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The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

  • Guoxin Ying
    Correspondence
    To whom correspondence may be addressed: Dept. of Ophthalmology and Visual Sciences, University of Utah Health Science Center, 65 Mario Capecchi Dr., Salt Lake City, UT 84132. Tel.:801-585-7621.
    Affiliations
    Department of Ophthalmology and Visual Sciences, University of Utah Health Science Center, Salt Lake City, Utah 84132
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  • Karsten Boldt
    Affiliations
    Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Elfriede-Aulhorn-Strasse 7, D-72076 Tübingen, Germany
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  • Marius Ueffing
    Affiliations
    Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Elfriede-Aulhorn-Strasse 7, D-72076 Tübingen, Germany
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  • Cecilia D. Gerstner
    Affiliations
    Department of Ophthalmology and Visual Sciences, University of Utah Health Science Center, Salt Lake City, Utah 84132
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  • Jeanne M. Frederick
    Affiliations
    Department of Ophthalmology and Visual Sciences, University of Utah Health Science Center, Salt Lake City, Utah 84132
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  • Wolfgang Baehr
    Correspondence
    Recipient of an RPB Senior Investigator award and an RPB Nelson Trust award. To whom correspondence may be addressed: Dept. of Ophthalmology and Visual Sciences, University of Utah Health Science Center, 65 Mario Capecchi Dr., Salt Lake City, UT 84132. Tel.:801-585-6643
    Affiliations
    Department of Ophthalmology and Visual Sciences, University of Utah Health Science Center, Salt Lake City, Utah 84132

    Departments of Neurobiology and Anatomy

    Biology, University of Utah, Salt Lake City, Utah 84112
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  • Author Footnotes
    3 The abbreviations used are: OSouter segmentISinner segmentONLouter nuclear layerOPLouter plexiform layerRPEretinal pigmented epitheliumROSrod outer segment(s)COScone outer segment(s)PSphosphatidylserineGTgene trapGC1guanylate cyclase 1Ppostnatal dayIPimmunoprecipitationPNApeanut agglutininLCALeber congenital amaurosisESembryonic stemcdcandelaANOVAanalysis of varianceDAPI4′,6-diamidino-2-phenylindoleEGFPenhanced GFPCRDcone-rod dystrophyERGelectroretinography.
Open AccessPublished:September 18, 2018DOI:https://doi.org/10.1074/jbc.RA118.005484
      RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human RAB28 gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated Rab28−/− mice by deleting exon 3 and truncating RAB28 after exon 2. We found that Rab28−/− mice recapitulate features of the human dystrophy (i.e. they exhibited reduced cone and rod ERG responses and progressive retina degeneration). Cones of Rab28−/− mice extended their outer segments (OSs) to the RPE apical processes and formed enlarged, balloon-like distal tips before undergoing degeneration. The visual pigment content of WT and Rab28−/− cones was comparable before the onset of degeneration. Cone phagosomes were almost absent in Rab28−/− mice, whereas rod phagosomes displayed normal levels. A protein–protein interaction screen identified several RAB28-interacting proteins, including the prenyl-binding protein phosphodiesterase 6 δ-subunit (PDE6D) and voltage-gated potassium channel subfamily J member 13 (KCNJ13) present in the RPE apical processes. Of note, the loss of PDE6D prevented delivery of RAB28 to OSs. Taken together, these findings reveal that RAB28 is required for shedding and phagocytosis of cone OS discs.

      Introduction

      Mammalian photoreceptors renew one-tenth of the OS
      The abbreviations used are: OS
      outer segment
      IS
      inner segment
      ONL
      outer nuclear layer
      OPL
      outer plexiform layer
      RPE
      retinal pigmented epithelium
      ROS
      rod outer segment(s)
      COS
      cone outer segment(s)
      PS
      phosphatidylserine
      GT
      gene trap
      GC1
      guanylate cyclase 1
      P
      postnatal day
      IP
      immunoprecipitation
      PNA
      peanut agglutinin
      LCA
      Leber congenital amaurosis
      ES
      embryonic stem
      cd
      candela
      ANOVA
      analysis of variance
      DAPI
      4′,6-diamidino-2-phenylindole
      EGFP
      enhanced GFP
      CRD
      cone-rod dystrophy
      ERG
      electroretinography.
      membrane daily by adding new disc membrane at the OS base and removing senescent components distally through disc shedding and phagocytosis by the adjacent retina pigmented epithelium (
      • Young R.W.
      The renewal of photoreceptor cell outer segments.
      ,
      • Young R.W.
      • Bok D.
      Participation of the retinal pigment epithelium in the rod outer segment renewal process.
      ). Rod outer segment (ROS) disc shedding and phagocytosis are synchronized by the circadian clock, with the peak of activity shortly after light onset (
      • LaVail M.M.
      Rod outer segment disk shedding in rat retina: relationship to cyclic lighting.
      ). Mammalian cone photoreceptors undergo similar OS shedding and phagocytosis (
      • Steinberg R.H.
      Phagocytosis by pigment epithelium of human retinal cones.
      ,
      • Anderson D.H.
      • Fisher S.K.
      • Erickson P.A.
      • Tabor G.A.
      Rod and cone disc shedding in the rhesus monkey retina: a quantitative study.
      ), but peak activity varies, depending on species (
      • Anderson D.H.
      • Fisher S.K.
      • Erickson P.A.
      • Tabor G.A.
      Rod and cone disc shedding in the rhesus monkey retina: a quantitative study.
      • Long K.O.
      • Fisher S.K.
      • Fariss R.N.
      • Anderson D.H.
      Disc shedding and autophagy in the cone-dominant ground squirrel retina.
      ,
      • Fisher S.K.
      • Pfeffer B.A.
      • Anderson D.H.
      Both rod and cone disc shedding are related to light onset in the cat.
      • Kocaoglu O.P.
      • Liu Z.
      • Zhang F.
      • Kurokawa K.
      • Jonnal R.S.
      • Miller D.T.
      Photoreceptor disc shedding in the living human eye.
      ). Much progress has been achieved in identifying molecular components of the phagocytosis pathway, including the OS-located “eat me” signal phosphatidylserine (PS) (
      • 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.
      ), RPE membrane receptors αVβ5 integrin (
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization.
      ) and receptor tyrosine kinase MerTK (
      • D'Cruz P.M.
      • Yasumura D.
      • Weir J.
      • Matthes M.T.
      • Abderrahim H.
      • LaVail M.M.
      • Vollrath D.
      Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat.
      ), and scavenger receptor CD36 (
      • Ryeom S.W.
      • Sparrow J.R.
      • Silverstein R.L.
      CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.
      ). The vast majority of data derives from study of rodent rods, as mouse and rat retinas are ∼97% rod-dominant (
      • Carter-Dawson L.D.
      • LaVail M.M.
      Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy.
      ).
      Prenylated Rab GTPases are master regulators of membrane vesicular trafficking, and mutations in human RAB genes are associated with multiple human inherited diseases (
      • Seixas E.
      • Barros M.
      • Seabra M.C.
      • Barral D.C.
      Rab and Arf proteins in genetic diseases.
      ). Rab5 isoforms have been shown to be present in RPE phagosomes, where their function is elusive (
      • Shelby S.J.
      • Feathers K.L.
      • Ganios A.M.
      • Jia L.
      • Miller J.M.
      • Thompson D.A.
      MERTK signaling in the retinal pigment epithelium regulates the tyrosine phosphorylation of GDP dissociation inhibitor alpha from the GDI/CHM family of RAB GTPase effectors.
      ). Absence of geranylgeranylation at the Rab27A C terminus, caused by a defect in REP1 (Rab escort protein 1), is associated with choroideremia, a rare X-linked retina degeneration (
      • Donnelly P.
      • Menet H.
      • Fouanon C.
      • Herbert O.
      • Moisan J.P.
      • Le Roux M.G.
      • Pascal O.
      Missense mutation in the choroideremia gene.
      ,
      • Tolmachova T.
      • Ramalho J.S.
      • Anant J.S.
      • Schultz R.A.
      • Huxley C.M.
      • Seabra M.C.
      Cloning, mapping and characterization of the human RAB27A gene.
      ). RAB28 is a distal member of the Rab family carrying a C-terminal CAAX box motif instead of the common Rab geranylgeranylation motif (
      • Brauers A.
      • Schürmann A.
      • Massmann S.
      • Mühl-Zürbes P.
      • Becker W.
      • Kainulainen H.
      • Lie C.
      • Joost H.G.
      Alternative mRNA splicing of the novel GTPase Rab28 generates isoforms with different C-termini.
      ,
      • Lee S.H.
      • Baek K.
      • Dominguez R.
      Large nucleotide-dependent conformational change in Rab28.
      ) and the first Rab GTPase linked directly to human retina disease (
      • Roosing S.
      • Rohrschneider K.
      • Beryozkin A.
      • Sharon D.
      • Weisschuh N.
      • Staller J.
      • Kohl S.
      • Zelinger L.
      • Peters T.A.
      • Neveling K.
      • Strom T.M.
      • van den Born L.I.
      • Hoyng C.B.
      • Klaver C.C.
      • et al.
      European Retinal Disease Consortium
      Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy.
      ). Nonsense mutations in Rab28 cause cone-rod dystrophy 18 (CRD18), presenting with foveal hyperpigmentation and atrophy, with diminished cone and rod electroretinography (ERG) responses (
      • Roosing S.
      • Rohrschneider K.
      • Beryozkin A.
      • Sharon D.
      • Weisschuh N.
      • Staller J.
      • Kohl S.
      • Zelinger L.
      • Peters T.A.
      • Neveling K.
      • Strom T.M.
      • van den Born L.I.
      • Hoyng C.B.
      • Klaver C.C.
      • et al.
      European Retinal Disease Consortium
      Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy.
      ,
      • Riveiro-Álvarez R.
      • Xie Y.A.
      • López-Martínez M.Á.
      • Gambin T.
      • Pérez-Carro R.
      • Ávila-Fernández A.
      • López-Molina M.I.
      • Zernant J.
      • Jhangiani S.
      • Muzny D.
      • Yuan B.
      • Boerwinkle E.
      • Gibbs R.
      • Lupski J.R.
      • Ayuso C.
      • Allikmets R.
      New mutations in the RAB28 gene in 2 Spanish families with cone-rod dystrophy.
      • Lee G.I.
      • Lee C.
      • Subramanian S.
      • Kim N.K.D.
      • Ki C.S.
      • Park W.Y.
      • Kim B.J.
      • Kim S.J.
      A novel likely pathogenic variant in the RAB28 gene in a Korean patient with cone-rod dystrophy.
      ).
      Whereas the molecular basis underlying CRD18 has been obscure, our study suggests that RAB28 is an essential factor in cone-specific disc shedding and phagocytosis. In Rab28 germ line knockout mice, which recapitulate the human CRD18 phenotype, we found that mutant cones elongate and form enlarged tips as early as P14, suggesting a disc shedding impairment. RAB28 traffics to the photoreceptor OS in a complex with PDE6D. Further, RAB28 interacts with KCNJ13, a potassium channel in the RPE associated with Leber congenital amaurosis (LCA16); KCNJ13 may collaborate with RAB28 to facilitate COS phagocytosis.

