Wake-sleep cycles are severely disrupted by diseases affecting cytoplasmic homeostasis.

The circadian clock is based on a transcriptional feedback loop with an essential time delay before feedback inhibition. Previous work has shown that PERIOD (PER) proteins generate circadian time cues through rhythmic nuclear accumulation of the inhibitor complex and subsequent interaction with the activator complex in the feedback loop. Although this temporal manifestation of the feedback inhibition is the direct consequence of PER's cytoplasmic trafficking before nuclear entry, how this spatial regulation of the pacemaker affects circadian timing has been largely unexplored. Here we show that circadian rhythms, including wake-sleep cycles, are lengthened and severely unstable if the cytoplasmic trafficking of PER is disrupted by any disease condition that leads to increased congestion in the cytoplasm. Furthermore, we found that the time delay and robustness in the circadian clock are seamlessly generated by delayed and collective phosphorylation of PER molecules, followed by synchronous nuclear entry. These results provide clear mechanistic insight into why circadian and sleep disorders arise in such clinical conditions as metabolic and neurodegenerative diseases and aging, in which the cytoplasm is congested.

Circadian rhythm shifts and alcohol access in adolescence synergistically increase alcohol preference and intake in adulthood in male C57BL/6 mice.

BACKGROUND: Adolescents have a natural tendency to be night owls, maintaining delayed circadian rhythms, and this rhythm is in direct conflict with the early wake times required during the school year. This leads to 'social jetlag', chronic circadian stress or desynchrony (CD) in which the rhythm of the intrinsic body clock is out of sync with behavior. CD increases alcohol intake in adolescents and adults, yet it is unknown whether adolescent CD also increases long-term addiction risk. The goal of this study was to determine whether adolescent alcohol intake in CD would increase adult alcohol preference and intake in male C57BL/6 J mice. METHODS: We measured free access alcohol intake, water intake, and wheel-running activity during a normal 12 h (h) baseline photoperiod and then during shifting lighting schedules (Experiment 1) or a shortened circadian day (Experiment 2). RESULTS: In Experiment 1, altered lighting produced a persistent increase in adolescent alcohol intake and in binge-like drinking (drinking at least 5 licks per minute, with no more than a 1 min break in drinking) in adulthood, but only a transient increase in total alcohol intake for the first week after alcohol was reintroduced in adulthood. In Experiment 2, the circadian shift produced a significant increase in alcohol intake in both adolescence and adulthood. Molecular analysis demonstrated changes in plasma corticosterone and neuronal markers of stress and addiction at the conclusion of these experiments in the CD and alcohol-exposed groups. CONCLUSIONS: Thus, we conclude that circadian stress during adolescence is sufficient to produce a long-lasting susceptibility to alcohol use.

DISC1 and reelin interact to alter cognition, inhibition, and neurogenesis in a novel mouse model of schizophrenia.

The etiology of schizophrenia (SCZ) is multifactorial, and depending on a host of genetic and environmental factors. Two putative SCZ susceptibility genes, Disrupted-in-Schizophrenia-1 (DISC1) and reelin (RELN), interact at a molecular level, suggesting that combined disruption of both may lead to an intensified SCZ phenotype. To examine this gene-gene interaction, we produced a double mutant mouse line. Mice with heterozygous RELN haploinsufficiency were crossed with mice expressing dominant-negative c-terminal truncated human DISC1 to produce offspring with both mutations (HRM/DISC1 mice). We used an array of behavioral tests to generate a behavioral phenotype for these mice, then examined the prefrontal cortex and hippocampus using western blotting and immunohistochemistry to probe for SCZ-relevant molecular and cellular alterations. Compared to wild-type controls, HRM/DISC1 mice demonstrated impaired pre-pulse inhibition, altered cognition, and decreased activity. Diazepam failed to rescue anxiety-like behaviors, paradoxically increasing activity in HRM/DISC1 mice. At a cellular level, we found increased α1-subunit containing GABA receptors in the prefrontal cortex, and a reduction in fast-spiking parvalbumin positive neurons. Maturation of adult-born neurons in the hippocampus was also altered in HRM/DISC1 mice. While there was no difference in the total number proliferating cells, more of these cells were in immature stages of development. Homozygous DISC1 mutation combined with RELN haploinsufficiency produces a complex phenotype with neuropsychiatric characteristics relevant to SCZ and related disorders, expanding our understanding of how multiple genetic susceptibility factors might interact to influence the variable presentation of these disorders.

