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Deletion of the phosphatase INPP5E in the murine retina impairs photoreceptor axoneme formation and prevents disc morphogenesis

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100529

ISSN

1083-351X

Autores

Ali S. Sharif, Cecilia D. Gerstner, Martha A. Cady, Vadim Y. Arshavsky, Christina A. Mitchell, Guoxin Ying, Jeanne M. Frederick, Wolfgang Baehr,

Tópico(s)

Microtubule and mitosis dynamics

Resumo

INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates the phosphoinositide composition of membranes. Mutations to or loss of INPP5E is associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans—a syndromic ciliopathy affecting multiple tissues including the brain, liver, kidney, and retina. In contrast to other primary cilia, photoreceptor INPP5E is prominently expressed in the inner segment and connecting cilium and absent in the outer segment, which is a modified primary cilium dedicated to phototransduction. To investigate how loss of INPP5e causes retina degeneration, we generated mice with a retina-specific KO (Inpp5eF/F;Six3Cre, abbreviated as retInpp5e−/−). These mice exhibit a rapidly progressing rod–cone degeneration resembling Leber congenital amaurosis that is nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. Although P10 mutant outer segments contain structural and phototransduction proteins, axonemal structure and disc membranes fail to form. Connecting cilia of retInpp5e−/− rods display accumulation of intraflagellar transport particles A and B at their distal ends, suggesting disrupted intraflagellar transport. Although INPP5E ablation may not prevent delivery of outer segment–specific proteins by means of the photoreceptor secretory pathway, its absence prevents the assembly of axonemal and disc components. Herein, we suggest a model for INPP5E–Leber congenital amaurosis, proposing how deletion of INPP5E may interrupt axoneme extension and disc membrane elaboration. INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates the phosphoinositide composition of membranes. Mutations to or loss of INPP5E is associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans—a syndromic ciliopathy affecting multiple tissues including the brain, liver, kidney, and retina. In contrast to other primary cilia, photoreceptor INPP5E is prominently expressed in the inner segment and connecting cilium and absent in the outer segment, which is a modified primary cilium dedicated to phototransduction. To investigate how loss of INPP5e causes retina degeneration, we generated mice with a retina-specific KO (Inpp5eF/F;Six3Cre, abbreviated as retInpp5e−/−). These mice exhibit a rapidly progressing rod–cone degeneration resembling Leber congenital amaurosis that is nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. Although P10 mutant outer segments contain structural and phototransduction proteins, axonemal structure and disc membranes fail to form. Connecting cilia of retInpp5e−/− rods display accumulation of intraflagellar transport particles A and B at their distal ends, suggesting disrupted intraflagellar transport. Although INPP5E ablation may not prevent delivery of outer segment–specific proteins by means of the photoreceptor secretory pathway, its absence prevents the assembly of axonemal and disc components. Herein, we suggest a model for INPP5E–Leber congenital amaurosis, proposing how deletion of INPP5E may interrupt axoneme extension and disc membrane elaboration. INPP5E is a farnesylated phosphatidylinositol polyphosphate 5-phosphatase (1Kisseleva M.V. Wilson M.P. Majerus P.W. The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase.J. Biol. Chem. 2000; 275: 20110-20116Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 2Kong A.M. Speed C.J. O'Malley C.J. Layton M.J. Meehan T. Loveland K.L. Cheema S. Ooms L.M. Mitchell C.A. Cloning and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network.J. Biol. Chem. 2000; 275: 24052-24064Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) catalyzing the hydrolysis of the 5 phosphate from PI (4,5)P2 (PIP2), and PI (3,4,5)P3 (reviewed in (3Conduit S.E. Dyson J.M. Mitchell C.A. Inositol polyphosphate 5-phosphatases; new players in the regulation of cilia and ciliopathies.FEBS Lett. 2012; 586: 2846-2857Crossref PubMed Scopus (56) Google Scholar, 4Balla T. 