ISX-9 potentiates CaMKIIδ-mediated BMAL1 activation to enhance circadian amplitude

Li, Huilin; Ou, Jiali; Li, Yaqun; Xu, Niannian; Li, Qing; Wu, Ping; Peng, Chao; Tang, Yun-Chi; Chang, Hung-Chun

doi:10.1038/s42003-022-03725-x

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Published: 28 July 2022

ISX-9 potentiates CaMKIIδ-mediated BMAL1 activation to enhance circadian amplitude

Huilin Li¹^na1,
Jiali Ou¹^na1,
Yaqun Li¹,
Niannian Xu^2,3,
Qing Li²,
Ping Wu⁴,
Chao Peng ORCID: orcid.org/0000-0002-6814-2676⁴,
Yun-Chi Tang ORCID: orcid.org/0000-0001-9385-2787¹ &
…
Hung-Chun Chang ORCID: orcid.org/0000-0003-0030-8323^2,3

Communications Biology volume 5, Article number: 750 (2022) Cite this article

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Abstract

Circadian dysregulation associates with numerous diseases including metabolic dysfunction, sleep disorder, depression and aging. Given that declined circadian amplitude is a trait commonly found with compromised health, interventions that design in precluding circadian amplitude from dampening will aid to mitigate complex, circadian-related diseases. Here we identify a neurogenic small molecule ISX-9 that is able to support persistent and higher amplitude of circadian oscillations. ISX-9 improves diurnal metabolic rhythms in middle-aged mice. Moreover, the ISX-9-treated mice show better sleep homeostasis with increased delta power during the day time and higher locomotive activity in the dark period. ISX-9 augments CaMKIIδ expression and increases BMAL1 activity via eliciting CaMKIIδ-mediated phosphorylation on BMAL1 residues S513/S515/S516, accordingly composes a positive feedback effect on enhancing circadian amplitude. CaMKIIδ-targeting, and the use of ISX-9 may serve as decent choices for treating circadian-related disorders.

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Introduction

Circadian clock is an intrinsic timekeeper that coordinates multiple physiological activities in an orderly manner^1,2,3. Environmental or genetic disruptions of circadian rhythms in model organisms have revealed a series of symptoms related to human diseases including cardiovascular dysfunction⁴, diabetic mellitus^2,5, cancer⁶, sleep disorders^7,8,9, depression^10,11,12,13, neurodegenerative diseases¹⁴, and aging^15,16,17,18. As circadian amplitude decay and phase mis-alignment are commonly associated with deteriorated health and aging^15,19,20, interventions with supporting circadian amplitude and correcting phase mis-alignment will assist to ameliorate circadian-associated diseases including aging²¹. Several compounds have been identified to modulate circadian oscillation and strength^22,23,24, and many of these small molecules target directly on circadian clock components such as REV-ERBs^25,26, RORs^27,28, and CRY1/2²⁹, to tune the circadian core loops transcriptionally or post-translationally. Agonists for REV-ERBs such as GSK4112 and the derivatives SR9009/SR9011 showed efficacies with increased energy expenditure hence preventing obesity from high-fat diet (HFD) feeding²⁵. SR9009/SR9011 were also demonstrated to act as anticancer agents³⁰, suggesting that the stimulating of REV-ERBs is a decent strategy to combat metabolic dysregulation and cancer. However, mice that were treated with SR9009/SR9011²⁵, or another potent derivative SR10067²⁶, all exhibited reduced wheel-running activities, implying that the treatment could lead to inactivity. Nobiletin is another small molecule that was shown to protect against obesity in HFD-fed Clock^D19/D19 mutant mice, and amend glucose intolerance in diabetic db/db mice in a RORα/γ-dependent manner²⁷. Nobiletin extended the median lifespan of mice under regular diet feeding and improved mitochondrial function via activating OXPHOS supercomplex-encoding genes²⁸, which suggested that Nobiletin is an adequate option for supporting circadian clock robustness and healthspan. Small molecules that affect CRY1/2 stability were also found to be applicable in tuning circadian rhythm and alleviating metabolic diseases. CRY1/2 stabilizer KL001 and the derivatives were shown to lengthen the period and decrease circadian amplitude in vitro²⁹, and proved to lower glucose intolerance in obese mice³¹. These results implied an intriguing point that both increasing and decreasing the circadian strength could be practical in treating glucose intolerance and obesity in genetic, or diet-provoked disease models. Nevertheless, reports of direct inspections of these circadian modulators in wild-type, aged mice for their rejuvenating activities remain limited. Also, it is important to note that the majority of these circadian-modulating compounds showed a clear phase-altering effect^32,33, so their applications would consider tentative due to long-term phase mis-alignment is known to be deleterious to health^34,35,36. For these reasons, we searched for small molecules that are able to promote circadian amplitude persistently without causing period change. From our chemical screen, we identified a small molecule ISX-9 that appeared to fulfill the pursuit. ISX-9 was originally found to facilitate neural stem cell conversion into neuron³⁷. Here we found that ISX-9 can enhance PER2::LUC amplitude and BMAL1 activity via triggering Ca²⁺ influx and CaMKIIδ-mediated phosphorylation on BMAL1. Together with other effects found in ISX-9-treated mice, including higher PER2 expression in the suprachiasmatic nucleus (SCN), and improved sleep homeostasis, our results pointed out that ISX-9 could help to mitigate age-associated circadian rhythm disorder and metabolic dysregulation.