Impact of Alcohol Abuse on Susceptibility to Rare Neurodegenerative Diseases.

Despite the prevalence and well-recognized adverse effects of prenatal alcohol exposure and alcohol use disorder in the causation of numerous diseases, their potential roles in the etiology of neurodegenerative diseases remain poorly characterized. This is especially true of the rare neurodegenerative diseases, for which small population sizes make it difficult to conduct broad studies of specific etiological factors. Nonetheless, alcohol has potent and long-lasting effects on neurodegenerative substrates, at both the cellular and systems levels. This review highlights the general effects of alcohol in the brain that contribute to neurodegeneration across diseases, and then focuses on specific diseases in which alcohol exposure is likely to play a major role. These specific diseases include dementias (alcohol-induced, frontotemporal, and Korsakoff syndrome), ataxias (cerebellar and frontal), and Niemann-Pick disease (primarily a Type B variant and Type C). We conclude that there is ample evidence to support a role of alcohol abuse in the etiology of these diseases, but more work is needed to identify the primary mechanisms of alcohol's effects.

The Molecular Clock and Neurodegenerative Disease: A Stressful Time.

Circadian rhythm dysfunction occurs in both common and rare neurodegenerative diseases. This dysfunction manifests as sleep cycle mistiming, alterations in body temperature rhythms, and an increase in symptomatology during the early evening hours known as Sundown Syndrome. Disruption of circadian rhythm homeostasis has also been implicated in the etiology of neurodegenerative disease. Indeed, individuals exposed to a shifting schedule of sleep and activity, such as health care workers, are at a higher risk. Thus, a bidirectional relationship exists between the circadian system and neurodegeneration. At the heart of this crosstalk is the molecular circadian clock, which functions to regulate circadian rhythm homeostasis. Over the past decade, this connection has become a focal point of investigation as the molecular clock offers an attractive target to combat both neurodegenerative disease pathogenesis and circadian rhythm dysfunction, and a pivotal role for neuroinflammation and stress has been established. This review summarizes the contributions of molecular clock dysfunction to neurodegenerative disease etiology, as well as the mechanisms by which neurodegenerative diseases affect the molecular clock.

Inhibition of casein kinase 1 δ/ε improves cognitive performance in adult C57BL/6J mice.

Time-of-day effects have been noted in a wide variety of cognitive behavioral tests, and perturbation of the circadian system, either at the level of the master clock in the SCN or downstream, impairs hippocampus-dependent learning and memory. A number of kinases, including the serine-threonine casein kinase 1 (CK1) isoforms CK1δ/ε, regulate the timing of the circadian period through post-translational modification of clock proteins. Modulation of these circadian kinases presents a novel treatment direction for cognitive deficits through circadian modulation. Here, we tested the potential for PF-670462, a small molecule inhibitor of CK1δ/ε, to improve cognitive performance in C57BL/6J mice in an array of behavioral tests. Compared to vehicle-treated mice tested at the same time of the circadian day, mice treated with PF-670462 displayed better recall of contextual fear conditioning, made fewer working memory errors in the radial arm water maze, and trained more efficiently in the Morris Water Maze. These benefits were accompanied by increased expression of activity-regulated cytoskeleton-associated protein (Arc) in the amygdala in response to an acute learning paradigm. Our results suggest the potential utility of CK1δ/ε inhibition in improving time-of-day cognitive performance.

Aberrant AZIN2 and polyamine metabolism precipitates tau neuropathology.

Tauopathies display a spectrum of phenotypes from cognitive to affective behavioral impairments; however, mechanisms promoting tau pathology and how tau elicits behavioral impairment remain unclear. We report a unique interaction between polyamine metabolism, behavioral impairment, and tau fate. Polyamines are ubiquitous aliphatic molecules that support neuronal function, axonal integrity, and cognitive processing. Transient increases in polyamine metabolism hallmark the cell's response to various insults, known as the polyamine stress response (PSR). Dysregulation of gene transcripts associated with polyamine metabolism in Alzheimer's disease (AD) brains were observed, and we found that ornithine decarboxylase antizyme inhibitor 2 (AZIN2) increased to the greatest extent. We showed that sustained AZIN2 overexpression elicited a maladaptive PSR in mice with underlying tauopathy (MAPT P301S; PS19). AZIN2 also increased acetylpolyamines, augmented tau deposition, and promoted cognitive and affective behavioral impairments. Higher-order polyamines displaced microtubule-associated tau to facilitate polymerization but also decreased tau seeding and oligomerization. Conversely, acetylpolyamines promoted tau seeding and oligomers. These data suggest that tauopathies launch an altered enzymatic signature that endorses a feed-forward cycle of disease progression. Taken together, the tau-induced PSR affects behavior and disease continuance, but may also position the polyamine pathway as a potential entry point for plausible targets and treatments of tauopathy, including AD.

