Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.     

Clock Genes and Behavioral Responses to Light Are Altered in a Mouse Model of Diabetic Retinopathy

There is increasing evidence that melanopsin-expressing ganglion cells (ipRGCs) are altered in retinal pathologies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing ipRGCs morphology and light-induced c-Fos and Period 1–2 clock genes in the central clock (SCN). The ability of STZ-diabetic mice to entrain to light was challenged by exposure animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-h advance of the LD cycle. Our results show that diabetes induces morphological changes of ipRGCs, including soma swelling and dendritic varicosities, with no reduction in their total number, associated with decreased c-Fos and clock genes induction by light in the SCN at 12 weeks post-onset of diabetes. In addition, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges.     

Glaucoma Alters the Circadian Timing System

Glaucoma is a widespread ocular disease and major cause of blindness characterized by progressive, irreversible damage of the optic nerve. Although the degenerative loss of retinal ganglion cells (RGC) and visual deficits associated with glaucoma have been extensively studied, we hypothesize that glaucoma will also lead to alteration of the circadian timing system. Circadian and non-visual responses to light are mediated by a specialized subset of melanopsin expressing RGCs that provide photic input to mammalian endogenous clock in the suprachiasmatic nucleus (SCN). In order to explore the molecular, anatomical and functional consequences of glaucoma we used a rodent model of chronic ocular hypertension, a primary causal factor of the pathology. Quantitative analysis of retinal projections using sensitive anterograde tracing demonstrates a significant reduction (∼50–70%) of RGC axon terminals in all visual and non-visual structures and notably in the SCN. The capacity of glaucomatous rats to entrain to light was challenged by exposure to successive shifts of the light dark (LD) cycle associated with step-wise decreases in light intensity. Although glaucomatous rats are able to entrain their locomotor activity to the LD cycle at all light levels, they require more time to re-adjust to a shifted LD cycle and show significantly greater variability in activity onsets in comparison with normal rats. Quantitative PCR reveals the novel finding that melanopsin as well as rod and cone opsin mRNAs are significantly reduced in glaucomatous retinas. Our findings demonstrate that glaucoma impacts on all these aspects of the circadian timing system. In light of these results, the classical view of glaucoma as pathology unique to the visual system should be extended to include anatomical and functional alterations of the circadian timing system.     

Analysis of immunohistochemical label of Fos protein in the suprachiasmatic nucleus: comparison of different methods of quantification

The induction of the proto-oncogene c-fos, and its phosphoprotein product Fos, has been extensively used to study the effects of light on the circadian pacemaker in the suprachiasmatic nucleus (SCN). Experimental approaches to the quantification of Fos induction have mainly been based on immunohistochemistry and subsequent measure of Fos immunoreactivity (IR) in sections of the SCN. In this study, the authors compare several methods of quantification using optical density image analysis or counts of Fos-IR labeled cells. To assess whether optical density measures using image analysis reflect the amount of Fos in brain tissue, the authors developed standards of known concentrations of Fos protein in an agar matrix. The agar standards were sectioned and treated simultaneously with sections of the SCN from animals exposed to different levels of irradiance. Optical density was found to be proportional to the quantity of Fos in the sections, indicating that this measure accurately reflects relative levels of Fos protein induction. Quantification by optical density analysis allows an objective measure in which the various parameters, conditions of illumination, and threshold can be maintained constant throughout the analysis. Counting cells by visual observation is more subjective because threshold values cannot be precisely defined and can vary according to the observer, illumination, degree of label, and other factors. In addition, cell counts involving direct visual observation, automated cell counts, or stereological methods do not take into account the difference in the density of label between cells, thus giving equal weight to lightly or densely stained cells. These measures are more or less weakly correlated with measures of optical density and thus do not accurately reflect the amount of bound Fos protein in the tissue sections. In contrast, labeled surface area as measured by image analysis shows a linear relationship with optical density. The main outcome of this study is that computer-assisted image analysis provides an accurate and rapid method to determine the relative amount of Fos protein in the SCN and the effects of light on intracellular signaling mechanisms involved in the circadian clock.     

Modeling the role of mid-wavelength cones in circadian responses to light

Nonvisual responses to light, such as photic entrainment of the circadian clock, involve intrinsically light-sensitive melanopsin-expressing ganglion cells as well as rod and cone photoreceptors. However, previous studies have been unable to demonstrate a specific contribution of cones in the photic control of circadian responses to light. Using a mouse model that specifically lacks mid-wavelength (MW) cones we show that these photoreceptors play a significant role in light entrainment and in phase shifting of the circadian oscillator. The contribution of MW cones is mainly observed for light exposures of short duration and toward the longer wavelength region of the spectrum, consistent with the known properties of this opsin. Modeling the contributions of the various photoreceptors stresses the importance of considering the particular spectral, temporal, and irradiance response domains of the photopigments when assessing their role and contribution in circadian responses to light.     

Alteration of Daily and Circadian Rhythms following Dopamine Depletion in MPTP Treated Non-Human Primates

Disturbances of the daily sleep/wake cycle are common non-motor symptoms of Parkinson''s disease (PD). However, the impact of dopamine (DA) depletion on circadian rhythms in PD patients or non-human primate (NHP) models of the disorder have not been investigated. We evaluated alterations of circadian rhythms in NHP following MPTP lesion of the dopaminergic nigro-striatal system. DA degeneration was assessed by in vivo PET ([¹¹C]-PE2I) and post-mortem TH and DAT quantification. In a light∶dark cycle, control and MPTP-treated NHP both exhibit rest-wake locomotor rhythms, although DA-depleted NHP show reduced amplitude, decreased stability and increased fragmentation. In all animals, 6-sulphatoxymelatonin peaks at night and cortisol in early morning. When the circadian system is challenged by exposure to constant light, controls retain locomotor rest-wake and hormonal rhythms that free-run with stable phase relationships whereas in the DA-depleted NHP, locomotor rhythms are severely disturbed or completely abolished. The amplitude and phase relations of hormonal rhythms nevertheless remain unaltered. Use of a light-dark masking paradigm shows that expression of daily rest-wake activity in MPTP monkeys requires the stimulatory and inhibitory effects of light and darkness. These results suggest that following DA lesion, the central clock in the SCN remains intact but, in the absence of environmental timing cues, is unable to drive downstream rhythmic processes of striatal clock gene and dopaminergic functions that control locomotor output. These findings suggest that the circadian component of the sleep-wake disturbances in PD is more profoundly affected than previously assumed.