Michael A. Rea

PML regulates PER2 nuclear localization and circadian function

Studies have suggested that the clock regulator PER2 is a tumour suppressor. A cancer network involving PER2 raises the possibility that some tumour suppressors are directly involved in the mammalian clock. Here, we show that the tumour suppressor promyelocytic leukaemia (PML) protein is a circadian clock regulator and can physically interact with PER2. In the suprachiasmatic nucleus (SCN), PML expression and PML–PER2 interaction are under clock control. Loss of PML disrupts and dampens the expression of clock regulators Per2, Per1, Cry1, Bmal1 and Npas2. In the presence of PML and PER2, BMAL1/CLOCK‐mediated transcription is enhanced. In Pml−/− SCN and mouse embryo fibroblast cells, the cellular distribution of PER2 is primarily perinuclear/cytoplasmic. PML is acetylated at K487 and its deacetylation by SIRT1 promotes PML control of PER2 nuclear localization. The circadian period of Pml−/− mice displays reduced precision and stability consistent with PML having a role in the mammalian clock mechanism.

Adenosine A1 receptors regulate the response of the mouse circadian clock to light

The purine nucleoside, adenosine, has been implicated in the regulation of circadian phase in the hamster. In the current report, we present pharmacological evidence supporting the involvement of adenosine A1 receptors in the regulation of the response of the circadian clock to light in mice. Systemic injection of the selective adenosine A1 receptor agonist, N(6)-cyclohexyladenosine (CHA; 0.3 mg/kg) resulted in a 49% reduction (P<0.05) in the magnitude of light-induced phase delays. The inhibitory effect of CHA on light-induced phase delays was dose dependent over a range of 0.1 to 5 mg/kg with an apparent EC(50) of 0.3 mg/kg. Prior administration of the selective adenosine A1 receptor antagonist dipropylcyclopentylxanthine (DPCPX; 1 mg/kg) completely blocked the effect of CHA on photic phase delays. Finally, CHA significantly attenuated light-induced phospho-extracellular signal-related kinases (ERK) immunoreactivity in the mouse suprachiasmatic nucleus (SCN), consistent with a mode of action involving events that occur early in the signaling cascade through which photic information is conveyed to the circadian clock. These data indicate that the role of adenosine in the regulation of circadian phase is similar in mice and hamsters.

5-HT1B receptor knockout mice exhibit an enhanced response to constant light.

Serotonin (5-HT) can act presynaptically at 5-HT1B receptors on retinal terminals in the suprachiasmatic nucleus (SCN) to inhibit glutamate release, thereby modulating the effects of light on circadian behavior. 5-HT1B receptor agonists (1) inhibit light-inducedphase shifts of circadian activity rhythms, (2) attenuate light-induced Fos expression in the SCN, and (3) reduce the amplitude of optic nerve–evoked excitatory postsynaptic currents in SCN neurons in vitro. To determine whether functional disruption of the 5-HT1B presynaptic receptors would result in an amplified response of the SCN to light, the period ([.tau]) of the circadian rhythm of wheel-running activity was estimated under several different conditions in 5-HT1B receptor knockout (KO) mice and genetically matched wild- typeanimals. Under constant light (LL) conditions, the [.tau] of 5-HT1B receptor KO mice was significantly greater than the [.tau] of wild-type mice. Aquantitative analysis of the wheel-running activity revealed no differences between wild-type and KO mice in either total activity or the temporal distribution of activity under LL conditions, suggesting that the observed increase in [.tau] was not a function of reduced activity. Under constant dark conditions, the period of the circadian rhythm of wheel-running activity of wild-type and 5-HT1B receptor KO mice was similar. In addition, no differences were noted between wild-type and 5-HT1Breceptor KO mice in the rate of reentrainment toa6h phase advance in the 12:12 light:dark cycle or in phase shifts in response to a 10 min light pulse presented at circadian time 16. The enhanced response of the SCN circadian clock of the 5- HT1B receptor KO mice to LLconditions is consistent with the hypothesis that the endogenous activation of 5-HT1B presynaptic receptors modulates circadian behavior by attenuating photic input to the SCN.

Adenosine A1 receptors regulate the response of the hamster circadian clock to light.

Circadian rhythms are synchronized to the environmental light-dark cycle by daily, light-induced adjustments in the phase of a biological clock located in the suprachiasmatic nucleus. Ambient light alters the phase of the clock via a direct, glutamatergic projection from retinal ganglion cells. We investigated the hypothesis that adenosine A1 receptors modulate the phase adjusting effect of light on the circadian clock. Systemic administration of the selective adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA), significantly (p<0.05) attenuated light-induced phase delays and advances of the circadian activity rhythm. Selective agonists for the adenosine A2A and adenosine A3 receptors were without effect. The inhibitory effect of CHA on light-induced phase advances was dose-dependent (0.025-1.0 mg/kg, ED(50)=0.3 mg/kg), and this effect was blocked in a dose-dependent (0.005-1.0 mg/kg) manner by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Injection of CHA (10 microM) into the region of the suprachiasmatic nucleus significantly attenuated light-induced phase advances, and this effect was also blocked by DPCPX (100 microM). The results suggest that adenosine A1 receptors located in the region of the suprachiasmatic nucleus regulate the response of the circadian clock to the phase-adjusting effects of light.