Results

Screen of circadian amplitude enhancers

To explore circadian modulators that exert amplitude-enhancing activity with better persistence and minimal disturbance to the period, we carried out a compound screen that included 8060 small molecules covering kinases, epigenetic factors, and GPCRs as targets in mPer2^Luc fibroblasts³⁸ (Fig. 1a). To validate amplitude versus phase change in the highthroughput primary screen, we measured the luminescence level at 24-h and 30-h, respectively, in mPer2^Luc fibroblasts post drug treatment (Fig. 1b and Supplementary Fig. 1a). The two reads allowed to indicate amplitude modulators if finds consistent increasing or decreasing effects on luminescence level in both time points, or phase modulators if the two values are reversing with related to the untreated control, as represented by BS-181 for the latter case (Supplementary Fig. 1b, c). After eliminating those with a clear cell toxicity effects, we focused on the remaining 6777 compounds (Fig. 1b and Supplementary Data 1) and identified 270 amplitude enhancers that were able to increase PER2::LUC amplitude by 1.2-fold (Supplementary Data 1). Among them, ISX-9, Atrasentan, HhAntag, OSI-930, GW4064, and 2-NP were clear examples of raising PER2::LUC luminescence in the secondary screen (Fig. 1c). Real-time recording of PER2::LUC luminescence confirmed that the six candidates were able to increase PER2::LUC amplitudes without affecting the circadian period (Fig. 1d and Supplementary Fig. 1d). Notably, most candidates enhanced PER2::LUC amplitude evidently only in day 1, then showed substantial efficacy decline in the remaining 3 days. ISX-9 acted as the sole endurable amplitude enhancer over 4 days of testing (Fig. 1e). The amplitude-enhancing effect of ISX-9 was consistent, and applicable to other circadian gene promoter-driven luciferase reporters, including Dbp:Luc and Rev-Erbα:Luc, in both cases revealed with the best efficacy among the six candidates (Fig. 1f).

**Fig. 1: Small molecule screen identified ISX-9 as a circadian amplitude enhancer.**

ISX-9 augments circadian amplitude

ISX-9 is an isoxazole compound that was initially identified with neurogenic activity³⁷ (Fig. 2a). In our test, we found ISX-9 elicited PER2::LUC amplitude in a dose-dependent manner (Fig. 2b), and showed low cell toxicity at 10 μM, the highest concentration in our examination (Fig. 2c). Agreed with the luciferase assays, ISX-9 augmented the levels of several clock transcripts including Bmal1, Per2, and Dbp (Fig. 2d and Supplementary Fig. 2a). DBP protein level was highly responsive to the ISX-9 dosage in comparison to BMAL1 and REV-ERBα (Fig. 2e), thus appeared to be a suitable marker to report ISX-9 effect in vitro. Interestingly, we found ISX-9 activity on DBP and PER2 inductions lessened in Bmal1^+/−, and blunted in Bmal1^−/− MEFs, indicating that the amplitude-enhancing activity of ISX-9 is BMAL1-dependent (Supplementary Fig. 2b, c). ISX-9 also elicited higher circadian PER2::LUC oscillations in explants, including the central circadian oscillator suprachiasmatic nucleus (SCN) and pituitary (Fig. 2f), and mice that were administrated with ISX-9 revealed higher PER2 level in the SCN (Fig. 2g), collectively telling a dependable amplitude-enhancing effect of ISX-9 in vitro, ex vivo, and in vivo.

**Fig. 2: ISX-9 enhanced circadian amplitudes in culture cells and tissues.**

ISX-9 ameliorates age-associated circadian disorders

To explore the metabolic-adjusting effect of ISX-9 in vivo, we further examined the metabolic rate in juvenile (2-month-old) and middle-aged (14-month-old) mouse cohorts. The juvenile mice exhibited clear respiratory exchange ratio (RER) oscillations diurnally and showed lower RER representing a resting/fasting state during daytime (Fig. 3a)^39,40. The middle-aged group showed a less distinctive diurnal RER phenotype in the metabolic cage, with only a brief elevated RER indicating a feeding-associated carbohydrate utilization in the early night (Fig. 3a). ISX-9 administration increased the amplitude of RER in the middle-aged group, but did not alter RER amplitude in the juvenile group (Fig. 3a). Moreover, we found ISX-9 corrected the RER period from 26 to 24 h (Fig. 3b), and slightly promoted water-assessment and food intake during the dark time, but not affect body weight in the middle-aged group (Fig. 3c), and the effect can be extended to another test in an older 18-month-old cohort (Supplementary Fig. 3a–e). As ISX-9 treatment did not affect the juvenile cohort (Fig. 3a), the results indicated that ISX-9 acted selectively in ameliorating metabolic dysregulations associated with aging. The ISX-9 treatment was conducted before the metabolic cage housing, confirming a sustainable activity of ISX-9 in promoting circadian amplitude in vivo.

