Unsupervised learning of aging principles from longitudinal data.

Age is the leading risk factor for prevalent diseases and death. However, the relation between age-related physiological changes and lifespan is poorly understood. We combined analytical and machine learning tools to describe the aging process in large sets of longitudinal measurements. Assuming that aging results from a dynamic instability of the organism state, we designed a deep artificial neural network, including auto-encoder and auto-regression (AR) components. The AR model tied the dynamics of physiological state with the stochastic evolution of a single variable, the "dynamic frailty indicator" (dFI). In a subset of blood tests from the Mouse Phenome Database, dFI increased exponentially and predicted the remaining lifespan. The observation of the limiting dFI was consistent with the late-life mortality deceleration. dFI changed along with hallmarks of aging, including frailty index, molecular markers of inflammation, senescent cell accumulation, and responded to life-shortening (high-fat diet) and life-extending (rapamycin) treatments.

Circadian Rhythm Disruption Increases Tumor Growth Rate and Accumulation of Myeloid-Derived Suppressor Cells.

Circadian rhythm disruption is implicated in the initiation and progression of many diseases, including cancer. External stimuli, such as sunlight, serve to synchronize physiological processes and cellular functions to a 24-h cycle. The immune system is controlled by circadian rhythms, and perturbation of these rhythms can potentially alter the immune response to infections and tumors. The effect of circadian rhythm disruption on the immune response to tumors remains unclear. Specifically, the effects of circadian disruption (CD) on immunosuppressive cell types within the tumor, such as myeloid-derived suppressor cells (MDSCs), are unknown. In this study, a shifting lighting schedule is used to disrupt the circadian rhythm of mice. After acclimation to lighting schedules, mice are inoculated with 4T1 or B16-F10 tumors. Tumor growth is increased in mice housed under circadian disrupting lighting conditions compared to standard lighting conditions. Analysis of immune populations within the spleen and tumor shows an increased accumulation of MDSCs within these tissues, suggesting that MDSC mediated immunosuppression plays a role in the enhanced tumor growth caused by circadian disruption. This paves the way for future studies of the effects of CD on immunosuppression in cancer.

Immune checkpoint protein VSIG4 as a biomarker of aging in murine adipose tissue.

Adipose tissue is recognized as a major source of systemic inflammation with age, driving age-related tissue dysfunction and pathogenesis. Macrophages (Mφ) are central to these changes yet adipose tissue Mφ (ATMs) from aged mice remain poorly characterized. To identify biomarkers underlying changes in aged adipose tissue, we performed an unbiased RNA-seq analysis of ATMs from young (8-week-old) and healthy aged (80-week-old) mice. One of the genes identified, V-set immunoglobulin-domain-containing 4 (VSIG4/CRIg), encodes a Mφ-associated complement receptor and B7 family-related immune checkpoint protein. Here, we demonstrate that Vsig4 expression is highly upregulated with age in perigonadal white adipose tissue (gWAT) in two mouse strains (inbred C57BL/6J and outbred NIH Swiss) independent of gender. The accumulation of VSIG4 was mainly attributed to a fourfold increase in the proportion of VSIG4+ ATMs (13%-52%). In a longitudinal study, VSIG4 expression in gWAT showed a strong correlation with age within a cohort of male and female mice and correlated strongly with physiological frailty index (PFI, a multi-parameter assessment of health) in male mice. Our results indicate that VSIG4 is a novel biomarker of aged murine ATMs. VSIG4 expression was also found to be elevated in other aging tissues (e.g., thymus) and was strongly induced in tumor-adjacent stroma in cases of spontaneous and xenograft lung cancer models. VSIG4 expression was recently associated with cancer and several inflammatory diseases with diagnostic and prognostic potential in both mice and humans. Further investigation is required to determine whether VSIG4-positive Mφ contribute to immunosenescence and/or systemic age-related deficits.

Superior cancer preventive efficacy of low versus high dose of mTOR inhibitor in a mouse model of prostate cancer.

