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11. Colegio, O.R., Chu, N.Q., Szabo, A.L., Chu, T., Rhebergen, A.M., Jairam, V., Cyrus, N., Brokowski, C.E., Eisenbarth, S.C., Phillips, G.M., et al. (2014). Functional polarization of tumourassociated macrophages by tumour-derived lactic acid. Nature 513, 559–563. 12. Huang, Q., Li, F., Liu, X., Li, W., Shi, W., Liu, F.F., O’Sullivan, B., He, Z., Peng, Y., Tan, A.C., et al. (2011). Caspase 3-mediated stimulation of tumor cell repopulation during cancer radiotherapy. Nat. Med. 17, 860–866. 13. Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell 144, 646–674. 14. Beroukhim, R., Mermel, C.H., Porter, D., Wei, G., Raychaudhuri, S., Donovan, J., Barretina, J., Boehm, J.S., Dobson, J., Urashima, M., et al. (2010). The landscape of somatic copy-number alteration across human cancers. Nature 463, 899–905.

15. Fesik, S.W. (2005). Promoting apoptosis as a strategy for cancer drug discovery. Nat. Rev. Cancer 5, 876–885. 16. Marin, M.C., Hsu, B., Stephens, L.C., Brisbay, S., and McDonnell, T.J. (1995). The functional basis of c-myc and bcl-2 complementation during multistep lymphomagenesis in vivo. Exp. Cell Res. 217, 240–247. 17. Lauber, K., Munoz, L.E., Berens, C., Jendrossek, V., Belka, C., and Herrmann, M. (2011). Apoptosis induction and tumor cell repopulation: the yin and yang of radiotherapy. Radiation Oncol. 6, 176. 18. Ni Chonghaile, T., and Letai, A. (2008). Mimicking the BH3 domain to kill cancer cells. Oncogene 27 (Suppl 1 ), S149–S157. 19. Kroemer, G., Galluzzi, L., Kepp, O., and Zitvogel, L. (2013). Immunogenic cell death in cancer therapy. Annu. Rev. Immunol. 31, 51–72.

Circadian Biology: Rhythms Leave Their Imprint A recent study has revealed that loss of neuronal expression of the paternally imprinted gene Ube3a in Angelman syndrome results in selective neuronal loss of robust circadian oscillations, with a resulting behavioural phenotype, and adipose tissue accumulation. David W. Ray Life evolved on Earth under conditions of marked environmental oscillation: cyclical transitions from day to night. Under these conditions complex life apportioned specific activities to particular phases, for example photosynthesis during the light phase, or sleep during the dark phase. However, such adaptations were driven by internal rhythms, rather than being a mere reaction to the change in environment; i.e., a biological clock. In a recent study reported in this issue of Current Biology from Shi et al. [1] new insights have been gained into how the biological clock is controlled, and with what consequences for behavior. The molecular basis of the circadian (circa; about, diem; day) clock has been elucidated, and the current model is of a transcriptional and translational feedback loop. This now includes many different molecular components, some of which are rhythmically expressed, or modified, and which participate in feedback loops to modulate one another’s expression. The core circuit involves an activating arm provided by the bHLH-PAS transcription factor BMAL1/ARNTL, which dimerises with CLOCK, or NPAS2. The heterodimeric transcription factor activates

expression of components of the repressive feedback arm of the clock, namely, PERIOD and CRYPTOCHROME. A core requirement for the clock to work is that at least one central clock component shows a rhythmic change in abundance of activity. Indeed, both PERIOD and CRYPTOCHROME proteins shows a rhythmic oscillation. Ubiquitinylation of CRYPTOCHROME, with resulting effects on protein turnover and circadian period, has been discovered to be due to the F-box proteins Fbxl3 and Fbxl21. However, it was not thought that BMAL1 oscillation was required for core clock function, or that such a regulatory pathway existed. However, a ubiquitinylation pathway mediated by Ube3a that regulates BMAL1 abundance and affects clock function was revealed recently in vitro. Ube3a is encoded on human chromosome 15q11-q13. This is of interest as deletions of the maternal copy of chromosome 15 in this region are causative for the complex neurodevelopmental disorder Angelman syndrome (AS). AS is characterized by a neurodevelopmental defect resulting in motor impairments, learning difficulties, epilepsy, and sleep disorders [2,3]. There is no treatment,