      Results

      The murine Rab28 gene and splice variants

      The mouse Rab28 gene consists of nine exons and expresses three splice variants, V1–V3. The variants share amino acids 1–191 encoded by exons 1–6 but differ in their C-terminals, encoded by exons 8, 7, and 9, respectively (Fig. 1A). RAB28V1 (221 amino acids) and V2 (220 amino acids) carry a CAAX box sequence, where X = Q predicts farnesylation. RAB28V3 (191 amino acids) has a shorter C-terminal region and is not prenylated. The polyclonal RAB28 antibody used in this study recognized both V1 and V2 recombinant proteins (Fig. 1B) and thus should also recognize V3. Recombinant variants V1 and V2 are present in the HEK293 cell body, and V2 appears to be translocated selectively into the nucleus (Fig. 1C).
      Figure thumbnail gr1
      Figure 1Generation of Rab28−/− mice. A, schematic representation of the mouse Rab28 gene and three splice variants, V1–V3. The mouse Rab28 gene consists of nine exons (yellow rectangles). Dashed gray, red, and green lines indicate three types of mRNA splicing. V1, V2, and V3 share exons 1–6 but differ in the C terminus, which is encoded by exons 8, 7, and 9, respectively. aa, amino acids. B, Western blotting of HEK293 lysates expressing mCherry-RAB28V2 (lane 1) and EGFP-RAB28V1 (lane 2). The polyclonal antibody used in this study recognizes both variants (red arrow); the faster moving polypeptide is nonspecific. C, mCherry-RAB28V2 and EGFP-RAB28V1 expression pattern in HEK293 cells. V1 and V2 colocalize in the cytoplasm, and V2 is also present in the nucleus. Top and right small panels, orthogonal views of y and z planes at the lines indicated. Scale bar, 20 μm. D, schematic representation of the Rab28 gene, the location of the gene trap in intron 2, the floxed allele, and the knockout allele. The En2SA-IRES-LacZ-pGK-Neo GT cassette is flanked by two FRT sites (green triangle) and is inserted into intron 2 by homologous recombination. Exon 3 is flanked by two LoxP sites (red triangle). Small arrows represent the approximate position of primers for genotyping. E, PCR genotyping of the 3′ and 5′ recombination arm, third LoxP, second FRT/LoxP, and first FRT sites in ES cells. D1 and A5, two ES cell clones and the derived mice with identical genotyping results. The right-most panel represents a PCR genotyping result of one litter of Rab28+/− × Rab28+/− pups. Primer sequences are listed in . F, retinal lysate Western blotting. Left lane, RAB28 with a mobility of 25 kDa; right lane, RAB28 is absent in Rab28−/− retina lysate. The identity of the slower moving species is unknown. G, immunohistochemistry of WT (left) and knockout retina cryosections (right) with anti-RAB28 antibody. RAB28 is present in WT photoreceptors and RPE and absent in Rab28−/− sections (right). H, expression of mCherry-RAB28V2 in rod photoreceptors by neonatal electroporation. Scale bars, 20 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

      Generation of Rab28 gene trap (GT) and knockout mice

      Rab28GT/GT mutant mice carrying a GT cassette in intron 2 and a floxed exon 3 were generated using a EUCOMM cell line. Correct gene targeting was verified by sequencing of PCR-amplified 3′ and 5′ recombination arms and FRT and LoxP sites (Fig. 1, D and E). To avoid interference of the GT cassette on nearby gene expression, we generated a Rab28 floxed allele (Rab28fl/+) by crossing Rab28GT/GT with flippase mice; the knockout allele (Rab28+/−) was generated subsequently by mating Rab28fl/+ with CMV-Cre mice. Rab28−/− mice are viable and fertile. RAB28 protein variants of 25-kDa mobility are undetectable in Rab28−/− retina lysates by immunoblotting (Fig. 1F); variant V3 is not expressed at detectable levels in WT samples. In WT retina cryosections, RAB28 is expressed in the RPE and all photoreceptor compartments, most strongly in outer segments (Fig. 1G, left); no variant is detectable in Rab28−/− retina cryosections (Fig. 1G, right). When electroporated into Rab28−/− rods, mCherry-RAB28V2 distributes throughout the cell (Fig. 1H).