The mechanistic target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation and survival in response to the availability of energy sources and growth factors. Cancer development and progression is often associated with constitutive activation of the mTOR pathway, thus justifying mTOR inhibition as a promising approach to cancer treatment and prevention. However, development of previous rapamycin analogues has been complicated by their induction of adverse side effects and variable efficacy. Since mTOR pathway regulation involves multiple feedback mechanisms that may be differentially activated depending on the degree of mTOR inhibition, we investigated whether rapamycin dosing could be adjusted to achieve chemopreventive efficacy without side effects. Thus, we tested the efficacy of two doses of a novel, highly bioavailable nanoformulation of rapamycin, Rapatar, in a mouse prostate cancer model (male mice with prostate epithelium-specific Pten-knockout). We found that the highest efficacy was achieved by the lowest dose of Rapatar used in the study. While both doses tested were equally effective in suppressing proliferation of prostate epithelial cells, higher dose resulted in activation of feedback circuits that reduced the drug's tumor preventive efficacy. These results demonstrate that low doses of highly bioavailable mTOR inhibitor, Rapatar, may provide safe and effective cancer prevention.

Daily Time of Radiation Treatment Is Associated with Subsequent Oral Mucositis Severity during Radiotherapy in Head and Neck Cancer Patients.

BACKGROUND: Limited treatment options are available for oral mucositis, a common, debilitating complication of cancer therapy. We examined the association between daily delivery time of radiotherapy and the severity of oral mucositis in patients with head and neck cancer. METHODS: We used electronic medical records of 190 patients with head and neck squamous cell carcinoma who completed radiotherapy, with or without concurrent chemotherapy, at Roswell Park Comprehensive Cancer Center (Buffalo, NY) between 2015 and 2017. Throughout a 7-week treatment course, patient mouth and throat soreness (MTS) was self-reported weekly using a validated oral mucositis questionnaire, with responses 0 (no) to 4 (extreme). Average treatment times from day 1 until the day before each mucositis survey were categorized into seven groups. Multivariable-adjusted marginal average scores (LSmeans) were estimated for the repeated- and maximum-MTS, using a linear-mixed model and generalized-linear model, respectively. RESULTS: Radiation treatment time was significantly associated with oral mucositis severity using both repeated-MTS (n = 1,156; P = 0.02) and maximum-MTS (n = 190; P = 0.04), with consistent patterns. The severity was lowest for patients treated during 8:30 to <9:30 am (LSmeans for maximum-MTS = 2.24; SE = 0.15), increased at later treatment times and peaked at early afternoon (11:30 am to <3:00 pm, LSmeans = 2.66-2.71; SEs = 0.16/0.17), and then decreased substantially after 3 pm. CONCLUSIONS: We report a significant association between radiation treatment time and oral mucositis severity in patients with head and neck cancer. IMPACT: Although additional studies are needed, these data suggest a potential simple treatment time solution to limit severity of oral mucositis during radiotherapy without increasing cost.

OT-82, a novel anticancer drug candidate that targets the strong dependence of hematological malignancies on NAD biosynthesis.

Effective treatment of some types of cancer can be achieved by modulating cell lineage-specific rather than tumor-specific targets. We conducted a systematic search for novel agents selectively toxic to cells of hematopoietic origin. Chemical library screenings followed by hit-to-lead optimization identified OT-82, a small molecule with strong efficacy against hematopoietic malignancies including acute myeloblastic and lymphoblastic adult and pediatric leukemias, erythroleukemia, multiple myeloma, and Burkitt's lymphoma in vitro and in mouse xenograft models. OT-82 was also more toxic towards patients-derived leukemic cells versus healthy bone marrow-derived hematopoietic precursors. OT-82 was shown to induce cell death by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD synthesis. In mice, optimization of OT-82 dosing and dietary niacin further expanded the compound's therapeutic index. In toxicological studies conducted in mice and nonhuman primates, OT-82 showed no cardiac, neurological or retinal toxicities observed with other NAMPT inhibitors and had no effect on mouse aging or longevity. Hematopoietic and lymphoid organs were identified as the primary targets for dose limiting toxicity of OT-82 in both species. These results reveal strong dependence of neoplastic cells of hematopoietic origin on NAMPT and introduce OT-82 as a promising candidate for the treatment of hematological malignancies.

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation.

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.

