the leber congenital amaurosis gene product aipl1 is localized

© 2002 Oxford University Press Human Molecular Genetics, 2002, Vol. 11, No. 7 823–831

The Leber congenital amaurosis gene product AIPL1is localized exclusively in rod photoreceptors of theadult human retinaJacqueline van der Spuy, J. Paul Chapple, Brian J. Clark, Philip J. Luthert,Charanjit S. Sethi and Michael E. Cheetham*

Department of Pathology, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK

Received December 12, 2001; Revised and Accepted January 31, 2002

Leber congenital amaurosis (LCA) is the most severe inherited retinal dystrophy resulting in markedlyimpaired vision or blindness at birth. LCA is characterized by an extinguished electroretinogram in infancy,which is thought to be indicative of an early and severe impairment of both the rod and cone photoreceptorsin the human retina. Recently, the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene wasidentified as the fourth causative gene of LCA. AIPL1 encodes a 384 amino acid protein of unknown function.We have generated a polyclonal antibody against a peptide from a unique region within the primate AIPL1 protein,which detects a protein of ∼43 kDa in human retinal extracts. A screen of human tissues and immortalized celllines with this antibody reveals AIPL1 to be specific to human retina and cell lines of retinal origin (Y79 retino-blastoma cells). Within the retina, AIPL1 was detected only in the rod photoreceptor cells of the peripheral andcentral human retina. The AIPL1 staining pattern extended within the rod photoreceptor cells from the innersegments, through the rod nuclei to the rod photoreceptor synaptic spherules in the outer plexiform layer.AIPL1 was not detected in the cone photoreceptors of peripheral or central human retina. This study is thefirst to suggest that AIPL1 performs a function essential to the maintenance of rod photoreceptor function.

INTRODUCTION

Leber congenital amaurosis (LCA), first described by TheodoreLeber in 1869 (1), is the most severe form of early-onset inheritedretinal dystrophy responsible for congenital blindness orseverely impaired vision. Clinically, patients affected by LCApresent in infancy with pendular nystagmus, unusual rovingeye movements and absent ocular pursuit upon ophthalmicexamination. LCA patients may habitually rub their eyes withthe fist or fingers (the oculodigital reflex) (2) and have a higherincidence of keratoconus than in the normal population. In earlyinfancy, the optic discs and fundus are normal in appearanceshowing no abnormality upon fundoscopy. However, progressiveabnormalities in the fundus appear with time, including attenuationof the retinal vasculature, optic nerve pallor, bone corpuscularpigmentation, atrophy of the retinal pigment epithelium (RPE)and occasionally irregular yellow pigmentation within theperipheral and mid-peripheral retina. In the absence of fundusabnormalities in early infancy, LCA is differentiated from otherearly-onset retinal dystrophies by electroretinographic (ERG)testing. The ERG recordings in LCA patients are markedlyattenuated or absent. Additional systemic disorders, of whichpsychomotor retardation is the most frequent, have beenreported in association with LCA (3–5).

LCA is a genetically heterogeneous disorder with an autosomalrecessive mode of inheritance (6). To date, six causative genes havebeen identified in LCA, the first of which maps to chromosome

17p13.1 in humans (7,8) and translates to a photoreceptor-specificguanylate cyclase (retGC-1 or GUCY2D) (9). RetGC-1 is anessential component of the phototransduction cascade, andmutations in retGC-1 impair the recovery of the dark state afterphoto-excitation of the photoreceptor cells. The situation isequivalent to sustained photo-excitation of the photoreceptorsand patients with LCA caused by mutations in retGC-1 presentwith severe hyperopia and photophobia (10). The second locusfor LCA was assigned to chromosome 1p31 (11) and theaffected gene encodes a microsomal membrane protein (RPE65)of the RPE (12). RPE65 is a crucial component of the visualcycle in that it facilitates the isomerization of all-trans-retinylesterto 11-cis-retinol and hence the regeneration of the universalchromatophore 11-cis-retinal (13,14). Mutations in the RPE65gene decrease the production of functional visual pigmentsleading to a situation equivalent to the sustained absence ofphoto-excitation of photoreceptor cells. Patients with LCAcaused by mutations in RPE65 present with moderate or nohyperopia and sometimes low myopia upon ophthalmicexamination (10). The third causative gene of LCA is a cone–rod homeobox (CRX) gene, which maps to chromosome19q13.3 in humans (15,16). The CRX protein is a photore-ceptor-specific homeodomain transcription factor that plays anessential role in the differentiation, development and mainte-nance of photoreceptor cells through the transactivation ofseveral photoreceptor-specific gene promoters, including

*To whom correspondence should be addressed. Tel: +44 207 6086944; Fax: +44 207 6086862; Email: [email protected]

824 Human Molecular Genetics, 2002, Vol. 11, No. 7

rhodopsin (16–18). Recently, two other LCA causative geneshave been identified, CRB1 (19,20) and RPGRIP1 (21). CRB1is a retina and CNS expressed protein that is homologous to theDrosophila crumbs protein that is important in cell polarityand cell–cell contact, although the role of CRB1 in the retinaremains to be identified. RPGRIP1 was identified as an inter-acting partner of the X-linked retinitis pigmentosa proteinRPGR and is thought to be localized to the photoreceptorconnecting cilium where it may play a role in transportbetween the inner and outer segments. The diverse nature ofthese LCA causative genes illustrates the complexity of retinalcell biology and highlights that many, as yet unstudied, path-ways are essential for the normal function of the retina.