      Rab28−/− mice recapitulate human CRD18

      Photoreceptor function was evaluated by scotopic and photopic ERG testing over a period of 13 months. At 3 months of age, both scotopic (rod) and photopic (cone) responses are significantly reduced in Rab28−/− mice, with greater reduction in photopic b-wave amplitudes. Mutant scotopic a-wave amplitudes are reduced to ∼70% of control (Fig. 2, A and C), whereas the photopic b-wave amplitude is <50% (Fig. 2, B and D). Retina dystrophy of human CRD18 patients is typically diagnosed in early childhood (
      • Roosing S.
      • Rohrschneider K.
      • Beryozkin A.
      • Sharon D.
      • Weisschuh N.
      • Staller J.
      • Kohl S.
      • Zelinger L.
      • Peters T.A.
      • Neveling K.
      • Strom T.M.
      • van den Born L.I.
      • Hoyng C.B.
      • Klaver C.C.
      • et al.
      European Retinal Disease Consortium
      Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy.
      ).
      Figure thumbnail gr2
      Figure 2Reduced function of Rab28−/− photoreceptors. A and B, representative scotopic ERG (A) traces at −1.6, 0.4, and 2.4 log cd s/m2 and photopic ERG traces (B) at 0.4, 0.9, and 1.4 photopic log cd s/m2 from 3-month-old (3m) Rab28+/+ and Rab28−/− mice. C and D, scotopic a-wave amplitude (C) and photopic b-wave amplitude (D) as a function of light intensity. Significant differences were observed for the scotopic a-wave amplitude at 0.4, 1.4, and 2.4 log cd s/m2 and for the photopic b-wave amplitude at all light intensities of −0.01, 0.4, 0.9, 1.4, and 1.9 log cd s/m2. Shown are mean ± S.D. (error bars), n ≥ 5 for each group, one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
      Immunolabeling of Rab28−/− retina with antibodies directed against guanylate cyclase 1 (GC1) or mouse cone arrestin revealed fast degeneration of cones, accompanied by progressive ROS shortening and a mild reduction of ONL thickness (Fig. 3A). The cone number is slightly reduced (88% of control) at 1 month (Fig. 3, A and F), but severely reduced at 3 months, and very few cones are detectable at 13 months (Fig. 3A, bottom panels). Accordingly, the scotopic a-wave amplitude declines slowly starting at 1 month, and by 13 months, it is 42% of WT controls (Fig. 3B). Photopic ERGs decline much faster and are attenuated at all tested time points, at 65% of control at 1 month and undetectable at 13 months (Fig. 3C). ONL thickness in the central retina is reduced to 87, 78, and 73% of controls by 3, 6, and 13 months of age (Fig. 3D), respectively, whereas mutant retina OS lengths are reduced to 69, 65, and 50% at 3, 6, and 13 months (Fig. 3E). Cone densities are reduced to 88, 71, 35, and 12% of control levels at 1, 3, 6, and 13 months, respectively (Fig. 3F).
      Figure thumbnail gr3
      Figure 3Progressive retina degeneration in Rab28−/− mice. A, immunostaining of retina frozen sections with anti-GC1 antibody (top panels) and with anti-cone arrestin (bottom panels) at 1, 3, 6, and 13 months of age. Note the gradual reduction of ROS length and ONL thickness and the more rapid cone degeneration. Scale bar, 20 μm. B and C, decline of scotopic a-wave (B) and photopic b-wave amplitudes (C) at 1, 3, 6, and 13 months of age. D–F, average ONL thickness (D), ROS length (E), and cone density (F) of WT and Rab28−/− retina near optic nerve at 1, 3, 6, and 13 months of age. All are significantly reduced at 3, 6, and 13 months. Shown are mean ± S.D. (error bars), n = 4, one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

      Rab28−/− cones have elongated OS and enlarged OS tips

      All examined rod proteins (rhodopsin, transducin, PDE6, and CNGA1) were correctly localized to the ROS of 1-month-old mutants (Fig. 4A, bottom panels). By contrast, visual pigments ML-opsin and S-opsin mislocalized in part to the IS, ONL, and OPL of mutant cones (Fig. 4 (C and D), red arrows). We further observed that mutant COS are elongated, stretching toward the apical RPE edge, and frequently display a bulge at the distal tip revealed by CNGA3 and cone opsin immunolabeling (Fig. 4 (B–D), white arrows). Whereas WT COS taper toward their tips (Fig. 4F), mutant COS exhibit enlarged, balloon-like tips carried by elongated and slender outer segments (Fig. 4, G and H). One-month-old mutant COS (21.7 ± 4.9 μm) average 43% longer than control COS (15.1 ± 1.6 μm) (Fig. 4I). Slight elongation of mutant COS and occasional COS tip enlargement are first observed shortly after eye opening (P12), which suggests a defect in COS membrane shedding and RPE phagocytosis (Fig. 4E).
      Figure thumbnail gr4
      Figure 4Cone outer segment elongation and tip enlargement in Rab28−/− retina. A, immunohistochemistry of 1-month-old WT (top panels) and Rab28−/− (bottom panels) retina frozen sections with antibodies against rhodopsin, CNGA1/A3, PDE6, rod Tα, and ROM1, as indicated. The immunostaining showed correct localization of ROS proteins in Rab28−/− rod photoreceptors. B–D, immunostaining of 1-month-old WT (top panels) and Rab28−/− (bottom panels) retina sections with anti-CNGA3 (B), ML-opsin (C), and S-opsin (D). White arrows point to the bulged COS tips at the apical RPE (B–D, bottom panels). Red arrows, cone opsin mislocalization in the IS, ONL, and OPL (C and D, bottom panels). E, ML-opsin staining of P14 WT (top) and Rab28−/− (bottom) retina sections. Mutant COS are slightly elongated, and enlarged COS tips are occasionally observed (dashed arrow in E). F–H, high-magnification confocal images of two WT COS (F) and two mutant COS (G and H). WT COS taper toward the RPE, but mutant COS have a balloon-like enlargement at the distal tip (red arrows). Scale bars, 20 μm (A–D), 10 μm (E), and 5 μm (F–H). I, average length of 1-month-old mutant COS (21.7 ± 4.9 μm) is significantly longer than seen in WT COS (15.1 ± 1.6 μm). Shown are mean ± S.D. (error bars). More than 80 cones from four animals were measured; one-way ANOVA. ***, p < 0.001.
      A second observation gleaned from immunohistochemistry was that levels of prenylated OS proteins (G protein–coupled receptor kinase 1 (GRK1), cone PDE α-subunit (cPDEα′), and cone transducin γ-subunit (cTγ)) appear reduced in mutant cones (Fig. 5 (A–C), bottom panels). Reduction of GRK1 levels in mutant cones could be observed as early as P14 (Fig. 5D), whereas GRK1 levels in ROS appear comparable at both P14 and 1 month of age (Fig. 5, A and D). Western blotting using 7-week-old retina lysates showed that Rab28−/− COS protein levels, including cPDEα′ and GRK1, are comparable with heterozygous controls (Fig. 5, E and F). The reduction of cTγ and cPDEγ was statistically significant (Fig. 5F). Western blotting with 3-week-old retina lysates did not display significant differences in COS protein levels (results not shown).
      Figure thumbnail gr5
      Figure 5Reduction of prenylated protein levels in Rab28−/− cone OS. A–C, immunostaining of WT and GRK1 (A), cPDEα′ (B), and cTγ (C) in 1-month WT (top) and Rab28−/− (bottom) retinas. All three cone OS prenylated proteins showed a reduction in intensity in Rab28−/− compared with WT, whereas GRK1 in rod OS is not reduced. D, GRK1 (red) and cone arrestin (green) staining in P14 WT (top) and Rab28−/− (bottom) retina. GRK1 reduction in cone OS is already obvious at this stage. Scale bars, 20 μm. E, Western blotting of 7-week-old Rab28+/− and Rab28−/− retina lysate with anti-cPDEγ, anti-cTγ, anti-cPDEα′, anti-cArrestin, anti-CNGA3, anti-GRK1, and anti-M- and S-opsin antibodies. α-Tubulin was used as loading control. F, quantification of Western blotting signal intensity (mean ± S.D. (error bars)), normalized to α-tubulin. Only the reduction of cPDEγ and cTγ is statistically significant. *, p < 0.05, n = 3, one-way ANOVA.

