Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.

Inherited neurodegenerative diseases: the one-hit model of neurodegeneration.

The clinical manifestations of inherited neurodegenerative diseases are often delayed for periods from years to decades. This observation has led to the idea that, in these disorders, neurons die from cumulative damage. A critical prediction of the cumulative damage hypothesis is that the probability of neuronal death increases with age. However, we recently demonstrated, in 17 examples of neurodegeneration, that the kinetics of neuronal death appear to be exponential. These examples include both monogenic disorders, such as photoreceptor degenerations, as well as others that are partly or entirely acquired (such as the clinical phase of parkinsonism and retinal detachment). Exponential kinetics indicate that (i) the risk of death is constant, (ii) death occurs randomly in time and (iii) the death of each neuron is independent of other neurons. We use the term 'one-hit model' to refer to the single catastrophic intracellular biochemical event, analogous to radioactive decay, which leads to neuronal death in the diseases we analyzed. Here, we examine the major features and implications of the one-hit model and provide preliminary evidence that amyotrophic lateral sclerosis also appears to fit this model. We also discuss a testable biochemical hypothesis, the mutant steady-state hypothesis, that we proposed to account for the one-hit model. Finally, we explore six unresolved issues that appear to challenge this model. The one-hit model appears to capture a novel principle underlying many neurodegenerations. Our findings suggest that any consideration of the biochemical basis of neurodegeneration must include a meticulous examination of the kinetics of cell death.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is approximately 60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

Human microphthalmia associated with mutations in the retinal homeobox gene CHX10.

Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.

A one-hit model of cell death in inherited neuronal degenerations.

In genetic disorders associated with premature neuronal death, symptoms may not appear for years or decades. This delay in clinical onset is often assumed to reflect the occurrence of age-dependent cumulative damage. For example, it has been suggested that oxidative stress disrupts metabolism in neurological degenerative disorders by the cumulative damage of essential macromolecules. A prediction of the cumulative damage hypothesis is that the probability of cell death will increase over time. Here we show in contrast that the kinetics of neuronal death in 12 models of photoreceptor degeneration, hippocampal neurons undergoing excitotoxic cell death, a mouse model of cerebellar degeneration and Parkinson's and Huntington's diseases are all exponential and better explained by mathematical models in which the risk of cell death remains constant or decreases exponentially with age. These kinetics argue against the cumulative damage hypothesis; instead, the time of death of any neuron is random. Our findings are most simply accommodated by a 'one-hit' biochemical model in which mutation imposes a mutant steady state on the neuron and a single event randomly initiates cell death. This model appears to be common to many forms of neurodegeneration and has implications for therapeutic strategies.

Recent advances in the molecular basis of inherited photoreceptor degeneration.

To date, 118 loci have been associated with photoreceptor degenerative disease. In this review, we will discuss recent advances in the identification of genes that cause progressive photoreceptor cell death when mutated. We will focus on 12 genes isolated within the last two years that have been shown to be photoreceptor-specific, or that have provided insight into photoreceptor biology and the mechanisms of photoreceptor cell death. To aid in understanding the biologic basis for these diseases, we also briefly review photoreceptor biology. Finally, we report on recent advances towards the treatment of these disorders.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

Retinal stem cells in the adult mammalian eye.

The mature mammalian retina is thought to lack regenerative capacity. Here, we report the identification of a stem cell in the adult mouse eye, which represents a possible substrate for retinal regeneration. Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Müller glia. Adult retinal stem cells are localized to the pigmented ciliary margin and not to the central and peripheral retinal pigmented epithelium, indicating that these cells may be homologous to those found in the eye germinal zone of other nonmammalian vertebrates.

PHR1 encodes an abundant, pleckstrin homology domain-containing integral membrane protein in the photoreceptor outer segments.

We cloned human and murine cDNAs of a gene (designated PHR1), expressed preferentially in retina and brain. In both species, PHR1 utilizes two promoters and alternative splicing to produce four PHR1 transcripts, encoding isoforms of 243, 224, 208, and 189 amino acids, each with a pleckstrin homology domain at their N terminus and a transmembrane domain at their C terminus. Transcript 1 originates from a 5'-photoreceptor-specific promoter with at least three Crx elements ((C/T)TAATCC). Transcript 2 originates from the same promoter but lacks exon 7, which encodes 35 amino acids immediately C-terminal to the pleckstrin homology domain. Transcripts 3 and 4 originate from an internal promoter in intron 2 and either include or lack exon 7, respectively. In situ hybridization shows that PHR1 is highly expressed in photoreceptors, with lower expression in retinal ganglion cells. Immunohistochemistry localizes the PHR1 protein to photoreceptor outer segments where chemical extraction studies confirm it is an integral membrane protein. Using a series of PHR1 glutathione S-transferase fusion proteins to perform in vitro binding assays, we found PHR1 binds transducin betagamma subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.

