Rab Escort Protein Research Articles

AAV-Mediated Gene Therapy for Choroideremia: Preclinical Studies in Personalized Models

Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1st or 2nd decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.

224. Induced Pluripotent Stem Cells as a Model To Test Gene Augmentation Therapy for Choroideremia

Choroideremia (CHM) is an X-linked, blinding, inherited retinal degeneration that is symptomatic in childhood and leads to total blindness in midlife. The disease is characterized by the degeneration of photoreceptors, retinal pigment epithelium (RPE) and choriocapillaris. The causative factor for CHM was identifi ed as a defect in the Rab Escort Protein 1 (REP1) which is involved in prenylation of Rab proteins. The nature and onset of the disease and the size of the gene makes CHM an excellent target for recombinant adeno-associated virus (rAAV) mediated gene therapy. However, the animal model of this disease is not an exact replica of the condition and is not readily available. Therefore, we generated and evaluated induced pluripotent stems cells (iPSCs) from CHM subjects as in vitro models to explore the potential of gene augmentation therapy. A recombinant adeno-associated virus (AAV2) was generated that delivers the wild-type human CHM cDNA driven by a constitutive promoter. Infection of iPSCs with AAV2-CHM resulted in the expression of the CHM protein and rescue of enzymatic function in the defective cells. The gene transfer is effi cient and appears to be safe in the short-term, as shown by studies in affected cells and in normalsighted animal models. These studies not only aid in deciphering the pathogenetic mechanisms of the disease, but also pave the way for a human gene therapy clinical trial for CHM.

Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (ChmFlox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the ChmFlox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

Copy Number Variant Analysis in CHM to Detect Duplications Underlying Choroideremia

Purpose: To investigate the possibility of duplications or deletions within the CHM gene as a cause of choroideremia (CHM).Materials and Methods: Eight males and one female subject were identified in whom clinical features were consistent with a clinical diagnosis of CHM. In all cases, sequencing of the coding region and adjacent intronic splice sites did not identify a mutation. In some cases, supplemental immunoblot analysis of protein from peripheral blood leukocytes with anti-REP-1 antibody confirmed absence of Rab escort protein-1, REP-1. A multiplex ligation-dependent probe amplification assay (MLPA) for the CHM gene was developed to test for deletions and duplications within the CHM gene.Results: A duplication of exons 3–8 of the CHM gene (NM_000390.2) was identified in one case.Discussion: While likely an uncommon event, duplication within the CHM gene could be considered as an explanation for CHM cases in which no mutation is found by sequence analysis.

Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic β cells

A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet β-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and β-subunits of RGGT in clonal INS 832/13 β-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or β-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.

Structure, regulation and cellular functions of Rab geranylgeranyl transferase and its cellular partner Rab Escort Protein

Rab geranylgeranyl transferase is an enzyme responsible for double geranylgeranylation of Rab proteins in all eukaryotic cells. In the present article we would like to focus on new findings concerning the holoenzyme structure and mechanism of catalytic activity, its mode of regulation and consequences of RGGT deficiency in different eucaryotic model organisms and patients.

