Implications of retinal effects observed in chronic toxicity studies on the clinical development of a CNS-active drug candidate.

The development path described for JNJ-26489112 provides perspectives on interpretation of retinal effects observed in nonclinical studies and their implications for clinical development. JNJ-26489112 is a CNS-active investigational drug that has potential as a novel treatment for treatment-resistant and bipolar depression, epilepsy, and neuropathic/inflammatory pain. In a 6-month toxicity study in albino rats, retinal atrophy was observed at supratherapeutic exposures to JNJ-26489112. The histopathological changes and topography of the lesions were characteristic of light-induced damage specific to albino rats. The species/strain specificity is supported by an absence of any ocular effects in dogs and in pigmented and albino rats, housed under standard and reduced lighting, respectively. To further evaluate its potential to cause ocular effects, in vivo functional and structural ocular analyses were included in a 9-month monkey toxicity study. Reductions in rod- and cone-mediated electroretinograms were observed at supratherapeutic exposures but without any histopathologic changes. These data suggested that the effects of JNJ-26489112 in monkeys were neuromodulatory and not neurotoxic. Taken together, data related to the light-induced atrophy in albino rats and reversible neuromodulatory effects in monkeys, supported the safe evaluation of JNJ-26489112 in a clinical proof-of-concept study that included comprehensive functional and structural ocular monitoring.

Gene therapy restores vision in a canine model of childhood blindness.

The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.

rAAV2/5 gene-targeting to rods:dose-dependent efficiency and complications associated with different promoters.

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken ß actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 10(11)vg ml(-1) were used.

Identification of genetic variation and haplotype structure of the canine ABCA4 gene for retinal disease association studies.

Over 200 mutations in the retina specific member of the ATP-binding cassette transporter superfamily (ABCA4) have been associated with a diverse group of human retinal diseases. The disease mechanisms, and genotype-phenotype associations, nonetheless, remain elusive in many cases. As orthologous genes are commonly mutated in canine models of human blinding disorders, canine ABCA4 appears to be an ideal candidate gene to identify and study sequence changes in dogs affected by various forms of inherited retinal degeneration. However, the size of the gene and lack of haplotype assignment significantly limit targeted association and/or linkage approaches. This study assessed the naturally observed sequence diversity of ABCA4 in the dog, identifying 80% of novel variations. While none of the observed polymorphisms have been associated with blinding disorders to date, breed and potentially disease specific haplotypes have been identified. Moreover, a tag SNP map of 17 (15) markers has been established that accurately predicts common ABCA4 haplotypes (frequency > 5%) explaining >85% (>80%) of the observed genetic diversity and will considerably advance future studies. Our sequence analysis of the complete canine ABCA4 coding region will clearly provide a baseline and tools for future association studies and comparative genomics to further delineate the role of ABCA4 in canine blinding disorders.

Comparative genomic mapping of uncharacterized canine retinal ESTs to identify novel candidate genes for hereditary retinal disorders.

PURPOSE: To identify the genomic location of previously uncharacterized canine retina-expressed expressed sequence tags (ESTs), and thus identify potential candidate genes for heritable retinal disorders. METHODS: A set of over 500 retinal canine ESTs were mapped onto the canine genome using the RHDF(5000-2) radiation hybrid (RH) panel, and the resulting map positions were compared to their respective localization in the CanFam2 assembly of the canine genome sequence. RESULTS: Unique map positions could be assigned for 99% of the mapped clones, of which only 29% showed significant homology to known RefSeq sequences. A comparison between RH map and sequence assembly indicated some areas of discrepancy. Retinal expressed genes were not concentrated in particular areas of the canine genome, and also were located on the canine Y chromosome (CFAY). Several of the EST clones were located within areas of conserved synteny to human retinal disease loci. CONCLUSIONS: RH mapping of canine retinal ESTs provides insight into the location of potential candidate genes for hereditary retinal disorders, and, by comparison with the assembled canine genome sequence, highlights inconsistencies with the current assembly. Regions of conserved synteny between the canine and the human genomes allow this information to be extrapolated to identify potential positional candidate genes for mapped human retinal disorders. Furthermore, these ESTs can help identify novel or uncharacterized genes of significance for better understanding of retinal morphology, physiology, and pathology.

