Molecular mechanism of phase-resetting by temperature

The molecular mechanism underlying the resetting of circadian clock by temperature in mammals is not known. In principle, temperature changes could act directly on the molecular oscillator by affecting the activity, synthesis or degradation rates of clock components.

Alternatively they could act indirectly, by changing the activity of temperature sensitive factors, which in turn could act on the expression and/or activity of clock constituents.

A direct action of temperature on clock component has already been described in N.crassa (see section 2.3.3). Thus, temperature changes directly modulate the ability of the 5’UTR of frq messenger to be scanned by ribosomes for translation initiation, resulting in temperature-dependent changes in FRQ protein abundance. However, analogous mechanisms have not yet been described in mammalian cells, although they may be relevant in this system as well.

A more indirect action of temperature on clock component has been described in Drosophila (see section 2.3.4). Indeed, PLC (phospholipase C) protein was shown to be involved in the temperature-dependent regulation of alternative splicing in the 3’ untranslated region of per mRNA. This splicing exhibits daily fluctuations and may influence transcript stability. Similarly, in mammals, temperature sensitive factors may act on clock components.

Interesting candidates that could fulfill such functions include heat shock transcription factors (HSFs), thermosensitive transient receptor potential (TRP) cation channels, and cold-induced RNA-binding protein (CIRP).

Previous results from our laboratory have shown that Per2 and Hsp genes are expressed in a cyclic fashion with very similar phases. Interestingly, they continue to be transcribed rhythmically with the same phase angle in a liver with an inactive clock, suggesting that their transcription is driven by systemic cues, possibly temperature rhythms (Kornmann et al., 2007a). In addition, the DNA-binding activity of HSF1 was shown to oscillate in a robust daily fashion, following the increase in body temperature in the evening (Reinke et al., 2008). Furthermore, Buhr and co-workers recently reported that KNK437, a pharmacological inhibitor of heat shock factors, compromised the phase-shifting by abrupt temperature changes of circadian clocks operative in tissue explants (Buhr et al., 2010). These findings raise the possibility that HSF1 might serve as an immediate early transcription factor in the temperature–dependent entrainment of circadian clocks.

TRP channels, involved in thermal transduction, are found in thermosensory neurons (see above), but they are also expressed in non-neuronal tissues, such as skin, tongue, trachea, liver or heart (Dhaka et al., 2006). This opens the possibility that non-neuronal cells could also directly sense temperature variations. Many substances can trigger Per1 and/or Per2 expression, and the synchronization of oscillators in cultured cells, and these include calcium ionophors that augment intracellular Ca2+ levels (Balsalobre et al., 2000b). Such increases in intracellular Ca2+ concentrations may also result from the activation of thermo TRPs in

peripheral cell types and may elicit a characteristic immediate early transcription program, similar to the clock resetting signaling pathway induced by light in SCN neurons. Moreover, the observation that the temperature pulse-induced PRC obtained with chicken pineal cells is qualitatively similar to the light-induced PRC in the same cells (Barrett and Takahashi, 1995), suggests that positive temperature changes and light signals might act through similar processes on molecular oscillators. Thus, we consider thermo TRPs channels as valid candidates in the temperature-dependent phase resetting of peripheral clocks.

Cirp has also been found by Kornmann and colleagues to be rhythmically expressed in liver cells with arrested clocks. However Cirp expression oscillates with a phase angle opposite to that observed for Per2 and Hsp expression. As insinuated by its name, cold-inducible RNA-binding protein, Cirp expression is maximal when mouse body temperature is minimal. In fact, Cirp has been identified in a screen for genes whose expression is induced in cultured cells by a temperature reduction (Fujita, 1999). It is thus conceivable that the cyclic expression of this RNA-binding protein is driven by body temperature rhythm in the intact animal, and that in turn it acts on circadian transcripts.

Clearly, the elucidation of the molecular mechanism responsible for the synchronization of mammalian circadian clocks by temperature still necessitates many more investigations.

2.6 Technical approaches for studying circadian rhythms

The study of circadian clock was initially limited to observations of physiological and behavioral manifestations, such as plant leaf movements, separation of nitrogen fixation and oxygenic photosynthesis in the cyanobacteria, formation of conidia in N.crassa, or locomotor activity in flies and mice. Forward genetic analysis to identify components of circadian clocks began in the 1970s. The identification of mutations conferring altered period length and the observation that the abundance of some transcripts exhibited a circadian rhythm represented crucial discoveries in the functional dissection of circadian clocks. Circadian clock function

could, therefore, be studied directly by monitoring circadian gene expression rhythms in tissues and cells from transgenic animals.

