Arylalkylamine N-acetyltransferase (AANAT): Blue light induction, nuclear translocation, and potential role in the survival of chicken retina neuronal cells.

In vertebrates, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is the time-keeping and key regulatory enzyme in melatonin (Mel) biosynthesis. AANAT is present in the pineal gland, retina, and other regions where it is controlled by light, cyclic adenosine monophosphate (cAMP) levels, and the molecular clock. AANAT converts serotonin to N-acetyl serotonin (NAS) and the last enzyme in the pathway, hydroxy-o-methyltransferase (HIOMT), forms Mel by NAS methylation. We have previously shown that AANAT is expressed in chicken retinal ganglion cells (RGCs) during daytime at the level of mRNA and enzyme activity. Here we investigated the presence of AANAT protein and mRNA throughout development in the chicken embryonic retina as well as AANAT expression, phosphorylation, and its sub-cellular localization in primary cultures of retinal neurons from E10 embryonic retinas exposed to blue light (BL) and controls kept in the dark (D). From embryonic days 7-10 (E7-10) AANAT mRNA and protein were visualized mainly concentrated in the forming ganglion cell layer (GCL), while from E17 through postnatal days, expression was detectable all through the different retinal cell layers. At postnatal day 10 (PN10) when animals were subjected to a 12:12 h LD cycle, AANAT was mainly expressed in the GCL and inner nuclear layer cells at noon (Zeitgeber Time (ZT 6)) and in the photoreceptor cell layer at night (ZT 21). Primary cultures of retinal neurons exhibited an induction of AANAT protein when cells were exposed to BL for 1 h as compared with D controls. After BL exposure, AANAT showed a significant change in intracellular localization from the cytoplasm to the nucleus in the BL condition, remaining in the nucleus 1-2 h in the D after BL stimulation. BL induction of nuclear AANAT was substantially inhibited when cultures were treated with the protein synthesis inhibitor cycloheximide (CHD). Furthermore, the phosphorylated form of the enzyme (pAANAT) increased after BL in nuclear fractions obtained from primary cultures as compared with D controls. Finally, the knockdown of AANAT by sh-RNA in primary cultures affected cell viability regardless of the light condition. AANAT knockdown also affected the redox balance, sh-AANAT treated cultures showing higher levels of reactive oxygen species (ROS) than in the sh-control. Our results support the idea that AANAT is a BL-sensing enzyme in the inner retina of diurnal vertebrates, undergoing phosphorylation and nuclear importation in response to BL stimulation. Moreover, it can be inferred that AANAT plays a novel role in nuclear function, cell viability, and, likely, through redox balance regulation.

"Disruption of the molecular clock severely affects lipid metabolism in a hepatocellular carcinoma cell model".

Involved in triglyceride (TG) and glycerophospholipid metabolism, the liver plays a crucial physiological role in the human body both as a major metabolic integrator and a central hub for lipid and energy homeostasis. Metabolic disorders can be caused by various factors that promote abnormal lipid accumulation in storage organelles called lipid droplets (LDs), as in hepatic steatosis, a metabolic syndrome manifestation that can progress to a hepatocellular carcinoma, the most common primary liver malignancy worldwide. Modern life involves conditions that disrupt the biological clock, causing metabolic disorders and higher cancer risk. A circadian clock is present in the liver and in immortalized cell lines and temporally regulates physiological processes by driving transcriptional and metabolic rhythms. Here we investigated metabolic rhythms in HepG2 cells, a human hepatocellular carcinoma-derived cell line, and the link between these rhythms and the circadian clock in control (Bmal1-wildtype) and Bmal1-disrupted (B-D) cells having their molecular clock impaired. Rhythms in the expression of lipid-synthesizing enzymes ChoKα, Pcyt2, and Lipin1, in the metabolism of particular glycerophospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine, and in the phosphatidylcholine/phosphatidylethanolamine ratio and TG and LD content were observed in Bmal1-wildtype cells. By contrast, in the B-D model, the whole hepatic metabolism was severely altered with a significant reduction in the TG and LD content as well as in ChoKα and other related lipid enzymes. Together, our results suggest a very strong crosstalk between the molecular clock and lipid metabolism, which exhibits an exacerbated pathological condition in B-D cells.