      Phagocytosis defects of Rab28−/− cones

      To evaluate COS membrane shedding defects, we harvested P24 WT and mutant retinas 1.5 h after light onset and labeled fixed cryosections with combined ML- and S-opsin antibodies. At this time, cone opsin–positive phagosomes (green) are located within the apical RPE microvilli or RPE somata (Fig. 6A, white arrows; cone sheaths identified using peanut agglutinin (PNA; red)). The number of COS phagosomes is reduced in mutant retina where the COS tip enlargement is already obvious (Fig. 6B, arrows). Rab28−/− COS phagosome counts (Fig. 6C) revealed a 5-fold reduction (7.5 ± 2.6 per 2-mm retina) compared with WT controls (39.8 ± 6.0 per 2-mm retina). In contrast, the rod phagosome density is comparable in P24 WT and Rab28−/− retinas (Fig. 6, D and E), by statistical evaluation, amounting to 56.3 ± 7.1 per 200-μm control RPE versus 51.4 ± 3.5 per 200-μm Rab28−/− RPE (Fig. 6F). These results are interpreted to indicate that Rab28−/− COS membrane shedding is defective, whereas ROS phagocytosis proceeds normally.
      Figure thumbnail gr6
      Figure 6Reduced COS phagosomes in P24 Rab28−/− retina. A and B, immunohistochemistry of P24 WT (A) and Rab28−/− (B) retina cryosections with anti-ML- and anti-S-opsin antibodies (mixed 1:1) (green). Retina samples were collected at 1.5 h after light onset. Cone opsin-positive COS phagosomes (arrows) in mutant (bottom) were diminished compared with WT control (top). At the right of each panel is an overlay with PNA (red) and the nuclear marker, DAPI (blue). C, quantitative evaluation of COS phagosomes at P24. Mutant COS phagosome count is significantly reduced compared with WT (Rab28−/− 7.5 ± 2.6 versus WT 39.8 ± 6.0 per 2-mm retina). Phagosomes of 12 sections from three animals (four sections for each retina) were counted. Shown are mean ± S.D. (error bars), one-way ANOVA. ***, p < 0.001. D and E, rhodopsin immunostaining of 1-month WT (D) and Rab28−/− (E) retina sections collected 1.5 h after light onset. Both the density and size of rhodopsin-positive phagosomes (green dots at RPE) are comparable between WT and Rab28−/−. F, quantitative evaluation of ROS phagosomes at P24. Mutant ROS phagosomes are comparable with WT (Rab28−/− 51.4 ± 5.5 versus WT 56.3 ± 7.1 per 200-μm RPE). Phagosomes of 12 sections from three animals (four sections for each retina) were counted. ns, not significant. G and H, S-opsin immunostaining of P24 Nrl−/− (G) and Rab28−/−; Nrl−/− (H) retina sections collected at 1.5 h after light onset. S-opsin-positive phagosomes are barely detected in the RPE of both genotypes. However, the OS tips are larger in Rab28−/−; Nrl−/− cones compared with Nrl−/− cones (arrows). Scale bars, 20 μm (A, B, D, and E) and 10 μm (G and H).
      The Nrl−/− mouse expresses “S-cone–like” photoreceptors and is a model for enhanced S-cone syndrome in which cone phagocytosis is impaired (
      • Mears A.J.
      • Kondo M.
      • Swain P.K.
      • Takada Y.
      • Bush R.A.
      • Saunders T.L.
      • Sieving P.A.
      • Swaroop A.
      Nrl is required for rod photoreceptor development.
      ). We generated Rab28−/−; Nrl−/− double knockouts to analyze COS phagocytosis. We observed that Rab28−/−; Nrl−/− cone tips were larger than in Nrl−/− controls (Fig. 6G, arrows) and that cone phagosomes could not be recorded in single (Fig. 6G) or double knockout mutants (Fig. 6H), consistent with another study (
      • Mustafi D.
      • Kevany B.M.
      • Genoud C.
      • Okano K.
      • Cideciyan A.V.
      • Sumaroka A.
      • Roman A.J.
      • Jacobson S.G.
      • Engel A.
      • Adams M.D.
      • Palczewski K.
      Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration.
      ). Larger tips in Rab28−/−; Nrl−/− cones suggest a more dramatic accumulation of unshed membrane relative to Nrl−/− S-cones.

      Accumulation of material in Rab28−/− COS tip

      Transmission EM of 1-month-old Rab28−/− and heterozygous control retinas documented elongated cones with disorganized distal membranes in mutants (Fig. 7, A and B). At the Rab28−/− RPE/photoreceptor interface, we observed RPE microvilli-wrapped structures filled with distorted membrane stacks or vacuolar membranes (Fig. 7, C and D). These structures appear in different shapes (round and oblique) at least 2 μm in size (Fig. 7, F and G), indicating buildup of materials at the OS tip. The size of the vacuoles (Fig. 7 (F and G), yellow arrows) and the shapes of membrane stacks (Fig. 7 (F and G), arrowheads) within these bulbous structures also vary. As these structures correspond to the bulges of COS tips seen in Rab28−/− immunostaining (Fig. 4), the variable-size vacuoles and distorted membranes stacks may represent different stages of COS degeneration. Additionally, there are increased RPE pigment deposits in Rab28−/− compared with Rab28+/− controls (Fig. 7 (C and D), dashed arrows). By contrast, ROS disc morphology appears to be normal in Rab28−/− rods (Fig. 7H).
      Figure thumbnail gr7
      Figure 7Ultrastructure. A, longitudinal image of 1-month-old Rab28−/− outer segments, showing an elongated COS (arrow) wedged between two ROS and reaching the apical RPE. A swollen, degenerating COS tip (red asterisk), belonging to the same cone, is membrane-bound and filled with vacuoles. B, enlargement of yellow boxed area in A, showing the partially disorganized distal COS (yellow arrows). C and D, representative electron micrographs of 1-month-old (1m) Rab28+/− (C) and Rab28−/− retinas (D). The asterisk in C marks a normal phagosome. Arrows in D indicate three enlarged mutant COS tips filled with membrane stacks or vacuoles at the RPE/photoreceptor interface. Note the increased pigment density in the mutant RPE (arrows). E–H, magnified images of three enlarged tips, ∼2 μm in the shortest dimension, in mutants with variable-size vacuoles (dashed arrows) and distorted membranes (arrowheads). Scale bars, 5 μm (A), 1 μm (B), 5 μm (C and D), 2 μm (E–G), and 100 nm (H).

      RAB28 interacts with KCNJ13, an RPE microvillus protein

      Co-immunoprecipitation (co-IP) using bovine retina lysates followed by MS was performed to identify interacting proteins. We identified dozens of potential interactors (t test, p < 0.05, RAB28 IP/control IP ratio >3, Table S2). Top candidates include the prenyl-binding protein PDE6D, which has been reported previously to interact with RAB28 (
      • Humbert M.C.
      • Weihbrecht K.
      • Searby C.C.
      • Li Y.
      • Pope R.M.
      • Sheffield V.C.
      • Seo S.
      ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting.
      ). Three other notable candidates are KCNJ13, mutations of which are associated with Leber congenital amaurosis (LCA16) (
      • Sergouniotis P.I.
      • Davidson A.E.
      • Mackay D.S.
      • Li Z.
      • Yang X.
      • Plagnol V.
      • Moore A.T.
      • Webster A.R.
      Recessive mutations in KCNJ13, encoding an inwardly rectifying potassium channel subunit, cause Leber congenital amaurosis.
      ); MEF2D, a transcription factor required for photoreceptor development and maintenance (
      • Omori Y.
      • Kitamura T.
      • Yoshida S.
      • Kuwahara R.
      • Chaya T.
      • Irie S.
      • Furukawa T.
      Mef2d is essential for the maturation and integrity of retinal photoreceptor and bipolar cells.
      ); and GLUT1 (glucose transporter 1), mediating RdCVF (rod-derived cone viability factor)-induced cone survival (
      • Aït-Ali N.
      • Fridlich R.
      • Millet-Puel G.
      • Clérin E.
      • Delalande F.
      • Jaillard C.
      • Blond F.
      • Perrocheau L.
      • Reichman S.
      • Byrne L.C.
      • Olivier-Bandini A.
      • Bellalou J.
      • Moyse E.
      • Bouillaud F.
      • Nicol X.
      • et al.
      Rod-derived cone viability factor promotes cone survival by stimulating aerobic glycolysis.
      ). In selective Western blotting verification, we successfully verified the interaction of RAB28 with KCNJ13, MEF2D, GLUT1, VAC14, and PDE6D, but failed to validate GRK1 and CAND1 (Fig. 8A), indicating high reliability of our co-IP results.
      Figure thumbnail gr8
      Figure 8Rab28-interacting proteins. A, immunoblot verification of co-IP () between RAB28 and select candidate proteins. Verified interactants include PDE6D, KCNJ13, MEF2D, GLUT1, and VAC14. GRK1 and CAND1 failed verification. Lane 1, input; lane 2, normal goat IgG control IP; lane 3, RAB28 antibody IP. Correctly verified proteins (red arrows) and two proteins that failed verification (yellow arrows) are indicated. B and C, representative immunohistochemistry using anti-KCNJ13 antibody (green) and PNA (red) in P24 WT (B) and Rab28−/− (C) retinas. KCNJ13 localization at RPE apical microvilli is comparable between WT and Rab28−/−. D and E, immunohistochemistry with anti-KCNJ13 antibody (red) to show relative delivery of KCNJ13 in P21 Nrl−/− (D) and Rab28−/−; Nrl−/− (E) double-knockout retinas/RPE. KCNJ13 is partially trapped at the base of RPE apical microvilli in double-knockout mice (arrows in E). Scale bars, 20 μm (B and C) and 10 μm (C and D).
      KCNJ13 is located at the root of WT apical RPE microvilli (
      • Kusaka S.
      • Inanobe A.
      • Fujita A.
      • Makino Y.
      • Tanemoto M.
      • Matsushita K.
      • Tano Y.
      • Kurachi Y.
      Functional Kir7.1 channels localized at the root of apical processes in rat retinal pigment epithelium.
      ) and overlaps with the cone sheath marker, PNA (Fig. 8B). This pattern is preserved in Rab28−/− (Fig. 8C). Because the possible alteration of KCNJ13 localization on cone-associated microvilli could be masked by the overwhelming signal of rod-associated microvilli, we determined the KCNJ13 localization in Rab28−/−; Nrl−/− mice. KCNJ13 is located at the RPE microvilli that span the entire short OS in Nrl−/− control animals (Fig. 8D). However, in Rab28−/−; Nrl−/− double knockout mice, KCNJ13 is partially trapped at the base of apical RPE microvilli (Fig. 8E), suggesting a role of RAB28 in KCNJ13 microvilli trafficking.