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Cell. 2015; 34: 400-409Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). INPP5E is associated with Joubert syndrome (JBTS) and mental retardation, truncal obesity, retinal dystrophy, and micropenis syndrome (7Jacoby M. Cox J.J. Gayral S. Hampshire D.J. Ayub M. Blockmans M. Pernot E. Kisseleva M.V. Compere P. Schiffmann S.N. Gergely F. Riley J.H. Perez-Morga D. Woods C.G. Schurmans S. INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse.Nat. Genet. 2009; 41: 1027-1031Crossref PubMed Scopus (259) Google Scholar, 8Travaglini L. Brancati F. Silhavy J. Iannicelli M. Nickerson E. Elkhartoufi N. Scott E. Spencer E. Gabriel S. Thomas S. Ben-Zeev B. Bertini E. Boltshauser E. Chaouch M. Cilio M.R. et al.Phenotypic spectrum and prevalence of INPP5E mutations in Joubert syndrome and related disorders.Eur. J. Hum. Genet. 2013; 21: 1074-1078Crossref PubMed Scopus (52) Google Scholar) caused by missense mutations in its phosphatase domain. JBTS is a syndromic ciliopathy affecting the brain, eyes, kidneys, and liver (9Bielas S.L. Silhavy J.L. Brancati F. Kisseleva M.V. Al-Gazali L. Sztriha L. Bayoumi R.A. Zaki M.S. Abdel-Aleem A. Rosti R.O. Kayserili H. Swistun D. Scott L.C. Bertini E. Boltshauser E. et al.Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.Nat. Genet. 2009; 41: 1032-1036Crossref PubMed Scopus (311) Google Scholar, 10Hardee I. Soldatos A. Davids M. Vilboux T. Toro C. David K.L. Ferreira C.R. Nehrebecky M. Snow J. Thurm A. Heller T. Macnamara E.F. Gunay-Aygun M. Zein W.M. Gahl W.A. et al.Defective ciliogenesis in INPP5E-related Joubert syndrome.Am. J. Med. Genet. A. 2017; 173: 3231-3237Crossref PubMed Scopus (17) Google Scholar) and presenting with ataxia, hyperpnea, polydactyly, molar tooth sign in the brain and Retinitis pigmentosa or Leber congenital amaurosis. Ten phosphatidylinositol 5-phosphatases (INPP5A, B, D (SHIP), E, F, G (synaptojanin 1), H (synaptojanin 2), J, K (SKIP), and INPPL1) are present in mammals, and of these, three (A, B, and E) are farnesylated (11Ooms L.M. Horan K.A. Rahman P. Seaton G. Gurung R. Kethesparan D.S. Mitchell C.A. The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease.Biochem. J. 2009; 419: 29-49Crossref PubMed Scopus (178) Google Scholar). Germline mouse KOs of INPP5E and INPP5K in mice are embryonically lethal, suggesting nonredundant roles for some phosphatase isoforms in various cells or subcompartments (7Jacoby M. Cox J.J. Gayral S. Hampshire D.J. Ayub M. Blockmans M. Pernot E. Kisseleva M.V. Compere P. Schiffmann S.N. Gergely F. Riley J.H. Perez-Morga D. Woods C.G. Schurmans S. INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse.Nat. Genet. 2009; 41: 1027-1031Crossref PubMed Scopus (259) Google Scholar, 9Bielas S.L. Silhavy J.L. Brancati F. Kisseleva M.V. Al-Gazali L. Sztriha L. Bayoumi R.A. Zaki M.S. Abdel-Aleem A. Rosti R.O. Kayserili H. Swistun D. Scott L.C. Bertini E. Boltshauser E. et al.Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.Nat. Genet. 2009; 41: 1032-1036Crossref PubMed Scopus (311) Google Scholar, 12Dyson J.M. Conduit S.E. Feeney S.J. Hakim S. DiTommaso T. Fulcher A.J. Sriratana A. Ramm G. Horan K.A. Gurung R. Wicking C. Smyth I. Mitchell C.A. INPP5E regulates phosphoinositide-dependent cilia transition zone function.J. Cell Biol. 2017; 216: 247-263Crossref PubMed Scopus (73) Google Scholar). Inpp5e−/− mice (deletion of exons 7 and 8) died soon after birth; E18.6 Inpp5e−/− embryos showed developmental arrest at the optic vesicle stage before appearance of the optic cup. Inpp5e−/− E18.5 embryos displayed multiple cysts, polydactyly, and skeletal abnormalities (7Jacoby M. Cox J.J. Gayral S. Hampshire D.J. Ayub M. Blockmans M. Pernot E. Kisseleva M.V. Compere P. Schiffmann S.N. Gergely F. Riley J.H. Perez-Morga D. Woods C.G. Schurmans S. INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse.Nat. Genet. 