Clyde K. Yamashita

Retinal Degeneration in Mice Lacking the γ 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 α and β subunits associated with two inhibitory γ subunits. A gene-targeting approach was used to disrupt the mouse PDEγ 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 PDEαβ dimer was formed but lacked hydrolytic activity. Thus, the inhibitory γ subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.

Cyclic GMP-phosphodiesterase of rd retina: Biosynthesis and content

Deficient cGMP-phosphodiesterase (cGMP-PDE) activity results in elevated levels of cGMP in the rd retina before any pathological signs are observed. Since the enzyme is present in rd retina, although it is barely activated by light, we determined whether its synthesis starts at the same time as that of cGMP-PDE in normal retina, or if either its synthesis is halted or degradation of the enzyme increases before the degeneration of the visual cells. We found that synthesis of cGMP-PDE in rd retina is comparable with that in normal retina while the photoreceptors are viable but that cGMP-PDE content is lower than that of normal retina before the visual cells begin to degenerate. Our results suggest that cGMP-PDE is more susceptible to degradation in rd than in normal photoreceptors or, alternatively, that proteolytic enzyme(s) involved in the degradation of cGMP-PDE are in higher concentration or more active in the defective than in the normal retina.

[47] Construction of encapsidated (gutted) adenovirus minichromosomes and their application to rescue of photoreceptor degeneration

Publisher Summary This chapter includes all the protocols required to construct and test EAMs with any transgene of interest. A discussion of the main features of the different types of EAMs and of the modified protocols, developed for analysis of EAM-mediated expression of transgenes in the retina has been provided. The construction of EAMs depends on two seminal observations regarding the nature of the origin of replication and encapsidation signals of adenovirus (Ad). EAMs were constructed to address the issue of the strong host immune response observed during clinical trials using first-generation Ad vectors. These are vectors that have the adenoviral region E1 deleted and replaced with the transgene of interest. Also included in the first-generation vectors are usually those Ad vectors with deletions in region E3. First-generation Ad vectors are propagated in human embryonic kidney 293 cells, which provide the E1 proteins in trans. To reduce the number of viral proteins being expressed in infected cells, second-generation viral vectors have been constructed, usually with deletions in genes coding for the viral replication machinery, further crippling these viruses in vivo. However, it is unclear if such viruses result in prolonged expression when compared with first-generation Ad vectors.

The positive role of the carboxyl terminus of the γ subunit of retinal cGMP-phosphodiesterase in maintaining phosphodiesterase activity in vivo☆

The inhibitory rod cyclic GMP-phosphodiesterase gamma subunit, PDEgamma, is a key component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice that lack PDEgamma due to a targeted disruption of the gene encoding PDEgamma, (Pdeg) suffer from a very rapid and severe photoreceptor degeneration. Previously, deletions in the carboxyl-terminal domain of PDEgamma blocked its ability to inhibit trypsin-activated PDE activity, in vitro. In other words, these mutations eliminated PDEgammas control on the catalytic activity of PDEalpha and PDEbeta. To study the in vivo effects resulting from the deletion of the last seven amino acids of the PDEgamma carboxyl terminal, this PDEgamma allele (Del7C) was introduced as a transgene Pdeg(tm1)/Pdeg(tm1) mice. These animals could only synthesize transgenic mutant PDEgamma. The mutant retinas were expected to display a higher basal level of PDE activity and lower cGMP levels in light and darkness than the PDEgamma knockout mice, which would allow the rescue of their photoreceptors. Instead, our results showed that the Del7C transgene could not complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. In fact, animals carrying the Del7C transgene have low PDE activity as well as reduced PDEalpha and PDEbeta content.