      RAB28 is PDE cargo targeted for the OS

      To elucidate the nature of PDE6D–RAB28 interaction, we co-expressed PDE6D-EGFP and mCherry-RAB28V2 in HEK293 cells and repeated co-immunoprecipitation using mCherry antibody. As expected, mCherry-RAB28V2 co-precipitated PDE6D-EGFP (Fig. 9A). However, mCherry-RAB28V2-C217A, in which the cysteine of the CAAX box motif is mutated to alanine, preventing farnesylation, is unable to interact with PDE6D-EGFP, indicating that the interaction is RAB28 prenylation-dependent (Fig. 9A). Nonfarnesylated mCherry-Rab29V2-C217A distributed evenly within the cell due to the loss of membrane association (Fig. 9B). In PDE6d−/− mice, RAB28 is absent in the OS but accumulates in the IS (Fig. 9C), verifying that RAB28 is a PDE6D cargo, as observed for other prenylated OS proteins (
      • Zhang H.
      • Li S.
      • Doan T.
      • Rieke F.
      • Detwiler P.B.
      • Frederick J.M.
      • Baehr W.
      Deletion of PrBP/δ impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments.
      ). The PDE6D level is unchanged in Rab28−/− retina (Fig. 9D).
      Figure thumbnail gr9
      Figure 9Rab28 interaction with PDE6D is Rab28 prenylation–dependent. A, co-IP and Western blotting of PDE6D and RAB28 interaction in vitro. PDE6D-EGFP and mCherry-RAB28V2 or mCherry-RAB28V2-C217A were co-expressed in HEK293 cells. Co-IP was performed using monoclonal anti-mCherry antibody or normal mouse IgG, and the blot was probed with anti-EGFP antibody. mCherry-RAB28V2, but not nonfarnesylated mCherry-RAB28V2-C217A, pulls down PDE6D-EGFP. mCherry was used as the co-IP control. B, confocal images of proteins expressed in vitro. mCherry-RAB28V2 (red) and PDE6D-EGFP (green) colocalize in the ER and cytoplasm when co-expressed (top panels); mCherry-RAB28V2-C217A distributes uniformly throughout the cytoplasm due to the loss of membrane association (bottom panels). C, WT (left) and PDE6D−/− (right) retina cryosections probed with anti-RAB28 antibody. RAB28 fails to enter the Pde6d−/− OS and accumulates primarily in the IS. D, immunoblot of Rab28+/− and Rab28−/− retina homogenates probed with antibodies directed against PDE6D and α-tubulin; PDE6D is not down-regulated in Rab28−/− retinas. Scale bars, 40 μm (B) and 20 μm (C).

