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Peter Humphries

Publications and source records attributed to Peter Humphries.

13 recordsLinked to original sources
PubMed2006

Synaptic plasticity in CNGA3(-/-) mice: cone bipolar cells react on the missing cone input and form ectopic synapses with rods.

In the mammalian retina, rods and cones connect to distinct sets of bipolar cells. Rods are presynaptic to a single type of rod bipolar cell, whereas cones connect to different types of cone bipolar cells. Synaptic rewiring between cone photoreceptor terminals and rod bipolar cell dendrites has been described as a general result of photoreceptor degeneration. To investigate whether cone bipolar cells also show synaptic plasticity in the absence of cone input, we studied the connectivity of cone bipolar cell dendrites in CNGA3(-/-) mice, a model with specific loss of cone photoreceptor function. Dendritic connections of ON and OFF cone bipolar cells were visualized using specific cell markers or by intracellular injection with fluorescent dyes. The results show that cone bipolar cells in CNGA3(-/-) mice form ectopic synapses with rods. In contrast, cone bipolar cells do not form ectopic synapses with rods in CNGA3(-/-)Rho(-/-) mice, in which both types of photoreceptors are nonfunctional. In analogy with these results, we found that input-deprived rod bipolar cells form ectopic synapses with functional cones in Rho(-/-) mice but not with inoperable cones in the CNGA3(-/-)Rho(-/-) mouse. Our data indicate that the formation of ectopic bipolar cell synapses in the outer plexiform layer requires a functional presynaptic photoreceptor.

Animals↗
PubMed2006

RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture.

Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS-peripherin-linked autosomal-dominant retinitis pigmentosa (RP), for which more than 50 disease-related mutations have been identified to date. Mutation-independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r-rds, which has been altered to escape suppression but nevertheless encodes wild-type protein) has been explored in COS-7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r-rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT-PCR) and protein (ELISA) levels in COS-7 cells. "Dual-" and "triple-expression" constructs carrying the shRNA suppressor and the marker EGFP with or without the r-rds cassette were electroporated in vitro into retinal explants from 1-day-old pups. The retinae were dissociated at day 14, and transduced cells were FACS-sorted using the coexpressed EGFP marker and analyzed by RT-PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r-rds was protected from suppression in COS-7 cells. Similarly, efficient RNAi-mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r-rds was also achieved. These data validate the concept of RNAi-based suppression coupled with replacement technology for the development of therapies targeting RDS-linked autosomal-dominant RP, and suggest that such approaches could potentially be used for other autosomal-dominant diseases with similarly extensive intragenic heterogeneity.

Animals↗
PubMed2005

Cone opsin mislocalization in Rpe65-/- mice: a defect that can be corrected by 11-cis retinal.

PURPOSE: In retinal degenerative diseases, rod photoreceptors typically deteriorate more rapidly than cone photoreceptors. In the Rpe65(-/-) mouse, a model for Leber's congenital amaurosis, cones degenerate much more rapidly than rods. In this model, the retinoid processing pathway in the retinal pigment epithelium is disrupted, and 11-cis retinal is not generated. This study was designed to investigate the feasibility of restoring functional cones with exogenous 11-cis retinal. METHODS: Rpe65(-/-)::Rho(-/-) mice were used to remove any interference of rods and compared with wild-type (wt) mice. Pups were injected intraperitoneally with 11-cis retinal, starting at postnatal day (P)10, and were maintained in complete darkness. At P25, cone function was assessed with photopic single-flash and flicker ERGs. Cone survival was determined immunohistochemically with cone-specific antibodies, and cone opsin levels were obtained by quantitative RT-PCR. RESULTS: At P25, cone density and transcript levels of cone opsins were drastically reduced, but a minute cone electroretinogram was detected, indicating that the cones were functional. Confocal microscopy revealed that the cone opsins were mislocalized, suggesting that their transport to the outer segments was impaired. Intraperitoneal administrations of 11-cis retinal before P25 led to increased transport of cone opsins to the outer segments and preserved cones anatomically and functionally. CONCLUSIONS: The results suggest that the ligand is required during cone opsin synthesis for successful opsin trafficking and that without 11-cis retinal, cones may degenerate because of opsin mislocalization. These results may have important consequences for the treatment of cone dystrophies.

