicing of the mRNAs for the main phagocytosis receptors and ligands is not altered in the Prpf-mutant mice, perhaps secondary factors are disrupted, such as signaling pathways or circadian regulation of cellular functions or gene expression in general. The neural retina and RPE function integrally, so it is possible that a process important for adhesion and phagocytosis is disrupted in the neural retina as well.
      • Nandrot E.F.
      • Kim Y.
      • Brodie S.E.
      • Huang X.
      • Sheppard D.
      • Finnemann S.C.
      Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.
      • Nandrot E.F.
      • Anand M.
      • Sircar M.
      • Finnemann S.C.
      Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak.
      With the era of retinal gene therapy, it will be important to fully answer these questions so that effective therapies can be designed for the RNA splicing factor forms of RP.

      Acknowledgments

      We thank Stéphane Fouquet (Imaging Facility, Institut de la Vision, Moreau, Paris) for help with confocal microscopy, Marie-Laure Niepon (Histology Facility, Institut de la Vision, Moreau, Paris) for treatment of paraffin samples, and Bertrand Calippe for help with peritoneal macrophages isolation.

      Supplemental Data

      • Supplemental Figure S1

        A: Phagocytosis assays performed in macrophage cells. shRNA-mediated knockdown of Prpf31 in the mouse macrophage cell line, J774A.1, produced between 40% and 75% knockdown relative to the NTC. B: Phagocytosis assays performed on the J774A.1 knockdown lines showed no loss of phagocytic activity relative to the NTC. C: Phagocytosis assays performed on peritoneal macrophage cells isolated from Prpf31 mutant and control mice showed no differences in either Bind or Intern. Data are expressed as means ± SD. Bind, binding; Intern, internalization; NTC, nontargeted control.

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