Targeting gene expression to cones with human cone opsin promoters in recombinant AAV.

Specific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter.

A second-generation genetic linkage map of the domestic dog, Canis familiaris.

Purebred strains, pronounced phenotypic variation, and a high incidence of heritable disease make the domestic dog uniquely suited to complement genetic analyses in humans and mice. A comprehensive genetic linkage map would afford many opportunities in dogs, ranging from the positional cloning of disease genes to the dissection of quantitative differences in size, shape, and behavior. Here we report a canine linkage map with the number of mapped loci expanded to 276 and 10-cM coverage extended to 75-90% of the genome. Most of the 38 canine autosomes are likely represented in the collection of 39 autosomal linkage groups. Eight markers were sufficiently informative to detect linkage at distances of 10-13 cM, yet remained unlinked to any other marker. Taken together, the results suggested a genome size of about 27 M. As in other species, the genetic length varied between sexes, with the female autosomal distance being approximately 1.4-fold greater than that of male meioses. Fifteen markers anchored well-described genes on the map, thereby serving as landmarks for comparative mapping in dogs. We discuss the utility of the current map and outline steps necessary for future map improvement.

Cloning and characterization of the cDNA and gene encoding the gamma-subunit of cGMP-phosphodiesterase in canine retinal rod photoreceptor cells.

Rod photoreceptor cyclic GMP-phosphodiesterase (cGMP-PDE) is one of the key enzymes of the visual phototransduction cascade in the vertebrate retina. The enzyme is composed of alpha- and beta-catalytic subunits and two identical inhibitory gamma-subunits. A defect in any of the subunits may potentially alter the activity of the enzyme, leading to aberration in the visual phototransduction. We have cloned and sequenced both the cDNA and gene for the canine gamma-subunit of cGMP-PDE (PDE gamma). The 952-bp cDNA has a coding region of 261 bp which is very similar to those of the PDE gamma cDNAs from human, mouse and bovine retinas. Among the 87 amino acids encoded by the transcribed region, differences in only three residues located within the first 17 amino acids were identified. The carboxyl terminus of PDE gamma, involved in interaction with the catalytic subunits of cGMP-PDE and the alpha-subunit of transducin, is conserved through evolution. The single polyadenylation signal (AATAAA) present in human and bovine PDE gamma cDNAs is replaced by AGTAAA in the canine sequence. The canine gene (2.8 kb) consists of four exons and is much smaller than the human gene (6 kb). The larger size of the human gene is primarily due to the presence of AluI repetitive elements in its first two introns.

TIMP-1 expression is increased in X-linked progressive retinal atrophy despite its exclusion as a candidate gene.

X-linked progressive retinal atrophy (XLPRA) is the only known natural animal model for X-linked retinitis pigmentosa (XLRP), a blinding disorder in man. The tissue inhibitor metalloproteinase 1 gene (TIMP-1), present in close proximity to one of the two XLRP loci, was tested as a candidate for XLPRA, by first characterizing the cDNA and gene from a normal dog. The cloned canine TIMP-1 cDNA is predicted to encode a protein of 207 amino acids with 66-83% identity in the deduced aa sequence with homologous mammalian genes. No sequence difference in the coding sequence of TIMP-1 was observed between normal and XLPRA-affected dogs. TIMP-1 was found to be expressed in all of the canine tissues examined by reverse transcription and polymerase chain reaction. The canine TIMP-1 spans 3.5kb and is interrupted by five introns with sizes comparable to those observed in the human and mouse homologues of the gene. The proximal promoter region of canine TIMP-1 contains sequence motifs shown to have regulatory significance in transcription of human TIMP-1. Linkage analysis between XLPRA and TIMP-1 using a newly identified intragenic polymorphism identified recombinants, which conclusively excluded the gene as a candidate for the disease. TIMP-1 is overexpressed several months before retinal degeneration is histologically evident in XLPRA dogs, implying that alterations in interphotoreceptor matrix composition precede retinal degeneration by a significant time period.