These initial experiments were quite labor-intensive, since clock-controlled gene expression had to be assayed over the course of one or multiple days. Population of cells, large heterogeneous tissue explants, or whole organisms had to be harvested at frequent intervals over fairly lengthy time courses for RNA extraction, RNA gel blotting, and nuclear run-on analyses. Such experiments inevitably became exercises in sleep deprivation for the experimenters and slightly impeded the recruitment of graduate students and postdocs to the field. Moreover, such approaches have at least three major limitations:

1) They are labor-intensive and therefore not ideal for achieving high temporal resolution of circadian gene expression.

2) They are not suitable for high throughput forward genetic screening of molecular circadian phenotypes.

3) Such assays do obviously not allow the recording of molecular clock components in the same individual (animal or cell) over time, making it difficult to separate defects in synchrony from defects in cell-autonomous clock functions.

Luciferases reporter genes offered a versatile class of non-invasive recording tools, providing an alternative method of assaying circadian gene expression that does not suffer from the above-mentioned limitations. The construction of reporters with the luciferase coding sequence placed under the control of a circadian promoter or fused to the sequence encoding a circadian protein allowed the isolation of a variety of clock mutants. Firefly luciferase mRNA (Luc) and protein (LUC) are sufficiently unstable (half-life of the protein

~2h) so that LUC activity can reliably be linked to gene expression, provided that the substrate luciferin is made available in unlimiting amounts. LUC catalyzes the ATP-dependent oxidative decarboxylation of luciferin, with the concomitant release of a photon at 560 nm. The use of luciferase reporter genes is now widespread in the circadian field (and

many other fields) and became a prevailing and powerful tool towards the understanding of circadian clocks.

Light emission (bioluminescence) can be readily quantified with luminometers, which are light-tight boxes equipped either with photons detectors called photomultiplier tubes (PMTs) or charge coupled device (CCD) cameras (Figure 21). PMTs are highly sensitive and respond very quickly to a flux of varying light intensity - however they do not provide any spatial information. The elucidation of many important questions, such as the role of intercellular communication within cells in circadian gene expression, the subcellular localization of circadian components, or the distinction between synchronization versus clock functions required tissue or cell imaging. This could be accomplished using microscopy together with sensitive (CCD) cameras for bioluminescence imaging. Long-term luciferase imaging of single cells has been elusive until recently; one major reason for this being the difficulty of maintaining cell cultures mounted on the microscope stage alive for long periods of time (Welsh et al., 2005). However, investigators of circadian clocks have begun to achieve single-cell resolution, leading to some very exciting results. Indeed it revealed important new insights about the nature of SCN and peripheral clocks. While bioluminescence from SCN slices exhibit robust self-sustained circadian oscillations for many days, the rhythms of clock gene expression in peripheral tissues explants or cultured fibroblasts tend to dampen after several cycles in vitro (Balsalobre et al., 1998; Yamazaki et al., 2000). This raised the question of whether peripheral cells might be incapable of self-sustained oscillations. Single cell analyses of SCN slices and cultured fibroblasts revealed that SCN neurons remain synchronized with one another, whereas independent fibroblasts oscillators progressively loose phase coherence (Nagoshi et al., 2004; Welsh et al., 2004; Yamaguchi et al., 2003). This demonstrated that the damping at the level of the fibroblasts population was the result of gradual loss of synchrony among individual oscillators, rather than the damping of individual cellular rhythms. Hence, individual fibroblasts, like SCN neurons, contain self-sustained and

Figure 21. Bioluminescence can be quantified using photomultiplier tubes (PMTs) or charge coupled device (CCD) cameras. (Top) The two most popular photomultiplier designs have configurations that place the sensitive photocathode element either on the side (side-on, left) or at the end (head-on, right) of the PMT. The photocathode is able to transfer the energy of an absorbed photon to an electron. The general anatomy of a PMT consists of a classical vacuum tube in which a glass or quartz window encases the photocathode and a chain of electron multipliers, known as dynodes, followed by an anode to complete the electrical circuit. Each time an electron impacts the inner wall of a dynode, multiple secondary electrons are emitted. These ejected photoelectrons have trajectories angled at the next bend in the dynode chain, which in turn emits a larger quantity of electrons angled at the next bend in the dynode chain. The effect occurs repeatedly, leading to an avalanche effect, with a gain exceeding 100 million. (Bottom) In a charged coupled device (CCD) camera, incoming photons generate electric charges that once in the detection area are rapidly transferred to the storage area, and then accumulated charge is transferred line by line to the horizontal serial register for readout in a normal FT-CCD (frame transfer CCD). At this point in an EM-CCD (electron multiplication CCD), a multiplication (charge multiplier) register is built into the horizontal register (left). When a signal electron charge is transferred from stage to stage, the signal charge is accelerated by applying high electric field. This high voltage is much greater than the normal horizontal transfer electrode voltage, and it generates an occasional extra electron-hole pair. Electrons are multiplied from stage to stage repeatedly in the gain register and high multiplication gain is achieved (right). These Figures are adapted from Olympus web site http://www.olympusfluoview.com/theory/pmtintro.html and Hamamatsu EM-CCD CAMERA technical note.