The aryl hydrocarbon receptor interacting protein-like 1(AIPL1) gene was the fourth gene found to cause LCA. AIPL1maps to within 2.5 Mb distal to the retGC-1 locus on chromo-some 17p13.1 in humans (22). The AIPL1 gene encompassessix exons encoding a protein of 384 amino acids in length. Theprotein sequence includes three consecutive 34 amino acidtetratricopeptide repeat (TPR) motifs, which are thought tomediate specific protein interactions. TPR motifs are ubiquitouslyconserved in structurally unrelated proteins that participate indiverse biological functions, including the co-ordination ofmultiprotein complex assembly and protein translocation (23).A 56 amino acid polyproline-rich sequence of high flexibilityencompassing multiple O-glycosylation sites and putativephosphorylation sites is present at the C-terminus of the AIPL1protein in humans. The function of AIPL1 in normal vision isunknown. However, AIPL1 is similar (49% identity) to thearyl hydrocarbon receptor-interacting protein (AIP) (24), alsoknown as the aryl hydrocarbon receptor-activated protein(ARA9) (25) or the X-associated protein (XAP2) (26). Thearyl hydrocarbon receptor is a cytosolic ligand-activatedtranscription factor that mediates adaptive and toxic responsesto environmental pollutants such as dioxin by increasing thetranscription of xenobiotic metabolizing enzymes. AIP facilitatesthe transactivation activity of the cognate transcription factorby regulating its nuclear translocation (24–31 and reviewed in32). The inclusion of the TPR motifs in the AIPL1 protein andthe similarity of AIPL1 to AIP collectively suggest that AIPL1may be involved in retinal protein folding or cellular translocation.

Northern blot hybridization and in situ hybridization demon-strated the expression of AIPL1 in the pineal gland and retina(22). This study is the first description of the localization ofAIPL1 protein in the adult human retina and photoreceptorcells. Our results are surprising in that AIPL1 is producedexclusively in the rod photoreceptor system of the adult humanretina and is absent from cones, and suggest that AIPL1performs an essential function in the maintenance of rodphotoreceptors.

RESULTS

Characterization of AIPL1 expression

A rabbit polyclonal antiserum was raised against a keyholelimpet haemocyanin (KLH)-conjugated peptide AEPATEPPP-SPGHSLQH comprising amino acid residues 368–384 of thepredicted human AIPL1 protein sequence. The polyclonalantiserum, Ab-hAIPL1, was characterized against humantissue homogenates and AIPL1 recombinant proteins. A single

protein of ∼43 kDa, corresponding to the predicted molecularweight of AIPL1 (43.865 kDa), was detected by the Ab-hAIPL1antiserum in human retinal tissue extracts under reducingconditions (Fig. 1A). The 43 kDa protein in human retinaltissue was not detected by Ab-hAIPL1 in the presence ofcompeting peptide (1 µg/ml) (Fig. 1A). The pre-immune serumshowed no reactivity with the 43 kDa protein or any otherprotein species (Fig. 1A), indicating that the reactivity againstthe 43 kDa retinal protein was specific. The Ab-hAIPL1antiserum was also able to specifically react against hetero-logously produced AIPL1 recombinant proteins (Fig. 1B).Single bands of ∼71 and 50 kDa were detected in immunoblotsof purified glutathione S-transferase (GST)-tagged AIPL1(GST-AIPL1) and polyhistidine-tagged AIPL1 (His-AIPL1),respectively, corresponding to the predicted molecular weightof each of these proteins. Ab-hAIPL1 showed no reactivityagainst any proteins of bacterial origin or GST on its own.Hence, the Ab-hAIPL1 antiserum was able to react with theAIPL1 epitope in both recombinant AIPL1 and AIPL1 inretinal extracts. The Ab-hAIPL1 antiserum was characterizedagainst a panel of eight other human tissues includingneocortex, cerebellum, spinal cord, kidney, lung, liver, testesand heart (Fig. 1C). The 43 kDa protein was only detected inhuman retina. This confirmed previous analyses using multi-tissue northern blot hybridization, which demonstrated mRNAexpression in adult human retina (22). Expression of AIPL1 inthe Y79 retinoblastoma cell line was investigated by immuno-blotting with Ab-hAIPL1, as retinoblastoma cells share manyphotoreceptor characteristics (33) (Fig. 1D). Ab-hAIPL1detected a specific band of ∼43 kDa in whole cell preparationsof Y79 cells, demonstrating that Y79 retinoblastoma cells alsoexpress AIPL1. Ab-hAIPL1 failed to detect a specific productin five other cell lines of non-retinal origin, including two celllines of human corneal origin (HCEF and EK1B) (a gift ofMr N.D.Ebenezer).

