Joseph Fogerty

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.

Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1-μm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.

Regulation of photoreceptor Per1 and Per2 by light, dopamine and a circadian clock.

In the Xenopus laevis retina, a principal model for retinal circadian organization, photoreceptors have all the properties of circadian oscillators. However, rhythmic oscillations of Per1 gene expression in the inner retina (but not photoreceptors) have been reported in mice with the suggestion that mice and frogs have a different retinal circadian organization. Although it is known that two period genes (xPer1 and xPer2) exhibit different temporal patterns of expression in the Xenopus retina, and that one (xPer2) is directly responsive to light and dopamine, it is not known whether this reflects the properties of period genes within photoreceptor oscillators or among distinct retinal cell populations. We addressed this by determining the cellular site of light and dopamine regulated xPer2 expression, and the diurnal expression of both xPer1 and xPer2 using in situ hybridization. Our data show that both xPer1 and xPer2 are expressed in most cell types in the retina, including inner nuclear neurons and ganglion cells. However, light and quinpirole, a dopamine agonist, increase xPer2 levels specifically in photoreceptors, and the effect of quinpirole, but not light, is blocked by pCPT‐cAMP. Furthermore, antiphasic diurnal expression of xPer1 and xPer2 also occurs in photoreceptors. Our analysis does not provide insight into the near constitutive expression of period genes in the inner retina, but supports a model in which light‐ and dopamine regulated‐xPer2 and rhythmic xPer1 play critical roles in entrainment and circadian oscillations within photoreceptors.

The de-ubiquitinylating enzyme, USP2, is associated with the circadian clockwork and regulates its sensitivity to light.

We have identified a novel component of the circadian clock that regulates its sensitivity to light at the evening light to dark transition. USP2 (Ubiquitin Specific Protease 2), which de-ubiquitinylates and stabilizes target proteins, is rhythmically expressed in multiple tissues including the SCN. We have developed a knockout model of USP2 and found that exposure to low irradiance light at ZT12 increases phase delays of USP2−/− mice compared to wildtype. We additionally show that USP2b is in a complex with several clock components and regulates the stability and turnover of BMAL1, which in turn alters the expression of several CLOCK/BMAL1 controlled genes. Rhythmic expression of USP2 in the SCN and other tissues offers a new level of control of the clock machinery through de-ubiqutinylation and suggests a role for USP2 during circadian adaptation to environmental day length changes.

Spatial Distribution of Intraflagellar Transport Proteins in Vertebrate Photoreceptors

Intraflagellar transport (IFT) of a approximately 17S particle containing at least 16 distinct polypeptides is required for the assembly and maintenance of cilia and flagella. Although both genetic and biochemical evidence suggest a role for IFT in vertebrate photoreceptors, the spatial distribution of IFT proteins within photoreceptors remains poorly defined. We have evaluated the distribution of 4 IFT proteins using a combination of immunocytochemistry and rod-specific overexpression of GFP tagged IFT proteins. Endogenous IFT proteins are most highly concentrated within the inner segment, around the basal body, and within the outer segment IFT proteins are localized in discrete particles along the entire length of the axoneme. IFT52-GFP and IFT57-GFP mimicked this pattern in transgenic Xenopus.

174delG Mutation in Mouse MFRP Causes Photoreceptor Degeneration and RPE Atrophy

PURPOSE The authors have identified a recessive mutation causing progressive retinal degeneration, white fundus flecks, and eventual retinal pigment epithelium (RPE) atrophy. The goal of these studies was to characterize the retinal phenotype, to identify the causative locus, and to examine possible functions of the affected gene. METHODS SNP mapping, DNA sequencing, and genetic complementation were used to identify the affected locus. Histology, electroretinography, immunohistochemistry, Western blot analysis, fundus photography, electron microscopy, and in vitro phagocytosis assays were used to characterize the phenotype of the mouse. RESULTS Gene mapping identified a single base pair deletion in membrane-type frizzled related protein (MFRP), designated Mfrp(174delG). MFRP is normally expressed in the RPE and ciliary body but was undetectable by Western blot in mutants. CTRP5, a binding partner of MFRP, was upregulated at the mRNA level and at the protein level in most patients. Assays designed to test the integrity of retinoid cycling and phagocytic pathways showed no deficits in Mfrp(174delG) or rd6 animals. However, the RPE of both Mfrp(174delG) and rd6 mice exhibited a dramatic increase in the number of apical microvilli. Furthermore, evidence of RPE atrophy was evident in Mfrp(174delG) mice by 21 months. CONCLUSIONS The authors have identified a novel null mutation in mouse Mfrp. This mutation causes photoreceptor degeneration and eventual RPE atrophy, which may be related to alterations in the number of RPE microvilli. These mice will be useful to identify a function of MFRP and to study the pathogenesis of atrophic macular degeneration.