cell-autonomous circadian oscillators. In addition, single cell analysis of SCN slices unveiled a stable pattern of phase differences among cells: dorsal cells tended to peak earlier when compared to ventral cells. When dorsomedial or ventrolateral portions of the SCN were isolated from each other, cells in both portions remained rhythmic, but dorsomedial cells drifted out of synchronization, while ventrolateral cells remained synchronized (Yamaguchi et al., 2003). Similarly dissociated SCN neurons become desynchronized, whereas individual cells within an intact SCN remain tightly synchronized (Liu et al., 2007; Welsh et al., 1995).

These results suggest that intercellular coupling, mostly originating form the ventrolateral SCN, is required to allow for a tight synchronization among SCN neurons, and that peripheral tissues, whose circadian oscillations damp after several days in culture, are devoid of such coupling interactions (Figure 22). None of these results would have been possible without the single-cell resolution and long-term monitoring of rhythms.

In parallel to the luciferase boom, successful work has also been accomplished with fluorescent proteins. Fluorescence results from a process that occurs when certain molecules called fluorophores, fluorochromes or fluorescent dyes absorb energy provided by an external source, such as a lamp or a laser. This creates an excited unstable electronic state. When the fluorophore decays from this excited state, it emits fluorescent light. Each fluorescent molecule has its own spectral properties with excitation and emission spectra of characteristic wavelengths. Fluorescence is widely used in molecular biology and biochemistry laboratories for a variety of applications, including the study of circadian rhythms (LeSauter et al., 2003;

Nagoshi et al., 2004). However, the use of fluorescent reporters exhibits some disadvantages over the luciferase reporters for circadian purposes:

1) It requires exogenous illumination of relatively high energy that can be deleterious for the cells because of cytotoxic effects. Indeed, in their excited state, fluorescent molecules tend to react with molecular oxygen to produce free radicals, and these can be genotoxic or provoke damages of subcellular components. Hence, the accumulation of fluorecent proteins

Figure 22. Bioluminescence single cell imaging in SCN slices, dissociated SCN neurons from Cry2-/-

Per2::luc mice or primary fibroblasts from Per2::luc mice reveals the importance of intercellular coupling for stable and synchronized oscillations. Neurons within SCN cultured slices (left) are highly rhythmic and remain tightly synchronized, while dissociated individual neurons (center) or primary fibroblasts (right) show cell-autonomous, but gradually desynchronized oscillations. For SCN slices and SCN dissociated neurons, raster plots of bioluminescence shown at the bottom present 40 cells, with each horizontal line representing a single cell. For primary fibroblasts, luminescence rhythms of 75 cells are represented in the raster plot. Luminescence intensity data from all cells were normalized for amplitude, and then color-coded: higher than average values are red, and lower than average values are green. The cells are sorted in order of start phase, so that the emergence of desynchrony can be more easily appreciated. These Figures are taken from (Liu et al., 2007) and (Welsh et al., 2004)

can be monitored during about two to three days at a low exposure frequency, but such experiments cannot be performed during significantly longer time periods.

2) Although fluorescent signals are usually much stronger than luciferase-generated bioluminescence signals, autofluorescence background levels can be very high, sometimes even masking the fluorecence signal of the reporter itself. By contrast, background emission of bioluminescnece is generally negligeable.

3) Fluorescent proteins have usually relatively long half-lives. This renders the detection of circadian expression problematic or even impossible, and thus requires the molecular engineering of short-lived fluorescent protein, for example by fusion with destabilizing sequences or intrinsically unstable proteins.

Fluorescence is however more appropriate for:

1) Multiple target detection. Indeed, multicolor fluorescence allows the detection and resolution of multiple targets using fluorescent proteins that can be spectrally resolved. The ability to detect and analyze two or more labeled targets in the same sample eliminates sample-to-sample variation, is time-effective, and allows to visualize and track separate events or molecules within a single cell. In this context, recent efforts have been done to couple luminescence with fluorescence imaging to monitor circadian bioluminescence at single-cell resolution in complex tissues or cell populations. The use of a CCD camera, combined with epifluorescence microscopy, allows the identification of the cells of interest by fluorescence detection and subsequent luminescence recording of circadian events (Sellix et al., 2010).