Molecular cloning, characterization and expression of a novel retinal clusterin-like protein cDNA.

A novel gene expressed predominantly in retina, but detected at a conspicuously lower level in retina of canine progressive rod cone degeneration (prcd), has been identified by suppression subtractive hybridization and retinal cDNA library screening. The characterized region of cDNA of the novel gene includes 1017 nucleotides of coding sequence predicted to encode a protein of 338 amino acids (M(r) 39389), 791 nucleotides of 5'-untranslated region (UTR), and 300 nucleotides of 3'-UTR including the poly(A)(+) tail. Multiple transcripts were detected in retina by Northern blot analysis, and a lower level of expression was observed in brain and liver by RT-PCR. The transcript appears to be developmentally regulated with a burst in gene expression at a time period (34 postnatal days) that coincides with the photoreceptor differentiation phase of retinal development. The deduced amino acid sequence from the cDNA of the novel gene has 24% identity and 48% similarity with the multifunctional glycoprotein clusterin. Hence, the putative gene product from the novel transcript has been named clusterin-like protein 1 (CLUL1). The human homologue of CLUL1 cDNA has 84 and 70% identity at the level of nucleotides and amino acids, respectively, with the characterized canine cDNA. The presence of a stretch of 128 amino acids in the putative human CLUL1, not detected in canine CLUL1, suggests alternate splicing events. An STS database search revealed that the human homologue of CLUL1 maps to chromosome 18p, a location not yet reported to harbor an RP locus. Tissue-specific expression of CLUL1 in retina, and its lower abundance in different forms of PRA suggest that this novel gene may represent an as-yet unidentified locus for a retinal disorder.

Characterization of canine photoreceptor phosducin cDNA and identification of a sequence variant in dogs with photoreceptor dysplasia.

Photoreceptor dysplasia (pd) is an autosomal recessive disease of miniature schnauzer dogs causing retinal degeneration. The disease is a homologue of retinitis pigmentosa, a group of genetically heterogeneous diseases, causing blindness in humans. A subtraction library was prepared from retinas of pd affected and age-matched normal control dogs to isolate de novo candidate genes for further examination. From the subtraction library, cDNA for phosducin (PDC), a member of the phototransduction pathway, was isolated as a transcript expressed at a higher level in the affected retina. First, the normal canine PDC cDNA was characterized to evaluate the PDC gene in the pd-affected retina. The characterized region of normal PDC cDNA spans 1258 nucleotides (nt) that include 738 nt of coding sequence predicted to encode a protein (Mr=28 209) of 245 amino acids (aa). Over the coding region, PDC shares 86-95% nt sequence identity and 90-95% identity in the deduced aa sequence with homologous mammalian sequences. A major transcript (1.9 kb) was observed only in retina by Northern analysis, but low levels of transcript were detected in brain, liver and kidney by reverse transcription and polymerase chain reaction. Retinal immunocytochemistry showed that PDC was detected only in rod photoreceptors, mainly in the inner segment and perinuclear region. By Northern blot analysis, increased PDC expression was observed in pre-degenerate affected retina relative to the age-matched normal. In pd- affected miniature schnauzer pedigree, a missense mutation was detected in codon 82 (CGA to GGA) that would create a non-conservative substitution (Arg to Gly) in close vicinity to the residue (Glu 85) which directly interacts with the betagamma-subunits of transducin. Only pd-affected dogs were found to be homozygous for the mutant allele, and none among 48 dogs tested from 20 other dog breeds had this allele, suggesting that the mutation could be causally associated with pd in miniature schnauzers. However, since some affected dogs are heterozygous for the mutant allele, and some are homozygous for the wild-type allele, this putative PDC missense mutation, if it is indeed a disease causing mutation, does not account entirely for the genetics of inherited retinal degeneration in the miniature schnauzer breed.