Immunocytochemistry of AIPL1 in Y79 cells

Undifferentiated Y79 retinoblastoma cells grown in suspen-sion were attached to glass slides by cytospin centrifugation,and the subcellular distribution of AIPL1 was determined byfluorescence immunocytochemistry using Ab-hAIPL1 (Fig. 2).AIPL1 was detected both in the nucleus (n) and in thecytoplasm (c) of Y79 cells, although immunostaining withAb-hAIPL1 was more intense in the cytoplasm and in somecells was perinuclear. The staining pattern throughout the Y79cells was granular in appearance. No immunostaining wasdetected either with the pre-immune serum or in the presenceof competing peptide (data not shown).

Immunohistochemistry of AIPL1 in human retina

In 10% neutral-buffered formalin-fixed, paraffin-embeddedsections of adult human eye, specific immunolabelling withAb-hAIPL1 was detected only in the retina, and was excludedfrom all other parts of the eye including the lens, cornea, sclera,choroid and optic nerve (data not shown). Within the retina,immunolabelling with Ab-hAIPL1 was detected in thephotoreceptor inner segments, the outer nuclear layer (ONL)and the outer plexiform layer (OPL) extending to the rodspherules and cone pedicles in the OPL (Fig. 3). Immuno-labelling with Ab-hAIPL1 was absent from the RPE, the inner

Human Molecular Genetics, 2002, Vol. 11, No. 7 825

nuclear layer (INL) and the inner plexiform layer (IPL)(Fig. 3). The connecting cilia of the photoreceptor innersegments appeared to be more intensely labelled with theAb-hAIPL1 antiserum. Towards the outer limit of the ONL, aclear absence of Ab-hAIPL1 immunolabelling was noticeablein the region of the cone photoreceptor cell bodies. Rodphotoreceptor cell bodies of the ONL were intensely labelledby Ab-hAIPL1, and the distribution of Ab-hAIPL1 immu-nolabelling around the rod photoreceptor nuclei appeared to beasymmetric with more intense labelling apically. The immuno-labelling of the OPL with the Ab-hAIPL1 antiserum waspunctate in appearance, with intense labelling visible in theregion of the photoreceptor spherules and pedicles. The absenceof Ab-hAIPL1 immunostain in the cone photoreceptor cell bodieswas confirmed at high titres (1:50) of Ab-hAIPL1 antiserum andlong chromophore development times (data not shown).Furthermore, immunolabelling of the central retina (macula)

with Ab-hAIPL1 demonstrated a similar distribution of AIPL1and confirmed the apparent exclusion of AIPL1 labelling fromthe cone photoreceptor cell bodies (data not shown). Noreaction product was observed when the primary antibody wasomitted or when immunolabelling was performed in the presenceof the peptide against which Ab-hAIPL1 was raised (data notshown).

Immunofluorescence confocal microscopy of AIPL1

Double immunofluorescent labelling was performed with theAb-hAIPL1 antiserum and a panel of retinal markers inparaformaldehyde-fixed agarose-embedded human retina. Inthe peripheral human retina, AIPL1 (Fig. 4, AIPL1) wasdetected in the rod photoreceptor inner segments, with intenselabelling in the region of the photoreceptor connecting cilia,but was absent from the rod photoreceptor outer segments.

Figure 1. (A) Immunoblot of human retinal proteins reacted with Ab-hAIPL1. A single band of ∼43 kDa is detected in human retina by Ab-hAIPL1 antiserum.Rabbit pre-immune serum did not detect the 43 kDa species and the band was not detected when the Ab-hAIPL1 antiserum had been pre-adsorbed by the peptideagainst which it was generated prior to immunoblotting. (B) Immunoblot of AIPL1 recombinant proteins. Ab-hAIPL1 specifically detects heterologously producedGST-AIPL1 (70 kDa) and His-AIPL1 (50 kDa). Ab-hAIPL1 showed no reactivity against GST on its own or against proteins of bacterial origin (JM109).(C) Immunoblot of proteins from a panel of human tissues using the Ab-hAIPL1 antiserum. The Ab-hAIPL1 antiserum specifically detects a protein of ∼43 kDaonly in the human retina. (D) Immunoblot of proteins from a panel of different cell lines. Ab-AIPL1 antiserum specifically detects a protein of ∼43 kDa only in theY79 retinoblastoma cell line.