Retinal degenerations with truncation mutations in the cone-rod homeobox (CRX) gene.

PURPOSE: To define the phenotypes of retinal degenerations associated with mutations in the gene encoding CRX (cone-rod homeobox), a photoreceptor-specific transcription factor. METHODS: Heterozygotes with the E168 [delta1 bp], E168 [delta2 bp], or G217 [delta1 bp] CRXgene mutation were studied clinically, with visual function tests, including rod and cone perimetry and electroretinography (ERG), and with optical coherence tomography (OCT). RESULTS: Clinical diagnoses included autosomal dominant cone-rod dystrophy in one family (E168 [delta1 bp] mutation) and simplex Leber congenital amaurosis in two families (E168 [delta2 bp], G217 [delta1 bp] mutations). In the family with the E168 [delta1 bp] mutation, two siblings had relatively mild disease expression in the third decade of life. The central retinas of these two patients had profound loss of rod and short wavelength cone function; long/middle wavelength cone thresholds were elevated at fixation, but there were greater paracentral than central abnormalities. Peripheral retinal dysfunction was evident by psychophysics and by maximum amplitude loss for rod- and cone-isolated ERG photoreceptor responses. OCT cross-sectional reflectance images showed decreased central retinal thickness consistent with photoreceptor loss. An additional member of this family (E168 [delta1 bp] mutation) and two other patients (representing E168 [delta2 bp] and G217 [delta1 bp] mutations) had a severe phenotype with retina-wide loss of function and islands of function remaining only in the temporal periphery. CONCLUSIONS: Truncation mutations in CRX are associated with retinopathies that share phenotypic features but vary in disease severity. The disease mechanism could involve abnormal photoreceptor development compounded by a disturbance in the maintenance of photoreceptors in the mature retina.

A range of clinical phenotypes associated with mutations in CRX, a photoreceptor transcription-factor gene.

Mutations in the retinal-expressed gene CRX (cone-rod homeobox gene) have been associated with dominant cone-rod dystrophy and with de novo Leber congenital amaurosis. However, CRX is a transcription factor for several retinal genes, including the opsins and the gene for interphotoreceptor retinoid binding protein. Because loss of CRX function could alter the expression of a number of other retinal proteins, we screened for mutations in the CRX gene in probands with a range of degenerative retinal diseases. Of the 294 unrelated individuals screened, we identified four CRX mutations in families with clinical diagnoses of autosomal dominant cone-rod dystrophy, late-onset dominant retinitis pigmentosa, or dominant congenital Leber amaurosis (early-onset retinitis pigmentosa), and we identified four additional benign sequence variants. These findings imply that CRX mutations may be associated with a wide range of clinical phenotypes, including congenital retinal dystrophy (Leber) and progressive diseases such as cone-rod dystrophy or retinitis pigmentosa, with a wide range of onset.

Association of the TLX-2 homeodomain and 14-3-3eta signaling proteins.

Homeodomain proteins play important roles in various developmental processes, and their functions are modulated by polypeptide cofactors. Here we report that both in vitro and in vivo, 14-3-3eta is associated with the TLX-2 homeodomain transcription factor that is required for mouse embryogenesis. Expression of 14-3-3eta shifts the predominant localization of TLX-2 in COS cells from the cytoplasm to the nucleus. Tlx-2 and 14-3-3eta are expressed in the developing peripheral nervous system with spatially and temporally overlapping patterns, and they are also coexpressed in PC12 cells. Increased expression of either gene by transfection considerably inhibited nerve growth factor-induced neurite outgrowth of PC12 cells, and cotransfection of both genes led to a synergistic effect of suppression. These findings define 14-3-3eta as a functional modulator of the TLX-2 homeodomain transcription factor and suggest that the in vivo function of TLX-2 in neural differentiation is likely regulated by signaling mediated by 14-3-3eta.

Intragenic complementation at the argininosuccinate lyase locus: reconstruction of the active site.

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus and the ASL alleles in the ASL-deficient cell strains of two complementation phenotypes have been identified. The frequent complementers, strains that participate in the majority of the complementation events, were found to be either homozygous or heterozygous for the Q286R allele, while the high-activity complementers, those strains in which complementation is associated with a high restoration of activity, were found to be either homozygous or heterozygous for the D87G allele. Direct proof of the intragenic complementation observed at the ASL locus has been obtained with the co-expression of the D87G and Q286R alleles in COS cells. A significant increase in the ASL activity was observed when the two alleles were co-expressed relative to the expression of each mutant allele alone. The increase in activity was comparable to that observed previously in the fibroblast complementation studies. The structure determinations of ASL and the homologous eye lens protein, duck delta II crystallin, have revealed that the active site of ASL is made up of residues from three different monomers. The structural mapping of the Q286 and D87 residues shows that both are located near the active site but that, in any one active site, each is contributed by a different monomer. The molecular symmetry of the ASL protein is such that when mutant monomers combine randomly, one active site will contain both mutations and at least one active site will contain no mutations at all. It is these 'native' active sites in the hybrid Q286R/D87G proteins that give rise to the partial recovery of enzymatic activity observed during intragenic complementation.