2009; 41: 1027-1031Crossref PubMed Scopus (259) Google Scholar). Inpp5e−/− embryos were anophthalmic, suggesting severe consequences of INPP5E deletion during early eye development (7Jacoby M. Cox J.J. Gayral S. Hampshire D.J. Ayub M. Blockmans M. Pernot E. Kisseleva M.V. Compere P. Schiffmann S.N. Gergely F. Riley J.H. Perez-Morga D. Woods C.G. Schurmans S. INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse.Nat. Genet. 2009; 41: 1027-1031Crossref PubMed Scopus (259) Google Scholar). A second Inpp5e−/− mouse model (deletion of exons 2–6) confirmed the JBTS phenotype and identified disordered sonic hedgehog–dependent patterning during embryonic development (12Dyson J.M. Conduit S.E. Feeney S.J. Hakim S. DiTommaso T. Fulcher A.J. Sriratana A. Ramm G. Horan K.A. Gurung R. Wicking C. Smyth I. Mitchell C.A. INPP5E regulates phosphoinositide-dependent cilia transition zone function.J. Cell Biol. 2017; 216: 247-263Crossref PubMed Scopus (73) Google Scholar). Conditional deletion of INPP5E in kidneys resulted in severe polycystic kidney disease and hyperactivation of PI3K/Akt and mTORC1 signaling (13Hakim S. Dyson J.M. Feeney S.J. Davies E.M. Sriratana A. Koenig M.N. Plotnikova O.V. Smyth I.M. Ricardo S.D. Hobbs R.M. Mitchell C.A. Inpp5e suppresses polycystic kidney disease via inhibition of PI3K/Akt-dependent mTORC1 signaling.Hum. Mol. Genet. 2016; 25: 2295-2313Crossref PubMed Scopus (29) Google Scholar). Similarly, deletion of the Inpp5e gene in the zebrafish led to cystic kidneys (14Xu W. Jin M. Hu R. Wang H. Zhang F. Yuan S. Cao Y. The Joubert syndrome protein Inpp5e controls ciliogenesis by regulating phosphoinositides at the apical membrane.J. Am. Soc. Nephrol. 2017; 28: 118-129Crossref PubMed Scopus (28) Google Scholar). Using transfection of IMCD3 cells with plasmids encoding FLAG–INPP5E or EGFP–INPP5E, INPP5E localized predominantly in the primary cilia (15Humbert 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.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 19691-19696Crossref PubMed Scopus (155) Google Scholar, 16Kosling S.K. Fansa E.K. Maffini S. Wittinghofer A. Mechanism and dynamics of INPP5E transport into and inside the ciliary compartment.Biol. Chem. 2018; 399: 277-292Crossref PubMed Scopus (16) Google Scholar). As a C-terminally farnesylated protein, INPP5E is chaperoned to the cilium by the prenyl-binding protein PDEδ (16Kosling S.K. Fansa E.K. Maffini S. Wittinghofer A. Mechanism and dynamics of INPP5E transport into and inside the ciliary compartment.Biol. Chem. 2018; 399: 277-292Crossref PubMed Scopus (16) Google Scholar, 17Thomas 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.Hum. Mutat. 2014; 35: 137-146Crossref PubMed Scopus (93) Google Scholar, 18Fansa E.K. Kosling S.K. Zent E. Wittinghofer A. Ismail S. PDE6delta-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity.Nat. Commun. 2016; 7: 11366Crossref PubMed Scopus (60) Google Scholar). Ciliary localization in hTERT-RPE1, 293T, and IMCD3 cells was confirmed using a polyclonal anti-INPP5E antibody (14Xu W. Jin M. Hu R. Wang H. Zhang F. Yuan S. Cao Y. The Joubert syndrome protein Inpp5e controls ciliogenesis by regulating phosphoinositides at the apical membrane.J. Am. Soc. Nephrol. 2017; 28: 118-129Crossref PubMed Scopus (28) Google Scholar, 19Plotnikova O.V. Seo S. Cottle D.L. Conduit S. Hakim S. Dyson J.M. Mitchell C.A. Smyth I.M. INPP5E interacts with AURKA, linking phosphoinositide signaling to primary cilium stability.J. Cell Sci. 2015; 128: 364-372Crossref PubMed Scopus (60) Google Scholar, 20Nozaki S. Katoh Y. Terada M. Michisaka S. Funabashi T. Takahashi S. Kontani K. Nakayama K. Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E.J. Cell Sci. 2017; 130: 563-576Crossref PubMed Scopus (56) Google Scholar, 21Roberson E.C. Dowdle W.E. Ozanturk A. Garcia-Gonzalo F.R. Li C. Halbritter J. Elkhartoufi N. Porath J.D. Cope H. Ashley-Koch A. Gregory S. Thomas S. Sayer J.A. Saunier S. Otto E.A. et al.TMEM231, mutated in orofaciodigital and Meckel syndromes, organizes the ciliary transition zone.J. Cell Biol. 2015; 209: 129-142Crossref PubMed Scopus (69) Google Scholar). Inpp5e−/− mouse embryonic fibroblasts developed primary cilia, suggesting that INPP5E activity is not required for ciliogenesis, but mutant cilia were more sensitive to resorption during the cell cycle (5Chavez M. Ena S. Van Sande J. de Kerchove d'Exaerde A. Schurmans S. Schiffmann S.N. Modulation of ciliary phosphoinositide content regulates trafficking and sonic Hedgehog signaling output.Dev. Cell. 2015; 34: 338-350Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 6Garcia-Gonzalo F.R. Phua S.C. Roberson E.C. Garcia 3rd, G. Abedin M. Schurmans S. Inoue T. Reiter J.F. Phosphoinositides regulate ciliary protein trafficking to modulate hedgehog signaling.Dev. Cell. 2015; 34: 400-409Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 9Bielas S.L. Silhavy J.L. Brancati F. Kisseleva M.V. Al-Gazali L. Sztriha L. Bayoumi R.A. Zaki M.S. Abdel-Aleem A. Rosti R.O. Kayserili H. Swistun D. Scott L.C. Bertini E. Boltshauser E. et al.Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.Nat. Genet. 2009; 41: 1032-1036Crossref PubMed Scopus (311) Google Scholar). In contrast to primary cilia, EGFP–INPP5E introduced to rod photoreceptors by neonatal electroporation distributed to inner segments (ISs) and partially colocalized with the Golgi apparatus (22Hanke-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.J. Biol. Chem. 2016; 291: 7142-7155Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Immunolabeling of dissociated rods similarly showed prominent IS signal for INPP5E and an additional nonuniform signal of the outer segment (OS) (23Rao K.N. Zhang W. Li L. Anand M. Khanna H. Prenylated retinal ciliopathy protein RPGR interacts with PDE6delta and regulates ciliary localization of Joubert syndrome-associated protein INPP5E.Hum. Mol. Genet. 2016; 25: 4533-4545PubMed Google Scholar). In this study, we confirm that INPP5E localizes to the WT photoreceptor IS by multiple means. We also deleted INPP5E from the retina by mating Inpp5eF/F mice (12Dyson J.M. Conduit S.E. Feeney S.J. Hakim S. DiTommaso T. Fulcher A.J. Sriratana A. Ramm G. Horan K.A. Gurung R. Wicking C. Smyth I. Mitchell C.A. INPP5E regulates phosphoinositide-dependent cilia transition zone function.J. Cell Biol. 2017; 216: 247-263Crossref PubMed Scopus (73) Google Scholar) with Six3Cre transgenic mice and observed that retInpp5e−/− rod outer segments (ROSs) initiate degeneration by postnatal day 10 (P10). While P10 connecting cilia (CC) of P10 mutant rods are nearly normal in length, shortened ROS are devoid of recognizable discs. Intraflagellar transport (IFT)-A and IFT-B particles accumulate in the proximal retInpp5e−/− OS, suggesting defective IFT, a bidirectional transport system powered by molecular motors (24Scholey J.M. Kinesin-2 motors transport IFT-particles, dyneins and tubulin subunits to the tips of Caenorhabditis elegans sensory cilia: Relevance to vision research?.Vis. Res. 2012; 75: 44-52Crossref PubMed Scopus (10) Google Scholar). Thus, deletion of INPP5E primarily impairs axoneme extension and disc morphogenesis of both rods and cones. INPP5E is a 72-kDa protein carrying a proline-rich domain in the N-terminal region, a large phosphatase active site encoded by exons 3 to 9, a coiled-coil domain, and a CAAX motif for C-terminal farnesylation (Fig. 1A). Mutations in human INPP5E associated with JBTS are located predominantly in the phosphatase domain. The mouse Inpp5e gene consists of 10 exons, producing two splice variants (Fig. 1B). The full-length variant is predicted to be farnesylated and membrane-anchored; the shorter variant lacking exon 10 and the CAAX box is predicted to be soluble. To generate retina-specific KO (retInpp5e−/−) mice, Inpp5eF/F mice were bred with Six3Cre mice (25Furuta Y. Lagutin O. Hogan B.L. Oliver G.C. Retina- and ventral forebrain-specific Cre recombinase activity in transgenic mice.Genesis. 