Figure 2. Immunocytochemistry in Y79 retinoblastoma cells using Ab-hAIPL1 antiserum. Confocal Z section shows that immunolabelling with Ab-hAIPL1 antiserum isgranular in appearance and is predominantly cytoplasmic (c), though also present in the nucleus (n). Scale bar, 10 µm.


The rod photoreceptor cell bodies in the ONL were intenselylabelled with Ab-hAIPL1. Though Ab-hAIPL1 labelling wasnot absent from the nuclei of the rod photoreceptors, labellingwas distributed in the rod photoreceptor cell bodies in anasymmetric, perinuclear fashion. Immunolabelling with theAb-hAIPL1 antiserum was absent from the cone photoreceptorcell bodies. Punctate labelling of the OPL could be detectedwith Ab-hAIPL1, which terminated with intense labelling ofthe rod photoreceptor synaptic spherules. The distribution ofAIPL1 in the peripheral adult human retina by confocal micro-scopy thus confirmed that previously shown by immunohisto-chemistry (Fig. 3). Immunofluorescent labelling with themonoclonal antibody 1D4 detected rhodopsin in the rod

photoreceptor outer segments (Fig. 4, 1D4). The signals forAb-hAIPL1 and 1D4 did not co-localize (Fig. 4, MERGE),demonstrating the exclusion of AIPL1 from the rod photo-receptor outer segments. An enlargement of the merged image(Fig. 4, ZOOM) emphasizes the intense Ab-hAIPL1 immuno-signal detected in the connecting cilia of the rod photoreceptorcells as well as the absence of Ab-hAIPL1 signal from the rodphotoreceptor outer segments.

The lectin peanut agglutinin (PNA) was used as a marker forthe extracellular matrix (ECM) sheaths of the cone photoreceptorouter segments and cone photoreceptor inner segments (Fig. 5A,PNA). PNA labelling did not co-localize with the Ab-hAIPL1immunosignal (Fig. 5A, MERGE), demonstrating the absenceof AIPL1 from the cone inner and outer segments. The absenceof Ab-hAIPL1 immunosignal from the cone photoreceptorouter and inner segments is highlighted in an enlargement(Fig. 5A, ZOOM) of the merged image.

The monoclonal antibody 7G6 was used as a marker for conephotoreceptors extending from the cone inner segments to thecone synaptic pedicles in the OPL (Fig. 5B, 7G6). 7G6 label-ling did not co-localize with the Ab-hAIPL1 immunosignal(Fig. 5B, MERGE), demonstrating the absence of AIPL1 fromthe cone photoreceptor cells, including the cell bodies, outerand inner segments and the cone synaptic pedicles in the OPL.In an enlargement of the merged image (Fig. 5B, ZOOM), theabsence of Ab-hAIPL1 immunosignal from the cone photo-receptor inner segments, cone photoreceptor cell bodies andcone photoreceptor spherules is clearer. Collectively, theseresults demonstrate the exclusion of AIPL1 from the conephotoreceptor cells in the peripheral retina, and confine theAIPL1 distribution to the rod photoreceptors extending fromthe connecting cilia of the rod photoreceptor inner segments to thesynaptic terminals of the rod photoreceptor spherules in the OPL.In each experiment, the Ab-hAIPL1 immunosignal was absentfrom cone photoreceptor cells, even at high titres of theAb-hAIPL1 antiserum, suggesting the absence of low levels ofAIPL1 expression in cone photoreceptors. Furthermore, noAIPL1 reaction product was observed when the Ab-hAIPL1antibody was omitted or when immunolabelling wasperformed in the presence of competing peptide (data not

Figure 3. Immunohistochemistry in the peripheral region of the adult humanretina using the Ab-hAIPL1 antiserum. The section is labelled as follows: RPE,retinal pigment epithelium; OLM, outer limiting membrane; ONL, outernuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, innerplexiform layer; os, photoreceptor outer segments; cc, connecting cilia; is,photoreceptor inner segments; ccb, cone photoreceptor cell bodies; rcb, rodphotoreceptor cell bodies; sp, spherules and pedicles. Immunolabelling is found inthe photoreceptor inner segments, ONL and OPL extending to the photoreceptorsynaptic terminals (sp). Scale bar, 20 µm.

Figure 4. Double immunofluorescent confocal microscopy in the peripheral adult human retina using the Ab-hAIPL1 antiserum (AIPL1) and the monoclonalantibody 1D4 (1D4) directed against rhodopsin. The confocal Z sections are labelled as follows: ROS, rod photoreceptor outer segments; RIS, rod photoreceptorinner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; cc, connecting cilia; ccb, cone photoreceptor cell bodies; rcb, rodphotoreceptor cell bodies; s, rod photoreceptor spherules. The Ab-hAIPL1 immunolabel does not co-localize with the 1D4 marker (MERGE). AIPL1 is thus absentfrom the rod photoreceptor outer segments, as seen in an enlargement of the merged image (ZOOM). Scale bars, 10 µM.


shown). No cross-reaction was detected between the primaryand secondary antibodies of each double labelling experimentand no immunosignals were detected in the presence of eitherprimary or secondary antibodies on their own (data notshown).