CHM/REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice

Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers: photoreceptors, retinal pigment epithelium (RPE) and choroid, caused by the loss of Rab Escort Protein-1 (REP1). As a recessive monogenic disorder, CHM is potentially curable by gene addition therapy. The present study aimed to evaluate the potential use of lentiviral vectors carrying CHM/REP1 cDNA transgene for CHM treatment. We generated lentiviral vectors carrying either CHM/REP1 cDNA or EGFP transgene under the control of the elongation factor-1α promoter (EF-1α) or its shortened version EFS. We transduced human (HT1080) and dog (D17) cells, CHM patient's fibroblasts and mouse primary RPE cells in vitro, as well as wild-type and CHM mouse retinas in vivo by subretinal injections. Transgene expression was confirmed by immunoblotting, fluorescence-activated cell sorting, immunofluorescence and confocal microscopy. CHM/REP1 transgene functionality was assessed by an in vitro prenylation assay. Lentiviral vectors with CHM/REP1 and EGFP transgenes efficiently transduced HT1080, D17 and CHM fibroblast cells; CHM/REP1 transgene lead to an increase in prenylation activity. Subretinal injections of lentiviral vectors into mouse retinas resulted in efficient transduction of the RPE (30-35% of total RPE cells transduced after a 1-µl injection), long-term expression for at least 6 months and a decrease in amount of unprenylated Rabs in the CHM RPE. Transduction of neuroretinal cells was restricted to the injection site. Lentiviral CHM/REP1 cDNA transgene rescues the prenylation defect in CHM mouse RPE and thus could be used to restore REP1 activity in the RPE of CHM patients.

Intein‐Mediated Construction of a Library of Fluorescent Rab GTPase Probes

Rab GTPases play a key role in the regulation of membrane trafficking. Post-translational geranylgeranylation is critical for their biological activity and is conferred by Rab geranylgeranyl transferease (RabGGTase), together with an accessory factor, Rab escort protein (REP). Mechanistic studies of Rab prenylation and identification of RabGGTase inhibitors require sensitive reporters of Rab prenylation. In the present work, a combination of protein engineering and expressed protein ligation was used to construct a library of semisynthetic Rab7 fluorescent conjugates. In order to avoid synthesis of a large number of fluorescently labeled peptides, we developed a strategy that combined thiol-reactive dye-labeling of cysteine with in vitro protein ligation. Application of this strategy required optimization of labeling and ligation conditions to promote thiol labeling and disfavor intramolecular cyclization. Using this approach, we constructed 46 fluorescent sensors with different spectral properties that reported on the interaction of Rab7 with RabGGTase, REP-1, and the overall prenylation reaction. Two constructs, Rab7Δ3CCK(NBD) and Rab7Δ2SCCC-dans, displayed 2.5- and 1.5-fold increase in fluorescence, respectively, upon prenylation. Moreover, dansyl-, NBD (4-nitro-benzofurazan)-, I-BA-, and I-SO-labeled Rab7 conjugates exhibited two- to tenfold change in fluorescence upon binding to REP or RabGGTase. These fluorescent sensors allowed us to monitor Rab prenylation in real time and to investigate the assembly of Rab-REP binary and Rab-REP-RabGGTase ternary complexes.

Comprehensive mutation analysis (20 families) of the choroideremia gene reveals a missense variant that prevents the binding of REP1 with rab geranylgeranyl transferase

Choroideremia (CHM), an X-linked degeneration of the retinal pigmented epithelium (RPE), photoreceptors, and choroid, ultimately leads to blindness. It is caused by loss-of-function of the CHM gene product, the Rab escort protein 1 (REP1) that is involved, together with its homologue REP2, in prenylation of Rab GTPases, key regulators of intracellular vesicular traffic. Here, we report the molecular characterization of 20 unrelated Italian families affected by CHM. We identified 19 different mutations, nine of which are new. In most cases, we analyzed the effect of the mutations at the mRNA level. Furthermore, we demonstrated, by in vitro trancription/translation assays, that the mutated mRNAs produced truncated proteins in all cases but one. In fact, we also identified a novel REP1 missense variant (c.1520A>G; p.H507R) associated to CHM. Thus far, only two other CHM-associated missense mutations have been identified, one of which was a splicing alteration. We investigated the impact of the p.H507R amino acid change on REP1 structure and function, thus providing the first experimental demonstration that correlates a missense mutation in CHM with a functional impairment of REP1. Overall, our results indicate that the REP1-Rab geranyl-geranyl transferase interaction and consequently REP1-mediated Rab prenylation is essential for RPE and photoreceptor function.