Animals↗
PubMed2004

RNAi of COL1A1 in mesenchymal progenitor cells.

Given that mutant COL1A1 is known to cause Osteogenesis Imperfecta (OI), tools to modulate COL1A1 expression are likely to be of significant therapeutic value. In this context, we have evaluated RNA interference (RNAi) as a means to downregulate COL1A1 expression in Cos-7 cells and in human mesenchymal progenitor stem cells (MPCs), the latter cells giving rise to bone and therefore representing a target cell type for collagen-related disorders. In addition, allele-specificity, a key factor to the success of RNAi-based suppression, was explored with a view to developing a mutation-independent RNAi-based therapeutic for OI by targeting an intragenic SNP within transcripts derived from the COL1A1 gene. Preferential suppression of individual polymorphic alleles that differed by a single nucleotide was observed.

Alleles↗
PubMed2004

Morphological characterization of the retina of the CNGA3(-/-)Rho(-/-) mutant mouse lacking functional cones and rods.

PURPOSE: To assess the structural changes in the retina caused by a functional blockade of rods and cones and to document the time course of their degeneration. METHODS: Double knockout mice were generated by cross-breeding CNGA3(-/-) mice with Rho(-/-) mice. Retinas of mutant and wild-type mice from 3 weeks up to 12 months of age were studied by confocal light and electron microscopy. The retinas were immunostained with cell-type-specific markers and with antibodies against synapse-associated proteins and transmitter receptors. RESULTS: In 3-week-old CNGA3(-/-)Rho(-/-) mice, retinal layers showed normal structural organization, and photoreceptors established normal synaptic contacts. Until postnatal week (Pw)7, presynaptic markers and postsynaptic glutamate receptors were well expressed at the photoreceptor terminals. Photoreceptor degeneration started at Pw4, progressing to an almost complete loss by 3 months. Rod spherules showed an increase in the number of synaptic ribbons and postsynaptic elements during this early stage of degeneration, and horizontal cell processes grew into the outer nuclear layer. At later stages of retinal degeneration, the inner plexiform layer (IPL) was also affected. Rod bipolar cell axon terminals showed morphologic alterations, but the stratification pattern of cone bipolar cell axons and amacrine cell processes appeared unaffected. Transmitter receptors (GlyRalpha3, GABA(A) alpha2, GluR2/3) showed no obvious changes in the distribution and density of their synaptic clusters throughout the IPL at postnatal month 12. CONCLUSIONS: The normal structural and synaptic organization of the mutant retina at Pw3 suggests that photoreceptor light responses are not essential for the development of the retinal circuitry. However, functional photoreceptors are necessary for the maintenance of rods and cones and their contacts in the OPL, because they degenerate almost completely by 3 months after birth. Degenerative changes can also be observed in the IPL; however, they appear to have a slower time course and by 12 months of age the IPL circuitry appears to be surprisingly intact.

Animals↗
PubMed2003

Characterization of RP1L1, a highly polymorphic paralog of the retinitis pigmentosa 1 (RP1) gene.

PURPOSE: To determine the full-length sequence of a gene with similarity to RP1 and to screen for mutations in this newly characterized gene, named retinitis pigmentosa 1-like 1(RP1L1). Since mutations in the RP1 gene cause autosomal dominant retinitis pigmentosa, it is possible that mutations in RP1's most sequence similar relative, RP1L1, may also be a cause of inherited retinal degeneration. METHODS: A combination of cDNA clone sequencing, RACE, and database analysis were used to determine the RP1L1 mRNA sequence and its genomic organization. PCR analysis, semi-quantitative RT PCR, and in situ hybridization were used to determine the expression pattern of RP1L1. Single-strand conformational analysis and automated sequencing were used to screen probands from 60 adRP families for potential disease-causing mutations in RP1L1. RESULTS: The human RP1L1 gene is encoded in 4 exons, which span 50 kb on chromosome 8p. The length of the RP1L1 mRNA is large, over 7 kb, but its exact length is variable between individuals due to the presence of several length polymorphisms, including a 48 bp repeat. RP1L1 encodes a protein with a minimal length of 2,400 amino acids and a predicted weight of 252 kDa. Expression of RP1L1 is limited to the retina and appears to be specific to photoreceptors. Mutational analysis of 60 autosomal dominant retinitis pigmentosa probands revealed the presence of 38 sequence substitutions in RP1L1. Over half of these substitutions result in alteration of the RP1L1 protein, but none of these substitutions appear to be pathogenic. CONCLUSIONS: The RP1L1 gene encodes a large, highly polymorphic, retinal-specific protein. No RP1L1 disease-causing mutations were identified in any of the samples tested, making it unlikely that mutations in RP1L1 are a frequent cause of autosomal dominant retinitis pigmentosa. Additional experiments will be needed to determine if mutations in RP1L1 cause other forms of inherited retinal degeneration.