Double immunofluorescent labelling was performed withthe Ab-hAIPL1 antiserum and the 7G6 monoclonal antibody inthe macula and fovea of the human retina. Within the rim of thefoveal pit (Fig. 6), the Ab-hAIPL1 immunosignal was limitedto the small population of rod photoreceptor cells in this region(white arrowheads) and did not co-localize with the 7G6immunosignal observed for the population of densely packedcone photoreceptor cells. Within the foveal pit, which containsexclusively cone photoreceptor cells, the Ab-hAIPL1 immuno-signal was absent (data not shown). These results demonstratethe exclusion of AIPL1 from the cone photoreceptor cells inthe central human retina.

DISCUSSION

Within the adult human retina, our studies show that AIPL1protein is produced exclusively in the rod photoreceptors and

is not present in the cone photoreceptors. This result isintriguing in light of the fact that LCA is thought to be theconsequence of either the impaired function, development orextremely early degeneration of both the rod and cone photo-receptor systems based on the absence of ERG recordings inLCA patients. The sustained expression of AIPL1 in adultphotoreceptors suggests that AIPL1 performs an essentialfunction in the maintenance of the rod photoreceptors in theadult human retina. It is possible that AIPL1 is expressed inboth rod and cone photoreceptor precursors and other celltypes during eye development. The early degeneration ordysfunction of both rod and cone photoreceptor progenitorcells in the absence of functional AIPL1 may explain why thecone cells fail to function in AIPL1-LCA. Alternatively,AIPL1 may be expressed exclusively in developing rodphotoreceptor cells as well as in the adult retina. In thisscenario, an extremely early degeneration of rod photoreceptorprecursors due to the absence of functional AIPL1 could leadto the dysfunction or degeneration of cone photoreceptors. Theimportance of rod derived cone survival factors in retinaldegeneration is well established (34), although the effects ofsuch early rod cell loss upon cone cell survival are unknown.

Figure 5. (A) Double immunofluorescent confocal microscopy in the peripheral adult human retina using the Ab-hAIPL1 antiserum (AIPL1) and peanut agglutinin(PNA). The confocal Z sections are labelled as follows: ROS, rod photoreceptor outer segments; RIS, rod photoreceptor inner segments; COS, cone photoreceptorouter segments; CIS, cone photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; cc, connecting cilium;ccb, cone photoreceptor cell body; rcb, rod photoreceptor cell body; s, rod photoreceptor spherules; p, cone photoreceptor pedicles. The Ab-hAIPL1 immunolabeldoes not co-localize with the PNA marker (MERGE). AIPL1 is absent from the COS and CIS, as seen in the enlargement of the merged image (ZOOM). Scalebars, 10 µm. (B) Double immunofluorescent confocal microscopy in the peripheral adult human retina using the Ab-hAIPL1 antiserum (AIPL1) and the mono-clonal antibody 7G6 against cones (7G6). The confocal Z sections are labelled as in (A). The Ab-hAIPL1 immunolabel does not co-localize with the 7G6 marker(MERGE). AIPL1 is thus absent from the cone photoreceptor cells including the COS, CIS, ccb and p, as shown in an enlargement of the merged image (Fig. 6,ZOOM). The Ab-hAIPL1 antiserum exclusively labels the rod photoreceptors from the inner segment connecting cilia to the rod photoreceptor spherules. Scale bars,10 µm.


Another possibility is that the loss of AIPL1 function in rodprecursors may result in an inhibition of the final maturation ofcone precursors due to the absence of an inductive or permissivesignal (34). The determination of the spatial and temporalexpression of AIPL1 in the developing human retina, therefore,will be fundamental in clarifying the pathogenesis of LCAcaused by mutations in AIPL1.

The proteins encoded by the different LCA causative geneseach participate in distinct and essential physiopathologicalpathways in the retina, including phototransduction (retGC-1),retinoid metabolism (RPE65), photoreceptor development (CRX),cell polarity (CRB1) and intracellular transport (RPGRIP1).Attempts have been made to correlate the genetic hetero-geneity of LCA with clinically heterogeneous features (10).For example, mutations in the retGC-1 gene are responsible forLCA characterized by congenital severe cone–rod dystrophy,whilst mutations in the RPE65 gene cause LCA characterizedby congenital severe but progressive rod–cone dystrophy andCRX mutations are associated with LCA characterized bycone–rod dystrophy (10). The exclusive localization of AIPL1to rod photoreceptors suggests that mutations in AIPL1 shouldresult in LCA characterized by congenital severe rod–conedystrophy. In addition to the heterogeneity in LCA, the LCAcausative genes also display a degree of allelism with somemutations resulting in clinical physiopathological diagnosesdistinct from LCA. Heterozygous mutations in exon 13 of theretGC-1 gene result in progressive autosomal dominant cone–roddystrophy (CORD6) (35–37). Similarly, a four amino acid deletionmutation (P351∆12) in the C-terminal polyproline-rich regionof AIPL1 has been identified in two probands given the clinicaldiagnosis of autosomal dominant cone–rod dystrophy andjuvenile RP, respectively (38), though all other AIPL1 mutationsare associated with LCA. This diagnosis is surprising in thelight of our findings, as we would predict that dominant mutations

