Disrupted circadian clock leads to an increased risk of retinal degeneration as we age


While bright light helps us see better, our eyes need darkness for better vision. Light breaks down the sensitive machinery of our eyes every day, and during the darkness of night, key pieces are rebuilt.

The clock of our circadian rhythms runs this process, and researchers have found that if the clock is disrupted, our eyes may be at greater risk of retinal degeneration as we age.

“Imagine if we could slow or prevent vision loss from retinal degeneration,” said Vikki Weake, associate professor of biochemistry in Purdue University’s College of Agriculture, who led the study.

“To do this, we need to understand the molecular mechanisms that drive age-associated changes and the external and internal factors that influence them. In this study, we discovered the circadian clock plays a surprisingly significant role in age-related changes in the retina. This internal clock may be critical in advanced age to prevent retinal degeneration and maintain eye health.”

The team studied the eyes of Drosophila flies, a common model for the human eye. However, the study was uncommon in its use of multiple time points during aging, focus on photoreceptor neurons and new data analysis approaches.

The findings are detailed in a paper in PLOS Genetics.

“In our earlier studies, just focusing on gene expression, we were missing part of the story,” Weake said. “By looking at changes in chromatin that alter access to the underlying DNA during aging, we were able to identify some of the transcription factors that drive these gene expression changes in the aging eye.”

Weake acknowledges doctoral student Juan “Jupa” Jauregui-Lozano for the idea for and application of the bioinformatics technique used.

“I came across a powerful bioinformatics technique that can identify changes in transcription factor activity, helping us to understand gene regulation,” Jauregui-Lozano said.

“The results revealed that the transcription factors Clock and Cycle – known for their role in circadian rhythmshowed progressive changes in activity with age. This fits with what we know about eye biology, and this unbiased approach led us to identify Clock and Cycle as interesting targets to study.”

The technique, called diffTF, looks at changes in DNA accessibility in chromatin between different conditions. It generates a panel of potential candidates to pursue, as opposed to a research team beginning with a target gene in mind.

“Clock and Cycle were known for being master regulators of circadian rhythms, but we saw they also regulate nearly all of the genes involved in sensing light in the retina,” Jauregui-Lozano said.

“When the Clock:Cycle complex is disrupted, flies are susceptible to light-dependent retinal degeneration, and light-independent increase of oxidative stress. In humans, disruption of circadian rhythms has been associated with the onset of several age-related eye diseases. This is another piece of the puzzle.”

Regulating the time at which these proteins are made is important to protect the light-sensing neurons and retain vision, Weake said.

“The proteins involved in sensing light are delicate and degrade during the day when they are exposed to light,” she said. “If the circadian clock is off and these proteins aren’t made at the right time, it’s a problem.”

The study found this complex controlled gene expression of nearly 20% of the active genes in Drosophila photoreceptors. The study also found the complex was responsible for maintaining global levels of chromatin accessibility in photoreceptors, a critical step in transcription of genes.

Co-author Hana Hall, research assistant professor of biochemistry at Purdue, performed light and dark experiments to see the effect on gene transcription when she was a researcher in Weake’s lab.

Unlike most cells in the human body, neurons don’t divide and replicate. The death of neurons lead to degenerative disease, Hall said. Because of this the cellular processes involved in repairing and regulating them are especially important. Proteins achieve this, and genes control which proteins are produced.

“Aging is the main risk factor for neurodegenerative disease,” Hall said.

“If we can understand the mechanics of how things get off track or become misregulated in our later years, we may be able to prevent or slow down the progression of these diseases. Vision loss affects a person’s lifespan, independence and quality of life. Even delaying onset by five years could make a tremendous difference. We have ideas, and we are going to seek the answers.”

Circadian rhythms have been observed in animals, plants, fungi and even cyanobacteria. In mammals, including humans, the master pacemaker controlling 24-hour rhythms is localized in the suprachiasmatic nuclei of the hypothalamus (SCN). The SCN is responsible for orchestrating circadian clocks in peripheral organs to regulate physiological functions such as behavior, sleep, body temperature, blood pressure and hormone release (Herzog and Tosini 2001). Accumulating evidence indicates that dysfunction of the circadian rhythms due to genetic mutations or environmental factors (i.e., jet-lag or shift work) may contribute to the development of many serious diseases, including cancer and type-2 diabetes (Evans and Davidson 2013).