Structure and analysis of the transducin beta3-subunit gene, a candidate for inherited cone degeneration (cd) in the dog.

The cDNA for the beta3-subunit of cone-specific transducin (Tbeta3) was cloned and characterized from wild type dogs, and used in linkage studies as a candidate gene for cone degeneration. Sequence analysis of the Tbeta3 cDNA revealed an open reading frame of 1020 bp, potentially coding for a protein of 340 amino acids (aa). The deduced aa sequence of canine Tbeta3 shares 97% identity with the previously identified human Tbeta3, and 82% identity with bovine rod-specific transducin (Tbeta1). RT-PCR and sequencing of the amplified products demonstrated that the retinal canine Tbeta3 gene is expressed in two different transcripts which can be generated by alternative splicing of the intron in the 3'-untranslated region (UTR). The short and the long mRNAs differ in the length of their 3'-UTR by 456 nt. We have also determined the genomic organization of the canine Tbeta3 gene; it consists of ten exons and the first exon is in the 5'-UTR. The cDNA encoding Tbeta3 from cd-affected dogs was also cloned and sequenced. We found no differences at the nucleotide level between the cDNAs isolated from normal and diseased retinas. The level of transcription of Tbeta3 mRNA in the cd dog retina appeared to be normal. Linkage analysis of a crossbred informative pedigree showed five obligate recombinants out of nine informative offspring. These results suggest that Tbeta3 is not a candidate gene for the cone degeneration of the cd mutant.

Molecular characterization and mapping of canine cGMP-phosphodiesterase delta subunit (PDE6D).

cGMP-phosphodiesterase (PDE) is composed of two catalytic (alpha and beta) and two identical inhibitory (gamma) subunits. The human gene (PDE6D) encoding a new subunit (delta) has been characterized and mapped to the long arm of chromosome 2 (HSA2q35-q36) where a new autosomal recessive retinitis pigmentosa (arRP) locus (RP26) has been localized. Characterization of the canine PDE6D shows the gene is about 4.2kb containing four exons interrupted by three introns; the size of the cDNA is 1059bp with an open reading frame (ORF) of 453bp. A single transcript of identical size (1.43kb) was detected in all tissues examined (liver, lung, spleen, kidney, heart, brain and retina), with the highest abundance in the retina. Canine PDE6D has been localized to canine radiation hybrid group 14-a, which extends conserved synteny between the dog, human chromosome 2q and mouse chromosome 1. The characterization of the canine PDE6D gene and its mapping provide important information for testing causal association of the gene with canine retinal degenerations, in particular rod-cone dysplasia 2 (rcd2) in collie dogs. This disease is characterized by abnormal retinal cGMP metabolism due to a deficiency in cGMP-PDE activity, yet the alpha, beta and gamma subunits of PDE have been excluded as candidate gene loci.

Rod and cone specific domains in the interphotoreceptor matrix.

The insoluble matrix domain of the interphotoreceptor matrix (IPM) from normal dog, cat, and mouse retinae were characterized using lectin cytochemistry. The lectins WGA (wheat germ agglutinin) and PNA (peanut agglutinin) were used to label interphotoreceptor matrix microdomains in cryosections of retinal tissue and in extracted insoluble matrix. Retinal cryosections and extracted matrix were examined by epifluorescence microscopy and scanning confocal laser microscopy, the latter allowed for the removal of all background fluorescence and gave increased resolution. The insoluble matrix was extracted as a continuous sheet that was comprised of two photoreceptor-specific matrix domains distinguished both by the size of the domains, and by differential binding of WGA and PNA lectins. Each domain encloses a photoreceptor inner and outer segment. Individual rod-associated domains were connected into a hexagonal lattice and this pattern was regularly interrupted by the larger cone-associated domains which have 8-10 surrounding rod domains. The PNA lectin primarily labeled the cone-associated matrix with faint binding to the rod matrix; the WGA lectin labeled both the rod- and cone-associated matrix.