in AIPL1 should result in rod–cone dystrophy. Fundoscopicand electrodiagnostic analyses of dominant AIPL1 cases andheterozygous ‘carriers’ of AIPL1 mutations combined withcellular and molecular studies of AIPL1 function may clarifythe role of AIPL1 in rod–cone and cone–rod dystrophies.

The epitope for our antiserum Ab-hAIPL1 resides in the56 amino acid C-terminal polyproline-rich region of thepredicted human AIPL1 sequence. A comparative analysis ofthe AIPL1 gene in different species demonstrated that the poly-proline-rich region is conserved only in primates (human,chimpanzee, baboon, rhesus monkey and squirrel monkey) andis absent in the mouse, rat and cow (39). Ab-hAIPL1, directedagainst this C-terminal extension, does not cross-react withAIPL1 in these species (data not shown) confirming theapparent primate specificity of this region. The results of ourstudy are based on the expression of this C-terminal domainand we cannot exclude the potential expression of an AIPL1variant lacking the polyproline-rich region, and thus our anti-body epitope, in cone photoreceptors. However, the poly-proline-rich region is encoded by exon 6 of the AIPL1 gene,which also encodes the third TPR motif (22), and it is thusunlikely that alternative splicing produces an AIPL1 isoformlacking the polyproline-rich region. Furthermore, it is evidentthat the polyproline-rich region performs an important if notessential function in normal primate vision, arguing against theexistence of an AIPL1 variant lacking the polyproline-richregion. Three mutations (R302L, A336∆2 and P376S) havebeen identified in the AIPL1 polyproline-rich region, whichcause the severe phenotype of LCA (22,38). In addition, anLCA-causing homozygous nonsense mutation at codon 278 ofAIPL1 (W278X), if expressed, truncates the C-terminal 107 aminoacids of AIPL1, including the entire polyproline-rich region(22,38) and once again emphasizing the importance of this regionin normal primate vision. The observed rod cell exclusivity ofAIPL1 expression in our study could be a consequence ofcone-specific epitope masking. We feel that this is highlyunlikely, as we have observed the same pattern of expressionunder a wide range of tissue fixations and antigen retrievalprocedures. Such a cell-specific epitope masking would,however, still indicate fundamental differences in AIPL1 functionbetween rods and cones.

There is a higher propensity for LCA patients to developkeratoconus, a chronic non-inflammatory corneal thinningdisorder. There is evidence for a genetic role in the etiology ofkeratoconus (40,41) and it has been demonstrated that AIPL1is a locus for the combined phenotype of LCA and kerato-conus, which cosegregate in an autosomal recessive manner(42,43). However, in a study of 12 LCA patients from fourconsanguineous pedigrees, there was a striking phenotypicvariability with respect to keratoconus even though theW278X mutation in AIPL1 was the underlying cause of LCAin each affected subject (43). Three of the affected subjects haddefinite keratoconus, and two were suspected of developingkeratoconus based on mild cone formation in the cornea of atleast one eye (43). Our results demonstrate that AIPL1 proteinis expressed only in immortalized cells of retinal origin and inthe retina of the adult human eye. AIPL1 is not expressedeither in immortalized cells of corneal origin or in the cornea ofthe adult human eye. Therefore, there is no simple relationshipbetween AIPL1 expression and the development of kerato-conus. Mutations in RetGC1 have also been associated with

Figure 6. Double immunofluorescent confocal microscopy in the foveal pit rim ofadult human retina using the Ab-hAIPL1 antiserum (AIPL1) and the monoclonalantibody 7G6. The confocal Z section is labelled as follows: COS, conephotoreceptor outer segments; CIS, cone photoreceptor inner segments; ONL,outer nuclear layer; OPL, outer plexiform layer. The Ab-hAIPL1 immunosignalis shown by white arrowheads, and is absent from the densely packed conephotoreceptor cell population of the foveal pit rim. Scale bar, 10 µm.


LCA and keratoconus with no obvious mechanistic linkbetween the photoreceptor protein and corneal disease (44). Itis possible that the combined phenotype of LCA and anteriorkeratoconus may arise due to unknown developmental effects ofAIPL1 functional ablation or combined genetic and environmentalfactors such as repeated eye rubbing (45).