Differential Expression of Rod Photoreceptor cGMP-Phosphodiesterase α and β Subunits mRNA AND PROTEIN LEVELS

The catalytic core of photoreceptor-specific cGMP-phosphodiesterase (PDE) consists of two subunits, PDEα and PDEβ, that are homologous and have similar domain organization but are encoded by different genes. We have examined the PDEα and PDEβ mRNA steady-state and protein levels as well as the biosynthesis rate of these proteins in developing and fully differentiated retinas. We have also determined the translational efficiency of PDE subunits and the role of their mRNA structures in regulating protein synthesis. In mature retinas, PDEα and PDEβ are represented by ∼1.5 × 108 and 7.5 × 108 copies/μg retinal mRNA, respectively. The levels of these transcripts in developing photoreceptors (P10) are approximately 75% of those at P30. Quantification of protein concentration indicated that PDEα and PDEβ are equally expressed in developing and fully differentiated photoreceptors. Furthermore, the PDEα/PDEβ ratios obtained throughout a 2-h pulsechase period revealed a similar turnover rate for both subunits. The observed discordance between the mRNA and protein levels of PDEα and PDEβ suggested post-transcriptional regulation of their expression. We found that PDEα mRNA is translated more efficiently than either of the two PDEβ transcripts expressed in retina. Therefore, the lower level of PDEα mRNA is compensated by its more efficient translation to achieve equimolar expression with PDEβ. We also analyzed the effect of PDEα and PDEβ mRNA 5′- and 3′-untranslated regions as well as that of their coding regions on protein synthesis. We determined that the PDE-coding regions play a critical role in the differential translation of these subunits.

ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

PURPOSE To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina. METHODS The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina. Flow cytometry sorted peanut agglutinin (PNA)-labeled cones from dissociated mouse retinas, and quantitative RT-PCR (QPCR) was used to measure ZBED4 mRNA levels in these cone cells. Immunohistochemistry localized ZBED4 in human retinas. Expression of ZBED4 mRNA transiently transfected into HEK293 cells was analyzed by immunofluorescence. ZBED4 subcellular localization was determined with Western blot analysis. RESULTS One of 80 cDNAs differentially expressed in normal and cd dog retinas corresponded to a novel gene, ZBED4, which is also expressed in human and mouse retinas. ZBED4 mRNA was found to be present in cone photoreceptors. When ZBED4 cDNA was transfected into HEK293 cells, the expressed protein showed nuclear localization. However, in human retinas, ZBED4 was localized to cone nuclei, inner segments, and pedicles, as well as to Müller cell endfeet. Confirming these immunohistochemical results, the 135-kDa ZBED4 was found in both the nuclear and cytosolic extracts of human retinas. ZBED4 has four predicted DNA-binding domains, a dimerization domain, and two LXXLL motifs characteristic of coactivators/corepressors of nuclear hormone receptors. CONCLUSIONS ZBED4 cellular/subcellular localization and domains suggest a regulatory role for this protein, which may exert its effects in cones and Müller cells through multiple ways of action.

Dynamics of the rhomboid-like protein RHBDD2 expression in mouse retina and involvement of its human ortholog in retinitis pigmentosa.

Background: RHBDD2 is distantly related to rhomboids, membrane-bound proteases. Results: In retina, RHBDD2 exists as a monomer in all cells throughout life and a homotrimer only in cone outer segments; a mutation in RHBDD2 possibly leads to retinitis pigmentosa. Conclusion: RHBDD2 plays important roles in development and normal retinal function. Significance: This is the first characterization of RHBDD2 and its association with retinal disease. The novel rhomboid-like protein RHBDD2 is distantly related to rhomboid proteins, a group of highly specialized membrane-bound proteases that catalyze regulated intramembrane proteolysis. In retina, RHBDD2 is expressed from embryonic stages to adulthood, and its levels show age-dependent changes. RHBDD2 is distinctly abundant in the perinuclear region of cells, and it localizes to their Golgi. A glycine zipper motif present in one of the transmembrane domains of RHBDD2 is important for its packing into the Golgi membranes. Its deletion causes dislodgment of RHBDD2 from the Golgi. A specific antibody against RHBDD2 recognizes two forms of the protein, one with low (39 kDa; RHBDD2L) and the other with high (117 kDa; RHBDD2H) molecular masses in mouse retinal extracts. RHBDD2L seems to be ubiquitously expressed in all retinal cells. In contrast, RHBDD2H seems to be present only in the outer segments of cone photoreceptors and may correspond to a homotrimer of RHBDD2L. This protein consistently co-localizes with S- and M-types of cone opsins. We identified a homozygous mutation in the human RHBDD2 gene, R85H, that co-segregates with disease in affected members of a family with autosomal recessive retinitis pigmentosa. Our findings suggest that the RHBDD2 protein plays important roles in the development and normal function of the retina.

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.