**Fig. 3: ISX-9 rejuvenated metabolic oscillations in middle-aged mice.**

To study whether ISX-9 could ameliorate sleep disorder, another phenotype that is associated with compromised circadian regulation^7,8, we conducted a telemetric recording of electroencephalogram (EEG) to score sleep status in a young versus old manner. The differences in wake and NREM levels were found most evidently during night time by comparing 2-month-old and 14-month-old mice. The latter exhibited partial awake, which correlated well with higher NREM sleep in the night time (Supplementary Fig. 4a). Together with the data including largely reduced physical activity, shallowed body temperature fluctuation (Supplementary Fig. 4b), lowered beta/gamma, and blunted delta/theta oscillations (Supplementary Fig. 4c), the results pointed out a collectively depressed situation in circadian amplitudes during aging. To test whether ISX-9 could entrain the circadian-programmed sleep cycle, we dosed the same group of mice with 20 mg/Kg ISX-9 for 7 days, then stopped the treatment for another 7 days before the EEG recoding. The treatment sharply promoted wake in the early night and sustained a higher wake state in the remaining dark period, at the same time lowered both REM and NREM sleep in the active period (Fig. 4a). The treated middle-aged mice also displayed higher physical activities during night time, conversely exhibited lower body temperature during the day (Fig. 4b, c), indicating that ISX-9 has tuned the middle-aged mice to a more alert, active status during awake, and stayed better rested during sleep. We noted the treated middle-aged mice with higher delta power in the resting period, and higher gamma power in the awake period, both in advance, revealed improved circadian homeostasis in brain oscillations (Fig. 4d). Indeed, by counting the transitions in the early stages of the awake-sleep cycle at light-on (ZT0-1) and light-off (ZT12–13), we found ISX-9 markedly suppressed dysregulated switches in the middle-aged group, by which rendered more stable sleep and wake homeostatic states, as observed in healthy juvenile mice (Fig. 4e).

**Fig. 4: Diurnal electroencephalogram and physical activities prior to and post ISX-9 treatment in aged mice.**

CaMKIIδ mediates ISX-9 effect via BMAL1

ISX-9 was identified to promote neuronal differentiation through Ca²⁺ signaling³⁷. We first followed the intracellular Ca²⁺ level by Fluo-4 AM binding assay in MEFs, and confirmed that ISX-9 was able to trigger Ca²⁺ influx rapidly in our tests (Fig. 5a). Consistent with the previous report, we also found that the ISX-9 effect was mediated by CaMKII activity, as the CaMKII-specific antagonist KN93 fully abolished the PER2::LUC enhancing effect of ISX-9 (Fig. 5b). To test whether CaMKII agonist could augment the amplitude of circadian oscillations without period change, we applied another small molecule, Isoproterenol, that was reported to stimulate β1-adrenergic receptor thus trigger Ca²⁺ influx and CaMKII activity⁴¹. We noted that Isoproterenol can also increase PER2::LUC amplitude, though with subtle efficacy when compared to ISX-9 (Supplementary Fig. 5a). Similar to ISX-9, Isoproterenol did not show period altering effect (Supplementary Fig. 5b). Among the four CaMKII isoforms⁴², we noted that Camk2b and Camk2d knockdowns retracted the ISX-9 effect evidently in real-time PER2::LUC recordings, but not in Camk2a and Camk2g knockdowns (Fig. 5c). Given that ISX-9 showed BMAL1 dependency in prompting DBP and PER2::LUC levels (Supplementary Fig. 2b, c), and triggered BMAL1 phosphorylation (Fig. 5d), we next examined the potential CaMKII activities on BMAL1 phosphorylation biochemically, and compared the differences among CaMKII isoforms. CaMKIIδ exhibited the highest activity on purified BMAL1 in the kinase assay (Fig. 5e). Together with the Camk2d knockdown result on PER2::LUC expression, the data implicated that CaMKIIδ was critical to mediate the ISX-9 effect through BMAL1. These collectively proposed an intriguing loop of connecting ISX-9, Ca²⁺ signaling, and BMAL1 phosphorylation for modulating the circadian amplitude. Interestingly, single-molecule RNA fluorescence in situ hybridization (smFISH) indicated that Camk2d distribution in the mouse brain was uniquely more abundant in subcortical regions and the hypothalamus (Supplementary Fig. 6), and showed a clear rhythmic expression that peaked at ZT18 in the SCN (Fig. 5f), jointly fit well with the anticipated role in circadian regulation. Other CaMKII isoform transcripts, such as Camk2a and Camk2b showed subtle oscillations and lower expressions in the SCN, and distributed more abundantly in the striatum and cortex (Supplementary Fig. 6). Furthermore, Camk2d was considerably reduced in the SCN of aged mice (Supplementary Fig. 7a), and ISX-9 improved Camk2d expression (Supplementary Fig. 7b), the results put forward the situation that ISX-9 helped to re-boost the CaMKIIδ level, accordingly the CaMKIIδ-mediated phosphorylation of BMAL1, in turn, reverted the circadian amplitude decline resulted from aging. Expression levels of Camk2a and Camk2b did not alter in old SCN, nor respond to the ISX-9 treatment (Supplementary Fig. 7). Of note, it seemed Camk2g expression was anti-phasic (peaked at ZT6) to Camk2d (Fig. 5f), and Camk2g showed slight up-regulation in the old SCN (Supplementary Fig. 7a). Whether the later indicated a marker of aging SCN or a compensatory effect to respond the decay of Camk2d would be intriguing questions to investigate afterward.