Rab-dependent vesicular traffic in Arabidopsis thaliana – role of the Rab Escort Protein

Rab-dependent vesicular traffic in Arabidopsis thaliana – role of the Rab Escort Protein

Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic protein probes

Post-translationally isoprenylated proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these protein probes, we have demonstrated that RabGDI and the related Rab escort protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab-GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.

Arabidopsis RAB geranylgeranyl transferase β-subunit mutant is constitutively photomorphogenic, and has shoot growth and gravitropic defects

RAB GTPases are important directional regulators of intracellular vesicle transport. Membrane localization of RAB GTPases is mediated by C-terminal double geranylgeranylation. This post-translational modification is catalyzed by the alpha-beta-heterodimer catalytic core of RAB geranylgeranyl transferase (RAB-GGT), which cooperates with the RAB escort protein (REP) that presents a nascent RAB. Here, we show that RAB-geranylgeranylation activity is significantly reduced in two homozygous mutants of the major Arabidopsis beta-subunit of RAB-GGT (AtRGTB1), resulting in unprenylated RAB GTPases accumulation in the cytoplasm. Both endocytosis and exocytosis are downregulated in rgtb1 homozygotes defective in shoot growth and morphogenesis. Root gravitropism is normal in rgtb1 roots, but is significantly compromised in shoots. Mutants are defective in etiolation and show constitutive photomorphogenic phenotypes that cannot be rescued by brassinosteroid treatment, similarly to the det3 mutant that is also defective in the secretory pathway. Transcriptomic analysis revealed an upregulation of specific RAB GTPases in etiolated wild-type plants. Taken together, these data suggest that the downregulation of the secretory pathway is interpreted as a photomorphogenic signal in Arabidopsis.

Loss-of-Function Mutations in Rab Escort Protein 1 (REP-1) Affect Intracellular Transport in Fibroblasts and Monocytes of Choroideremia Patients

BackgroundChoroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion in peripheral cells of CHM patients.Methodology/Principal FindingsTo evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. With the use of pHrodo™ BioParticles® conjugated with E. coli, collagen I coated FluoSpheres beads and fluorescent DQ™ ovalbumin with BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased in monocytes of CHM patients and, as a consequence, the rates of proteolytic degradation were slowed. Microarray analysis of gene expression revealed that some genes involved in the immune response, small GTPase regulation, transcription, cell adhesion and the regulation of exocytosis were significantly up and down regulated in cells from CHM patients compared to controls. Finally, CHM fibroblasts secreted significantly lower levels of cytokine/growth factors such as macrophage chemoattractant protein-1 (MCP-1), pigment epithelial derived factor (PEDF), tumor necrosis factor (TNF) alpha, fibroblast growth factor (FGF) beta and interleukin (lL)-8.Conclusions/SignificanceWe demonstrated for the first time that peripheral cells of CHM patients had increased pH levels in lysosomes, reduced rates of proteolytic degradation and altered secretion of cytokines. Peripheral cells from CHM patients expose characteristics that were not previously recognized and could used as an alternative models to study the effects of different mutations in the REP-1 gene on mechanism of CHM development in human population.

Silencing of theCHMGene Alters Phagocytic and Secretory Pathways in the Retinal Pigment Epithelium

