Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.

The retinal cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of alpha and beta subunits associated with two inhibitory gamma subunits. A gene-targeting approach was used to disrupt the mouse PDEgamma gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEalphabeta dimer was formed but lacked hydrolytic activity. Thus, the inhibitory gamma subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.

The human cGMP-PDE beta-subunit promoter region directs expression of the gene to mouse photoreceptors.

PURPOSE: We previously demonstrated that 350 bp of the human rod cGMP phosphodiesterase beta-subunit (beta-PDE) gene promoter are sufficient to direct high levels of gene expression in human Y-79 retinoblastoma cells in vitro. In this study the cell specificity and expression pattern conferred by the short beta-PDE 5' flanking sequence in vivo were examined. METHODS: A construct containing the bacterial LacZ gene driven by a fragment of the beta-PDE 5' flanking region (-297 to +53) was used to generate transgenic mice. Gene expression was analyzed by measuring beta-galactosidase activity in tissue homogenates or visualizing enzymatic activity or protein production at a cellular level by in situ histochemistry or immunocytochemistry. RESULTS: Three independently derived transgenic lines were generated carrying the -297 to +53 beta-PDE 5' flanking region fragment. Within the retina, the reporter gene was specifically expressed in photoreceptors, consistent with the localization of endogenous beta-PDE. Significant expression of LacZ was not observed in other ocular or peripheral tissues. CONCLUSIONS: Photoreceptor-specific reporter gene expression is driven in vivo by a 350-bp segment of the beta-PDE 5' flanking sequence. This study demonstrates the utility of the human beta-PDE promoter for directing the expression of foreign genes to photoreceptors and suggests that the -297 to +53 beta-PDE 5' flanking region fragment may have important implications for therapeutic gene delivery to the visual cells.

Estrogen receptor in the human eye: influence of gender and age on gene expression.

PURPOSE: Many epidemiologic studies indicate an increased incidence of certain vision threatening conditions in postmenopausal women. These data suggest that changes in sex steroid homeostasis may affect the physiology of the eye. To provide support to this hypothesis, the expression of estrogen receptor alpha (ERalpha) in human eye tissues was investigated. METHODS: Complementary studies including RNA analysis by reverse transcription polymerase chain reaction, western blot analysis, and immunocytochemistry were used to provide evidence of ERalpha expression. Protein detection was carried out using a mouse monoclonal antibody raised against an epitope located in the ligand binding domain of the human receptor. Cellular localization was studied on formalin-fixed paraffin-embedded eye sections using conventional immunohistochemical techniques. RESULTS: Gender and age differences in ERalpha mRNA expression were observed in retina. The 65-kDa ERalpha protein was detected in the retina and retinal pigment epithelium (RPE) of young female eyes but not in eye tissues dissected from men and postmenopausal women. Immunocytochemistry corroborated ERalpha staining of a young female neurosensory retina and RPE. In addition, ERalpha could be detected in the ciliary body, in the iris, and in the epithelium of the lens. CONCLUSIONS: The presence of the ERalpha in the human eye suggests that the sex steroid hormone axis may play a role in the pathogenesis of certain ocular diseases.

The effects of light on cyclic nucleotide metabolism of isolated cone photoreceptors.

A procedure has been developed for the isolation of cone photoreceptors from the retina of the lizard Anolis carolinensis in order to study the effects of dark- and light-adaptation on the cyclic nucleotide metabolism of these visual cells. Incubation of the retina in N2 medium with hyaluronidase and DNAase allows us to obtain intact photoreceptors with good purity (85-90%), yields (greater than 2 x 10(5) cells per retina), and more than 95% of them excluding Trypan blue. cAMP levels are 20-fold higher than cGMP levels in cells from dark-adapted animals and are decreased by 35% upon exposure to light, whereas cGMP levels show no apparent change. However, both cAMP- and cGMP-phosphodiesterases, as well as adenylate and guanylate cyclase, are activated several-fold by light, but the enzymes involved in cGMP metabolism have higher Vmaxs than the cAMP related enzymes. The apparent constant levels of cGMP found in cone photoreceptors may result from our inability to detect the very fast changes that occur in these cells when they are exposed to light.

In vivo studies of the gamma subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84.

The inhibitory rod cGMP phosphodiesterase gamma subunit (PDEgamma) is a major component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice (which lack PDEgamma owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr(84)-->Gly) allele of PDEgamma has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEalphabeta) exerted by the wild-type gamma subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdeg(tm1) mice of a mutant Y84G and a wild-type PDEgamma control transgene. The transgenic mice were crossed with Pdeg(tm1)/Pdeg(tm1) mice to generate animals able to synthesize only the transgenic PDEgamma. Our results showed that wild-type PDEgamma and Y84G transgenes could complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEgamma on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much 'milder' phenotype in vivo than was predicted from the biochemical assays in vitro.