**Fig. 5: ISX-9 triggered Ca²⁺ influx and BMAL1 phosphorylation in a CaMKIIδ-dependent manner.**

CaMKIIδ regulates circadian amplitude via phosphorylating BMAL1

Given that Camk2d expression was rhythmic and could be triggered by ISX-9, we searched for putative E-box sequences in the Camk2 promotors. We found all isoforms contain E-box elements, including the canonical E-box sequence (CACGTG) identified in Camk2d and Camk2g promotors (Fig. 6a). Using promotor reporting assay in neuroblastoma N2a cells with comparing CLOCK:BMAL1 co-expression to non-supplemented basal condition, we found Camk2d promotor was most responsive to CLOCK:BMAL1 activation with a 6.8-fold increase in Camk2d:Luc expression (Supplementary Fig. 8a), and the induction was not due to dexamethasone synchronization in the test (Supplementary Fig. 8b). ISX-9 could further boost the expression of Camk2d:Luc to the highest level with CLOCK:BMAL1 co-expression (Fig. 6b), the results indicated a preferable expressional response of Camk2d to ISX-9 that was consistent with the observation in aged SCN (Supplementary Fig. 7b). Furthermore, we found Camk2d:Luc expressed rhythmically in MEFs (Supplementary Fig. 8c), collectively the data verified that Camk2d is an authentic clock-controlled gene. Mass spectrometry identified BMAL1 serine 513, 515, 516, and 519 (Fig. 6c), four highly conserved serine residues among different species (Fig. 6d), to be major phosphorylated sites that responded to ISX-9 treatment. Mutation of either S513, S515, or S516 residue to alanine led to 40–60% decrease in BMAL1 activity of driving the expression of BMAL1-dependent Dbp:Luc, but not S519A in the same test (Fig. 6e). The S513A/S515A/S516A triple mutation produced an even substantial impact on BMAL1 activity, suggesting that the three serine residues are important for BMAL1 activation. Consistent with the idea, the CaMKIIδ catalytic mutation K43M (an equivalent design to mimic CaMKIIα-K42M) (Supplementary Fig. 9a)^42,43 with confirmed blunt kinase activity on a substrate peptide (Supplementary Fig. 9b)⁴⁴ and purified BMAL1 (Fig. 6f), led to similar downgrade effect in Dbp:Luc expression in N2a cells (Fig. 6g). Under CLOCK/BMAL1/CaMKIIδ co-expression conditions, BMAL1-S513A/S515A/S516A and CaMKIIδ-K43M showed a similar defect in driving Dbp:Luc expression in comparison to their wild-type counterparts (Supplementary Fig. 9c), these results highlighted again a CaMKIIδ-associated regulation of circadian amplitude via BMAL1. All told, our data suggested that CaMKIIδ, as a clock output, comprised a positive feedback loop in modulating BMAL1 activity, and their decay caused by aging could be rejuvenated by CaMKII agonist ISX-9 (Fig. 7).

**Fig. 6: ISX-9 prompted BMAL1 activity through S513/515/516 phosphorylation.**

**Fig. 7: Model of ISX-9 effect through CaMKIIδ-mediated activation of BMAL1 for circadian amplitude enhancement.**

Discussion

ISX-9 was shown to exert interesting activities, initially identified as a neurogenic chemical that could convert neural stem cells into neurons³⁷. In a later work, ISX-9 was further revealed to be able to reprogram fibroblasts into functional neurons along with a BET family bromodomain inhibitor, I-BET151⁴⁵. ISX-9 was then proposed in a few applications related to neurogenesis, for instance, to potentiate cell proliferation and neuronal commitment in the dentate gyrus in vivo⁴⁶. Interestingly, besides the primary role as a neurogenesis inducer, ISX-9 was reported to be useful in treating neuropsychiatric conditions, such as lowering the relapse of methamphetamine abuse⁴⁷ and fear response in ethanol-dependent rat model⁴⁸. In an age-related perspective, ISX-9 was shown to rescue the loss of cortico-striatal synapse, thus improving cognition in Huntington’s disease model R6/2 strain⁴⁹. ISX-9 application can be extended to other peripheral tissues in addition to the brain, for example, to promote pancreatic β-cell survival against apoptosis⁵⁰.