      Discussion

      RAB28 has been suggested to participate in lysosomal delivery of endocytosed surface protein in unicellular trypanosomes (
      • Lumb J.H.
      • Leung K.F.
      • Dubois K.N.
      • Field M.C.
      Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways.
      ), angiotensin-induced NF-κB nuclear delivery and activation in rat endothelia (
      • Jiang J.
      • Qi Y.X.
      • Zhang P.
      • Gu W.T.
      • Yan Z.Q.
      • Shen B.R.
      • Yao Q.P.
      • Kong H.
      • Chien S.
      • Jiang Z.L.
      Involvement of Rab28 in NF-κB nuclear transport in endothelial cells.
      ), glucose transporter 4 plasma membrane trafficking in mouse skeletal muscle (
      • Zhou Z.
      • Menzel F.
      • Benninghoff T.
      • Chadt A.
      • Du C.
      • Holman G.D.
      • Al-Hasani H.
      Rab28 is a TBC1D1/TBC1D4 substrate involved in GLUT4 trafficking.
      ), and intraflagellar transport in Caenorhabditis elegans (
      • Jensen V.L.
      • Carter S.
      • Sanders A.A.
      • Li C.
      • Kennedy J.
      • Timbers T.A.
      • Cai J.
      • Scheidel N.
      • Kennedy B.N.
      • Morin R.D.
      • Leroux M.R.
      • Blacque O.E.
      Whole-organism developmental expression profiling identifies RAB-28 as a novel ciliary GTPase associated with the BBSome and intraflagellar transport.
      ).
      In this study, we show that Rab28 germ line knockout mice recapitulate the human CRD18 phenotype. In human CRD patients, the RAB28 gene carries nonsense mutations in exons 5 and 6, truncating all known splice variants (
      • Roosing S.
      • Rohrschneider K.
      • Beryozkin A.
      • Sharon D.
      • Weisschuh N.
      • Staller J.
      • Kohl S.
      • Zelinger L.
      • Peters T.A.
      • Neveling K.
      • Strom T.M.
      • van den Born L.I.
      • Hoyng C.B.
      • Klaver C.C.
      • et al.
      European Retinal Disease Consortium
      Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy.
      ). CRD may be accompanied by visual acuity deterioration, absence of photopic ERG responses, and myopia as early as age 16. In the Rab28−/− mouse model, photopic b-waves are reduced at 1 month and nearly absent by 1 year of age, which is roughly consistent with human CRD (Figure 2, Figure 3).
      We found that Rab28−/− COS are elongated to the length of rod outer segments, and the mutant tips are expanded to balloon-like structures and appear unable to shed membranes to be phagocytosed by the RPE. In view of impaired disc shedding at the distal cone outer segment, continued overproduction of new disc membranes at the proximal end may provide a jammed gate impenetrable for prenylated proteins (see the model in Fig. 10). The absence of membrane shedding is supported by dramatic reduction of cone pigment–containing phagosomes (Fig. 6). Bulbous OS tips and lack of phagosome production was also observed in the Nrl−/− mouse, a model for enhanced S-cone syndrome (
      • Mustafi D.
      • Kevany B.M.
      • Genoud C.
      • Okano K.
      • Cideciyan A.V.
      • Sumaroka A.
      • Roman A.J.
      • Jacobson S.G.
      • Engel A.
      • Adams M.D.
      • Palczewski K.
      Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration.
      ). Rab28−/− COS elongation and enlarged COS tips were observed shortly after eye opening (Fig. 4). We propose that CRD18 is caused by impaired membrane shedding at the distal COS and/or failed phagocytosis by the RPE. The resulting accumulation is detrimental for cone survival. The molecular identities of components involved in human and mouse disc shedding are currently unclear. Human cone membrane shedding, observed in vivo by adaptive optics optical coherence tomography (
      • Kocaoglu O.P.
      • Liu Z.
      • Zhang F.
      • Kurokawa K.
      • Jonnal R.S.
      • Miller D.T.
      Photoreceptor disc shedding in the living human eye.
      ), was characterized by abrupt and transient loss of the COS tip. Average duration of tip loss was ∼9 min, and average length loss was reported as 2.1 μm.
      Figure thumbnail gr10
      Figure 10Model of COS disc shedding failure in Rab28−/− mouse retina. In WT retina (left), COS length is maintained by the daily addition of new disc membranes at the proximal COS and shedding of aged discs at the distal tip. Shed discs are phagocytized by RPE processes. In Rab28−/− (right), COS shedding at the distal tip fails, elongating COS toward the RPE and accumulating membrane material. Membrane incorporation at the base continues, jamming the ciliary gate and blocking the entry of prenylated proteins. CC, connecting cilium; BB, basal body; CIS, cone inner segment.
      In 1-month-old Rab28−/− retina, COS are elongated, and COS distal tips are filled with vacuoles and/or distorted membranes (Fig. 7). The accumulated membranes resemble material observed in degenerating Nrl−/− mouse cones at 8 weeks of age (
      • Mustafi D.
      • Kevany B.M.
      • Genoud C.
      • Okano K.
      • Cideciyan A.V.
      • Sumaroka A.
      • Roman A.J.
      • Jacobson S.G.
      • Engel A.
      • Adams M.D.
      • Palczewski K.
      Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration.
      ). We did not detect organized COS discs in the enlarged Rab28−/− tips similar to those observed in 4-week Nrl−/− retina (
      • Mustafi D.
      • Kevany B.M.
      • Genoud C.
      • Okano K.
      • Cideciyan A.V.
      • Sumaroka A.
      • Roman A.J.
      • Jacobson S.G.
      • Engel A.
      • Adams M.D.
      • Palczewski K.
      Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration.
      ), indicating that Rab28−/− COS degeneration starts earlier and is more severe. Another notable observation is that both Rab28−/− and Nrl−/− showed COS disorganization at the disc periphery. The Rab28−/− disc edge shows merger/swelling or disassembly (Fig. 7B), whereas the Nrl−/− disc edge reveals interconnection (
      • Mustafi D.
      • Kevany B.M.
      • Genoud C.
      • Okano K.
      • Cideciyan A.V.
      • Sumaroka A.
      • Roman A.J.
      • Jacobson S.G.
      • Engel A.
      • Adams M.D.
      • Palczewski K.
      Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration.
      ), suggesting that the COS disc edge is vulnerable to the accumulation of oxidative materials.
      Bulbous Rab28−/− OS tips are only observed in mutant cones, as mutant rods are initially unaffected and shed discs exhibit a normal phagosome count (Fig. 6). The reason for this specificity is unknown. ROS phagocytosis is triggered by diurnal surface exposure of the anionic phospholipid phosphatidylserine at the OS tip (
      • 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.
      ), which is recognized by multiple RPE membrane receptors and their ligands, including integrin αVβ5 and ligand MFG-E8 (
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization.
      ,
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for αvβ5 integrin in diurnal retinal phagocytosis.
      ), MerTK and ligand proteins S and Gas6 (
      • D'Cruz P.M.
      • Yasumura D.
      • Weir J.
      • Matthes M.T.
      • Abderrahim H.
      • LaVail M.M.
      • Vollrath D.
      Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat.
      ,
      • 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.
      ,
      • Hall M.O.
      • Prieto A.L.
      • Obin M.S.
      • Abrams T.A.
      • Burgess B.L.
      • Heeb M.J.
      • Agnew B.J.
      Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6.
      • 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.
      ), and CD36, which directly binds PS (
      • Ryeom S.W.
      • Sparrow J.R.
      • Silverstein R.L.
      CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.
      ). Following substrate–receptor binding, the shed ROS tip is engulfed and ingested by the RPE apical microvillus. MerTK signaling plays a critical role in initiating this process (
      • D'Cruz P.M.
      • Yasumura D.
      • Weir J.
      • Matthes M.T.
      • Abderrahim H.
      • LaVail M.M.
      • Vollrath D.
      Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat.
      ). Whether COS phagocytosis is subjected to similar molecular regulation is unknown. Phagocytosis requires that internalized COS phagosomes move along the microvillus before entering the RPE soma (
      • Steinberg R.H.
      • Wood I.
      • Hogan M.J.
      Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina.
      ). However, as both αVβ5 integrin and CD36 localize at the RPE soma (
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization.
      ,
      • Ryeom S.W.
      • Sparrow J.R.
      • Silverstein R.L.
      CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.
      ), their participation in COS recognition and binding is unlikely. A recent large-cohort genetic study of inherited retina diseases found that MerTK mutations exist in a small percentage of CRD patients (
      • Audo I.
      • Mohand-Said S.
      • Boulanger-Scemama E.
      • Zanlonghi X.
      • Condroyer C.
      • Démontant V.
      • Boyard F.
      • Antonio A.
      • Méjécase C.
      • El Shamieh S.
      • Sahel J.A.
      • Zeitz C.
      MERTK mutation update in inherited retinal diseases.
      ), suggesting a conserved MerTK-dependent mechanism between ROS and COS phagocytosis. In agreement with this, MerTK is expressed at apical RPE microvilli (
      • 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.
      ). As RPE signals actively participate in delineating the position of disc shedding (e.g. the localized PS exposure) at ROS tips (
      • 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.
      ), the failed Rab28−/− shedding phenotype may indicate a communication defect between RPE microvilli and mutant COS tips.
      Our protein–protein interaction screen identified the RPE microvilli–resident protein, KCNJ13, as a RAB28 interactant. Recessive mutations of KCNJ13 are associated with LCA16 in humans, with an undefined mechanism (
      • Sergouniotis P.I.
      • Davidson A.E.
      • Mackay D.S.
      • Li Z.
      • Yang X.
      • Plagnol V.
      • Moore A.T.
      • Webster A.R.
      Recessive mutations in KCNJ13, encoding an inwardly rectifying potassium channel subunit, cause Leber congenital amaurosis.
      ). Kcnj13 mutant mice generated with the CRISPR-Cas9 system showed mosaic expression of KCNJ13 in RPE cells and photoreceptor degeneration resembling LCA (
      • Zhong H.
      • Chen Y.
      • Li Y.
      • Chen R.
      • Mardon G.
      CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes.
      ). RPE cells null for KCNJ13 can survive, but KCNJ13 expression is required for RPE cells to maintain photoreceptor survival (
      • Zhong H.
      • Chen Y.
      • Li Y.
      • Chen R.
      • Mardon G.
      CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes.
      ). We found that KCNJ13 delivery to RPE microvilli in Rab28−/−; Nrl−/− double knockout is partially reduced (Fig. 8), providing a clue that RPE phagocytosis may be impaired as well. We speculate that absence of RAB28 may alter the KCNJ13 activity, which in turn affects the function of RPE microvilli and cone phagocytosis.
      A second important RAB28 interactant is PDE6D, a prenyl-binding protein. A Pde6d−/− mouse develops recessive CRD in which prenylated OS proteins (cPDEα′, GRK1) are absent in the COS (
      • Zhang H.
      • Li S.
      • Doan T.
      • Rieke F.
      • Detwiler P.B.
      • Frederick J.M.
      • Baehr W.
      Deletion of PrBP/δ impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments.
      ). We showed previously that RAB28 trafficking to the ROS is ARL3-GTP–dependent (
      • Hanke-Gogokhia C.
      • Wu Z.
      • Gerstner C.D.
      • Frederick J.M.
      • Zhang H.
      • Baehr W.
      Arf-like protein 3 (ARL3) regulates protein trafficking and ciliogenesis in mouse photoreceptors.
      ). Our finding that farnesylated RAB28 is unable to traffic to the OS in the absence of PDE6D (Fig. 9C) raises the interesting point that the cone-rod dystrophy in PDE6d mutant mice may result partly from the absence of RAB28 in cones. A PDE6D null allele in human patients was associated with Joubert syndrome and shown to impair trafficking of INPP5E, a phosphatidylinositol polyphosphate phosphatase involved in phosphatidylinositol metabolism (
      • Thomas S.
      • Wright K.J.
      • Le Corre S.
      • Micalizzi A.
      • Romani M.
      • Abhyankar A.
      • Saada J.
      • Perrault I.
      • Amiel J.
      • Litzler J.
      • Filhol E.
      • Elkhartoufi N.
      • Kwong M.
      • Casanova J.L.
      • Boddaert N.
      • et al.
      A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium.
      ).
      The Rab28−/− mouse model provides evidence that CRD18 is caused by a defect in cone disc shedding. Owing to continuous renewal of membranes containing phototransduction components at the proximal OS and the absence of disc shedding, mutant OSs stretch toward the RPE and expand their tips to a balloon-like structure. Cone pigment–containing phagosomes are dramatically reduced. However, because RPE and cones collaborate in phagocytosis and RAB28 is normally expressed in the RPE, a role of the Rab28−/− in defective RPE phagocytosis cannot be excluded.