20. Bondanza, A., Zimmermann, V.S., RovereQuerini, P., Turnay, J., Dumitriu, I.E., Stach, C.M., Voll, R.E., Gaipl, U.S., Bertling, W., Poschl, E., et al. (2004). Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo. J. Exp. Med. 200, 1157–1165. 1Clinic

for Radiotherapy and Radiation Oncology, Ludwig-Maximilians-University Munich, Germany. 2Department of Internal Medicine 3, Friedrich-Alexander-University Erlangen-Nuremberg, Germany. *E-mail: [email protected] http://dx.doi.org/10.1016/j.cub.2015.01.040

and management is targeted at symptom control, with most patients requiring medication to control epilepsy. AS results from genomic imprinting, as the paternal allele is imprinted, and so not expressed. Therefore, loss of the maternal allele results in loss of Ube3a expression in tissues affected by the imprinting. In AS, paternal imprinting is only seen in the brain, so loss of Ube3a expression is seen only in brain neurons, and not glia or peripheral tissues [4]. These observations suggest a fascinating mechanistic link between loss of neuronal Ube3a expression, leading to altered BMAL1 ubiquitinylation [5] and stability, and circadian disruption, leading to sleep disturbance, specifically short sleep duration and increased sleep onset latency. In the study reported in this issue Shi et al. [1] identify a causal link between Ube3a expression and circadian rhythm in mouse models of AS. They report weakened rhythms in vivo, which is manifest by more rapid re-setting of the circadian oscillation to changes in environmental light timing, an experimental model of jet lag or shift work. Similarly, under constant light conditions the AS model animals show an accelerated decay in rhythmic behavior. Analyses of tissue ex vivo by Shi et al. provided new insight into the causative mechanisms of AS in their model animals. While no detectable differences in circadian oscillations in peripheral tissues were seen in AS animals compared to controls, the central brain clock located in the suprachiasmatic nucleus had a

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Figure 1. The imprinted gene Ube3a controls BMAL1 protein stability and thereby circadian control of sleep and activity. The paternally imprinted gene Ube3a encodes the Ubiquitin ligase E6-AP, which targets BMAL1 for degradation. Loss of neuronal Ube3a expression resulting from maternal allele loss causes BMAL1 stabilisation, disruption of circadian oscillation, and disturbance of sleep. As is often seen with circadian disruption there is a fat accumulation phenotype, possibly due to reduced energy expenditure.

significantly prolonged period, with an accompanying reduction in damping rate. These data thus suggest a core circadian oscillator problem, and one restricted to neural structures. As mentioned, the defect in Ube3a expression results from silencing of the paternal allele. But by using a topoisomerase inhibitor, which opens the targeted chromatin and makes it permissive for transcription, it is possible to reverse this silencing. Indeed, Shi et al. showed that such treatment restored circadian period in brain slice cultures from AS mice [1]. Such a novel pharmacological approach to targeting clock defects opens up attractive new avenues for both scientific exploration, but also therapeutic application.