2) Imaging of subcellular structures and rapid spatial dynamics (<1min). Indeed, bioluminescence signal is very dim so exposure times for sufficient detection may be relatively long, thus limiting spatial and/or temporal resolution.

During the recent years, the availability and improvement of instruments, analysis software and reagents, as well as the enterprise of creative approaches led to growing development and interest in whole body imaging in small animal models (Zinn et al., 2008).

Indeed, imaging in intact living animals can greatly enhance our understanding of biological processes in the context of functional tissues and organs within the complexity of the whole organism. Imaging in small animals is for example becoming an important component of biomedical research for studying diverse processes, such as the detection and tracking of tumors and metastases (Yang et al., 2000a), the identification and quantification of immune or stem cells after an adoptive transfer (Azadniv et al., 2007), the progression of bacteria, viruses or other pathogens infection over time (Ploemen et al., 2009), or the monitoring of therapeutic interventions (Katz et al., 2003). This approach is also of great interest to follow the tissue- or time-specific expression of target genes or to monitor biological processes such as signaling,

protein stability, or protein interactions in real time. Different methods can be employed for such purposes, including positron emission tomography (PET), magnetic resonance imaging (MRI), single photon emission tomography (SPECT), fluorescence or bioluminescence.

Fluorescence and bioluminescence recording are beginning to be widely now used for such aims (Contag and Bachmann, 2002).

In the circadian field, different transgenic animals expressing circadian reporters have already been established and studied in vivo (as mentioned above). However until now most of these analyses have been done on cultured tissues explants. Circadian whole body imaging would be extremely important for a better understanding of circadian clock function and synchronization in the context of the entire organism. The establishment of such approaches is in progress but remains hampered by several technical constraints. The mating of wild type rats females to Per1-luc transgenic males allowed the visualization of diurnal oscillation of Per1 expression several days prior to birth in pups in utero (Saxena et al., 2007). However, this study unveiled several problematic issues of bioluminescence whole body imaging in the circadian field. Indeed, the requirement of repeated anesthesia to obtain satisfactory temporal resolution of measures can be a source of troubles. First, it can perturb animal rhythms because of the anesthetic itself and/or because of the stress provoked by repeated handling. In addition, such experiment also requires repeated administration of the luciferase’s substrate, luciferin, and this manipulation can introduce variability into the results. A study designed to resolve this problem described the implantation of a micro-osmotic pump into the intraperitoneal cavity for the continuous delivery of luciferin in Per1-luc transgenic rats (Gross et al., 2007). However, the animals were still subjected to repeated anesthesia for the imaging of the olfactive bulb. Remarkably, using Per1-luc transgenic mice, the group of H.

Okamura was able to continuously record light emission from the SCN in vivo in mice kept in constant darkness (Yamaguchi et al., 2001). To avoid repeated anesthesia and luciferin injection, a polymer optical fiber was inserted just above the SCN using a stereotaxic frame

(the other end of the optical fiber was connected to a PMT) and luciferin dissolved in artificial cerebrospinal fluid was continuously infused through a lateral ventricle. Although this model system enables real-time gene expression to be monitored in the intact brain under physiological conditions, it remains rather invasive, stressful and restricts the free movement of animals. Alternatively, the group of R.M. Hoffman succeeded in live monitoring of GFP expression in various organs and tissues of nude mice (Yang et al., 2000b). The GFP coding sequence, under the control of the strong CMV promoter, was inserted in an adenoviral vector. The resulting viral particles were injected into the tissues of interest, and a high GFP signals could be measured in real time in unanesthetized mice using a light box illuminated by blue light fiber optics and a cooled color CCD camera. The possibility to apply such technique with a circadian promoter would be very exciting. Nevertheless, the much lower strength of circadian promoters and the short half-lives of circadian mRNAs and proteins render the application of this approach difficult, in particular for deep tissues analysis (signal/noise ratio). Moreover, the strong light pulses necessary to excite the fluorescent proteins would result in phase-shifting, and would thus only be compatible with circadian fluorescence recording in blind animals.

Real-time whole body imaging will undoubtedly open new doors in the circadian field and allow to obtain temporal information with greatly reduced number of animals sacrificed per experiment. Moreover, it will provide comprehensive assessments of individual animals over the entire duration of experiments. Although the available approaches represent impressive progress toward studying gene expression in real time in the organs of small

Real-time whole body imaging will undoubtedly open new doors in the circadian field and allow to obtain temporal information with greatly reduced number of animals sacrificed per experiment. Moreover, it will provide comprehensive assessments of individual animals over the entire duration of experiments. Although the available approaches represent impressive progress toward studying gene expression in real time in the organs of small