      Experimental procedures

      Animals

      Animal procedures were approved by the University of Utah institutional animal care and use committee and were conducted in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

      Generation of Rab28−/− mouse

      Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany). ES cell blastocyst injection, generation of chimera, and generation/breeding of heterozygous (GT/+) mice were performed at the University of Utah transgenic core. The rd8 mutation (
      • Mattapallil M.J.
      • Wawrousek E.F.
      • Chan C.C.
      • Zhao H.
      • Roychoudhury J.
      • Ferguson T.A.
      • Caspi R.R.
      The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells and confounds ocular induced mutant phenotypes.
      ) was removed by crossing our Rab28GT/+ animals with C57BL/J WT mice. We crossed Rab28GT/GT with flippase mice (C57BL/6 background) to generate animals with a floxed allele (Rab28fl/+) (Fig. 1D). Exon 3 was deleted by crossing Rab28fl/fl with CMV-Cre mice (C57BL/6 background) to generate Rab28+/− and Rab28−/− mice. Mice were maintained under 12-h cyclic dark/light conditions. ES cell and mouse tail genomic DNA was extracted using a standard protocol (
      • Wu S.
      • Ying G.
      • Wu Q.
      • Capecchi M.R.
      A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond.
      ). Routine genotyping was performed using genomic DNA prepared with HotSHOT (
      • Truett G.E.
      • Heeger P.
      • Mynatt R.L.
      • Truett A.A.
      • Walker J.A.
      • Warman M.L.
      Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT).
      ). PCR genotyping primers and other primers (see below) are listed in Table S1.

      RNA extraction, RT-PCR, and plasmid construction

      Retina tissues were dissected from WT mice, and total RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription was performed using SuperScript II reverse transcriptase (with random primers) following the manufacturer's instructions. Full-length RAB28V1 and RAB28V2 were amplified by PCR and cloned into pEGFP-C1 and pmCherry-C1 (Clontech), respectively. PDE6D-EGFP was kindly provided by Dr. Houbin Zhang (University of Electronic Science and Technology of China, Chengdu). Nonprenylated RAB28V2-C217A was generated using the Phusion site-directed mutagenesis kit (Thermo Fisher Scientific). All plasmids were verified by Sanger sequencing.

      ERG

      ERG was performed on 1-, 3-, 6-, and 13-month-old Rab28−/− and WT control animals (n = 5 each group) using an UTAS E-3000 universal electrophysiological system (LKC Technologies) as described (
      • Ying G.
      • Avasthi P.
      • Irwin M.
      • Gerstner C.D.
      • Frederick J.M.
      • Lucero M.T.
      • Baehr W.
      Centrin 2 is required for mouse olfactory ciliary trafficking and development of ependymal cilia planar polarity.
      ). Briefly, mice were dark-adapted overnight, anesthetized by intraperitoneal injection of ketamine (100 μg/g body weight) and xylazine (10 μg/g body weight) in 0.1 m PBS, and loaded onto a recording platform with body temperature maintained at 37 ± 0.5 °C. After dilating pupils with 1% tropicamide solution (Bausch & Lomb Inc., Tampa, FL), ERG responses were recorded from five mice of each genotype per time point. For scotopic ERG, mice were tested at intensities ranging from −1.63 to 2.38 log cd s/m2. For photopic ERG, a rod saturating background light of 1.3979 log cd s/m2 was applied for 20 min before and during recording at −0.01 to 1.86 log cd s/m2. Bactitracin ophthalmic ointment (Perrigo, Minneapolis, MN) was routinely applied to the eye to prevent infection after ERG testing, and animals were kept on heating pads until fully recovered before being returned to cages. Peak amplitudes for both a- and b-waves were used for analysis using a one-way ANOVA test.

      Measurement of ONL thickness and OS length

      Average ONL thickness and OS length were measured based on DAPI and anti-GC1 antibody fluorescence (
      • Coleman J.A.
      • Zhu X.
      • Djajadi H.R.
      • Molday L.L.
      • Smith R.S.
      • Libby R.T.
      • John S.W.
      • Molday R.S.
      Phospholipid flippase ATP8A2 is required for normal visual and auditory function and photoreceptor and spiral ganglion cell survival.
      ). Cone density was measured based on cone arrestin staining, and COS length was measured based on CNGA3 staining.

      In situ phagosome counting

      Retina sections collected at 1.5 h after light onset were stained with anti-rhodopsin antibody or mixed anti-ML-opsin and S-opsin antibodies. Rhodopsin-positive phagosomes were counted as described (
      • Nandrot E.F.
      • Anand M.
      • Almeida D.
      • Atabai K.
      • Sheppard D.
      • Finnemann S.C.
      Essential role for MFG-E8 as ligand for αvβ5 integrin in diurnal retinal phagocytosis.
      ). Cone opsin–positive phagosomes were counted from entire retina sections.

      Immunofluorescence and confocal microscopy

      Staining of cryosections with antibodies and confocal imaging were performed as described (
      • Ying G.
      • Gerstner C.D.
      • Frederick J.M.
      • Boye S.L.
      • Hauswirth W.W.
      • Baehr W.
      Small GTPases Rab8a and Rab11a are dispensable for rhodopsin transport in mouse photoreceptors.
      ) with minor modifications. Eyecups were fixed by immersion in ice-cold 4% paraformaldehyde, cryoprotected in 30% sucrose, and embedded in OCT compound. Sections (12 μm thick) were cut using a Micron cryostat and mounted on charged Superfrost® Plus slides (Fisher). Sections were washed in 0.1 m PBS, blocked using 10% normal goat serum or 2% BSA, 0.3% Triton X-100 in PBS, and incubated with primary antibodies at 4 °C overnight. After washes in PBS, signals were detected with Cy3-conjugated or Alexa 488–conjugated goat anti-rabbit/mouse and/or donkey anti-goat/rabbit/mouse secondary antibody (Jackson ImmunoResearch, Inc.) and counterstained with 1 μl/ml DAPI (Invitrogen). Primary antibodies were as follows: goat anti-RAB28 (Biorbyt), mouse monoclonal anti-rhodopsin (1D4; 1:1000), anti-ROM-1 (1:25) (Dr. Robert Molday, University of British Columbia), anti-GC1 (IS4; 1:1000; Dr. Kris Palczewski, Case Western Reserve University), cyclic nucleotide–gated channel α 1 and 3 (CNGA1/A3) (1:1000; NeuroMab, University of California, Davis, CA), GRK1 (G8, Santa Cruz Biotechnology, Inc.; 1:300); rabbit polyclonal anti-ML- and S-opsin (Chemicon; 1:1500), CNGA3 (1:500; Dr. Xi-Qin Ding, University of Oklahoma), cTγ (Cytosignal; 1:500), cone arrestin (mCAR; 1:500) (
      • Zhu X.
      • Li A.
      • Brown B.
      • Weiss E.R.
      • Osawa S.
      • Craft C.M.
      Mouse cone arrestin expression pattern: light induced translocation in cone photoreceptors.
      ), PDE6 (MOE, Cytosignal; 1:1500), rod transducin α (Cytosignal; 1:500), and cone PDE6α′ (Dr. Tiansen Li, NEI, National Institutes of Health; 1:40,000). Images were captured using a Zeiss LSM-800 confocal microscope. Some images were adjusted for brightness and contrast using Adobe Photoshop CS3.