Physiological frailty index (PFI): quantitative in-life estimate of individual biological age in mice.

The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.

Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody.

Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated ß-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system. In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and MS analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging. Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation.

Metabolic clock generates nutrient anticipation rhythms in mTOR signaling.

The mTOR signaling pathway modulates metabolic processes with respect to nutrient availability and other growth-related cues. According to the existing paradigm, mTOR complex 1 (mTORC1) activityin vivo is induced by food and gradually decreases during fasting. We found that mTORC1 activity is controlled by an internal clock mechanism different from the known light-entrainable circadian clock. We observed 24-hr rhythms in phosphorylation of mTORC1 downstream targets, which were entrained by food, persisted during fasting and could be uncoupled from oscillating expression of the canonical circadian clock genes. Furthermore, these rhythms were present in tissues of mice with disrupted light-entrainable circadian clock. We propose tissue-specific rhythms in the expression of tor and its negative regulator deptor as the molecular mechanism of the mTORC1 activity oscillation. Our data demonstrate the existence of at least two independent molecular circadian clocks: one providing metabolic adaptation to periodic light/darkness and the other - to feeding.

Daily rhythms are retained both in spontaneously developed sarcomas and in xenografts grown in immunocompromised SCID mice.

The circadian clock generates and regulates many daily physiological, metabolic and behavioral rhythms as well as acute responses to various types of stresses including those induced by anticancer treatment. It has been proposed that modulatory function of the clock may be used for improving the therapeutic efficacy of established anti-cancer treatments. In order to rationally exploit this mechanism, more information is needed to fully characterize the functional status of the molecular clock in tumors of different cellular origin; however, the data describing tumor clocks are still inconsistent. Here we tested the status of clock in two models of tumors derived from connective tissue: sarcomas spontaneously developed in p53-deficient mice and human fibrosarcoma cells grown as xenografts in immunocompromised severe combined immunodeficient (SCID) mice. We show that both types of tumors retain a functional clock, which is synchronized in phase with normal tissues. We also show that spontaneously developed tumors are not only oscillating in the context of an organism where they receive hormonal and metabolic signals but continue oscillating ex vivo in tissue explants demonstrating that tumors have functional clocks capable of timing all their functions. We also provide evidence that similar to liver, tumors can be synchronized by food availability independent of the central pacemaker in the suprachiasmatic nuclei (SCN). These data provide the basis for the design of anticancer therapies that take into account the circadian metabolic and physiological patterns of both the tumor and normal tissues.

Tissue-specific changes in molecular clocks during the transition from pregnancy to lactation in mice.

Circadian clocks regulate homeostasis and mediate responses to stressors. Lactation is one of the most energetically demanding periods of an adult female's life. Peripartum changes occur in almost every organ so the dam can support neonatal growth through milk production while homeostasis is maintained. How circadian clocks are involved in adaptation to lactation is currently unknown. The abundance and temporal pattern of core clock genes' expression were measured in suprachiasmatic nucleus, liver, and mammary from late pregnant and early lactation mice. Tissue-specific changes in molecular clocks occurred between physiological states. Amplitude and robustness of rhythms increased in suprachiasmatic nucleus and liver. Mammary rhythms of core molecular clock genes were suppressed. Attenuated rhythms appeared to be a physiological adaptation of mammary to lactation, because manipulation of timing of suckling resulting in significant differences in plasma prolactin and corticosterone had no effect on amplitude. Analysis of core clock proteins revealed that the stoichiometric relationship between positive (CLOCK) and negative (PER2) components remained 1:1 in liver but was increased to 4:1 in mammary during physiological transition. Induction of differentiation of mammary epithelial cell line HC11 with dexamethasone, insulin, and prolactin resulted in similar stoichiometric changes among positive and negative clock regulators, and prolactin induced phase shifts in HC11 Arntl expression rhythm. Data support that distinct mechanisms drive periparturient changes in mammary clock. Stoichiometric change in clock regulators occurs with gland differentiation. Suppression of mammary clock gene expression rhythms represents a physiological adaptation to suckling cues. Adaptations in mammary clock are likely needed in part to support suckling demands of neonates.

BMAL1-dependent regulation of the mTOR signaling pathway delays aging.