2000; 26: 130-132Crossref PubMed Scopus (154) Google Scholar). LoxP sites placed in introns 1 and 6 specify deletion of most of the phosphatase domain (Fig. 1C). LoxP, Six3Cre, and KO genotyping is shown (Fig. 1D). Immunoblotting with two independent antibodies against human INPP5E (Proteintech [PT]) and a mouse N-terminal fragment of INPP5E (custom-made at Covance, Inc. [CO]) confirmed the presence of two splice variants in WT retinal lysates and deletion of both isoforms in the conditional KO line (Fig. 1E). We recorded whole-field scotopic (dark-adapted) and photopic (light-adapted) electroretinography (ERGs) at P15 and P21 at light intensities ranging from −1.63 to 2.38 log cd s/m2 (Fig. 2, A and B). Scotopic a-waves (Fig. 2, C and F) and scotopic b-waves (Fig. 2, D and G) of KO mice were barely recordable at P15 and P21. Scotopic a-waves of heterozygous littermates displayed normal a-wave amplitudes at P21 (Fig. 2F), indistinguishable from the WT, suggesting haplosufficiency. Surprisingly, the P15 retInpp5e−/− photopic ERG b-wave amplitudes were still recordable (Fig. 2E), presumably due in part to a slower degeneration of cones in the peripheral retina. At P21, retInpp5e−/− photopic ERG b-wave amplitudes were much reduced (Fig. 2H). The b-wave amplitudes of heterozygous littermates were somewhat reduced relative to the WT, but the reduction was statistically insignificant. Plastic sections of retInpp5e−/− and littermate WT control retinas collected at P10, P15, and P21 (Fig. 3, A–F) confirmed rapid photoreceptor degeneration, revealed by a progressively shrinking outer nuclear layer (ONL). At P10, retInpp5e−/− retinas (Fig. 3D) showed only a slightly reduced ONL thickness compared with the WT (Fig. 3A), but the distance between the retinal pigmented epithelium and ONL was substantially reduced, suggesting attenuation of the IS and/or OS volume. At P15, nuclear tiers were reduced by ∼30% (Fig. 3E), and at P21, only one row of nuclei, presumably cones, was present (Fig. 3F), consistent with rapid rd1-like degeneration. Evaluation of the ONL thickness across the entire retina (Fig. 3, G–I) showed 40% reduction in the central retina at P15 and nearly complete obliteration of the central ONL at P21. Persisting ONL thickness of the retinal periphery (Fig. 3I) is consistent with lower Cre expression in the peripheral retina under the Six3 promoter (25Furuta Y. Lagutin O. Hogan B.L. Oliver G.C. Retina- and ventral forebrain-specific Cre recombinase activity in transgenic mice.Genesis. 2000; 26: 130-132Crossref PubMed Scopus (154) Google Scholar, 26Hanke-Gogokhia C. Wu Z. Sharif A. Yazigi H. Frederick J.M. Baehr W. The guanine nucleotide exchange factor Arf-like protein 13b is essential for assembly of the mouse photoreceptor transition zone and outer segment.J. Biol. Chem. 2017; 292: 21442-21456Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The retInpp5e−/− inner retina, including the inner nuclear and ganglion cell layers, was practically unaffected as judged by near-normal morphology of P10, P15, and P21 cryosections (Fig. 3, D and E). INPP5E immunolocalization was performed using the well-characterized (PT) antibody raised against a His-tagged fusion protein corresponding to the N-terminal half of human INPP5E. WT cryosections analyzed at P15 (Fig. 4A) and P21 (Fig. 