Choroideremia (CHM) is an X-linked progressive degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid caused by mutations in the CHM gene, which encodes Rab escort-protein-1 (REP-1). REP-1 enables posttranslational isoprenyl modification of Rab GTPases, proteins that control vesicle formation, movement, docking, and fusion. The aim of this study was to determine the effect of REP-1 depletion on vesicular trafficking in phagocytic and secretory pathways of human RPE. In vitro, REP-1 expression was inhibited in human fetal RPE (hfRPE) cells by siRNA knockdown and its effects measured on the uptake of bovine photoreceptor outer segments (POS), proteolysis of POS rhodopsin, phagosomal pH, phagosome fusion with early and late endosomes/lysosomes, and polarized secretion of cytokines. Depletion of REP-1 in human RPE cells did not affect POS internalization but reduced phagosomal acidification and delayed POS protein clearance. REP-1 depletion also caused a decrease in the association of POS-containing phagosomes with late endosomal markers (Rab7, LAMP-1) and increases in the secretion of monocyte chemotactic protein (MCP-1) and interleukin (IL)-8 by hfRPE cells. Lack of REP-1 protein expression in hfRPE cells leads to reduced degradation of POS most likely because of the inhibition of phagosome-lysosome fusion events and increased constitutive secretion of MCP-1 and IL-8. These observations may explain the accumulation of unprocessed outer segments within the phagolysosomes of RPE cells and the presence of inflammatory cells in the choroid of patients with CHM.

A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis

The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1-Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis.

Choroideremia: New Findings from Ocular Pathology and Review of Recent Literature

Histopathology of young individuals affected by choroideremia is rarely available to allow correlation with the clinical presentation. A 30-year-old man with choroideremia died in a motor vehicle accident and one eye was subjected to histopathological examination. Immunoblot analysis of protein derived from white blood cells of a living brother, also affected with choroideremia, confirmed the absence of Rab escort protein-1, the normal CHM gene product. Direct sequencing of the coding region and adjacent splice sites of the CHM gene was undertaken on genomic DNA from the living brother and revealed a transition mutation, C to T, in exon 6 (R253X) which resulted in a stop codon and was predicted to truncate the protein product. Histopathological examination of the eye of the deceased brother showed relative independent degeneration of choriocapillaris, retinal pigment epithelium, and retina, similar to observations in the mouse model of choroideremia. In addition, mild T-lymphocytic infiltration was found within the choroid. The ophthalmic features and the pathology of choroideremia are discussed in light of new findings in the current case.

Fundus Autofluorescence in Carriers of Choroideremia and Correlation with Electrophysiologic and Psychophysical Data

To describe fundus autofluorescence (FAF) in carriers of choroideremia (CHM), and to compare FAF findings with ophthalmoscopy and electrophysiologic and psychophysical data. Prospective, observational case series and systematic review. Six unrelated carriers of CHM. Clinical examination included a comprehensive ophthalmic examination, fundus photography, FAF, kinetic perimetry, 2-color threshold perimetry (2CTP), full-field electroretinography (ERG), and multifocal ERG (mfERG). All 6 carriers (33-60 years of age) were screened for mutations in the coding region of Rab Escort Protein 1 gene (REP1) including close flanking intronic sequence and deletions within 2160 bp of 5' untranslated sequence. Intensity and distribution of FAF, rod sensitivity loss, cone sensitivity loss in 2CTP, amplitude and latency in full-field ERG, amplitude in mfERG, and correlation of all 3 parameters. Mutations in the coding region of REP1 were identified in 3 of 6 carriers. All 6 carriers had good visual acuity. Three carriers complained of photophobia and 1 of impaired vision in dim light. Ophthalmoscopy revealed peripapillary atrophy and retinal pigment epithelium (RPE) mottling mainly in the macular region, and additional RPE clumping and flecks of atrophy in the periphery. A very irregular pattern of low- and high-density FAF speckles was seen. Low-density FAF surrounding the optic nerve head corresponded with the peripapillary atrophy. In areas of major FAF changes, mfERG was deteriorated. The 2CTP images revealed functional disturbances in rods and cones. No general pattern was observed. On MfERG, reduced amplitudes in areas with normal cone sensitivity in 2CTP were seen. All 6 carriers of CHM showed a characteristic FAF pattern that can guide mutation analysis. Even when other functional testing is inconspicuous, FAF is a rapid, noninvasive indicator. The authors have no proprietary or commercial interest in any of the materials discussed in this article.