The role of AIPL1 within the rod photoreceptors of the retinahas yet to be established. However, it is interesting that withinthe highly polarized photoreceptor cell, AIPL1 is excludedfrom the rod photoreceptor outer segments and otherwiseextends throughout the cell from the inner segments to the rodspherules. The similarity of AIPL1 to AIP as well as the inclu-sion of the TPR motifs in the AIPL1 sequence suggests thatAIPL1 may participate in protein complex formation and/ortranslocation within the rod photoreceptor cells of the retina.Furthermore, proline-rich regions similar to those found inAIPL1 mediate protein interactions in which the participantsare involved in cellular processes that require the rapid recruit-ment or interchange of proteins, including cytoskeletal rear-rangements, signal transduction and transcriptional initiation(46). Hence, AIPL1 may well function in protein complexformation and translocation in the rod photoreceptors of thehuman retina, and it is important to identify the partner proteinsthat interact with AIPL1.

In conclusion, this study is the first to demonstrate thatAIPL1, a product of a gene that causes Leber congenitalamaurosis, is expressed exclusively in the rod photoreceptorsof the adult human retina. This result will impact on futurestudies of AIPL1, including the development of appropriateanimal models and potential therapies for LCA caused bymutations in AIPL1. Further evaluation of the expression ofAIPL1 in the developing human retina and the function ofAIPL1 within the rod photoreceptors will provide insight intothe biology of normal vision and the pathology of LCA.

MATERIALS AND METHODS

Antibody, SDS–PAGE and immunoblotting

Rabbit polyclonal antiserum, Ab-hAIPL1, was raised againstpeptide AEPATEPPPSPGHSLQH (amino acid residues 368–384)conjugated to KLH (Genosys Biotechnologies, Cambridge,UK). Total protein extracts were prepared by homogenizationof tissues or cells in sample buffer (0.0625 M Tris–HCl, 2.5%SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.25% bromophenolblue). Total protein concentrations in tissue and cell extracts wereassayed using the Bradford microassay (Bio-Rad Laboratories,Hercules, CA) to ensure even loading on gels. Protein sampleswere resolved by SDS–PAGE on 12% gels and electroblottedonto ECL nitrocellulose (Amersham, Little Chalfont, UK). Thenitrocellulose membrane was incubated overnight in blockingsolution [5% Marvel non-fat milk powder, 1× phosphate-bufferedsaline (PBS), 0.1% Tween-20], following which blots werehybridized with rabbit pre-immune serum or Ab-hAIPL1(1:2000) for 1 h at room temperature. In competition assays,peptide (1 µg/ml) against which the antiserum was generatedwas incubated with the Ab-hAIPL1 antiserum (1:2000) for 30 minon ice prior to incubation with the nitrocellulose membrane for1 h at room temperature. Subsequently, the membrane wasincubated with mouse anti-rabbit secondary antibody (1:20000)conjugated to horseradish peroxidase (Pierce, Rockford, IL)

for 1 h at room temperature. Bands were visualized usingenhanced chemiluminescence (Amersham).

Retinoblastoma cell culture and immunocytochemistry

Y79 human retinoblastoma cells (47) (a gift of Dr D.Trump,University of Cambridge, UK) were maintained in suspensionculture in RPMI 1640 medium (Gibco BRL, Paisley, UK)supplemented with 15% fetal bovine serum, 2 mM L-glutamineand 50 µg/ml gentamycin (Sigma, Poole, UK) with mediumchanges every 2–3 days. For western analysis of endogenousAIPL1, Y79 cells were washed three times with ice cold PBSbefore whole cell homogenization in sample buffer. For immuno-cytochemistry, Y79 cells were washed three times in ice coldPBS and fixed in 3.7% formaldehyde (TAAB Laboratories,Reading, UK) in PBS for 15 min at room temperature. TheY79 retinoblastoma cells were fixed to glass slides by cytospincentrifugation, permeabilized in 0.1% Triton X-100 (Sigma) inPBS for 5 min at room temperature and blocked with 3%bovine serum albumin (BSA) (Sigma) and 10% normal swineserum (NSS) (DAKO Ltd, Ely, UK) in PBS for 30 min at roomtemperature. Cells were subsequently incubated in rabbitpre-immune serum or Ab-hAIPL1 antiserum (1:250) inblocking solution for 1 h at room temperature, followed byswine anti-rabbit secondary antibody conjugated to the TRITCfluorophore (DAKO) in blocking solution for a further 1 h atroom temperature. 4′6-diamidino-2-phenylindole (DAPI)(Sigma) was included in the final PBS wash before the cellswere mounted in fluorescent mounting medium containing15 mM sodium azide (DAKO) and visualized with a Zeisslaser scanning confocal microscope.