Redistribution of insoluble interphotoreceptor matrix components during photoreceptor differentiation in the mouse retina.

The development of the nervous system is largely influenced by the extracellular matrix (ECM). In the neural retina, the photoreceptors are surrounded by a unique ECM, the interphotoreceptor matrix (IPM). The IPM plays a central and possibly crucial role in the development, maintenance and specific function of the photoreceptors. Therefore, the characterization of IPM components is necessary to understand the mechanisms regulating photoreceptor differentiation. The IPM in the mouse retina was examined during photoreceptor morphogenesis with the monoclonal antibody (MAb) F22, which recognizes a 250 kDa component of the interphotoreceptor matrix. The binding pattern of MAb F22 revealed a striking redistribution in the expression of the 250 kDa F22 antigen in late stage of postnatal photoreceptor differentiation in the mouse retina. The F22 staining was detectable in the IPM around the inner segments on the third postnatal day (P3). The MAb F22 initially labeled the region around inner segments, but as the outer segments elongated, the F22 distribution became concentrated to the matrix around the rod and cone outer segments until P16-17. At P17, the F22 label around rods began to disappear, while the label around cones became more defined. The shift in label distribution was largely completed by P20. Residual rod-associated label disappeared within a few days. In the adult animal, the F22 antibody labeled the cone-associated matrix only, and this labeling pattern remained stationary. The change in the distribution of MAb F22 demonstrated by immunolabeling was not accompanied by changes in the size of the molecule; F22 antigen isolated from the IPM of P13-15, and from adult IPM migrated with the same molecular weight on SDS gels. The distribution of MAb F22 was compared to that of chondroitin sulfate proteoglycans which are abundant in the IPM. The labeling patterns of MAbs CS-56, C6-S and C4-S were distinct from that of MAb F22. A general decrease of the label intensity was seen with two chondroitin sulfate MAbs (CS-56 and C4-S) between 16 days and 4 months, but a total loss of rod-associated label was not observed. All three chondroitin sulfate MAbs labeled the retina at embryonic day (E) 11.5-13.5, a time of outgrowth of ganglion cell axons, but the F22 antigen was not detected in the retina at this stage of development. The results demonstrate that the F22 and the chondroitin sulfate antibodies are recognizing different molecules that have distinct roles in retinal morphogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Reductions in taurine secondary to photoreceptor loss in Irish setters with rod-cone dysplasia.

These studies show that onset of photoreceptor cell degeneration preceded the loss of taurine in retinas of Irish setters with rod-cone dysplasia. The numbers of photoreceptor cell nuclei were within the normal adult range in affected setters at 10 through 26 days of age but declined rapidly between 26 and 45 days and more gradually thereafter; their numbers became reduced to 50% of normal at 45 days and then to 12-20% and 3-10% of normal at 192 and 346 days, respectively. Taurine concentrations increased within the photoreceptor cell layer during normal development to peak values (50 mM) at a time (45 days of age) corresponding to the development of adult photoreceptor function. In the affected setters, taurine levels increased as in the normal until 26 days of age and then remained at that value until 40-50% of the photoreceptor cells had degenerated. Thereafter, taurine levels declined gradually throughout the period of photoreceptor cell degeneration and were reduced to 30-40% of the normal adult level at the time (346 days) when the thickness of the outer nuclear layer was reduced to less than one complete row of nuclei. These observations agree with findings in retinal degeneration (rd) mice and RCS rats and indicate that in all three of these animal models of hereditary retinal degenerations, reductions in retinal taurine levels occur secondary to the loss of photoreceptor cells.