The functional effect of pathogenic mutations in Rab escort protein 1

Choroideremia (CHM) is a chorioretinal degeneration with an X-linked pattern of inheritance. Affected males experience progressive atrophy of the choroid, retinal pigment epithelium and retina leading to eventual blindness. The CHM gene encodes Rab escort protein 1 (REP-1). REP-1 is involved in trafficking of Rab proteins in the cell. To date, the majority of reported mutations in the CHM gene cause a complete loss of REP-1 function. Here we report pathogenic mutations: a novel missense mutation, L550P; a truncation c.1542T > A, STOP; and two deletions (c.525_526delAG and c.1646delC) in the CHM gene and their phenotypic effect. To analyze the effect of mutations, the 3D structure of human REP-1 and the proteins associated with REP-1 function were modeled using sequence homology with rat proteins. In silico analysis of the missense mutation L550P suggests that the proline residue at position 550 destabilizes the β-structural elements, and the REP-1 tertiary structure. Truncation and deletion mutants are associated with a partial or total loss of the REP-1 essential activity and protein–protein interactions as predicted by the analysis of the structure and stability of these protein products. The presumptive loss of protein was confirmed by Western Blot analysis of protein from mononuclear cells and fibroblasts (FB) from CHM patients.

Sfp1 Interaction with TORC1 and Mrs6 Reveals Feedback Regulation on TOR Signaling

Ribosome biogenesis drives cell growth, and the large transcriptional output underlying this process is tightly regulated. The Target of Rapamycin (TOR) kinase is part of a highly conserved signaling pathway linking nutritional and stress signals to regulation of ribosomal protein (RP) and ribosome biogenesis (Ribi) gene transcription. In Saccharomyces cerevisiae, one of the downstream effectors of TOR is Sfp1, a transcriptional activator that regulates both RP and Ribi genes. Here, we report that Sfp1 interacts directly with TOR complex 1 (TORC1) in a rapamycin-regulated manner, and that phosphorylation of Sfp1 by this kinase complex regulates its function. Sfp1, in turn, negatively regulates TORC1 phosphorylation of Sch9, another key TORC1 target that acts in parallel with Sfp1, revealing a feedback mechanism controlling the activity of these proteins. Finally, we show that the Sfp1-interacting protein Mrs6, a Rab escort protein involved in membrane trafficking, regulates both Sfp1 nuclear localization and TORC1 signaling.

SinglechoroideremiaGene in Nonmammalian Vertebrates Explains Early Embryonic Lethality of the Zebrafish Model of Choroideremia

Mutations of the CHM gene underlie the X-linked chorioretinal degeneration choroideremia (CHM). The affected gene product, Rab Escort Protein (REP)1, mediates the posttranslational prenyl modification of Rab GTPases. In patients with CHM, the related REP2 partially compensates for the loss of function of REP1. The objective of this investigation was to study the natural history of disease in a zebrafish model of CHM. Zebrafish chm(-/-) were bred and subjected to extensive histologic analysis and TUNEL assays, and cellular extracts were used for immunoblot and in vitro prenylation assays. A detailed evolutionary analysis was performed on the REP family. The retina of chm(-/-) zebrafish develops normally for the first 4 days postfertilization (dpf) but that catastrophic multilayer degeneration synchronous with severe multisystem disease follows. Mean survival time is 4.8 dpf. At the onset of generalized disease, a significant reduction in rep expression levels and activity, with unprenylated rabs accumulating in the cytosol was demonstrated. Extensive bioinformatic analysis of the REP family of proteins revealed a single rep isoform in fish and other nonmammalian vertebrates and invertebrates that is similar to mammalian REP1. REP1 appears to be the ancestral gene in the family, whereas the intronless REP2 gene is restricted to the mammalian lineage. The results of this study propose that in chm(-/-) zebrafish, maternally derived rep allows initial successful development of the embryo, but its gradual loss leads to multisystem disease and invariably to lethality. In its current form, the chm(-/-) zebrafish has limited usefulness.