Amino Acid Sequence↗
PubMed2003

Transplantation of syngeneic Schwann cells to the retina of the rhodopsin knockout (rho(-/-)) mouse.

PURPOSE: To determine whether subretinal Schwann cell transplantation can prolong the survival of photoreceptors in the rhodopsin knockout (rho(-/-)) mouse. METHODS: Schwann cells were prepared from postnatal day (PN) 5 to 7 mouse pups and grafted subretinally into the eyes of PN35 rho(-/-) mice. RT-PCR was performed on similarly prepared cells to determine growth factor production in vitro. Eyes were retrieved at PN70 for anatomic and statistical analysis. Control animals received grafts of fibroblasts or sham surgery. RESULTS: RT-PCR demonstrated the presence of message for ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic factor (GDNF) in the cultured Schwann cells. Schwann cell grafts produced a statistically significant rescue of photoreceptors in a restricted area of retina at PN70, but the effect was lost by PN140. Preserved inner segments could be identified, but outer segments were never present. Sham surgery also resulted in photoreceptor rescue but at a reduced level. Fibroblast grafts appeared to produce little or no rescue effect. Grafts of Schwann cells or fibroblasts and sham surgery induced a reactive Müller glial response. CONCLUSIONS: Schwann cells can prolong photoreceptor survival in the rhodopsin knockout mouse until at least PN70.

Animals↗
PubMed2002

Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta.

Over 100 dominant-negative mutations within the COL1A1 gene have been identified in osteogenesis imperfecta (OI). In terms of human therapeutics, targeting each of these mutations independently is unlikely to be feasible. Here we show that the hammerhead ribozyme Rzpol1a1, targeting a common polymorphism within transcripts from the COL1A1 gene, downregulates COL1A1 transcript in human mesenchymal progenitor cells at a ribozyme to transcript ratio of only 1:1. Downregulation was confirmed at the protein level. Transducing stem cells with Rzpol1A1 ex vivo followed by autologous transplantation could provide a gene therapy for a large proportion of OI patients with gain-of-function mutations using a single therapeutic.

Bone Marrow↗
PubMed2002

Murine model of autosomal dominant retinitis pigmentosa generated by targeted deletion at codon 307 of the rds-peripherin gene.

We introduced a targeted single base deletion at codon 307 of the rds-peripherin gene in mice, similar mutations being known to cause autosomal dominant retinitis pigmentosa (RP) in man. Histopathological and electroretinographic analysis indicate that the retinopathy in mice homozygous for the codon 307 mutation appears more rapid than that in the naturally occurring null mutant, the rds(-/-) mouse, suggesting that the rds-307 mutation displays a dominant negative phenotype in combination with that due to haplosufficiency. RP is the most prevalent cause of registered visual handicap in those of working age in developed countries, the 50 or so mutations so far identified within the RDS-peripherin gene accounting for up to 10% of dominant cases of the disease. Given the sequence homologies that exist between the murine rds-peripherin and the human RDS-peripherin gene, this disease model, the first to be generated for peripherin-based RP using gene targeting techniques, should in principle be of value in the work-up in mice of therapeutics capable of targeting transcripts derived from the human gene.

Amino Acid Sequence↗
PubMed2002

On the genetics of retinitis pigmentosa and on mutation-independent approaches to therapeutic intervention.