4C) revealed the presence of INPP5E in the IS with traces in the ONL, and absence in the OS. The INPP5E signal of the contemporaneously labeled KO IS was significantly reduced at P15 (Fig. 4B) and undetectable at P21 (Fig. 4D). To verify specificity of the human antibody in mouse, we generated an independent antibody (CO) against the N-terminal peptide (MPSKSASLRHTEAC) of mouse INPP5E. As its sequence is distinct from the human homolog, the antibody is predicted to be mouse-specific. This antibody showed strong immunoreactivity in the entire IS, along the outer limiting membrane, with traces in the ONL (Fig. 4E); immunoreactivity was erased when the antibody was saturated with the antigenic peptide (Fig. 4F). Anti-INPP5E immunoreactivity was still detectable in KO sections at P15 with the PT antibody (Fig. 4B) and at P13 with the CO antibody (Fig. 4H), suggesting ineffective clearing of INPP5E or nonspecific binding. Differences in IS antigen localization using the PT antibody (fixed sections, Fig. 4A) and CO antibody (slightly fixed sections, Fig. 4E) are likely attributable to different fixation protocols (see Experimental procedures). To further verify the IS localization of INPP5E, we performed the technique of combined serial tangential retina sectioning and immunoblotting (27Sokolov M. Strissel K.J. Leskov I.B. Michaud N.A. Govardovskii V.I. Arshavsky V.Y. Phosducin facilitates light-driven transducin translocation in rod photoreceptors. Evidence from the phosducin knockout mouse.J. Biol. Chem. 2004; 279: 19149-19156Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). A Western blot of proteins from serial sections cut through a WT rat retina starting from the photoreceptor tips was probed with antibodies against rhodopsin (OS marker) and INPP5E (Fig. 4, I and J). This analysis revealed the presence of a single INPP5E species in sections corresponding to the IS (the rat Inpp5e gene expresses no splice variants) and only trace protein amounts in the sections representing the OS. A rod photoreceptor cartoon (Fig. 4K) indicates the approximate positions of the ROS, CC, rod inner segment (RIS), nucleus, and synapse. The INPP5E localization was further confirmed in the retina of a macaque and macula of a 71-year-old human postmortem donor where INPP5E was detected in the IS of rods and cones (Fig. 4, L and M). In the perfusion-fixed macaque retina, INPP5E is prominent in the ellipsoid regions of the cone IS and RIS, respectively, with lesser amounts in perinuclear locales. Cone outer segment (COS) and ROS were mostly devoid of INPP5E. This pattern differs from INPP5E localization within the primary cilia where INPP5E is delivered with the aid of factors to achieve solubilization, PDEδ (PDE6D), and cargo displacement, ARL3–GTP (16Kosling S.K. Fansa E.K. Maffini S. Wittinghofer A. Mechanism and dynamics of INPP5E transport into and inside the ciliary compartment.Biol. Chem. 2018; 399: 277-292Crossref PubMed Scopus (16) Google Scholar). Dissociated mouse rods (23Rao K.N. Zhang W. Li L. Anand M. Khanna H. Prenylated retinal ciliopathy protein RPGR interacts with PDE6delta and regulates ciliary localization of Joubert syndrome-associated protein INPP5E.Hum. Mol. Genet. 2016; 25: 4533-4545PubMed Google Scholar) were probed with both PT and CO antibodies (Fig. 5, A–D). The OSs were identified by anti-CNGA1/3 (cyclic nucleotide-gated channel subunits) located in the OS cell membrane and the CC by anti-MAK (male germ cell–associated kinase) antibodies localizing to the CC (28Omori Y. Chaya T. Katoh K. Kajimura N. Sato S. Muraoka K. Ueno S. Koyasu T. Kondo M. Furukawa T. Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 22671-22676Crossref PubMed Scopus (92) Google Scholar). These results clearly showed the absence of INPP5E in the OS and its prominent presence in both the IS and CC. Another CC marker is centrin-2 (CETN2), a Ca2+-binding protein associated with the CC and basal body. To demonstrate localization of INPP5E at the CC in vivo, we perfused P10 Egfp-Cetn2+ retInpp5e+/− and retInpp5e−/− mice with 4% paraformaldehyde (PFA), postfixed overnight, generated 100-μm retina sections, performed antigen retrieval with 1% SDS, and analyzed the sections treated with anti-INPP5E antibody by confocal microscopy. The results show the presence of INPP5E specifically at the retInpp5e+/−CC (Fig. 5E) and the absence of INPP5E in retInpp5e−/− sections (Fig. 5F). The IS location of INPP5E was unchanged in cryosections obtained from Arl3−/− mice (22Hanke-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.J. Biol. Chem. 2016; 291: 7142-7155Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), suggesting that it is not delivered in a complex with the prenyl-binding protein PDEδ. We verified that cellular distribution of farnesylated INPP5E is not dependent on PDEδ by analyzing its localization patterns in Pde6d−/− photoreceptors (Fig. 6). The OS of the PDE6D+/- cones contain geranylgeranylated PDE6α' (cone PDE6 α-subunit) (Fig. 6A) and farnesylated GRK1 (G-protein receptor kinase) (Fig. 6B) whose transport to COS is completely impaired in the absence of PDEδ. (Fig. 6, D and E). In contrast, the localization of farnesylated INPP5E was insensitive to the absence of PDEδ (Fig. 6, C and F). In the WT and mutant retina at P6, the OS begin to expand and rhodopsin is present in nascent WT OS and smaller mutant OS (Fig. 7, A and B; P8 panels). At P10, mutant OSs are shorter than the corresponding WT OSs (Fig. 7, A and B; P10 panel) and rhodopsin begins to mislocalize and its CC (labeled with EGFP–CETN2) are located closer to the ONL, suggesting a shrinking, degenerating IS. At P12, the WT OSs are maturing, but the mutant OSs are stunted and rhodopsin accumulates throughout the ONL (Fig. 7, A and B, P12 panels). As judged by Western blotting (Fig. 7C, left panel), the level of rhodopsin in mutant rods is slightly reduced at P6 and about half at P10 and P12 (Fig. 7C, right panel). Prenylated peripheral OS proteins (PDE6, GRK1) are thought to traffic the OS by diffusion, using PDEδ as a solubilization factor (29Baehr W. Membrane protein transport in photoreceptors: The function of PDEdelta: The proctor lecture.Invest. Ophthalmol. Vis. Sci. 2014; 55: 8653-8666Crossref PubMed Scopus (42) Google Scholar, 30Zhang H. Constantine R. Frederick J.M. Baehr W. The prenyl-binding protein PrBP/delta: A chaperone participating in intracellular trafficking.Vis. Res. 2012; 75: 19-25Crossref PubMed Scopus (41) Google Scholar) and ARL3–GTP as a cargo dispensation factor (22Hanke-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.J. Biol. Chem. 2016; 291: 7142-7155Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 31Ismail S.A. Chen Y.X. Miertzschke M. Vetter I.R. Koerner C. Wittinghofer A. Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119.EMBO J. 2012; 31: 4085-4094Crossref PubMed Scopus (85) Google Scholar, 32Ismail S.A. Chen Y.X. Rusinova A. Chandra A. Bierbaum M. Gremer L. Triola G. Waldmann H. Bastiaens P.I. Wittinghofer A. Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo.Nat. Chem. Biol. 2011; 7: 942-949Crossref PubMed Scopus (201) Google Scholar). In WT and retInpp5e−/− at P6, trace amounts of PDE6 are present in the OS (Fig. 7, D and E). At P8 (just after onset of degeneration), the level of OS PDE6 in retInpp5e−/− rods is comparable with the WT rods, but it is significantly reduced at P10 (Fig. 7E), consistent with the reduction of their OS size. These results suggest that endoplasmic reticulum docking, for post-translational modifications of PDE6, is not affected by INPP5E ablation. The COSs begin to form at P4 (33Fei Y. Development of the cone photoreceptor mosaic in the mouse retina revealed by fluorescent cones in transgenic mice.Mol. Vis. 2003; 9: 31-42PubMed Go

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