Here we proposed ISX-9 as a small molecule-based intervention to persistently magnify the circadian amplitude, which is commonly tapered in an aging-sensitive fashion^19,21. The amplitude decline is partly due to, as we proposed here, the decay of CaMKIIδ, thus restricting its capacity to lift BMAL1 activity through S513/S515/S516 phosphorylation. BMAL1 phosphorylation has been demonstrated to play both promotive^51,52 and suppressive^53,54 roles, judged by the serine/threonine sites and the subsequent outcomes to regulate BMAL1 transcriptional activity of BMAL1 stability. Here we add S513/S515/S516 as a novel regulatory cluster on BMAL1 to respond to the CaMKIIδ-mediated activation. The mode is likely suitable for setting circadian rhythms in the central circadian oscillator SCN, as CaMKIIδ expresses more abundantly in the SCN, and SCN shows clear calcium level change diurnally⁵⁵. Future works regarding the understanding of BMAL1 phosphorylation, with details on the corresponding residues combining temporal information, would be important to support versatile designs for modulating circadian amplitude and phase.

Our finding is coherent with other evidence which demonstrated the importance of calcium signaling and CaMKII activity in regulating circadian rhythms. Calcium influx was recognized as an important signal for circadian rhythm generation in the SCN^{56,57,58,59,60}, and mice carrying CaMKIIα-K42R mutation was shown to exhibit a prolonged period phenotype⁶¹. Our study took a step forward to show that sensitizing Ca²⁺ influx by ISX-9 is a convenient way to replenish another CaMKII participant, CaMKIIδ oscillation, thus adjusting the circadian amplitude in aging cells. Together with other positive activities discussed earlier, the use of ISX-9 seemed to be promising in defying age-related diseases, markedly also in neurological disorders. Whether these beneficial effects were all associated with improved circadian functions, and whether the advantageous outcomes observed in the current study could be extended to female or older subjects (as we only investigated the ISX-9 effect in 14–18-month-old male mice), are interesting topics to examine in the future.

Methods

Reagent

Small molecules that were elected for further oscillation tests were purchased from the following companies: ISX-9 from Selleck; GW4064, OSI-930, Atrasentan, Forskolin, and KN93 from Med Chem Express; 2-NP and HhAntag from TargetMol. Antibodies were purchased from the following resources: anti-BMAL1 (14020, Cell Signaling Technology), anti-DBP (12662-1-AP, ProteinTech), anti-PER2 (13168, ABclonal), anti-REV-ERBα (13418, Cell Signaling Technology), anti-GAPDH (60004, ProteinTech), HRP-conjugated goat anti-rabbit secondary antibody (1706515, Bio-Rad), and HRP-conjugated goat anti-mouse secondary antibody (1706516, Bio-Rad).

Cell line

HEK293T, NIH-3T3, and N2a cell lines were obtained from ATCC. MEF cells were isolated from E13.5d embryos of wild-type, Bmal1^−/− or mPer2^Luc/+ mice. Cells were cultured in DMEM (Invitrogen) with 10% FBS and 100 U/ml penicillin/streptomycin, and maintained at 37 °C with 5% CO₂ in a humidified environment. All cell lines were examined with a PCR-based method (MP0035, Sigma) to ensure mycoplasma-free.

Animal strain

Reporter mouse strain mPer2^Luc (006852)³⁸ and Bmal1-floxed conditional strain (007668)⁶² were obtained from the Jackson Laboratory and maintained in a C57BL/6 J background. Mice were group housed in a specific pathogen-free facility under a 12:12 h light-dark cycle at 22 ± 2 °C, and animal experiments were conducted in male mice in accordance with protocols approved by the institutional Biomedical Research Ethics Committee, Shanghai Institute of Nutrition and Health and Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.

Plasmid

For luciferase-based expression assays, mouse Camk2a/b/d/g, Dbp, and Rev-erbα promoter fragments were PCR amplified from mouse genomic DNA and then cloned into pGL3-Basic-luc vector (E1751, Promega) via MluI and BglII sites (primer information see Supplementary Table 1). Constructs for overexpressing Bmal1, FLAG-Bmal1, FLAG-Camk2a, b, d, and g were cloned via NotI and MluI sites into a pCAGGS vector with FLAG-tag fusion. Bmal1 mutants (S513A, S515A, S516A, S519A, and S513A/S515A/S516A) and Camk2d K43M mutant were generated using PCR-based site-directed mutagenesis (primer information list in Supplementary Table 1), then subcloned into the same pCAGGS backbone.

Small molecule screen

Primary small molecule screen (Natural Products Library, NIH Clinical Collection LOPAC Collection and The Spectrum Collection from Sigma-Aldrich; New compound library, Epigenetics library and GPCR & G Protein library from Selleck; Approved drug screening library and Inhibitor library from TargetMol; Stem cell regulator library from Merck; Neurotransmitters library from Tocris Bioscience; Exclusive new compound library from MCE; and Nuclear Receptor Ligand agonists or antagonists Library from Enzo Life Sciences) was carried out in 384-well plates, with ~3000 mPer2^Luc MEF cells seeded 48 h prior to the chemical applications at 10 μM dosage. Luciferase activities were measured at two time points, 24-h and 30-h post chemical treatment, respectively, using the ONE-Glo™ luciferase assay system (E6120, Promega). After chemiluminescence measurement, cell viability was determined by alamarBlue assay (Invitrogen). The summary of the relative compound effect was listed in Supplementary Data 1. The screen was performed in the Chemical Biology Core Facility, Center for Excellence in Molecular Cell Science, CAS.