Glial cell response to constant low light exposure in rat retina.

To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5-6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.

Circadian Regulation and Clock-Controlled Mechanisms of Glycerophospholipid Metabolism from Neuronal Cells and Tissues to Fibroblasts.

Along evolution, living organisms developed a precise timekeeping system, circadian clocks, to adapt life to the 24-h light/dark cycle and temporally regulate physiology and behavior. The transcriptional molecular circadian clock and metabolic/redox oscillator conforming these clocks are present in organs, tissues, and even in individual cells, where they exert circadian control over cellular metabolism. Disruption of the molecular clock may cause metabolic disorders and higher cancer risk. The synthesis and degradation of glycerophospholipids (GPLs) is one of the most highly regulated metabolisms across the 24-h cycle in terms of total lipid content and enzyme expression and activity in the nervous system and individual cells. Lipids play a plethora of roles (membrane biogenesis, energy sourcing, signaling, and the regulation of protein-chromatin interaction, among others), making control of their metabolism a vital checkpoint in the cellular organization of physiology. An increasing body of evidence clearly demonstrates an orchestrated and sequential series of events occurring in GPL metabolism across the 24-h day in diverse retinal cell layers, immortalized fibroblasts, and glioma cells. Moreover, the clock gene Per1 and other circadian-related genes are tightly involved in the regulation of GPL synthesis in quiescent cells. However, under proliferation, the metabolic oscillator continues to control GPL metabolism of brain cancer cells even after molecular circadian clock disruption, reflecting the crucial role of the temporal metabolism organization in cell preservation. The aim of this review is to examine the control exerted by circadian clocks over GPL metabolism, their synthesizing enzyme expression and activities in normal and tumorous cells of the nervous system and in immortalized fibroblasts.

Adjusting the Molecular Clock: The Importance of Circadian Rhythms in the Development of Glioblastomas and Its Intervention as a Therapeutic Strategy.

Gliomas are solid tumors of the central nervous system (CNS) that originated from different glial cells. The World Health Organization (WHO) classifies these tumors into four groups (I-IV) with increasing malignancy. Glioblastoma (GBM) is the most common and aggressive type of brain tumor classified as grade IV. GBMs are resistant to conventional therapies with poor prognosis after diagnosis even when the Stupp protocol that combines surgery and radiochemotherapy is applied. Nowadays, few novel therapeutic strategies have been used to improve GBM treatment, looking for higher efficiency and lower side effects, but with relatively modest results. The circadian timing system temporally organizes the physiology and behavior of most organisms and daily regulates several cellular processes in organs, tissues, and even in individual cells, including tumor cells. The potentiality of the function of the circadian clock on cancer cells modulation as a new target for novel treatments with a chronobiological basis offers a different challenge that needs to be considered in further detail. The present review will discuss state of the art regarding GBM biology, the role of the circadian clock in tumor progression, and new chrono-chemotherapeutic strategies applied for GBM treatment.

Temporal regulation of tumor growth in nocturnal mammals: In vivo studies and chemotherapeutical potential.

Tumors of the nervous system including glioblastoma multiforme (GBM) are the most frequent and aggressive form of brain tumors; however, little is known about the impact of the circadian timing system on the formation, growth, and treatment of these tumors. We investigated day/night differences in tumor growth after injection of A530 glioma cells isolated from malignant peripheral nerve sheath tumor (MPNSTs) of NPcis (Trp53+/- ; Nf1+/- ) mice. Synchronized A530 cell cultures expressing typical glial markers were injected at the beginning of the day or night into the sciatic nerve zone of C57BL/6 mice subject to a 12:12 hours light/dark (LD) cycle or after being released to constant darkness (DD). Tumors generated in animals injected early at night in the LD cycle or in DD showed higher growth rates than in animals injected diurnally. No differences were found when animals were injected at the same time with cultures synchronized 12 hours apart. Similar experiments performed with B16 melanoma cells showed higher tumor growth rates in animals injected at the beginning of the night compared to those injected in the daytime. A higher tumor growth rate than that in controls was observed when mice were injected with knocked-down clock gene Bmal1 cells. Finally, when we compared day/night administration of different doses of the proteasome inhibitor Bortezomib (0.5-1.5 mg/kg) in tumor-bearing animals, we found that low-dose chemotherapy displayed higher efficacy when administered at night. Results suggest the existence of a precise temporal control of tumor growth and of drug efficacy in which the host state and susceptibility are critical.