Nonallelism of erd and prcd and exclusion of the canine RDS/peripherin gene as a candidate for both retinal degeneration loci.

PURPOSE: To determine whether early retinal degeneration (erd) and progressive rod cone degeneration (prcd), two canine hereditary retinal degenerations, are caused by allelic mutations; to determine the cDNA sequence of the canine RDS/peripherin homolog (CFRDSP); and to test whether mutations(s) in CFRDSP cause(s) either erd or prcd. METHODS: Three erd-affected dogs were crossbred to three prcd-affected dogs, and their progeny were tested by electroretinography and retinal morphology for evidence of retinal degeneration. Canine RDS/peripherin cDNA was cloned and sequenced after reverse-transcription-polymerase chain reaction (RT-PCR) of total retinal RNA. A set of overlapping fragments of CFRDSP cDNA amplified from normal and prcd-affected retinal RNA was examined by double-stranded conformational polymorphism analysis for evidence of any mutation in prcd-affected dogs. RDS/peripherin-specific restriction fragment length polymorphism (RFLP) allelic differences within informative prcd and erd pedigrees were sought by digestion of amplified regions of the CFRDSP gene with different restriction enzymes. A Hinf I RFLP was identified with alleles segregating in a set of prcd and erd informative pedigrees. Linkage of CFRDSP to either prcd or erd was tested using the LINKAGE analysis package. RESULTS: All progeny from the erd x prcd cross were phenotypically normal at ages beyond the age of diagnosis for both parental disorders. The sequence of CFRDSP cDNA is reported (Genbank accession U27349). It is, overall, 79% identical at the nucleotide level with the corresponding human sequence. The coding region shares 89% and 93% nucleotide identity with the corresponding human and feline sequences, respectively. No mutation has been identified in the coding region of CFRDSP in prcd-affected dogs. In prcd pedigrees informative for both prcd and the CFRDSP Hinf I RFLP, a minimum of six obligate recombinants were identified. Similarly, in erd pedigrees, 14 of 29 progeny informative for both erd and this RFLP were obligate recombinants. CONCLUSIONS: The canine erd and prcd mutations are nonallelic. The canine RDS/peripherin gene (CFRDSP) has been excluded as a candidate for both prcd and erd. The demonstrated informativeness of the canine pedigrees on which these studies were based will enable testing other candidate genes for prcd and erd. Sequence information of CFRDSP will enable testing this locus as a candidate in other canine hereditary retinal degenerations.

Structural changes of the interphotoreceptor matrix in an inherited retinal degeneration: a lectin cytochemical study of progressive rod-cone degeneration.

PURPOSE: In the retinal disorder progressive rod-cone degeneration (prcd) in miniature poodle dogs, the photoreceptor layer degenerates slowly in the course of 5 to 7 years. Components of the interphotoreceptor matrix form a continuous extracellular lattice around photoreceptors. The purpose was to study the photoreceptor cell-matrix interactions during the disease and degeneration phases. Because degeneration rate was slower in cones, the authors also wanted to investigate whether there was a link between the degeneration and the photoreceptor-specific interphotoreceptor matrix domains. METHODS: Rod- and cone-specific interphotoreceptor matrix domains were examined during two periods: before morphological signs of disease had appeared and during the degenerative stages. Two lectin probes were used; wheat germ agglutinin and peanut agglutinin. By their affinity for terminal carbohydrates, the lectins visually separated the two photoreceptor-specific domains and allowed follow-up of the fate of the rod and cone matrices separately. RESULTS: Before and during the course of disease, the lectin distribution in rod and cone domains remained normal, however, in the degenerative phase of the disease, there were structural changes in the matrix domains. The matrix connections between the individual domains was disrupted and single domains were formed. Cone domains and, to a lesser degree rod domains, were thickened around the inner and outer segments. CONCLUSIONS: The changes occurring in the photoreceptor-specific domains were indicative of structural adaptation to cell death and to degenerative conditions. There was no evidence of an active involvement of the interphotoreceptor matrix components studied in the disease process.