Real-time bioluminescence recording

For the real-time recording of PER2::LUC bioluminescence, ~2.5 × 10⁵ mPer2^Luc MEF cells were seeded and then cultured for 48 h with DMEM in 3.5 cm dishes until confluent. The cells were then synchronized with 200 nM dexamethasone for 1 h, before the bioluminescence recording in serum-free DMEM medium supplemented with 200 μM luciferin (A5030, Tokyo Chemical Industry) together with indicated small molecules at 10 μM or indicated concentration. For Camk2d:Luc oscillation recording, pGL3 reporter plasmid containing mouse Camk2d promoter was transfected (1 μg) via Lipofectamine 3000 (Invitrogen) into 2.5 × 10⁵ MEF cells, then cultured for 48 h before 200 nM dexamethasone synchronization then bioluminescence recording. The cultures were recorded in a light-tight chamber containing photon multiplier tube recording (PMT) detector assemblies (LumiCycle 32, Actimetrics) for continuous 4 days. Data including amplitude and period results, were processed with the LumiCycle Analysis software (Actimetrics).

Tissue culture for bioluminescence recording

SCN slices and recordings were conducted as described in ref. ⁶³. In brief, brains were harvested from 6-month-old mPer2^Luc mice, then the SCN slices were sectioned via McIlwain Tissue Chopper (TC752, Cavey Laboratory Engineering Co. Ltd) into 300-μm thickness. SCN slices were cultured on a membrane (PICM0RG50, Millipore) in a 1.5 ml recording medium (1x DMEM, 1x B27 supplement, 4.2 mM NaHCO₃, 10 mM HEPES, 1X penicillin/streptomycin and 200 μM luciferin) together with DMSO or ISX-9 (10 μM), then detected by continuous PMT recording (LumiCycle 32, Actimetrics). Pituitary was cultured and recorded under the same conditions as for SCN.

Quantitative RT-PCR analysis

Total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Approximately 3 μg RNA was applied for cDNA synthesis using Omniscript RT Kit (205111, Qiagen). Real-time PCR reactions were assembled with the use of the SYBR Green PCR Kit (208054, Qiagen), then analyzed in an ABI ViiATM 7. The QuantStudioTM Real-Time PCR Software was used to determine gene expression levels and normalized to a ribosomal reference gene, Rpl19. Primer sequences are listed in Supplementary Table 2.

Immunoblot

Cell cultures were lysed in chilled lysis buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5% Nonidet P-40 and 1% Triton X-100) supplemented with protease inhibitors (118735800, Sigma-Aldrich) and phosphatase inhibitors (4906837001, Roche). The lysates were measured for protein concentration by Bradford assay, then adjusted for equal protein content before immunoblot analysis with antibodies described above. To prepare the Phos-tag gel electrophoresis, cell lysates were resolved in 4% acrylamide gels containing 40 μM acrylamide Phos-tag ligand (F4002, APExBIO) and 80 μM MnCl₂.

Immunohistochemistry

Male mice at indicated ages were euthanized, then perfused with 4% paraformaldehyde, subsequently cryoprotected in 30% sucrose before being embedded in OCT (4583, SAKURA). Coronal brain sections were collected by using a cryostat (CM1850, Leica) at 30-μm thickness, then treated with 3% H₂O₂ to quench residual peroxidase activity in samples. Followed by antigen retrieval step via 0.05% trypsin treatment for 5 min at room temperature, brain sections were blocked with TNB blocking buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% FBS) prior to the incubation of primary anti-PER2 antibodies with 1:300 dilutions at 4 °C for overnight. After being washed with TNT buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) three times, HRP-conjugated anti-rabbit secondary antibodies (1:1000) were applied to the sections and proceeded incubation for 1.5 h at room temperature. After TNT buffer washes three times, brain sections were incubated with fluorophore-labeled tyramide substrate (TSA plus Cyanine3 kit, PerkinElmer) and DAPI (10 ng/ml, Sigma-Aldrich), then visualized by confocal microscopy (Cell Observer, ZEISS). The microscopy results were analyzed and quantified by ImageJ.

Indirect calorimetry

Indirect calorimetry in a metabolic cage was performed with the CLAMS-16 system (Columbus Instruments). 2-, 14-, and 18-month-old male mouse cohorts were treated with ISX-9 (20 mg/Kg) or DMSO for 7 days before housing in the metabolic cages. Mice were singly housed and allowed for accommodation for 2 days in the metabolic cage before measurements. Standard chow and water were provided ad libitum throughout the experiment. Measurements of VO₂, VCO₂, food intake, water access, and activity were recorded every 10 min for up to 3 consecutive days in a 25 °C cubicle, under a 12:12 h light-dark cycle. Physical activities and respiratory exchange ratio (RER = VCO₂/VO₂) were calculated via software CLAX (Columbus Instruments).

