BIOCHEMICAL CHARACTERISATION OF THE OSCILLATORY FUNCTION OF CLOCK

Circadian clocks are oscillator systems that drive rhythms in behavior and physiology in an organism to coordinate with the light-dark cycle. The molecular clock is made up of core clock genes acting in a transcriptional negative feedback loop. Transcriptional activators BMAL1 and CLOCK form a heterodimer which binds to cis E/E-box elements present in the promoter region of target genes including Per1,2,3 and Cry1,2. PER and CRY proteins then translocate to the nucleus to repress BMAL1/CLOCK-mediated transcription. This transcriptional negative feedback mechanism underlies circadian oscillations. BMAL1 and CLOCK are bHLH and PAS domain transcription factors and play a central role in the positive limb of the negative feedback loop. In cell-autonomous clock models, absence of Clock leads to loss in rhythmicity. Previous studies and our recent results have demonstrated that Npas2, a Clock paralog, cannot complement Clock function in tissue- or cell-autonomous clock models including peripheral tissues, cells and dissociated SCN neurons. Promoted by our previous studies on BMAL1 biochemistry, we systematically investigated the CLOCK protein. We use cell-based genetic complementation and real-time bioluminescence recording and demonstrate that Clock alone can enable and maintain cell-autonomous circadian rhythms, while Npas2 cannot. Despite similarities in sequence, domain structure and biochemical activity, they play distinct roles in the clock function. We demonstrate that the C-terminal regions of CLOCK, downstream from the core bHLH and PAS domains, therefore named here the C-terminal Regulatory Domain (CRD), is key in enabling circadian oscillation and rendering its core clock function. We further identify a ~60 amino acid residue region, encompassing two motifs, within the CLOCK CRD that are required for clock function and distinguish CLOCK from NPAS2. These findings provide novel insights into the evolution of the diverse functions of the bHLH/PAS family of proteins involved in the circadian cycle and offer new opportunities for mechanistic studies of CLOCK function

mTOR signaling regulates central and peripheral circadian clock function

The circadian clock coordinates physiology and metabolism. mTOR (mammalian/mechanistic target of rapamycin) is a major intracellular sensor that integrates nutrient and energy status to regulate protein synthesis, metabolism, and cell growth. Previous studies have identified a key role for mTOR in regulating photic entrainment and synchrony of the central circadian clock in the suprachiasmatic nucleus (SCN). Given that mTOR activities exhibit robust circadian oscillations in a variety of tissues and cells including the SCN, here we continued to investigate the role of mTOR in orchestrating autonomous clock functions in central and peripheral circadian oscillators. Using a combination of genetic and pharmacological approaches we show that mTOR regulates intrinsic clock properties including period and amplitude. In peripheral clock models of hepatocytes and adipocytes, mTOR inhibition lengthens period and dampens amplitude, whereas mTOR activation shortens period and augments amplitude. Constitutive activation of mTOR in Tsc2–/–fibroblasts elevates levels of core clock proteins, including CRY1, BMAL1 and CLOCK. Serum stimulation induces CRY1 upregulation in fibroblasts in an mTOR-dependent but Bmal1- and Period-independent manner. Consistent with results from cellular clock models, mTOR perturbation also regulates period and amplitude in the ex vivo SCN and liver clocks. Further, mTOR heterozygous mice show lengthened circadian period of locomotor activity in both constant darkness and constant light. Together, these results support a significant role for mTOR in circadian timekeeping and in linking metabolic states to circadian clock functions

mTOR signaling regulates central and peripheral circadian clock function.

The circadian clock coordinates physiology and metabolism. mTOR (mammalian/mechanistic target of rapamycin) is a major intracellular sensor that integrates nutrient and energy status to regulate protein synthesis, metabolism, and cell growth. Previous studies have identified a key role for mTOR in regulating photic entrainment and synchrony of the central circadian clock in the suprachiasmatic nucleus (SCN). Given that mTOR activities exhibit robust circadian oscillations in a variety of tissues and cells including the SCN, here we continued to investigate the role of mTOR in orchestrating autonomous clock functions in central and peripheral circadian oscillators. Using a combination of genetic and pharmacological approaches we show that mTOR regulates intrinsic clock properties including period and amplitude. In peripheral clock models of hepatocytes and adipocytes, mTOR inhibition lengthens period and dampens amplitude, whereas mTOR activation shortens period and augments amplitude. Constitutive activation of mTOR in Tsc2-/-fibroblasts elevates levels of core clock proteins, including CRY1, BMAL1 and CLOCK. Serum stimulation induces CRY1 upregulation in fibroblasts in an mTOR-dependent but Bmal1- and Period-independent manner. Consistent with results from cellular clock models, mTOR perturbation also regulates period and amplitude in the ex vivo SCN and liver clocks. Further, mTOR heterozygous mice show lengthened circadian period of locomotor activity in both constant darkness and constant light. Together, these results support a significant role for mTOR in circadian timekeeping and in linking metabolic states to circadian clock functions

<i>mTor</i>^{<i>flx/–</i>}mice have long period length of circadian locomotor activity rhythms.

<p><b>(A)</b> Representative double-plotted actograms of wheel-running activity rhythms in <i>mTor</i>^{<i>flxflx</i>} and <i>mTor</i>^{<i>flx/–</i>}mice. X axis: zeitgeber time (ZT) of the 12 h/12 h light/dark cycle (LD) indicated by the bar (top). Y axis: number of days during the experiment (left). Mice were first entrained to a regular LD cycle for 10 days and then released to constant darkness (DD) for 20 days. On the 31^st day, mice were released into constant light (LL) for 40 days. Grey and white areas indicate dark and light periods. <b>(B)</b> Circadian free-running period lengths of mice in DD and LL. Data are mean ± SEM (red) of individual values that are shown in black dots (n = 6 for <i>mTor</i>^{<i>flxflx</i>} mice and n = 9 for <i>mTor</i>^{<i>flx/–</i>}mice. <b>(C)</b> Average wheel-running activity of mice expressed in wheel revolutions per 10 min across 24 h in LD, DD and LL.</p

Pharmacological inhibition of mTOR alters circadian clock function in MMH-D3 hepatocytes.