The retinal circadian clock was the first extra-SCN circadian oscillator to be discovered in mammals (Tosini and Menaker 1996). The molecular clockwork mechanism of the retinal clock is similar to what has been reported for the SCN (Tosini et al. 2008), albeit it appears that the retinal clock is less robust (Ruan et al. 2012; Jaeger et al. 2015). Several studies have also established that many aspects of retinal physiology and function are under the control of retinal circadian clocks (see McMahon et al. 2014 for a review) and new experimental evidence suggests that other ocular structures (e.g., cornea, retinal pigment epithelium) also possess circadian clocks that control important physiological functions (Yoo et al. 2005; Baba et al. 2010; Baba et al. 2015, Buhr et al. 2015). Interestingly, as seen in the SCN, it appears that the neural retina communicates the photic information to the other ocular structures via humoral signals (e.g., melatonin and dopamine, Ruan et al. 2008, Baba et al. 2015) since most of these ocular structures are not capable of direct light transduction (Baba et al. 2010).

XX.2 The Retinal Circadian Clock and Ocular health
Similar to molecular circadian clock in SCN, the retinal clock also consists of auto-regulatory transcriptional/translational negative feedback loops involving several clock genes and their protein products which generate approximately 24 hours cycle. The primary core loop involves two basic helix-loop-helix-PAS domain transcription factors, BMAL1 and CLOCK, which heterodimerize and bind to E-box elements in promoter region to enhance transcription of Period 1 and 2, and Cryptochrome 1and 2. The protein products, PERIOD and CRYPTOCHROME together then inhibit their own transcription by blocking CLOCK/BMAL1-mediated transactivation (see Tosini et al. 2008, Figure 1 for a schematic illustration).

The second feedback loop involves the negative and positive transactivation of five other genes, Rev-erb α, β and Ror α, β, c via REV-ERB/ROR response element (RRE) promoter elements in promoter regions. REV-ERB as a negative element inhibits Clock and Bmal1 transcription whereas ROR as a positive element promotes Clock and Bmal1 transcription. The transcriptions of Rev-erbs and Rors are regulated via E-box elements in their promoter regions. REV-ERBs and RORs compete for binding to RRE in the Bmal1 promoter regions to regulate rhythmic expressions of Bmal1.

These intertwining oscillation signals also regulate transcription of other clock controlled genes via E-box or RRE elements, and the products of these genes serve as circadian clock outputs (Takahashi et al. 2008). In the mouse, clock genes are rhythmically expressed in the different retinal layers (Hiragaki et al. 2014). In the photoreceptor layer only the cones appear to express all the circadian clock proteins (Lui et al. 2012).

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Fig. 1
Morphometric analysis of retinae obtained from young (3 months old, black bars) and old (8 months old, white bars) of control, Bmal1−/− and Clock−/−/Npas2−/− (Cl/Np2) mice. The number of cells in the ONL of 8 months old Bmal1 (B) and Clock/Npas2 KOs (D) were significantly lower than the number of cells in 3 months old mice (A, C) of the same genotype (One-way ANOVA following post hoc test * p< 0.05) and age matched control group (# p<0.05). No differences in the number of cells were observed in the INL and/or GCL (n= 3-4). The number of cells in the ONL of young Bmal1 and Clock/Npas2 KO mice was not different from the number of cells in control mice of the same genotype (C57BL/6). The microphotographs in A, B, C and D represent a typical example of a section obtained from 3 months old Bmal1 KO (A), 8 months old Bmal1 KO (B), 3 months old Clock/Npas2 KO (C) and 8 months old Clock/Npas2 KO (D).

Emerging evidence suggests that retinal circadian clocks and their output signals contribute to retinal disease and pathology, as well as normal retinal function. For example, diabetic retinopathy is associated with reduced clock gene expression in the retina (Busik et al. 2009), circadian disruption recapitulates diabetic retinopathy in mice (Bhatwadekar et al. 2013), and removal of Period2 induces dysfunction in the retinal microvasculature (Jadhav et al. 2016).