Metastasis-associated protein 1 is an integral component of the circadian molecular machinery

The mammalian circadian clock regulates the daily cycles of many important physiological processes, but its mechanism is not well understood. Here we provide genetic and biochemical evidence that metastasis-associated protein 1 (MTA1), a widely upregulated gene product in human cancers, is an integral component of the circadian molecular machinery. Knockout of MTA1 in mice disrupts the free-running period of circadian rhythms under constant light and normal entrainment of behaviour to 12-h-light/12-h-dark cycles. The CLOCK-BMAL1 heterodimer activates MTA1 transcription through a conserved E-box element at its promoter. MTA1, in turn, interacts with and recruits CLOCK-BMAL1 at its own and CRY1 promoters and promotes their transcription. Moreover, MTA1 deacetylates BMAL1 at lysine 538 through regulating deacetylase SIRT1 expression, thus disturbing the CRY1-mediated negative feedback loop. These findings uncover a previously unappreciated role for MTA1 in maintenance of circadian rhythmicity through acting on the positive limb of the clock machinery.

Fast Multisite Optical Recording of Mono- and Polysynaptic Activity in the Hamster Suprachiasmatic Nucleus Evoked by Retinohypothalamic Tract Stimulation

Responses of the hamster suprachiasmatic nucleus (SCN) to retinohypothalamic tract (RHT) stimulation were studied in horizontal hypothalamic slices using fast multisite optical recording techniques. A 124-element photodiode detector array provided high-speed monitoring (0.5 ms/frame) of evoked neural activity in the SCN, while a larger 464-element photodiode array yielded improved spatial imaging with some loss in temporal resolution (1.6 ms/frame). Brief electrical stimulation of the optic nerves evoked a propagated compound action potential that was recorded optically as a single transient depolarization in many slice regions, including the SCN. Only within the SCN, however, was this optic tract signal followed by additional voltage-dependent optical responses which exhibited a fast and a slow depolarizing component. The initial upstroke of the fast component was Ca(2+)-insensitive and is presumed to reflect activity in presynaptic RHT afferents. The remainder of the fast depolarization and the slow depolarization were Ca(2+)-sensitive. These responses were labeled the early population excitatory postsynaptic potential (Early P.E.P.S.P.) and the Late P.E.P.S.P. respectively. The Late P.E.P.S.P. was not enhanced by K+ channel blockade, suggesting that glial depolarization is not the primary source of this component. Drugs known to suppress RHT-evoked SCN field potentials also suppressed the Early and Late P.E.P.S.P.s recorded optically in the SCN. Unexpectedly, the Early P.E.P.S.P. was also reduced by the GABAA antagonist, bicuculline. Surface plots of normalized peak amplitudes showed that both SCN components had similar spatial distributions within the SCN, although the Early P.E.P.S.P. tended to be slightly more prominent within the medial SCN in some preparations. It is suggested that the Early P.E.P.S.P. represents firing of monosynaptically activated SCN neurons, while the Late P.E.P.S.P. reflects polysynaptic activity within the intrinsic SCN neuronal network that may be involved in the light entrainment of the circadian oscillator.

Targeted reduction of the EGFR protein, but not inhibition of its kinase activity, induces mitophagy and death of cancer cells through activation of mTORC2 and Akt

The oncogenic epidermal growth factor receptor (EGFR) is commonly overexpressed in solid cancers. The tyrosine kinase activity of EGFR has been a major therapeutic target for cancer; however, the efficacy of EGFR tyrosine kinase inhibitors to treat cancers has been challenged by innate and acquired resistance at the clinic. Accumulating evidence suggests that EGFR possesses kinase-independent pro-survival functions, and that cancer cells are more vulnerable to reduction of EGFR protein than to inhibition of its kinase activity. The molecular mechanism underlying loss-of-EGFR-induced cell death remains largely unknown. In this study, we show that, unlike inhibiting EGFR kinase activity that is known to induce pro-survival non-selective autophagy, downregulating EGFR protein, either by siRNA, or by a synthetic EGFR-downregulating peptide (Herdegradin), kills prostate and ovarian cancer cells via selective mitophagy by activating the mTORC2/Akt axis. Furthermore, Herdegradin induced mitophagy and inhibited the growth of orthotopic ovarian cancers in mice. This study identifies anti-mitophagy as a kinase-independent function of EGFR, reveals a novel function of mTORC2/Akt axis in promoting mitophagy in cancer cells, and offers a novel approach for pharmacological downregulation of EGFR protein as a potential treatment for EGFR-positive cancers.

A chiming biological clock

Microscopists are in the unique position of being able to encapsulate great science with a single image. The data may be complex, but the visualization can be breathtaking.Now, Olympus has joined forces with Current Biology to offer a state-of-the-art digital camera for the best photomicrograph. We’re not just looking for bright colours and strong imagery – though they might help – we want good science simply visualized, in any application, on any system.The winning entry will receive one of the new Olympus CAMEDIA digital cameras, the first film-free image recording and storage system for the leisure user.Send your images, with a brief description of the subject and method, to: Peter Newmark, The Editor, Current Biology, Current Biology Ltd, 34–42 Cleveland Street, London W1p 6LB, UK.The closing date for entries is 1 December 1997.