The circadian clock, an internal time-keeping system, has been linked with control of aging, but molecular mechanisms of regulation are not known. BMAL1 is a transcriptional factor and core component of the circadian clock; BMAL1 deficiency is associated with premature aging and reduced lifespan. Here we report that activity of mammalian Target of Rapamycin Complex 1 (mTORC1) is increased upon BMAL1 deficiency both in vivo and in cell culture. Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%. Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling. We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

Deficiency in PER proteins has no effect on the rate of spontaneous and radiation-induced carcinogenesis.

There is a growing body of evidence that components of the circadian clock are involved in modulation of numerous signaling pathways, and that clock deregulation due to environmental or genetic factors contributes to the development of various pathologies, including cancer. Previous work performed in tissue culture and in in vivo mouse models defined mammalian PERIOD proteins as tumor suppressors, although some experimental inconsistencies (the use of mice on mixed genetic background, lack of sexual discrimination) did not allow a definitive conclusion. To address this issue in a systematic way, we performed a detailed analysis comparing the incidence of tumor development after low-dose ionizing radiation in male and female wild-type, Per1(-/-), and Per2(-/-) mice. We showed that in contrast to previous reports deficiency in either Per1 or Per2 genes by itself does not make mice more tumor-prone; moreover, some of the long-term effects of ionizing radiation in Per2-deficient mice are reminiscent more of accelerated aging rather than tumor-prone phenotype. Our histopathological analysis also revealed significant sexual dimorphism both in the rate of radiation-induced tumorigenesis and in the spectrum of tumors developed, which underscores the importance of using sex-matched experimental groups for in vivo studies. Based on our results, we suggest that the role of PER proteins as bona fide tumor suppressors needs to be reevaluated.

Pharmacological modulators of the circadian clock as potential therapeutic drugs: focus on genotoxic/anticancer therapy.

The circadian clock is an evolutionary conserved intrinsic timekeeping mechanism that controls daily variations in multiple biological processes. One important process that is modulated by the circadian clock is an organism's response to genotoxic stress, such as that induced by anticancer drug and radiation treatments. Numerous observations made in animal models have convincingly demonstrated that drug-induced toxicity displays prominent daily variations; therefore, undesirable side effects could be significantly reduced by administration of drugs at specific times when they are better tolerated. In some cases, these critical times of the day coincide with increased sensitivity of tumor cells allowing for a greater therapeutic index. Despite encouraging results of chronomodulated therapies, our knowledge of molecular mechanisms underlying these observations remains sketchy. Here we review recent progress in deciphering mechanistic links between circadian and stress response pathways with a focus on how these findings could be applied to anticancer clinical practice. We discuss the potential for using high-throughput screens to identify small molecules that can modulate basic parameters of the entire circadian machinery as well as functional activity of its individual components. We also describe the discovery of several small molecules that can pharmacologically modulate clock and that have a potential to be developed into therapeutic drugs. We believe that translational applications of clock-targeting pharmaceuticals are twofold: they may be developed into drugs to treat circadian-related disorders or used in combination with existing therapeutic strategies to improve therapeutic index of a given genotoxic treatment via the intrinsic clock mechanism.

Rapamycin extends lifespan and delays tumorigenesis in heterozygous p53+/- mice.

TOR (Target of Rapamycin) pathway accelerates cellular and organismal aging. Similar to rapamycin, p53 can inhibit the mTOR pathway in some mammalian cells. Mice lacking one copy of p53 (p53+/- mice) have an increased cancer incidence and a shorter lifespan. We hypothesize that rapamycin can delay cancer in heterozygous p53+/- mice. Here we show that rapamycin (given in a drinking water) extended the mean lifespan of p53+/- mice by 10% and when treatment started early in life (at the age less than 5 months) by 28%. In addition, rapamycin decreased the incidence of spontaneous tumors. This observation may have applications in management of Li-Fraumeni syndrome patients characterized by heterozygous mutations in the p53 gene.

New nanoformulation of rapamycin Rapatar extends lifespan in homozygous p53-/- mice by delaying carcinogenesis.

