Cyclic AMP signaling pathways play crucial roles in photoreceptor cells and other retinal cell types. Previous studies demonstrated a circadian rhythm of cyclic AMP level in chick photoreceptor cell cultures that drives the rhythm of activity of the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (Ivanova and Iuvone, 2003a) and the rhythm of affinity of the cyclic nucleotide-gated channel for cyclic GMP (Ko et al., 2004). Here we report that the photoreceptor circadian clock generates a rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase activity, which accounts for the temporal changes in the cyclic AMP levels in the photoreceptors. The circadian rhythm of cyclic AMP in photoreceptor cell cultures is abolished by treatment with the L-type Ca2+ channel antagonist nitrendipine, while the Ca2+ channel agonist, Bay K 8644, increased cyclic AMP levels with continued circadian rhythmicity in constant darkness. These results indicate that the circadian rhythm of cyclic AMP is dependent, in part, on Ca2+ influx. Photoreceptor cell cultures exhibit a circadian rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase enzyme activity with high levels at night and low levels during the day, correlating with the temporal changes of cyclic AMP in these cells. Both of the Ca2+/calmodulin-stimulated adenylyl cyclase genes, type 1 and type 8 (Adcy1 and Adcy8), displayed significant daily rhythms of mRNA expression under a light-dark cycle, but only the Adcy1 transcript rhythm persisted in constant darkness. Similar rhythms of Adcy1 mRNA level and Ca2+/calmodulin-stimulated adenylyl cyclase activity were observed in retinas of 2 week old chickens. These results indicate that a circadian clock controls the expression of Adcy1 mRNA and Ca2+/calmodulin-stimulated adenylyl cyclase activity; and calcium influx into these cells gates the circadian rhythm of cyclic AMP, a key component in the regulation of photoreceptor function.
Purpose
The interphotoreceptor retinoid-binding protein (IRBP) gene possesses an unusual structure, encoding multiple Repeats, each consisting of about 300 amino acids. Our goals were to gain insight into the function of IRBP, and to test the current model for the evolution of IRBP, in which Repeats were replicated from a simpler ancestral gene.
Methods
We employed a bioinformatics approach to analyze IRBP loci in recently completed or near-complete genome sequences of several vertebrates and nonvertebrate chordates. IRBP gene expression in zebrafish was evaluated by reverse transcriptase PCR (RT-PCR) and in situ mRNA hybridizations with gene-specific probes.
Results
Patterns of exons and introns in the IRBP genes of tetrapods were highly similar, as were predicted amino acid sequences and Repeat structures. IRBP gene structure in teleost fish was more variable, and we report a new gene structure for two species, the Japanese puffer fish (Takifugu rubripes) and the zebrafish (Danio rerio). These teleost genomes contain a two-gene IRBP locus arranged head-to-tail in which the first gene, Gene 1, is intronless and contains a single large exon encoding three complete Repeats. It is followed by a second gene, Gene 2, which corresponds to the previously reported gene consisting of two Repeats spread across four exons and three introns. Each of the two zebrafish genes is transcribed. Gene 2 is expressed in the photoreceptors and RPE, and Gene 1 is expressed in the inner nuclear layer and weakly in the ganglion cell layer.
Conclusions
The tetrapod IRBP gene structure is highly conserved while the teleost fish gene structure was a surprise: It appears to be a two-gene locus with distinct Repeat organization in each open reading frame. This gene structure and gene expression data are consistent with possible neofunctionalization or sub-function partitioning of Gene 1 and Gene 2 in the zebrafish. We suggest that the two-gene locus in teleost fish arose as a consequence of either the known whole genome duplication or single gene tandem duplication.
by
Nikita Pozdeyev;
Carla Taylor;
Rashidul Haque;
Shyam S. Chaurasia;
Amy Visser;
Aamera Thazyeen;
Yuhong Du;
Haian Fu;
Joan Weller;
David C. Klein;
P Michael Iuvone
14-3-3 proteins are a ubiquitous, highly conserved family of chaperone proteins involved in signal transduction, regulation of cell cycle, intracellular trafficking/targeting, cytoskeletal structure, and transcription. Although 14-3-3 proteins are among the most abundant proteins in the CNS, very little is known about their functional roles in the vertebrate retina. In the present study, we demonstrated that photoreceptors express 14-3-3 protein(s) and identified a 14-3-3 binding partner in photoreceptor cells, the melatonin-synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT). Importantly, our data demonstrate that the binding of 14-3-3 to AANAT is regulated by light, with dramatic functional consequences. During the night in darkness, retinal AANAT is phosphorylated and forms a complex with 14-3-3 proteins with an apparent molecular weight of ∼90 kDa. Phosphorylation of AANAT facilitates the binding of enzyme to 14-3-3 proteins. Within the complex, AANAT is catalytically activated and protected from dephosphorylation and degradation. Light disrupts the AANAT/14-3-3 complex, leading to catalytic inactivation, dephosphorylation, and proteolytic degradation of the enzyme. In the presence of the proteasome inhibitor, lactacystin, light results in the formation of a high molecular weight complex (>150 kDa), which may represent an intermediate in the AANAT degradation process. These findings provide new insight into the roles of 14-3-3 proteins in photoreceptor cells and to the mechanisms controlling melatonin synthesis in the vertebrate retina.
by
Jeffrey Boatright;
Anisha G. Moring;
Clinton McElroy;
Michael J. Phillips;
Vi T. Do;
Bo Chang ;
Norm L. Hawes;
Amber P. Boyd;
Sheree S. Sidney;
Rachael E. Stewart;
Steven C. Minear;
Rajashree Chaudhury;
Vincent Thomas Ciavatta;
Cecilia M.P. Rodrigues;
Clifford J. Steer;
John Nickerson;
Machelle Pardue
PURPOSE:
Bear bile has been used in Asia for over 3,000 years to treat visual disorders, yet its therapeutic potential remains unexplored in Western vision research. The purpose of this study was to test whether treatment of mice undergoing retinal degeneration with tauroursodeoxycholic acid (TUDCA), a primary constituent of bear bile, alters the course of degeneration.