      Retina protein extraction and Western blotting

      Retinas from WT and Rab28−/− mice or bovine were homogenized in radioimmune precipitation assay buffer (10 mm Tris-Cl, pH 8.0, 140 mm NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride) and centrifuged (13,000 × g, 4 °C, for 20 min), and supernatants were collected. Protein samples were separated by 10–12% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with primary antibodies followed by horseradish peroxidase–conjugated secondary antibody. Antigens were visualized using an ECL-plus kit (Pierce).

      RAB28 co-immunoprecipitation

      Bovine retinas (n = 5) were pooled and homogenized in 50 ml of lysis buffer (50 mm Tris-Cl, pH 7.6, 120 mm NaCl, 0.5% IGEPAL CA-630, 1 mm phenylmethylsulfonyl fluoride, 1× protease inhibitor) using a Dounce homogenizer. Cell debris was removed by centrifugation at 3000 × g, 4 °C, for 5 min. The cleaned samples were centrifuged at 20,000 × g, 4 °C, for 30 min, and the supernatants were collected. The supernatants were precleared with 500 μl of 50% protein G–Sepharose 4 fast flow beads (GE Healthcare) for 30 min. Precleared lysates were divided into 10 aliquots (5 ml each). Half of the aliquots were each incubated with goat anti-RAB28 primary antibody (10 μg), and the other half were incubated with normal goat IgG (10 μg) for 1 h 45 min at 4 °C with gentle agitation, followed by incubation with protein G–Sepharose beads for 45 min. Beads were collected by centrifugation at 2300 × g for 1 min and washed six times in lysis buffer without protease inhibitors and once in 1× PBS. Immunoprecipitated proteins were eluted using an improved soft elution method, which minimally co-elutes immunoglobulin (
      • Antrobus R.
      • Borner G.H.
      Improved elution conditions for native co-immunoprecipitation.
      ). The eluted proteins (200 μl) were incubated with 1 ml of acetone at −20 °C overnight, precipitated by centrifugation at 10,000 × g for 10 min at 4 °C, washed with 75% ethanol, and air-dried. Samples were subjected to quantitative MS.

      Quantitative MS

      Protein samples were subjected to in-solution tryptic cleavage, as described previously (
      • Gloeckner C.J.
      • Boldt K.
      • Schumacher A.
      • Ueffing M.
      Tandem affinity purification of protein complexes from mammalian cells by the Strep/FLAG (SF)-TAP tag.
      ). LC-MS/MS analysis was performed using an Ultimate3000 nano-RSLC system (Thermo Scientific) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) by a nanospray ion source. Tryptic peptide mixtures were injected automatically and loaded at a flow rate of 30 μl/min in 0.1% TFA in HPLC grade water onto a nano trap column (300-μm inner diameter × 5-mm precolumn, packed with Acclaim PepMap100 C18, 5 μm, 100 Å; Thermo Scientific). After 3 min, peptides were eluted and separated on the analytical column (75-μm inner diameter × 25 cm, Acclaim PepMap RSLC C18, 2 μm, 100 Å; Thermo Scientific) by a linear gradient from 2 to 30% of buffer B (80% actetonitrile and 0.08% formic acid in HPLC-grade water) in buffer A (2% acetonitrile and 0.1% formic acid in HPLC-grade water) at a flow rate of 300 nl/min over 85 min. Remaining peptides were eluted by a short gradient from 30 to 95% buffer B in 5 min. Analysis of the eluted peptides was performed by an LTQ Fusion mass spectrometer. From the high-resolution MS prescan at a resolution of 120,000 with a mass range of 335–1500, the most intense peptide ions were selected for fragment analysis if they were at least doubly charged. The normalized collision energy for higher-energy collisional dissociation was set to a value of 30, and the resulting fragments were detected in the ion trap. The lock mass option was activated; the background signal with a mass of 445.12003 was used as lock mass (
      • Olsen J.V.
      • de Godoy L.M.
      • Li G.
      • Macek B.
      • Mortensen P.
      • Pesch R.
      • Makarov A.
      • Lange O.
      • Horning S.
      • Mann M.
      Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.
      ). Every ion selected for fragmentation was excluded for 20 s by dynamic exclusion. MS/MS data were analyzed using the MaxQuant software (version 1.5.2.8) (
      • Cox J.
      • Mann M.
      MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
      ). As digesting enzyme, trypsin/P was selected with maximal 2 missed cleavages. Cysteine carbamidomethylation was set for fixed modifications, and oxidation of methionine and N-terminal acetylation were specified as variable modifications. The data were analyzed by label-free quantification with a minimum ratio count of 2. The first search peptide tolerance was set to 20, the main search peptide tolerance was set to 4.5 ppm, and the requantify option was selected. For peptide and protein identification, the Bos taurus subset of the Uniprot database (Release 2013_09) was used, and contaminants were detected using the MaxQuant contaminant search. A minimum peptide number of 2 and a minimum length of 7 amino acids was tolerated. Unique and razor peptides were used for quantification. The match between run options was enabled with a match time window of 0.7 min and an alignment time window of 20 min. The statistical analysis, including ratio, t test, and significance calculation, was done using the Perseus software (
      • Tyanova S.
      • Temu T.
      • Sinitcyn P.
      • Carlson A.
      • Hein M.Y.
      • Geiger T.
      • Mann M.
      • Cox J.
      The Perseus computational platform for comprehensive analysis of (prote)omics data.
      ).

      HEK293 cell culture and transfection

      HEK293 cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1× penicillin/streptomycin in a standard CO2 incubator. Cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Two days following transfection, cells were fixed and imaged for protein localization, lysed in radioimmune precipitation assay buffer for Western blotting, or lysed in co-IP lysis buffer (see above) for co-IP experiments.

      RAB28V2 neonatal electroporation

      For retina electroporation, 0.3 μl (1.5 μg) of pmCherry-RAB28V2 plasmid DNA was electroporated into the right eye subretinal space of P0 WT mice (
      • Matsuda T.
      • Cepko C.L.
      Electroporation and RNA interference in the rodent retina in vivo and in vitro.
      ). Protein localization was analyzed at P21.

      Transmission EM

      1- and 2.5-month-old WT and Rab28−/− retinas were fixed 2 h in fixative (2% glutaraldehyde, 1% paraformaldehyde in 0.1 m sodium cacodylate buffer, pH 7.4) at 4 °C, post-fixed for 1 h in 1% osmium tetroxide, and stained en bloc with uranyl acetate. Washed specimens were dehydrated through an ascending series of methanol, dried in propylene oxide, and infiltrated overnight with Epon resin mix/propylene oxide (1:1) mixture, followed by 100% Epon resin for 2 days. Specimens were embedded in plastic, and the plastic was cured by incubation in a 60 °C oven for 2 days. Blocks were trimmed, and 1-μm-thick sections were cut to identify and orient photoreceptors near the optic nerve. Ultrathin sections at 60 nm were cut, placed onto slot grids with carbon-coated Formvar film (EMS, Hatfield, PA), post-stained with uranyl acetate followed by lead citrate, and finally examined using a JOEL electron microscope at 75 kV.

      Statistics

      Data are presented as mean ± S.D., where n represents the number of mice analyzed (ERG, retina measurement, phagosome count), or experiment repeats (RAB28 co-IP). Statistical comparisons (significance level set at p < 0.05) were performed using one-way ANOVA for all experimental data.

      Author contributions

      G. Y. and W. B. conceptualization; G. Y., K. B., and J. M. F. data curation; G. Y. and J. M. F. formal analysis; G. Y., K. B., M. U., C. D. G., and J. M. F. methodology; G. Y. writing-original draft; K. B., M. U., and W. B. funding acquisition; K. B., M. U., and C. D. G. investigation; M. U. resources; W. B. project administration; W. B. writing-review and editing.

      Acknowledgments

      We thank the following investigators for providing antibodies directed against the named proteins: Dr. Xi-Qin Ding (University of Oklahoma) for CNGA3, Dr. Tiansen Li (NEI, National Institutes of Health) for cPDEα′, Dr. Robert Molday (University of British Columbia-Vancouver) for rhodopsin and Rom1, and Dr. Kris Palczewski (Case Western Reserve University) for GC1 IS4. Dr. Houbin Zhang (University of Electronic Science and Technology of China) provided PDE6D-EGFP plasmid.

      Supplementary Material

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