Trophic signaling by the retinal clock and its outputs seem to play a role in the regulation of eye growth and refractive errors (reviewed in: Stone et al. 2013). A recent study has also reported that mice lacking Period1 and Period2 show significant alteration in the distribution of cone photoreceptors (Ait-Hmyed et al. 2013) and mice lacking Rev-erb α show a significant alteration in photoreceptor response to light (Ait-Hmyed et al. 2016).

Finally, it is worthwhile mentioning that the retinal clock influences the susceptibility of photoreceptors to light induced damage (Organisciak et al. 2000) and recent genomic studies have also implicated the clock genes Rev-erbα and Rora in retinal functioning (Mollema et al. 2011) and age-related macular degeneration (Jun et al. 2011).

XX.3 Bmal1 and retinal cell viability
As previously mentioned, Bmal1 gene (also known as Arntl) is a key component of the mammalian circadian clock. Bmal1 knock-out mice (Bmal1−/−) do not show any circadian rhythmicity (Bunger et al. 2000) and develop several pathologies (Kondrakov et al. 2006). Bmal1−/− mice show premature aging and their lifespan is significantly reduced (about 9 months) (Kondratov et al. 2006). In the mouse retina, Bmal1 is expressed in many cell types, (Ruan et al. 2008), but within the photoreceptor layer BMAL1 was only detected in the cones (Liu et al. 2012). Storch et al. (2007) reported that many genes (more than a thousand) show a daily rhythm in mouse retina, but a large fraction of these genes are no longer rhythmically expressed or have reduced amplitude in Bmal1−/− mice. In Bmal1−/− mice, the day/night (circadian) changes in the amplitude of the photopic b-wave are no longer present (Storch et al. 2007). The same result has been also obtained from the mice lacking Bmal1 only in the retina (Chx10-Cre-ArntlloxP/loxP mice; Storch et al. 2007), thus indicating that retinal Bmal1 is required for the circadian rhythm in visual processing. Interestingly, the photoreceptors of these mice (2-3 months) appear to be normal and unaffected by the lack of Bmal1 (Storch et al. 2007). Additional studies have reported that in mice lacking the Bmal1 gene there is a significant increase in the rate of cataract development and corneal inflammation during aging (Kondratov et al. 2006).

Previous studies have shown that the effects of circadian disruption become evident during the aging process (Baba et al. 2009; Musiek et al. 2013). Hence we decided to investigate whether removal of the Bmal1 gene affects retinal cell viability during aging. Eyes from Bmal1−/− and control mice at two different ages (3 months and 7-8 months) were obtained and then the morphometric analysis of the retina was performed according to a well-established method in our laboratory (see Baba et al. 2009 for details). As expected, and previously reported by Storch et al. (2007), young Bmal1−/− did not show any significant variation in the number of cells in the outer nuclear layer or in any other retinal layers (Figure 1) whereas in older Bmal1−/− mice we observed a significant reduction in the number of photoreceptor cell nuclei (about 20-30%) with respect to control.

No changes were detected in the other retinal layers (Figure 1). Previous studies have shown that Bmal1 can interact with a large number of genes (more than 1000) and therefore the phenotypes observed in Bmal1 KOs may or may not be the consequence of a dysfunctional circadian clock (Rey et al. 2011). Thus we decided to investigate the retinal morphometry in another mouse model in which the circadian clock has been disabled. A previous investigation reported that Clock/Npas2 KO mice do not have a functional circadian clock (DeBruyne et al. 2007, Musiek et al. 2013).

Eyes from young (3months) and old (9 months) Clock/Npas2 KO mice were obtained from Dr. David Weaver’s laboratory (University of Massachusetts Medical School) and the retinal morphometry was investigated using the same method mentioned for Bmal1−/− mice retinas. As shown in Figure 1, Clock/Npas2 KO mice showed an almost identical phenotype as Bmal1−/− animals. The fact that almost identical results were obtained in Bmal1 and Clock/Npas2 KOs indicates that the reduced photoreceptor viability observed in Bmal1−/− is likely due lack of a functional circadian clock in these cells and not to a possible pleiotropic effect of Bmal1. Our preliminary data indicate that dysfunctions of circadian clock genes may affect the photoreceptor cell viability during aging.

reference link : https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC6003627/

Original Research: Open access.
The Clock: Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress” by Juan Jauregui-Lozano et al. PLOS Genetics


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