Proliferative Glioblastoma Cancer Cells Exhibit Persisting Temporal Control of Metabolism and Display Differential Temporal Drug Susceptibility in Chemotherapy.

Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, 32P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.

Identification and characterization of key circadian clock genes of tobacco hairy roots: putative regulatory role in xenobiotic metabolism.

The circadian clock is an endogenous system that allows organisms to daily adapt and optimize their physiology and metabolism. We studied the key circadian clock gene (CCG) orthologs in Nicotiana tabacum seedlings and in hairy root cultures (HRC). Putative genes involved in the metabolism of xenobiotic compounds (MXC) were selected and their expression profiles were also analyzed. Seedlings and HRC displayed similar diurnal variations in the expression profiles for the CCG examined under control conditions (CC). MXC-related genes also showed daily fluctuations with specific peaks of expression. However, when HRC were under phenol treatment (PT), the expression patterns of the clock and MXC-related genes were significantly affected. In 2-week-old HRC, PT downregulated the expression of NtLHY, NtTOC1, and NtPRR9 while NtFKF1 and NtGI genes were upregulated by phenol. In 3-week-old HRC, PT also downregulated the expression of all CCG analyzed and NtTOC1 was the most affected. Following PT, the expression of the MXC-related genes was upregulated or displayed an anti-phasic expression profile compared to the expression under CC. Our studies thus provide a glimpse of the circadian expression of clock genes in tobacco and the use of HRC as a convenient system to study plant responses to xenobiotic stresses.

Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina.

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+) mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+) levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+) mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

Differential responses of the mammalian retinal ganglion cell line RGC-5 to physiological stimuli and trophic factors.

The rat retinal ganglion cell (RGC) line RGC-5 constitutes a widely used model for studying physiological processes in retinal cells. In this paper we investigated the expression of clock and immediately early genes, and calcium mediated responses to physiological stimuli in differentiated and mitotically active RGC-5 cells. To this end, we attempted to differentiate the RGC-5 cells with a variety of effectors classically used to induce morphological differentiation. No sign of morphological differentiation was observed after 24 h of treatment with BDNF (80 ng/mL), NGF (100 ng/mL) and retinoic acid (20 ng/mL), among others. Only staurosporine (SSP) was able to promote neurite outgrowth at concentrations ranging from 53.5 to 214 nM. However, apoptotic nuclei were seen at 24 h of treatment using DNA staining, and a few cells remained at 72 h post-treatment. Concentrations of SSP lower than 214 nM were partially effective in inducing cell differentiation. Dividing RGC-5 cells express the RGC marker Thy-1 and different clock genes such as Per1, Clock and Bmal1. When characterizing the responsiveness of proliferative RGC-5 cells we found that in most of them, brief pulses of 50% FBS induced c-Fos and PER1 expression. Subsets of RGC-5 cells displayed significant changes in intracellular Ca2+ levels by ATP (100 microM) but not by glutamate (100-200 microM) stimulation. On the basis of cell morphology, size and complexity and effector responsiveness it was possible to distinguish different subpopulations within the cell line. The results demonstrate that only SSP is effective in promoting RGC-5 morphological differentiation, though the treatment provoked cell death. Proliferative cells expressing the RGC marker Thy-1 and a number of clock genes, responded differentially to diverse physiological stimuli showing a rapid c-Fos and PER1 induction by FBS stimulation, and an increase in intracellular Ca2+ by ATP.

Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day.

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.

c-Fos activated phospholipid synthesis is required for neurite elongation in differentiating PC12 cells.

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.