A consistent and robust connection has emerged between defects in the core circadian machinery, or environmental disruption of circadian function, and important physiological functions, such as energy homeostasis [6] and immunity [7]. It appears that the switch from fed to fasted states is tightly controlled by the central clock, and requires coordination between food seeking behaviours, digestion, and energy partitioning between liver, muscle, and adipose tissue. And as shown by Shi et al., disruption of brain Ube3a expression in the AS mouse model in turn results in increased adipose tissue deposition. This was not accompanied by changes in energy intake, and did not require a high-energy or high-fat diet. However, the AS mice did show

reduced overall activity with a change in the timing of peak movement to later in the dark period than typically seen in wild-type mice [1]. Therefore, fat accumulation may have resulted from reduced overall energy consumption, although the neural mechanisms underlying this change remain unclear. These new observations provide new insights into the operation of the transcriptional–translational core clock. The rhythmic abundance of PERIOD and CRYPTOCHROME proteins is essential for clock oscillation, but BMAL1 variation was not thought to be required. However, as loss of Ube3a results in stabilization of BMAL1 protein and prolongation of the circadian period with resulting weakening of the cellular circadian oscillator, this assumption is challenged. It is counter intuitive that stabilizing BMAL1 protein would result in a weak oscillation. However, important recent observations suggest that some nutritional inputs to the cellular clock also act by regulating BMAL1 protein stability [8]. Taken together these discoveries suggest that the positive arm of the clock is indeed sensitive to environmental change, and that such effects are not restricted to the negative arm (i.e., PERIOD and CRYPTOCHROME) as previously thought (see Figure 1 for a potential mechanism). A further intriguing suggestion is that the sleep disruption seen in AS patients may be a direct result of central circadian disruption and that appropriate environmental cues could offer an attractive means to therapeutically intervene. Such an initiative might be expected to not only offer low risk benefit to the management of sleep disruption for the affected patients and their careers, but also have an impact on the obesity phenotype seen in AS, and the related Ube3a imprinting disorder Prader-Willi syndrome. References 1. Shi, S.-Q., Bichell, T.J., Ihrie, R.A., and Johnson, C.H. (2015). Ube3a imprinting impairs circadian robustness in Angelman Syndrome models. Curr. Biol. 25, 537–545. 2. Robb, S.A., Pohl, K.R.E., Wilson, B.J., and Brett, E.M. (1989). The ‘happy puppet’ syndrome of Angelman: review of the clinical features. Arch. Dis. Child. 64, 83–86. 3. Takaesu, Y., Komada, Y., and Inoue, Y. (2012). Melatonin profile and its relation to circadian rhythm sleep disorders in Angelman syndrome patients. Sleep Med. 13, 1164–1170.

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4. Weeber, E.J., Jiang, Y.H., Elgersma, Y., Varga, A.W., Carrasquillo, Y., Brown, S.E., Christian, J.M., Mirnikjoo, B., Silva, A., et al. (2003). Derangements of hippocampal calcium/ calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome. J. Neurosci. 23, 2634–2644. 5. Gossan, N.C., Zhang, F., Guo, B., Jin, D., Yoshitane, H., Yao, A., Glossop, N., Zhang, Y.Q., Fukada, Y., and Meng, Q.J. (2014). The E3 ubiquitin ligase Ube3a is an integral component of the molecular circadian clock through regulating the BMAL1

transcription factor. Nucleic Acids Res. 42, 5765–5775. 6. Asher, G., and Schibler, U. (2011). Crosstalk between components of circadian and metabolic cycles in mammals. Cell Metab. 13, 125–137. 7. Gibbs, J., Ince, L., Matthews, L., Mei, J., Bell, T., Yang, N., Saer, B., Begley, N., Poolman, T., Pariollaud, M., et al. (2014). An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action. Nat. Med. 20, 919–926. 8. Zhang, L., Abraham, D., Lin, S.T., Oster, H., ek, L.J. (2012). Eichele, G., Fu, Y.H., and Pta´c

