Joseph C. Besharse

The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance

Approximately 10% of the photoreceptor outer segment (OS) is turned over each day, requiring large amounts of lipid and protein to be moved from the inner segment to the OS. Defects in intraphotoreceptor transport can lead to retinal degeneration and blindness. The transport mechanisms are unknown, but because the OS is a modified cilium, intraflagellar transport (IFT) is a candidate mechanism. IFT involves movement of large protein complexes along ciliary microtubules and is required for assembly and maintenance of cilia. We show that IFT particle proteins are localized to photoreceptor connecting cilia. We further find that mice with a mutation in the IFT particle protein gene, Tg737/IFT88, have abnormal OS development and retinal degeneration. Thus, IFT is important for assembly and maintenance of the vertebrate OS.

Circadian clock functions localized in xenopus retinal photoreceptors.

A circadian oscillator that regulates visual function is located somewhere within the vertebrate eye. To determine whether circadian rhythmicity is generated by retinal photoreceptors, we isolated and cultured photoreceptor layers from Xenopus retina. On average, 94% of the viable cells in these preparations were rod or cone photoreceptors. Photoreceptor layers produced melatonin rhythmically, with an average period of 24.3 hr, in constant darkness. The phase of the melatonin rhythm was reset by in vitro exposure of the photoreceptor layers to cycles of either light or quinpirole, a D2 dopamine receptor agonist. These data indicate that other parts of the eye are not necessary for generation or entrainment of retinal circadian melatonin rhythms and suggest that rod and/or cone photoreceptors are circadian clock cells.

Circadian rhythmicity in vertebrate retinas: Regulation by a photoreceptor oscillator

Abstract Circadian regulation of retinal function has now been shown in all classes of vertebrates and attempts are now under way to understand the mechanisms underlying this regulation. The functions and organization of retinal rhythm mechanisms seem to vary among species and types of rhythms. To some extent this reflects the experimental advantages of different systems, but it also seems to reflect real variation in control mechanisms among species. Many aspects of retinal function, from gene transcription to complex intercellular interactions, are regulated by circadian oscillators (Fig. 8). Circadian clock regulation of the visual system ranges from dramatic, spontaneous rhythmicity in melatonin synthesis and disc shedding in some species to subtle rhythmic regulation of responsiveness to light and dark signals in other species. Most of the known retinal rhythms, as well as a circadian oscillator, are localized in photoreceptors. This suggests that circadian rhythmicity is fundamental for normal photoreceptor function. Some of the intracellular and paracrine components of the pathways linking different retinal rhythms have been identified. However, because of the complex feedback interactions present in the system, it is still difficult to draw strong conclusions about the causal relationships among rhythms. For example, melatonin is rhythmic and regulates dopamine release, but it is not clear that the rhythms in dopamine are driven directly by melatonin rhythms. Dopamine can modulate melatonin rhythms, but it is not necessary for generation of those rhythms (Cahill and Besharse, 1993). The recently discovered circadian regulation of photoreceptor gene expression (Pierce et al. , 1993; Yoshida et al. , 1993; Green and Besharse, 1994) may underlie rhythmicity at higher levels of organization. It will be important to determine which other photoreceptor genes are also regulated by circadian mechanisms and whether common factors are involved. Mechanisms of the photoreceptor oscillator itself are now approachable in a few experimental preparations, including Xenopus eyecups and photoreceptor layers and chick retinal cell cultures (Cahill and Besharse, 1991, 1993; Pierce et al. , 1993). These in vitro preparations eliminate systemic influences from the interpretation of experimental results. Furthermore, by measuring the timing of rhythmicity, it is possible to distinguish oscillator responses to experimental manipulations from acute changes in rhythmic variables. This is the key to unraveling the mechanisms underlying retinal circadian rhythmicity.

Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity

The mammalian circadian system consists of a central oscillator in the suprachiasmatic nucleus of the hypothalamus, which coordinates peripheral clocks in organs throughout the body. Although circadian clocks control the rhythmic expression of a large number of genes involved in metabolism and other aspects of circadian physiology, the consequences of genetic disruption of circadian-controlled pathways remain poorly defined. Here we report that the targeted disruption of Nocturnin (Ccrn4l) in mice, a gene that encodes a circadian deadenylase, confers resistance to diet-induced obesity. Mice lacking Nocturnin remain lean on high-fat diets, with lower body weight and reduced visceral fat. However, unlike lean lipodystrophic mouse models, these mice do not have fatty livers and do not exhibit increased activity or reduced food intake. Gene expression data suggest that Nocturnin knockout mice have deficits in lipid metabolism or uptake, in addition to changes in glucose and insulin sensitivity. Our data support a pivotal role for Nocturnin downstream of the circadian clockwork in the posttranscriptional regulation of genes necessary for nutrient uptake, metabolism, and storage.

Rhythmic regulation of retinal melatonin: Metabolic pathways, neurochemical mechanisms, and the ocular circadian clock

Summary1.Current knowledge of the mechanisms of circadian and photic regulation of retinal melatonin in vertebrates is reviewed, with a focus on recent progress and unanswered questions.2.Retinal melatonin synthesis is elevated at night, as a result of acute suppression by light and rhythmic regulation by a circadian oscillator, or clock, which has been localized to the eye in some species.3.The development of suitablein vitro retinal preparations, particularly the eyecup from the African clawed frog,Xenopus laevis, has enabled identification of neural, cellular, and molecular mechanisms of retinal melatonin regulation.4.Recent findings indicate that retinal melatonin levels can be regulated at multiple points in indoleamine metabolic pathways, including synthesis and availability of the precursor serotonin, activity of the enzyme serotoninN-acetyltransferase, and a novel pathway for degradation of melatonin within the retina.5.Retinal dopamine appears to act through D2 receptors as a signal for light in this system, both in the acute suppression of melatonin synthesis and in the entrainment of the ocular circadian oscillator.6.A recently developedin vitro system that enables high-resolution measurement of retinal circadian rhythmicity for mechanistic analysis of the circadian oscillator is described, along with preliminary results that suggest its potential for elucidating general circadian mechanisms.7.A model describing hypothesized interactions among circadian, neurochemical, and cellular mechanisms in regulation of retinal melatonin is presented.

Intraflagellar transport and the sensory outer segment of vertebrate photoreceptors

Analysis of the other segments of rod and cone photoreceptors in vertebrates has provided a rich molecular understanding of how light absorbed by a visual pigment can result in changes in membrane polarity that regulate neurotransmitter release. These events are carried out by a large group of phototransduction proteins that are enriched in the outer segment. However, the mechanisms by which phototransduction proteins are sequestered in the outer segment are not well defined. Insight into those mechanisms has recently emerged from the findings that outer segments arise from the plasma membrane of a sensory cilium, and that intraflagellar transport (IFT), which is necessary for assembly of many types of cilia and flagella, plays a crucial role. Here we review the general features of outer segment assembly that may be common to most sensory cilia as well those that may be unique to the outer segment. Those features illustrate how further analysis of photoreceptor IFT may provide insight into both IFT cargo and the role of alternative IFT kinesins. Developmental Dynamics 237:1982–1992, 2008.

Regulation of indoleamine N-Acetyltransferase activity in the retina: Effects of light and dark, protein synthesis inhibitors and cyclic nucleotide analogs

The regulation of indoleamine N-acetyltransferase (NAT) in the posterior eye was investigated in vivo, and in vitro in cultured eye cups. Surgical separation of neural retina from the retinal pigment epithelium-choroid complex indicated that NAT was localized to neural retina. The activity of retinal NAT fluctuated in vivo in a rhythmic fashion, with peak activity in the dark phase of the light-dark cycle. The rhythm of NAT activity persisted for up to 3 days in constant darkness, with a rhythmic period of approximately 25 h. The rhythm was suppressed by constant light, and could be phase-shifted by exposure to a new light-dark cycle. These observations indicate that retinal NAT activity occurs as a circadian rhythm that is entrained by light and dark. Retinas also responded to light and dark in vitro with changes of NAT activity. A significant increase in retinal NAT activity occurred in eye cups cultured in darkness during the dark phase of the light-dark cycle. This increase was completely suppressed in eye cups cultured at the same time of day in light. The dark-induced increase in NAT was completely blocked by protein synthesis inhibitors, and mimicked in light by cyclic AMP analogs. The similarity of the regulation of NAT activity in retina to that in pineal, and the possible relationship of the retinal NAT rhythm to cyclic metabolism in photoreceptors are discussed.

