11-cis-retinal and Nerve-Degeneration

Many degenerative diseases involve apoptotic cell death--can they be treated with apoptosis inhibitors, while protecting the normal physiological function of the rescued cells? Reason for optimism comes from a recent study of mutant flies with an analogue of the human degenerative disease retinitis pigmentosa.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Cysteine Proteinase Inhibitors; Drosophila melanogaster; Drosophila Proteins; Eye Proteins; Humans; Inhibitor of Apoptosis Proteins; Nerve Degeneration; Phosphoprotein Phosphatases; Retinal Degeneration; Rhodopsin; Viral Proteins

Two outstanding unknowns in the biology of photoreceptors are the molecular determinants of cell size, which is remarkably uniform among mammalian species, and the mechanisms of rod cell death associated with inherited neurodegenerative blinding diseases such as retinitis pigmentosa. We have addressed both questions by performing an in vivo titration with rhodopsin gene copies in genetically engineered mice that express only normal rhodopsin or an autosomal dominant allele, encoding rhodopsin with a disease-causing P23H substitution. The results reveal that the volume of the rod outer segment is proportional to rhodopsin gene expression; that P23H-rhodopsin, the most common rhodopsin gene disease allele, causes cell death via a dominant-negative mechanism; and that long term survival of rod cells carrying P23H-rhodopsin can be achieved by increasing the levels of wild type rhodopsin. These results point to promising directions in gene therapy for autosomal dominant neurodegenerative diseases caused by dominant-negative mutations.

Topics: Alleles; Animals; Gene Expression; Genes, Dominant; Genetic Therapy; Mice; Mutation; Nerve Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

Although the photoreceptors cell death is the main cause of some retinopathies diseases, the mechanisms involved in this process are poorly understood. The neuroprotective effects of interleukin-4 (IL-4) have been shown in several tissues, including retina. We demonstrate that treatment of rat retinal explants with IL-4 completely inhibited the thapsigargin-induced rod photoreceptor cell death after 24 hr in culture. We also showed that IL-4 receptor alpha subunit (IL-4Ralpha) is abundantly present in retina. Colocalization of IL-4Ralpha and rhodopsin indicate a direct effect of this cytokine in rod photoreceptor cells. Moreover, IL-4 increased the intracellular levels of cAMP in 7.4-fold, indicating that the neuroprotective effect of this cytokine was completely blocked by RpcAMP, an inhibitor of protein kinase (PKA). Our data demonstrate, for the first time, the neuroprotective effect of IL-4 through cAMP/PKA pathway in thapsigargin-induced photoreceptor cell death.

Topics: Animals; Cell Death; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoprotection; Enzyme Inhibitors; Interleukin-4; Nerve Degeneration; Neuroprotective Agents; Organ Culture Techniques; Rats; Receptors, Interleukin-4; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Signal Transduction; Thapsigargin; Up-Regulation

This study used organ cultures to examine whether retinal ganglion cells (RGCs) retain their ability to regenerate axons in buphthalmos. A rat mutant with hereditary buphthalmos was used to (1) determine whether the extent of RGC loss corresponds to the severity and duration of elevated intraocular pressure (IOP), (2) examine whether RGCs exposed to an elevated IOP are able to regenerate their axons in a retina culture model, and (3) analyze the proteome of the regenerating retina in order to identify putative regeneration-associated proteins. Retrograde labeling of RGCs revealed a decrease in their numbers in the retinas of buphthalmic eyes that increased with age. Quantification of axons growing out of retinal explants taken at different stages of the disease demonstrated that buphthalmic RGCs possess a remarkable potential to regrow axons. As expected, immunohistochemistry and immunoblotting revealed that elevated IOP was associated with upregulation of certain known proteins, such as growth-associated protein 43, glial fibrillary acidic protein, and endothelin-1. In addition, two-dimensional polyacrylamide gel electrophoresis and mass spectrometry revealed several spots corresponding to proteins that were specifically regulated when buphthalmic RGCs were permitted to regrow their axons. Out of the proteins identified, heat-shock protein (HSP)-60 was constantly expressed during axonal growth at all stages of the disease. Antibodies against HSP-60 reduced axonal growth, indicating the involvement of this protein in regenerative axonal growth. These data are the first to show that diseased retinal neurons can grow their axons, and that HSP-60 supports neuritogenesis. This model may help to elucidate the fundamental mechanisms of optic neuropathy at stages preceding death caused by chronic injury, and aid in the development of neuroprotective strategies.