Retinitis pigmentosa (RP), the group of hereditary conditions involving death of retinal photoreceptors, represents the most prevalent cause of visual handicap among working populations in developed countries. Here we provide an overview of the molecular pathologies associated with such disorders, from which it becomes clearly apparent that RP is one of the most genetically heterogeneous of hereditary conditions for which molecular pathologies have so far been elucidated. While heterogeneity of such magnitude would appear to represent a major impediment to the development of therapeutics, mutation-independent approaches to therapy are being developed to effectively by-pass such diversity in genetic aetiology. The implications of such technologies in terms of therapeutic intervention in RP, and indeed other genetically heterogeneous conditions, will be addressed.

3' Untranslated Regions↗
PubMed2002

Sensitivity of photoreceptor-derived cell line (661W) to baculoviral p35, Z-VAD.FMK, and Fas-associated death domain.

PURPOSE: Rod, cone, cone-rod, and macular dystrophies eventually bring about the death of cone photoreceptor cells. The present study explores means of inhibiting apoptosis in addition to inducing a specific apoptotic pathway within a photoreceptor-derived cell line. METHODS: Retinal cell culture of murine 661W photoreceptor-derived cells was used to assess the effect of both a synthetic peptide inhibitor of caspases (benzyloxycarbonyl-Val-Ala-DL-Asp-[Ome] fluoromethylketone [Z-VAD.FMK]) and a natural inhibitor, baculoviral p35. In addition, the effect of transfection of Fas-associated death domain (FADD), a cellular protein implicated in receptor-induced apoptosis, was assessed. Assays were performed by transient transfection of cell cultures, and results were recorded by cell counting, Western blot, and spectrophotometry. RESULTS: Western blot analysis and chromogenic caspase substrate cleavage analysis confirmed the activation of caspases within 661W cells. At a concentration of 80 micro M, Z-VAD.FMK, 72.36% +/- 0.93% of 661W cells survived cytotoxic insult compared with 6.99% +/- 1.35% of control cells. Transient transfection of 1200 ng baculoviral p35 conferred a protection of 75.30% +/- 4.23%, compared with 19.61% +/-1.84% of control cells, and it was additionally observed that as little as 50 ng transfection of FADD was capable of inducing the death of 53.21% +/- 1.33% of cells in 661W cultures. CONCLUSIONS: Apoptotic cell death in 661W cells is caspase dependent and may be inhibited with both a synthetic and natural inhibitor of caspase function. Furthermore, 661W cells are highly sensitive to the FADD protein, which may suggest a number of novel therapeutic approaches to halt photoreceptor cell apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Residual photosensitivity in mice lacking both rod opsin and cone photoreceptor cyclic nucleotide gated channel 3 alpha subunit.

The mammalian retina contains three classes of photoreceptor. In addition to the rods and cones, a subset of retinal ganglion cells that express the putative sensory photopigment melanopsin are intrinsically photosensitive. Functional and anatomical studies suggest that these inner retinal photoreceptors provide light information for a number of non-image-forming light responses including photoentrainment of the circadian clock and the pupil light reflex. Here, we employ a newly developed mouse model bearing lesions of both rod and cone phototransduction cascades (Rho(-/-) Cnga3(-/-)) to further examine the function of these non-rod non-cone photoreceptors. Calcium imaging confirms the presence of inner retinal photoreceptors in Rho(-/-) Cnga3(-/-) mice. Moreover, these animals retain a pupil light reflex, photoentrainment, and light induction of the immediate early gene c-fos in the suprachiasmatic nuclei, consistent with previous findings that pupillary and circadian responses can employ inner retinal photoreceptors. Rho(-/-) Cnga3(-/-) mice also show a light-dependent increase in the number of FOS-positive cells in both the ganglion cell and (particularly) inner nuclear layers of the retina. The average number of cells affected is several times greater than the number of melanopsin-positive cells in the mouse retina, suggesting functional intercellular connections from these inner retinal photoreceptors within the retina. Finally, however, while we show that wild types exhibit an increase in heart rate upon light exposure, this response is absent in Rho(-/-) Cnga3(-/-) mice. Thus, it seems that non-rod non-cone photoreceptors can drive many, but not all, non-image-forming light responses.

Animals↗