Retinal Circadian Clocks and Control of Retinal Physiology

Retinas of all classes of vertebrates contain endogenous circadian clocks that control many aspects of retinal physiology, including retinal sensitivity to light, neurohormone synthesis, and cellular events such as rod disk shedding, intracellular signaling pathways, and gene expression. The vertebrate retina is an example of a “peripheral” oscillator that is particularly amenable to study because this tissue is well characterized, the relationships between the various cell types are extensively studied, and many local clock-controlled rhythms are known. Although the existence of a photoreceptor clock is well established in several species, emerging data are consistent with multiple or dual oscillators within the retina that interact to control local physiology. Aprominent example is the antiphasic regulation of melaton in and dopamine in photoreceptors and inner retina, respectively. This review focuses on the similarities and differences in the molecular mechanisms of the retinal versus the SCN oscillators, as well as on the expression of core components of the circadian clockwork in retina. Finally, the interactions between the retinal clock(s) and the master clock in the SCN are examined.

Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina.

Several lines of evidence indicate that retinal photoreceptors produce melatonin. However, there are other potential melatonin sources in the retina, and melatonin synthesis can be regulated by feedback from the inner retina. To analyze cellular mechanisms of melatonin regulation in retinal photoreceptors, we have developed an in vitro method for destruction of the inner retina that preserves functional photoreceptors in contact with the pigment epithelium. Eyecups, which include the neural retina, retinal pigment epithelium, choriod, and sclera were prepared. The vitreal surface of the retina in each eyecup was washed sequentially with 1% Triton X-100, water, and culture medium. This lysed the ganglion cells and neurons and glia of the inner nuclear layer, causing the retina to split apart within the inner nuclear layer. The damaged inner retina was peeled away, leaving photoreceptors attached to the pigment epithelium. The cell density of the inner nuclear layer was reduced 94% by this method, but there was little apparent damage to the photoreceptors. Lesioned eyecups produced normal melatonin levels in darkness at night, and melatonin production was inhibited by light. These results indicate that the inner retina is not necessary for melatonin production nor for regulation of photoreceptor melatonin synthesis by light. The lesion method used in this study may be useful for other physiological and biochemical studies of photoreceptors.

IFT20 Links Kinesin II with a Mammalian Intraflagellar Transport Complex That Is Conserved in Motile Flagella and Sensory Cilia

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism thought to be required for the assembly and maintenance of all eukaryotic cilia and flagella. Although IFT proteins are present in cells with sensory cilia, the organization of IFT protein complexes in those cells has not been analyzed. To determine whether the IFT complex is conserved in the sensory cilia of photo-receptors, we investigated protein interactions among four mammalian IFT proteins: IFT88/Polaris, IFT57/Hippi, IFT52/NGD5, and IFT20. We demonstrate that IFT proteins extracted from bovine photoreceptor outer segments, a modified sensory cilium, co-fractionate at ∼17 S, similar to IFT proteins extracted from mouse testis. Using antibodies to IFT88 and IFT57, we demonstrate that all four IFT proteins co-immunoprecipitate from lysates of mouse testis, kidney, and retina. We also extended our analysis to interactions outside of the IFT complex and demonstrate an ATP-regulated co-immunoprecipitation of heterotrimeric kinesin II with the IFT complex. The internal architecture of the IFT complex was investigated using the yeast two-hybrid system. IFT20 exhibited a strong interaction with IFT57/Hippi and the kinesin II subunit, KIF3B. Our data indicate that all four mammalian IFT proteins are part of a highly conserved complex in multiple ciliated cell types. Furthermore, IFT20 appears to bridge kinesin II with the IFT complex.