<p>Top panel: representative records of bioluminescence rhythms of MMH-D3 hepatocytes harboring the <i>Per2-dLuc</i> reporter in the presence of mTOR inhibitors: 50 nM rapamycin <b>(A)</b>, 20 nM Torin1 <b>(B)</b>, or 10 uM PP242 <b>(C)</b>. Real-time bioluminescence expression was recorded in a Synergy microplate luminometer on 96-well plates. Bottom panel: period length and amplitude are mean ± SD (n = 8 independent wells) for each treatment. All three inhibitors caused significantly longer period length and lower rhythm amplitude. ***p < 0.001 vs. DMSO.</p

Genetic manipulation of mTOR pathway alters circadian clock function.

<p>RNAi knockdown of <i>mTor</i> lengthens the period length of circadian bioluminescence rhythms in MMH-D3 hepatocytes. (<b>A</b>) and 3T3-L1 adipocytes (<b>B</b>). Hepatocytes and adipocytes harboring the <i>Per2-dLuc</i> reporter were infected with lentiviral non-specific (NS) shRNA or shRNA constructs against <i>mTor</i>. Left panel: real-time bioluminescence expression was recorded in a Lumicycle luminometer on 35-mm culture dishes and the bioluminescence data are representative of at least three independent experiments. Middle panel: <i>mTor</i> knockdown efficiency was determined by Western blot analysis (middle). Right panel: period length and rhythm amplitude are mean ± standard deviation (SD) (n = 3 independent dishes). * p < 0.05 vs. NS. <b>(C)</b> Elevated mTOR via constitutively active Rheb shortens circadian bioluminescence rhythms in MMH-D3 hepatocytes. Bioluminescence rhythms from MMH-D3 hepatocytes harboring the <i>Per2-dLuc</i> reporter and overexpressing either the empty vector or constitutively active Rheb (CA-Rheb). p-S6 as a proxy of mTOR activation was hyper-phosphorylated in cells expressing CA-Rheb relative to vector control cells, whereas S6 levels were similar in the two cell lines. * p < 0.05 vs. Vector; ** p < 0.01 vs. Vector.</p

mTOR perturbation alters the liver clock function.

<p><b>(A)</b> Bioluminescence rhythms of liver explants derived from <i>Per2</i>^<i>Luc</i> mice in the presence of DMSO or 20 nM Torin1. mTOR inhibition by Torin1 led to long period and low amplitude in liver explants cultured <i>ex vivo</i>. ** p < 0.01 vs. DMSO. <b>(B)</b> Bioluminescence rhythms of liver explants derived from <i>mTor</i>^{<i>flxflx</i>}<i>;Per2</i>^<i>Luc</i> (wt control) and <i>mTor</i>^{<i>flx/–</i>}<i>;Per2</i>^<i>Luc</i> (<i>mTor</i> heterozygous) mice. Heterozygous deletion of <i>mTor</i> reduced the rhythm amplitude in liver explants. * p < 0.05 vs. <i>mTor</i>^{<i>flxflx</i>}. <b>(C-E)</b> Western blots (C and D) and Q-PCR (E) of liver tissue samples from <i>mTor</i>^{<i>flxflx</i>} and <i>mTor</i>^{<i>flx/–</i>}mice. Mice were entrained to regular light/dark cycles and then released to constant darkness (DD), followed by tissue harvest at 4-hr intervals beginning at 52 hr in DD (CT52). While individual tissue samples were used for Q-PCR analysis, tissues from 3~5 mice were pooled at each time point for Western blotting. Relative Q-PCR values are presented for each gene and error bars represent SD of expression levels from 3 mice. Circadian time (CT): hours after animal release to constant darkness. Quantitation of the blots is shown in (D). * p < 0.05 vs. <i>mTor</i>^{<i>flxflx</i>}.</p

mTOR perturbation alters the central SCN clock function.

<p><b>(A)</b> SCN explants from <i>Per2</i>^<i>Luc</i> reporter mice were treated with either DMSO or 50 nM rapamycin as indicated by an arrow. Representative bioluminescence records are shown (left). Period and amplitude data (right) are mean ± SD (n = 4 mouse SCN slices). ** p < 0.01 vs. rapamycin pre-treatment. Rapamycin treatment altered tissue-autonomous circadian rhythms of SCN explants cultured <i>ex vivo</i>. <b>(B)</b> Bioluminescence record from one representative SCN explant treated with PP242. Period and amplitude data (right) are mean ± SD (n = 4 mouse SCN slices). ** p < 0.01 vs. PP242 pre-treatment. <b>(C)</b> Bioluminescence rhythms of SCN explants derived from <i>mTor</i>^{<i>flxflx</i>}<i>;Per2</i>^<i>Luc</i> and <i>mTor</i>^{<i>flx/–</i>}<i>;Per2</i>^<i>Luc</i> mice. Heterozygous deletion of <i>mTor</i> reduced tissue-autonomous circadian rhythm amplitude and lengthened the period length of SCN explants. Period length and amplitude data are mean ± SD (n = 5 mouse SCN slices). ** p < 0.01 vs. <i>mTor</i>^{<i>flxflx</i>}.</p

Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity

A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19