Cortical Control: Learning from the Lamprey The function of the motor cortex has been a persistent mystery. A recent study has found striking correspondence between the descending projections of lamprey pallium and mammalian motor cortex, encouraging comparative studies of the origin (and role) of forebrain motor control. Gonc¸alo Lopes and Adam R. Kampff* Motor cortex is a confusing part of cortex: over a hundred years of research [1–6] has implicated motor cortex in the control of movement, but when it is completely removed most mammals recover to perform much of their behavioural repertoire [7,8]. Some of this recovery can be attributed to the brain’s plasticity [9], but are there aspects of behaviour for which motor cortex is absolutely required? Some clarity has emerged from studying primates. When motor cortex is lesioned in primates (including humans), a long-lasting deficit in dexterous movement results [10,11]. However, these same dexterity deficits can be induced by severing the direct projection from motor cortex to spinal cord [4], which is by no means the only pathway from cortex to movement (Figure 1). Motor cortex targets many other brain regions that can themselves generate movement. In fact, the direct connection from cortex to spinal cord appeared only recently in vertebrate evolution, and was further elaborated to include a direct connection from cortex to motor neurons in only some primate species [12]. In other mammals, such as rodents, the cortex’s projection to spinal cord does not contact motor neurons [13] and largely avoids ventral (motor) spinal cord [6,14]. It thus seems likely that most mammals rely on ‘indirect’ pathways to convey cortical motor

commands to spinal cord. What movements do these pathways control? What is their function? These are important questions for which many patients suffering from motor cortical stroke, and experiencing more than just deficits of dexterity, are looking to neuroscientists for answers [15]. We can gain insight into the origin and role of cortical motor projections by studying the extant (current) form of mammalian relatives — birds, reptiles, amphibians and so on — and the homologous brain region, the pallium. But how ‘far back’ in evolution can we go? In a study published recently in Current Biology, Ocan˜a et al. [16] investigated whether a primordial pallial motor centre was already present in one of our most distant vertebrate relatives: the lamprey. The lamprey lineage diverged from its fellow vertebrates more than half a billion years ago [17]. Remarkably, these distant relatives retain many common structures, including those responsible for movement: spinal circuits for generating the basic dynamics [18], reticulospinal centres directing fixed action patterns [19], and midbrain nuclei — for example, the mesencephalic locomotor region (MLR) and tectum — responsible for the control of locomotion and orientation. In mammals, the forebrain has asserted itself in the control of movement, and the motor regions of cortex have long been considered necessary for the skilled, dexterous

PKCg participates in food entrainment by regulating BMAL1. Proc. Natl. Acad. Sci. USA 109, 20679–20684.

Faculty of Medical and Health Sciences, University of Manchester, Manchester, UK. E-mail: [email protected]

http://dx.doi.org/10.1016/j.cub.2014.12.045

movements that are characteristic of mammals. Does such a pallial (cortical) motor control structure exist in the lamprey? Ocan˜a et al. [16] set out to identify and characterize the forebrain motor centres of the lamprey pallium using a suite of functional and anatomical methods. By electrically stimulating specific regions of the lateral pallium, they could provoke a semi-restrained lamprey to produce movements involving the eyes, body and mouth. In mammals, the primary motor region of cortex is defined as the part of cortex that requires the least amount of electrical current stimulation to elicit movement. In the lamprey, the authors found multiple areas where stimulation with low currents could elicit movement of different body parts, and where the intensity of the movement could be controlled by varying the intensity and frequency of stimulation. Using fluorescent dyes that travel from the site of injection to target areas, Ocan˜a et al. [16] then identified neural pathways connecting this ‘movement eliciting’ region of the pallium to other motor centres of the lamprey brain. Specifically, motor pallial projections were found to innervate the tectum deep motor output layer, the midbrain tegmentum and reticulospinal cells, all of them major components in the mid- and hindbrain motor circuits of the lamprey and other vertebrates. Furthermore, pallial projections were found to target different subnuclei in the lamprey basal ganglia and thalamus, components of the classic cortico-striatal-thalamocortical loop that has been extensively characterized in mammals. The authors use these two lines of evidence to support their claim that the lamprey pallium is not only involved in movement control, but that the pattern of downstream projections is