Electroencephalography recording and EEG data analysis

Mouse EEG recording was carried out by HD-X02 telemetry implants (DSI PhysioTel™). Mice were deeply anesthetized by single intramuscular dosing of 5 mg/kg pentobarbital before implantation surgery. The two EEG electrodes were screwed into the skull 1 mm lateral, 1 and 3 mm posterior to the Bregma, respectively. After surgery, mice were intraperitoneally injected with 5% glucose solution (40 μl/gram body weight) for 7 days to facilitate recovery. Activity, body temperature, EEG, and EMG signals were collected by the RPC-1 receiver connected to an MX2 hub and a computer. The recordings were carried out under a normal light-dark cycle for 2 days as a pretreated baseline. Male mice at indicated age (2- and 14-month-old) were injected intraperitoneally with DMSO for 7 days, then waited for 7 days before the 3-day recording of EEG and EMG as the control data. Finally, the same cohorts of mice were injected with ISX-9 (20 mg/Kg) for 7 days, stayed untreated for 7 days, then proceeded to 3-day EEG and EMG recording. NeuroScore 3.2.0 (DSI) was applied for mouse EEG sleep analysis to summarize awake, REM, and NREM sleep stages, and identify the day-night sleep-wake transitions scored in 10-s epochs. EEG powers (δ: 0.5–4 Hz, θ: 4–8 Hz, α: 8–12 Hz, σ:12–16 Hz, β: 16–32 Hz, γ: >32 Hz) with 1-h average data points were shown^64,65.

In vitro Ca²⁺ imaging

MEF cells (2 × 10⁵) were seeded on glass bottom culture dishes (801002, NEST) 24 h prior to the incubation of Fluo-4 AM (2 μM, Molecular Probes) in serum-free DMEM for 45 min at 37 °C. After the cleaning of unbound dye by PBS rinse twice, the cells were measured for baseline emission (excitation 488 nm, emission 520 nm) for 15 min. The Ca²⁺-influx recording was started at DMSO or 10 μM ISX-9 addition (time 0). Images were collected at 1 min intervals for 15 min by fluorescence microscopy (Cell Observer, ZEISS).

siRNA knockdown

Silencer oligonucleotides for mouse Camk2a/b/d/g and scramble controls were purchased from GenePharma (Scramble: UUCUCCGAACGUGUCACGU; siCamk2a: CAACAUCGUCCGACUACAU; siCamk2b:GAAGAGCUGCGCCCUGGUU; siCamk2d: GAAGCCAAAGACCUCAUCA; siCamk2g: GUAGAGUGCUUACGCAAAU). MEF cells were transiently transfected with siRNA oligonucleotides using Lipofectamine 3000 (Invitrogen) and then subjected to real-time bioluminescence recording as described above.

Protein purification and kinase assay

For BMAL1 and CaMKII purifications, pCAGGS plasmids (5 μg) encoding FLAG-Bmal1, FLAG-Camk2 isoforms, and mutants were transfected individually into 2 × 10⁶ HEK293T cells. After expressing for 48 h, cells were harvested and lysed in 400 μl lysis buffer containing protease inhibitor (Roche). Approximately 3 mg of cell lysates were incubated with 20 μl anti-FLAG M2 magnetic beads (Sigma) for 4 h at 4 °C, then cleaned the impurities via TBS buffer (50 mM Tris at pH 7.5, 150 mM NaCl) washes three times, before the elution by 100 μl of 3X FLAG peptide (150 ng/μl, Sigma). Kinase assays were carried out with a Universal kinase activity kit (EA004, R&D Systems) at 1:100 CaMKII to Autocamtide-2 Peptide (sc-3029, Santa Cruz Biotechnology), or 1:4 CaMKII to BMAL1 ratio, according to the manufacturer’s instruction, finally measured the absorbance of reactions at 620 nm.

Single-molecule RNA in situ hybridization

Single-molecule in situ hybridization was performed with the RNAscope Multiplex Fluorescent v2-kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. Briefly, mice were euthanized, perfused with 4% paraformaldehyde, then the brains were carefully removed and post-fixed in 4% paraformaldehyde for overnight before the cryoprotection procedure in 30% sucrose-1X PBS at 4 °C. After embedding with OCT medium, 30-μm coronal brain sections were collected, followed by dehydration, peroxidase activity quenching, and target retrieval steps via Protease Plus solution (322381, Advanced Cell Diagnostics). After washes with H₂O, sections were incubated with either Camk2a, 2b, 2d, 2g target probe (445231, 453601, 508941, and 522071, respectively, Advanced Cell Diagnostics, 1:300 dilution) at 40 °C for 2 h in HybEZ oven. Amplification, horseradish peroxidase incorporation, and tyramide stain steps were conducted as described in the user manual (323100-USM, Advanced Cell Diagnostics), followed by DAPI counterstain and fluorescence microscopy (VS120, OLYMPUS). The images were analyzed and quantified by ImageJ.