Retinal gene therapy 1998: summary of a workshop.

The 1998 Workshop on Retinal Gene Therapy evaluated the potential of gene therapy in treatment of retinal disease. Academic, industry, and private foundation representatives attended. Topics included: determing which retinal diseases are likely candidates for gene therapy, specific retinal degenerations and nonspecific neuronal survival mechanisms, design and use of viral and retroviral vectors in achieving regulated gene expression, animal models of retinal degeneration and associated therapies, human trials, and alternatives to gene therapy. The discussion of human trials explored the justification for moving from animal models to human testing, patient population concerns, lessons learned from previous human gene therapy trials, and the role of industry in support of basic and clinical research.

The Tlx-2 homeobox gene is a downstream target of BMP signalling and is required for mouse mesoderm development.

TGFbeta-related factors are critical regulators of vertebrate mesoderm development. However, the signalling cascades required for their function during this developmental process are poorly defined. Tlx-2 is a homeobox gene expressed in the primitive streak of mouse embryos. Exogenous BMP-2 rapidly activates Tlx-2 expression in the epiblast of E6.5 embryos. A Tlx-2 promoter element responds to BMP-2 signals in P19 cells, and this response is mediated by BMP type I receptors and Smad1. These results suggest that Tlx-2 is a downstream target gene for BMP signalling in the primitive streak where BMP-4 and other TGFbeta-related factors are expressed. Furthermore, disruption of Tlx-2 function leads to early embryonic lethality. Similar to BMP4 and ALK3 mutants, the mutant embryos display severe defects in primitive streak and mesoderm formation. These experiments thus define a BMP/Tlx-2 signalling pathway that is required during early mammalian gastrulation.

Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor.

Genes associated with inherited retinal degeneration have been found to encode proteins required for phototransduction, metabolism, or structural support of photoreceptors. Here we show that mutations in a novel photoreceptor-specific homeodomain transcription factor gene (CRX) cause an autosomal dominant form of cone-rod dystrophy (adCRD) at the CORD2 locus on chromosome 19q13. In affected members of a CORD2-linked family, the highly conserved glutamic acid at the first position of the recognition helix is replaced by alanine (E80A). In another CRD family, a 1 bp deletion (E168 [delta1 bp]) within a novel sequence, the WSP motif, predicts truncation of the C-terminal 132 residues of CRX. Mutations in the CRX gene cause adCRD either by haploinsufficiency or by a dominant negative effect and demonstrate that CRX is essential for the maintenance of mammalian photoreceptors.

Human argininosuccinate lyase: a structural basis for intragenic complementation.

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits enzymatic activity that is greater than that found in the oligomeric proteins produced by each mutant allele alone. The mutations involved in the most successful complementation event observed in ASL deficiency were found to be an aspartate to glycine mutation at codon 87 of one allele (D87G) coupled with a glutamine to arginine mutation at codon 286 of the other (Q286R). To understand the structural basis of the Q286R:D87G intragenic complementation event at the ASL locus, we have determined the x-ray crystal structure of recombinant human ASL at 4. 0 A resolution. The structure has been refined to an R factor of 18. 8%. Two monomers related by a noncrystallographic 2-fold axis comprise the asymmetric unit, and a crystallographic 2-fold axis of space group P3121 completes the tetramer. Each of the four active sites is composed of residues from three monomers. Structural mapping of the Q286R and D87G mutations indicate that both are near the active site and each is contributed by a different monomer. Thus when mutant monomers combine randomly such that one active site contains both mutations, it is required by molecular symmetry that another active site exists with no mutations. These "native" active sites give rise to the observed partial recovery of enzymatic activity.

Expression analysis and chromosomal assignment of the human SFRS5/SRp40 gene.

Alternative splicing plays a major role in the regulation of gene expression. SFRS5/SRp40 is a member of the serine/arginine (SR) protein family of regulators of alternative pre-mRNA splicing. We cloned the human SFRS5 cDNA and observed two major SFRS5 transcripts, an approximately 1.8-kb short form and an approximately 3.3-kb long form, in both human and rat tissues. Both transcripts were detected in all human tissues examined, but there were notable tissue-specific differences in their relative abundance, the short form being most abundant in retina. Affinity-purified SFRS5 antisera recognized a single 40-kDa polypeptide in human and mouse retinal lysates. The abundant retinal expression of SFRS5 was not restricted to any specific cell type, since immunofluorescent labeling of human retinal sections identified the SFRS5 protein in nuclei of all three nuclear layers of the retina. The human SFRS5 gene was localized to human chromosome 14q24 by fluorescence in situ hybridization and PCR analysis of a human/hamster somatic cell hybrid panel.