Topics: Animals; Axons; Blotting, Western; Cell Count; Chaperonin 60; Electrophoresis, Gel, Two-Dimensional; Endothelin-1; GAP-43 Protein; Glial Fibrillary Acidic Protein; Hydrophthalmos; Immunohistochemistry; Intraocular Pressure; Nerve Degeneration; Nerve Regeneration; Organ Culture Techniques; Peptides; Rats; Rats, Mutant Strains; Retinal Ganglion Cells; Rhodopsin

Several mutations in the opsin gene have been associated with congenital stationary night blindness, considered to be a relatively nonprogressive disorder. In the present study, we examined the structural and functional changes induced by one of these mutations, i.e., substitution of aspartic acid for glycine at position 90 (G90D). Transgenic mice were created in which the ratio of transgenic opsin transcript to endogenous was 0.5:1, 1.7:1, or 2.5:1 and were studied via light and electron microscopy, immunocytochemistry, electroretinography (ERG), and spectrophotometry. Retinas with transgenic opsin levels equivalent to one endogenous allele (G0.5) appeared normal for a period of about 3-4 months, but at later ages there were disorganized, shortened rod outer segments (ROS), and a loss of photoreceptor nuclei. Higher levels of G90D opsin expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology. Despite these adverse effects, the mutation had a positive effect on the retinas of rhodopsin knockout (R-/-) mice, whose visual cells fail to form ROS and rapidly degenerate. Incorporation of the transgene in the null background (G+/-/R-/- or G+/+/R-/-) led to the development of ROS containing G90D opsin and prolonged survival of photoreceptors. Absorbance spectra measured both in vitro and in situ showed a significant reduction of more than 90% in the amount of light-sensitive pigment in the retinas of G+/+/R-/- mice, and ERG recordings revealed a >1 log unit loss in sensitivity. However, the histological appearances of the retinas of these mice show no significant loss of photoreceptors and little change in the lengths of their outer segments. These findings suggest that much of the ERG sensitivity loss derives from the reduced quantal absorption that results from a failure of G90D opsin to bind to its chromophore and form a normal complement of light-sensitive visual pigment.

Topics: Animals; Blotting, Northern; Blotting, Western; Disease Models, Animal; Electroretinography; Immunohistochemistry; Mice; Mice, Knockout; Mice, Transgenic; Microscopy, Electron; Nerve Degeneration; Night Blindness; Point Mutation; Retina; Rhodopsin; Rod Opsins; Spectrophotometry; Transgenes

In addition to rod photoreceptor loss, many mutations in rod photoreceptor-specific genes cause degeneration of other neuronal types. Identifying mechanisms of cell-cell interactions initiated by rod-specific mutations and affecting other retinal cells is important for understanding the pathogenesis and progression of retinal degeneration. Here we show in animals with rod and cone degeneration due to mutations in the genes encoding rhodopsin and cGMP phosphodiesterase beta-subunit (PDE-beta) respectively, that rod bipolar cells received ectopic synapses from cones in the absence of rods. Thus, synaptic plasticity links certain rod-specific mutations to retina-wide structural alterations that involve different types of neurons.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Animals, Genetically Modified; Cyclic Nucleotide Phosphodiesterases, Type 6; GTP-Binding Proteins; Mice; Mutation; Nerve Degeneration; Neuronal Plasticity; Phosphoric Diester Hydrolases; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Rhodopsin; Swine; Synapses

Rhodopsin regeneration requires attachment between the retinal pigment epithelium (RPE) and rod outer segments; however, in experimentally induced retinal detachment, rhodopsin regeneration can be restored partially upon addition of IRBP (interphotoreceptor retinoid binding protein). The mi(vit)/mi(vit) (vitiligo) mutant mouse, a model of slowly progressing photoreceptor cell degeneration, has a marked elevation of IRBP at 4 weeks as well as progressive detachment of the retina. The purpose of this study was to determine whether this mutant is capable of regenerating rhodopsin within a few hours following an intense light bleach. Rhodopsin regeneration was determined spectrophotometrically in mice after an intense one hour light bleach followed by 0,1,2,4 or 24 h of dark recovery. IRBP was localized immunohistochemically in fixed frozen tissue at the light microscopic level and in LR Gold embedded tissue at the ultrastructural level. Rhodopsin regeneration experiments indicated that rhodopsin levels following 0,1,2 and 4 h dark-recovery were significantly less in mi(vit)/mi(vit) mutants compared with controls. Immunohistochemical detection of IRBP indicated an altered distribution of the protein in the mutant mice compared with controls. There was accumulation in the region of the inner segments in mutant retinas rather than distribution only to the RPE/OS apical regions as in controls. The data suggest that regeneration of rhodopsin is reduced by 4 weeks postnatally in the mi(vit)/mi(vit) mouse. There is partial detachment of the retina at this age; and IRBP, thought to be essential for proper functioning of the visual cycle, is aberrantly distributed in this mutant.