Luciferase assay

N2a cells were seeded into 24-well dishes at 8 × 10⁴ cells per well density 24 h before transfections. Bmal1/Clock (200/200 ng), or Camk2 (200 ng) overexpression with reporter plasmids (100 ng) were transfected per well with Lipofectamine 3000 (Invitrogen). After 24 h, cells were synchronized with 100 nM dexamethasone for 15 min, followed by the treatment with DMSO or 10 μM ISX-9 in DMEM for 24 h. Cells were harvested and determined for cell number, then ~1 × 10⁵ cells were lysed in 100 μl 1X Glo Lysis Buffer (E266A, Promega). About 5 μl lysate was mixed with 100 μl substrate (ONE-Glo™ luciferase assay system, E6120, Promega) for luciferase measurement (Lumat LB 9508, BERTHOLD).

Immunoprecipitation and mass spectrometry

N2a cells were transfected with a pCAGGS-FLAG-Bmal1 plasmid for the expression of FLAG-BMAL1 for 24 h with treating DMSO or 10 μM ISX-9. About 3 × 10⁷ cells were then harvested and lysed with 1200 μl chilled lysis buffer containing protease and phosphatase inhibitors (Roche). Approximately 10 mg of cell lysates were incubated with 60 μl anti-FLAG M2 magnetic beads (Sigma) for 4 h at 4 °C. The beads were then washed with TBS buffer (50 mM Tris at pH 7.5, 150 mM NaCl) three times, before the elution by 300 μl of 3X FLAG peptide (150 ng/μl, Sigma). The eluted FLAG-BMAL1 from ISX-9-treated or DMSO control were compared via immunoblot, before the analysis via mass spectrometry (LC-MS/MS). The identification of BMAL1 phosphorylation sites was carried out by first enriching the phospho-peptides with TiO₂ beads, before the separation with an Easy-nLC 1200 nano HPLC (Thermo Fisher Scientific). Tandem mass spectrometry (MS/MS) analysis was performed with a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) in National Facility for Protein Science in Shanghai.

Statistics and reproducibility

An individual in vitro experiments were performed at least three times unless otherwise indicated. Animal experiments were independently repeated at least two times with consistent results. Numbers are indicated in the figure legends. Results are presented as mean ± SD or mean ± SEM, and statistical analyses were performed using Prism version 7.0 (GraphPad) with unpaired two-tailed Student’s t-test (statistical significances are labeled as ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001), one-way ANOVA, or two-way ANOVA with Bonferroni correction (statistical significances are labeled as ns, not significant, #p < 0.05, ##p < 0.01, ###p < 0.001) as indicated in legends.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The small molecule screen results are available in Supplementary Data 1. The source data for graphs, charts, and immunoblots with the paper is available in Supplementary Data 2. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) with the dataset ID PXD034339. The 18 plasmids associated with this study have been deposited to Addgene with the ID 186818-186835. All other data that support the finding of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Shujue Lan of the Chemical Biology Core Facility, Center for Excellence in Molecular Cell Science, CAS, for the assistance with the small molecule screen. This work was supported by grants from the Lingang Laboratory (LG202106-02-04 to Y.-C.T. and LG202106-02 to H.-C.C.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB32060200 to H.-C.C.), Shanghai Municipal Science and Technology Major Project (2018SHZDZX05 to H.-C.C.), Shanghai Municipal Government Bureau of Science and Technology (18JC1410100 to H.-C.C.), the National Natural Science Foundation of China (3167122 and 82021001 to H.-C.C.; 32171161 and 31671408 to Y.-C.T.).

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These authors contributed equally: Huilin Li, Jiali Ou.

Authors and Affiliations

CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
Huilin Li, Jiali Ou, Yaqun Li & Yun-Chi Tang
Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, 200031, China
Niannian Xu, Qing Li & Hung-Chun Chang
Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai, 201210, China
Niannian Xu & Hung-Chun Chang
National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai, 201210, China
Ping Wu & Chao Peng

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Huilin Li
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Contributions

Y.-C.T. and H.-C.C. supervised the project and wrote the manuscript. H.L. and J.O. performed most experiments and data analysis. H.L. and N.X. assisted with the EEG and metabolic cage data analysis. Y.L. assisted with biochemical assays. Q.L. assisted with animal experiments and husbandry. P.W. and C.P. performed mass spectrometry analysis. The manuscript was approved by all authors.

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Li, H., Ou, J., Li, Y. et al. ISX-9 potentiates CaMKIIδ-mediated BMAL1 activation to enhance circadian amplitude. Commun Biol 5, 750 (2022). https://doi.org/10.1038/s42003-022-03725-x

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Received: 13 October 2021
Accepted: 15 July 2022
Published: 28 July 2022
DOI: https://doi.org/10.1038/s42003-022-03725-x

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