Immunohistochemistry

Adult human retinae were fixed with 10% neutral-bufferedformalin within 2 min of enucleation for at least 24 h. Theretinae were dehydrated with increasing concentrations ofindustrial methylated spirits (IMS), equilibrated in xylene andembedded in paraffin wax. Retinal sections were cut with amicrotome at a setting of 8 µm, floated out onto 20% methanol,expanded on water pre-heated to 40°C and mounted on glassslides treated with 2% aminopropyltriethoxysilane (APES)(Sigma) in acetone. The retinal sections were deparaffinedsuccessively with xylene, 100 and 95% IMS before proceedingwith the immunohistochemistry. Endogenous peroxidaseactivity was blocked by incubating the retinal sections with0.5% hydrogen peroxide (Sigma) in methanol for 30 min atroom temperature. Antigen retrieval was accomplished bymicrowaving the retinal sections four times for 2.5 min each at800 W in Tris-buffered saline (TBS) (100 mM Tris–HCl,150 mM NaCl, pH 7.6) containing 5% urea. The retinalsections were blocked with 10% NSS (DAKO) and 2% BSA inTBS for 45 min at room temperature, incubated withAb-hAIPL1 antiserum (1:500 or 1:1000) in TBS containing0.1% BSA overnight at 4°C, and finally with biotin-conjugatedswine anti-rabbit IgG (1:300) (DAKO) in TBS containing0.1% BSA for 45 min at room temperature. The immuno-reaction was visualized with streptavidin biotinylated horseradishperoxidase complex (DAKO) and 3′,3′-diaminobenzidine(DAB) (Sigma), 0.03% hydrogen peroxide in TBS. The retinalsections were successively dehydrated with 70, 95 and 100%IMS, clarified with three successive changes of xylene and


mounted in dibutyl phthalate xylene (DPX) mounting medium(Merck, Lutterworth, UK). To verify the specificity of theimmunostaining, retinal sections were also stained with rabbitpre-immune serum and Ab-hAIPL1 antiserum pre-absorbedwith the peptide epitope (30 µg/µl). The retinal sections werevisualized with an Olympus BX50 light microscope using brightfield and differential interference contrast (Nomarski) optics.Photography was done using an integral Olympus DP10 digitalcamera.

Fluorescence scanning confocal microscopy

Adult human retinae were fixed with freshly prepared 4% para-formaldehyde in isotonic PBS pH 7.3 within 2 min of enucleationfor at least 2 h. The retinae were washed extensively withchilled PBS, embedded in 5% low melting temperature agaroseand cut with a vibratome at a setting of 100 µm. The retinalsections were blocked with 5% normal donkey serum (NDS)(Jackson ImmunoResearch Laboratories, West Grove, PA) and0.5% BSA in PBS overnight at 4°C and double labelled withAb-hAIPL1 antiserum (1:1000) in the presence of a secondretinal marker. Rhodopsin, a marker for rod photoreceptorouter segments, was detected with the mouse monoclonal anti-body 1D4 (National Cell Culture Centre, USA) (1:200). Conephotoreceptor outer and inner segment ECM oligosaccharideswere detected with PNA (1:200) (Vector laboratories, Burlin-game, CA). Cone photoreceptors were detected with the mousemonoclonal antibody 7G6 (1:100) (a generous gift ofDr P.MacLeish, Morehouse School of Medicine NeuroscienceInstitute, USA). Secondary antibody incubations were performedwith CY3-conjugated donkey anti-rabbit (Molecular Probes,Eugene, OR), CY2-conjugated donkey anti-mouse (MolecularProbes) and CY2-conjugated streptavidin (Molecular Probes).All antibody incubations were performed in block at 4°C over-night. DAPI was included in the final PBS wash and the retinalsections were mounted in fluorescent mounting mediumcontaining 15 mM sodium azide (DAKO) and visualized witha Zeiss laser scanning confocal microscope. Retinal sectionswere stained with rabbit pre-immune serum and Ab-hAIPL1antiserum pre-adsorbed with the peptide epitope (30 µg/ml) toverify the specificity of the immunostain. Retinal sections werealso incubated with either the primary or secondary antibodieson their own to confirm that the signal observed did not arisenon-specifically from any of the antibodies per se. In addition,rabbit primary antibodies were incubated with mousesecondary antibodies and vice versa to verify that immunosig-nals did not arise from cross-reaction between the antibodies inthe double label procedure.

ACKNOWLEDGEMENTS

We are grateful to Dr D.Trump for the provision of Y79retinoblastoma cells and Mr N.D.Ebenezer for the provision ofHCEF and EK1B cells. We would like to thank Dr P.MacLeishfor provision of the 7G6 cone marker. We are very grateful toDr V.Pearson for assistance in immunohistochemicaltechnique, and would also like to thank Mr R.Alexander andMiss R.Hall. We are grateful to the Brain Bank, Department ofNeuropathology, Institute of Psychiatry, for the provision ofhuman tissue samples. We are grateful for financial support

from the Wellcome Trust (J.v.d.S. is the recipient of aWellcome Trust Travelling Research Fellowship).

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the leber congenital amaurosis gene product aipl1 is localized

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