Topics: Animals; Eye Proteins; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Nerve Degeneration; Photoreceptor Cells; Retinol-Binding Proteins; Rhodopsin; Time Factors; Vitiligo

Expression of a mouse opsin transgene containing three point mutations (V20G, P23H, and P27L; termed VPP) causes a progressive photoreceptor degeneration that resembles in many important respects that seen in patients with autosomal dominant retinitis pigmentosa caused by a P23H point mutation. We have attempted to determine whether the degree of degeneration induced by expression of the transgene is influenced by albinism, a genetically mediated recessive trait that results in a deficiency in melanin formation in pigmented tissues throughout the body. Litters of albino and pigmented mice (normal as well as transgenic) were reared in either darkness or cyclic light. Retinal structure and function were evaluated by light microscopy, electroretinography (ERG), and retinal densitometry. The data were consistent in demonstrating that at similar ages, the extent of photoreceptor degeneration was greater in transgenic albino animals than in their pigmented counterparts. The albino VPP mice had significantly fewer cell bodies in the outer nuclear layer of the retina, a larger reduction in ERG amplitude, and a lower rhodopsin content in the rod photoreceptors. These structural and functional differences could not be attributed to the greater level of retinal illumination experienced by the albino retina under normal ambient conditions, because they persisted when pigmented and albino mice were reared in darkness from birth. Although the explanation remains unclear, our findings indicate that the rate of photoreceptor degeneration in VPP mice is adversely affected by the existence of the albino phenotype, a factor that may have implications for the counseling of human patients with retinitis pigmentosa and a familial history of other genetic disorders.

Topics: Albinism; Animals; Densitometry; Electroretinography; Gene Expression; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Degeneration; Photoreceptor Cells; Reference Values; Retina; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Five different, well-characterized mutants of the R1-6 rhodopsin gene (ninaE), which corresponds to the rod opsin gene of vertebrates, have been examined morphologically as a function of age (up to 9 weeks) to determine whether or not the photoreceptors degenerate and to assess the pattern of degeneration. Structural deterioration of R1-6 photoreceptors with age has been found in all five mutants. The structural pattern of degeneration is similar in the five mutants, but the time course of degeneration is allele dependent and varies greatly among the five, with the strongest alleles causing the fastest degeneration. The degeneration appears to be independent of either the illumination cycle to which the animals are exposed or the presence of screening pigments in the eye. Although the degeneration first appears in R1-6 photoreceptors, eventually R7/8 photoreceptors, which correspond to cones of vertebrates, are also affected. In many of these mutants, striking proliferations of membrane processes have been observed in the subrhabdomeric region of R1-6 photoreceptors. It is hypothesized that (1) this accumulation of membranes may be caused by the failure of newly synthesized membranes that are inserted into the base of microvilli to be assembled into R1-6 rhabdomeres and (2) this failure may be caused by the extremely low concentration of normal R1-6 rhodopsin in the ninaE mutants.

Topics: Aging; Animals; Cell Membrane; Drosophila melanogaster; Microscopy, Electron; Mutation; Nerve Degeneration; Photoreceptor Cells; Retina; Rhodopsin

The norpAH44 phototransduction mutant of Drosophila melanogaster, an allele that, on eclosion, does not exhibit a receptor potential was found, at later ages, to undergo light and temperature dependent degeneration of its photoreceptors as well as decreases in rhodopsin concentration. Pseudopupil measurements and light and electron microscopy were used to monitor the structure of the photoreceptors. When norpAH44 flies were maintained exclusively in the dark, no changes in structure or rhodopsin concentration were observed. When maintained on a 12 h light-12 h dark cycle, structural changes were first observed at 6 days of age for flies maintained at 24 degrees C or at 12 days of age for flies maintained at 19 degrees C. When the light-dark cycle was initiated after 10 days in the dark there was a more rapid loss of rhodopsin concentration and pseudopupil. The data suggest that even in the dark, although no obvious changes in structure or rhodopsin concentration were observed, certain processes that support these components had been affected. NorpAP12, an allele that exhibits small receptor potential amplitudes, also displayed age- and light-dependent photoreceptor degeneration and decreases in rhodopsin concentration, whereas no degeneration or decreases in rhodopsin were observed in norpAP16, an allele that exhibits receptor potential amplitudes similar to those of wild-type. The data suggest that the processes that affect phototransduction, such as the phosphatidylinositol cycle, have a long-term role in the maintenance of rhodopsin concentration and photoreceptor integrity.

Topics: Alleles; Animals; Darkness; Drosophila melanogaster; Light; Mutation; Nerve Degeneration; Photoreceptor Cells; Retinal Pigments; Rhodopsin; Temperature