The nutrient-sensing mTOR (mammalian Target of Rapamycin) pathway regulates cellular metabolism, growth functions, and proliferation and is involved in age-related diseases including cancer, type 2 diabetes, neurodegeneration and cardiovascular disease. The inhibition of mTOR by rapamycin, or calorie restriction, has been shown to extend lifespan and delays tumorigenesis in several experimental models suggesting that rapamycin may be used for cancer prevention. This requires continuous long-term treatment making oral formulations the preferred choice of administration route. However, rapamycin by itself has very poor water solubility and low absorption rate. Here we describe pharmacokinetic and biological properties of novel nanoformulated micelles of rapamycin, Rapatar. Micelles of Rapatar were rationally designed to increase water solubility of rapamycin to facilitate oral administration and to enhance its absorption. As a result, bioavailability of Rapatar was significantly increased (up to 12%) compared to unformulated rapamycin, which concentration in the blood following oral administration remained below level of detection. We also demonstrated that the new formulation does not induce toxicity during lifetime administration. Most importantly, Rapatar extended the mean lifespan by 30% and delayed tumor development in highly tumor-prone p53-/- mice. Our data demonstrate that water soluble Rapatar micelles represent safe, convenient and efficient form of rapamycin suitable for a long-term treatment and that Rapatar may be considered for tumor prevention.

Core circadian protein CLOCK is a positive regulator of NF-κB-mediated transcription.

The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB-mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB-responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box-containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB-responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

Selenium is a modulator of circadian clock that protects mice from the toxicity of a chemotherapeutic drug via upregulation of the core clock protein, BMAL1.

Selenium compounds are known as cancer preventive agents and are also able to ameliorate the toxicity associated with anti-cancer radiation and chemotherapy in mouse models. Sensitivity to the toxicity of chemotherapy is also modulated by the circadian clock, molecular time-keeping system that underlie daily fluctuations in multiple physiological and biochemical processes. Here we show that these two mechanisms are interconnected. By screening a library of small molecules in a cell-based reporter system, we identified L-methyl-selenocysteine as a positive regulator of the core clock protein, BMAL1. L-methyl-selenocysteine up-regulates BMAL1 at the transcriptional level both in cultured cells and in mice. We also show that in tissue culture selenium exerts its action by interfering with TIEG1-mediated repression of Bmal1 promoter. Selenium treatment fails to protect BMAL1-deficient mice from toxicity induced by the chemotherapeutic agent cyclophosphamide but does protect Clock mutant mice deficient in circadian rhythm control but having normal BMAL1. These findings define selenium as circadian modulator and indicate that the tissue protective effect of selenium results, at least in part, from up-regulation of BMAL1 expression and subsequent enhancement of CLOCK/BMAL1-mediated transcription.

Circadian clock proteins control adaptation to novel environment and memory formation.

Deficiency of the transcription factor BMAL1, a core component of the circadian clock, results in an accelerated aging phenotype in mice. The circadian clock regulates many physiological processes and was recently implicated in control of brain-based activities, such as memory formation and the regulation of emotions. Aging is accompanied by the decline in brain physiology, particularly decline in the response and adaptation to novelty. We investigated the role of the circadian clock in exploratory behavior and habituation to novelty using the open field paradigm. We found that mice with a deficiency of the circadian transcription factor BMAL1 display hyperactivity in novel environments and impaired intra- and intersession habituation, indicative of defects in short- and long-term memory formation. In contrast, mice double-deficient for the circadian proteins CRY1 and CRY2 (repressors of the BMAL1-mediated transcription) demonstrate reduced activity and accelerated habituation when compared to wild type mice. Mice with mutation in theClock gene (encoding the BMAL1 transcription partner) show normal locomotion, but increased rearing activity and impaired intersession habituation. BMAL1 is highly expressed in the neurons of the hippocampus - a brain region associated with spatial memory formation; BMAL1 deficiency disrupts circadian oscillation in gene expression and reactive oxygen species homeostasis in the brain, which may be among the possible mechanisms involved. Thus, we suggest that the BMAL1:CLOCK activity is critical for the proper exploratory and habituation behavior, and that the circadian clock prepares organism for a new round of everyday activities through optimization of behavioral learning.