Retinal pigment epithelial glycosaminoglycan metabolism: intracellular versus extracellular pathways. In vitro studies in normal and diseased cells.

The synthesis and turnover of glycosaminoglycans (GAGs) in different fractions of cultured feline retinal pigment epithelium (RPE) were characterized. In one method of fractionation, trypsin was used to separate the extracellular components (referred to as trypsin-soluble glycocalyx) from the intracellular components. As a second method, the basal extracellular matrix (basal ECM) was separated from the rest of the GAGs (cell-associated GAGs) by extracting the cell layer with NH4OH. The incorporation of 35SO4 into cetylpyridinium chloride-precipitable GAGs in the cell-associated and the intracellular fractions increased throughout the labeling period, while in the trypsin-soluble glycocalyx and the basal ECM incorporation approached a maximum. While heparan sulfate was the predominant GAG in all compartments, most was located extracellularly. The majority of dermatan sulfate was localized in the intracellular fraction. GAGs in the trypsin-soluble glycocalyx exhibited a rapid rate of turnover, while GAGs in the intracellular compartment and basal ECM turned over much more slowly. Ascorbic acid increased the incorporation of 35SO4 into ECM chondroitin sulfate/dermatan sulfate, but not heparan sulfate, on a per cell basis. Cycloheximide reduced incorporation of 35SO4-GAGs into both the cell-associated compartment and the basal ECM. In contrast, monensin caused a reduction in basal ECM GAGs while increasing the GAGs in the cell-associated compartment. The intracellular accumulation of GAGs and resultant pathology in alpha-L-iduronidase (alpha-L-id)-deficient RPE indicated that this pathway for the intracellular degradation of GAGs is important in normal RPE function. However, the turnover of GAGs in the trypsin-soluble glycocalyx was not affected by deficient alpha-L-id activity or by the subsequent intracellular accumulation of GAGs. Therefore, normal lysosomal activity in the RPE is not a prerequisite for maintaining the rate of extracellular GAG turnover within normal limits.

Retinal pathology of canine X-linked progressive retinal atrophy, the locus homologue of RP3.

PURPOSE: To describe the course of photoreceptor disease in canine X-linked retinal degeneration. METHODS: Retinas from 55 dogs (44 males, 8 carrier females, 3 homozygous females) were obtained by enucleation under general anesthesia. After fixation and dehydration, tissues were embedded in epoxy resin, sectioned at 1 microm for light microscopy and stained with azure II/methylene blue and a paraphenylenediamine counterstain. For electron microscopy, regions identified by light microscopy were selected and cut at 60 nm. Sections were stained with uranyl acetate-lead citrate. Electroretinography from an additional group of normal males, affected males, and carrier females was performed and the rod and cone responses evaluated. RESULTS: The earliest lesion detectable by electron microscopy was vesiculation of rod discs, followed by disruption of outer segments and death of rods. Loss of cones and progressive atrophy of inner retinal layers followed. Lesions were most severe in the peripheral retina and advanced toward the optic disc with disease progression. Significant variation in disease severity was present in males despite the presence of the same disease allele in all affected dogs. Carrier females displayed generalized reduction in photoreceptor density as well as multifocal areas of complete rod loss. The electroretinogram (ERG) findings were compatible with the histopathologic abnormalities. Homozygous females had lesions similar to those seen in affected males. CONCLUSIONS: X-linked retinal degeneration is characterized by initial degeneration of rod photoreceptors, followed by loss of cones and progressive atrophy of the inner retina. Carrier females display a phenotype consistent with random X-chromosome inactivation. Variation in genetic background may alter expression of the disease allele in affected animals, thus accounting for variation in phenotypic expression of the disease.