Redox-Mediated Rewiring of Signalling Pathways: The Role of a Cellular Clock in Brain Health and Disease.

Metazoan signalling pathways can be rewired to dampen or amplify the rate of events, such as those that occur in development and aging. Given that a linear network topology restricts the capacity to rewire signalling pathways, such scalability of the pace of biological events suggests the existence of programmable non-linear elements in the underlying signalling pathways. Here, we review the network topology of key signalling pathways with a focus on redox-sensitive proteins, including PTEN and Ras GTPase, that reshape the connectivity profile of signalling pathways in response to an altered redox state. While this network-level impact of redox is achieved by the modulation of individual redox-sensitive proteins, it is the population by these proteins of critical nodes in a network topology of signal transduction pathways that amplifies the impact of redox-mediated reprogramming. We propose that redox-mediated rewiring is essential to regulate the rate of transmission of biological signals, giving rise to a programmable cellular clock that orchestrates the pace of biological phenomena such as development and aging. We further review the evidence that an aberrant redox-mediated modulation of output of the cellular clock contributes to the emergence of pathological conditions affecting the human brain.

Using ALLIGATORs to Capture Circadian Bioluminescence.

Luciferases are a popular tool in circadian biology research as longitudinal reporters of gene expression. Here, we describe a short updated protocol for the use of an Automated Longitudinal Luciferase Imaging Gas and Temperature-Optimized Recorder (ALLIGATOR) to record cellular bioluminescence over many days. The ALLIGATOR has superior capacity and flexibility compared with traditional luminometers that employ photomultiplier tubes (PMTs), with high-throughput capability and spatial resolution. It can be readily adapted to a wide variety of applications, such as different sample types and plate sizes, under a wide range of physiologically relevant conditions.

CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.

The Use of Chemical Compounds to Identify the Regulatory Mechanisms of Vertebrate Circadian Clocks.

Circadian clocks are intrinsic, time-tracking processes that confer a survival advantage on an organism. Under natural conditions, they follow approximately a 24-h day, modulated by environmental time cues, such as light, to maximize an organism's physiological efficiency. The exact timing of this rhythm is established by cell-autonomous oscillators called cellular clocks, which are controlled by transcription-translation negative feedback loops. Studies of cell-based systems and wholeanimal models have utilized a pharmacological approach in which chemical compounds are used to identify molecular mechanisms capable of establishing and maintaining cellular clocks, such as posttranslational modifications of cellular clock regulators, chromatin remodeling of cellular clock target genes' promoters, and stability control of cellular clock components. In addition, studies with chemical compounds have contributed to the characterization of light-signaling pathways and their impact on the cellular clock. Here, the use of chemical compounds to study the molecular, cellular, and behavioral aspects of the vertebrate circadian clock system is described.

Post-translational Modifications are Required for Circadian Clock Regulation in Vertebrates.

Circadian clocks are intrinsic, time-tracking systems that bestow upon organisms a survival advantage. Under natural conditions, organisms are trained to follow a 24-h cycle under environmental time cues such as light to maximize their physiological efficiency. The exact timing of this rhythm is established via cell-autonomous oscillators called cellular clocks, which are controlled by transcription/translation-based negative feedback loops. Studies using cell-based systems and genetic techniques have identified the molecular mechanisms that establish and maintain cellular clocks. One such mechanism, known as post-translational modification, regulates several aspects of these cellular clock components, including their stability, subcellular localization, transcriptional activity, and interaction with other proteins and signaling pathways. In addition, these mechanisms contribute to the integration of external signals into the cellular clock machinery. Here, we describe the post-translational modifications of cellular clock regulators that regulate circadian clocks in vertebrates.

Cyclic Variation of Cellular Clock Proteins in the Mouse Estrous Ovary.

BACKGROUND: The mammalian ovary is controlled by a number of biological rhythms, which regulate the recruitment and release of mature oocytes. The main objective of this study was to investigate the role of cellular clock proteins during follicle maturation in the mouse estrous ovary. METHODS: Immunohistochemical (IHC) studies were performed on ovaries from 50 estrous staged mice culled at two time points of 09:00 [day] and 01:00 [mid-point of the dark cycle]. Six antibodies were used to identify the expression of core cellular clock proteins (BMAL1, CLOCK, CRY1, CRY2, PER1 and PER2) within the ovary and four follicle stages, primordial, primary, antral and corpus lutea. IHC data was scored using the Allred protocol and significance determined by Mann-Whitney tests. Differences were considered significant at p<0.05. RESULTS: All four follicle stages presented greater BMAL1 and CLOCK protein scores during the day and up regulation of CRY1-2 and PER1-2 at night. In primordial follicles, BMAL1 and CLOCK increases were significant (p<0.05) and CRY-1 and PER-1 were highly significant (p<0.001), and CRY-2 did not reach significance. Primary follicles demonstrated a similar response with BMAL1 and CLOCK, and CRY-1, PER-1-2 all reaching significant expression (p<0.05; p<0.001; p<0.001 respectively). CRY-2 expression was not significant. Antral follicles did not show significant BMAL1 or CLOCK expression, CRY-1 and PER-1 were highly significant (p<0.001) and CRY-2 had a small but significant increase (p<0.05). Corpus lutea demonstrated significant BMAL1 increase but CLOCK had no significant variation. CRY-1, PER1-2 increases were highly significant (p<0.001) and CRY-2 was up regulated but failed to reach significance. CONCLUSION: The ovary demonstrated a cellular clock response to the light: dark cycle and in addition, as the ovarian follicles mature changes in the positive and negative arms of both clock responsive proteins were observed.