METHODS:
Two retinal degeneration models were tested: the rd10 mouse, which has a point mutation in the gene encoding the beta subunit of rod phosphodiesterase, and light induced retinal damage (LIRD). For LIRD studies, albino Balb/C adult mice were subcutaneously injected with TUDCA (500 mg/kg body weight) or vehicle (0.15 M NaHCO(3)). Sixteen h later, each mouse received repeat injections. Half of each treatment group was then placed in bright light (10,000 lux) or dim light (200 lux) for seven h. At the end of exposure, animals were transferred to their regular housing. Electroretinograms (ERGs) were assessed 24 h later, mice sacrificed, eyes embedded in paraffin and sectioned, and retina sections assayed for morphology and apoptosis by TUNEL and anti-active caspase-3 immunoreactivity via fluorescent confocal microscopy. A subset of mice were sacrificed 8 and 15 days after exposure and retina sections analyzed for morphology and apoptosis. For rd10 studies, mice were injected subcutaneously with TUDCA or vehicle at postnatal (P) days 6, 9, 12, and 15. At p18, ERGs were recorded, mice were euthanized and eyes were harvested, fixed, and processed. Retinal sections were stained (toluidine blue), and retinal cell layers morphometrically analyzed by light microscopy. Consecutive sections were analyzed for apopotosis as above.
RESULTS:
By every measure, TUDCA greatly slowed retinal degeneration in LIRD and rd10 mice. ERG a-wave and b-wave amplitudes were greater in mice treated with TUDCA compared to those treated with vehicle. Retinas of TUDCA-treated mice had thicker outer nuclear layers, more photoreceptor cells, and more fully-developed photoreceptor outer segments. Finally, TUDCA treatments dramatically suppressed signs of apoptosis in both models.
CONCLUSIONS:
Systemic injection of TUDCA, a primary constituent of bear bile, profoundly suppressed apoptosis and preserved function and morphology of photoreceptor cells in two disparate mouse models of retinal degeneration. It may be that bear bile has endured so long in Asian pharmacopeias due to efficacy resulting from this anti-apoptotic and neuroprotective activity of TUDCA. These results also indicate that a systematic, clinical assessment of TUDCA may be warranted.
Purpose
The interphotoreceptor retinoid-binding protein (IRBP) gene possesses an unusual structure, encoding multiple Repeats, each consisting of about 300 amino acids. Our goals were to gain insight into the function of IRBP, and to test the current model for the evolution of IRBP, in which Repeats were replicated from a simpler ancestral gene.
Methods
We employed a bioinformatics approach to analyze IRBP loci in recently completed or near-complete genome sequences of several vertebrates and nonvertebrate chordates. IRBP gene expression in zebrafish was evaluated by reverse transcriptase PCR (RT-PCR) and in situ mRNA hybridizations with gene-specific probes.
Results
Patterns of exons and introns in the IRBP genes of tetrapods were highly similar, as were predicted amino acid sequences and Repeat structures. IRBP gene structure in teleost fish was more variable, and we report a new gene structure for two species, the Japanese puffer fish (Takifugu rubripes) and the zebrafish (Danio rerio). These teleost genomes contain a two-gene IRBP locus arranged head-to-tail in which the first gene, Gene 1, is intronless and contains a single large exon encoding three complete Repeats. It is followed by a second gene, Gene 2, which corresponds to the previously reported gene consisting of two Repeats spread across four exons and three introns. Each of the two zebrafish genes is transcribed. Gene 2 is expressed in the photoreceptors and RPE, and Gene 1 is expressed in the inner nuclear layer and weakly in the ganglion cell layer.
Conclusions
The tetrapod IRBP gene structure is highly conserved while the teleost fish gene structure was a surprise: It appears to be a two-gene locus with distinct Repeat organization in each open reading frame. This gene structure and gene expression data are consistent with possible neofunctionalization or sub-function partitioning of Gene 1 and Gene 2 in the zebrafish. We suggest that the two-gene locus in teleost fish arose as a consequence of either the known whole genome duplication or single gene tandem duplication.
A growing body of evidence suggests that plasticity at GABAergic synapses is of critical importance during development and aging. A balance between excitation and inhibition maintains homeostasis at the neuronal and circuit levels, and inhibitory plasticity can function to drive a perturbed system toward homeostasis. Activity-dependent modification of inhibitory synaptic strength must be non-Hebbian, however, because the interaction between an inhibitory neuron and its target prevents them from firing together. Mechanisms that may underlie inhibitory plasticity will be discussed, including the possibility that it is limited to the early period when GABA/glycine release is excitatory (Ben-Ari, 2002) or that corelease of another substance alters synapses that produce inhibition (Gillespie et al., 2005). Alternatively, inhibitory synapses may decline in strength through long-term depression (Kotak et al., 2001; Chang et al., 2003), or an as-yet undiscovered mechanism may be responsible. Whatever the mechanism, it is clear that inhibitory plasticity plays an important role in activity-dependent modification of developing circuits.