A phylogenetically conserved DNA damage response resets the circadian clock.

The mammalian circadian clock influences the timing of many biological processes such as the sleep/wake cycle, metabolism, and cell division. Environmental cues such as light exposure can influence the timing of this system through the posttranslational modification of key components of the core molecular oscillator. We have previously shown that DNA damage can reset the circadian clock in a time-of-day-dependent manner in the filamentous fungus Neurospora crassa through the modulation of negative regulator FREQUENCY levels by PRD-4 (homologue of mammalian Chk2). We show that DNA damage, generated with either the radiomimetic drug methyl methane sulfonate or UV irradiation, in mouse embryonic fibroblasts isolated from PER2::LUC transgenic mice or in the NIH3T3 cell line, elicits similar responses. In addition to induction of phase advances, DNA damage caused a decrease in luciferase signal in PER2::LUC mouse embryonic fibroblast cells that is indicative of PER2 degradation. Finally, we show that the activity of the BMAL1 promoter is enhanced during DNA damage. These findings provide further evidence that the DNA damage-mediated response of the clock is conserved from lower eukaryotes to mammals.

Racing the clock: The role of circadian rhythmicity in addiction across the lifespan.

Although potent effects of psychoactive drugs on circadian rhythms were first described over 30 years ago, research into the reciprocal relationship between the reward system and the circadian system - and the impact of this relationship on addiction - has only become a focus in the last decade. Nonetheless, great progress has been made in that short time toward understanding how drugs of abuse impact the molecular and physiological circadian clocks, as well as how disruption of normal circadian rhythm biology may contribute to addiction and ameliorate the efficacy of treatments for addiction. In particular, data have emerged demonstrating that disrupted circadian rhythms, such as those observed in shift workers and adolescents, increase susceptibility to addiction. Furthermore, circadian rhythms and addiction impact one another longitudinally - specifically from adolescence to the elderly. In this review, the current understanding of how the circadian clock interacts with substances of abuse within the context of age-dependent changes in rhythmicity, including the potential existence of a drug-sensitive clock, the correlation between chronotype and addiction vulnerability, and the importance of rhythmicity in the mesocorticolimbic dopamine system, is discussed. The primary focus is on alcohol addiction, as the preponderance of research is in this area, with references to other addictions as warranted. The implications of clock-drug interactions for the treatment of addiction will also be reviewed, and the potential of therapeutics that reset the circadian rhythm will be highlighted.

Alcohol Intake Increases in Adolescent C57BL/6J Mice during Intermittent Cycles of Phase-Delayed, Long-Light Conditions.

Adolescents naturally go to bed and awaken late, but are forced to awaken early for school and work. This leads to "social jetlag", a state of circadian desynchrony (CD), in which internal biological rhythms are out of sync with behavioral rhythms. CD is associated with increased alcohol intake in adults, but has been less well-studied in adolescents. The goal of this study was to model adolescent alcohol intake during similar CD conditions in male C57BL/6J mice. Free access alcohol intake, water intake and wheel-running activity were measured during a normal 12HR photoperiod or during alternating photoperiod (Experiment 1: 12 h light for 4 days followed by 18 h light for 3 days, with dark (activity onset) delayed 9 h during the 18HR photoperiod; Experiment 2: 12 h light for 4 days followed by 6 h light for 3 days, with dark onset delayed 3 h during the 6HR photoperiod). In Experiment 1, CD produced a small but significant increase in the total alcohol intake per day as well as in intake in bouts, with the greatest increase over controls in the hours following the 6HR dark period. Additionally, the pattern of alcohol intake in bouts shifted to increase alcohol intake during the shorter dark period. In Experiment 2, the opposite effect occurred-the longer dark cycle led to lower alcohol drinking in the second half of the dark period. However, in Experiment 2, CD produced no significant changes in either total alcohol intake or alcohol intake in bouts. CONCLUSION: shifts in the light cycle that disrupt the regular pattern of day and night, and increase the length of the night phase, are sufficient to increase both drinking in bouts and restricted drinking in adolescent mice, modeling increased alcohol intake in adolescents during CD.

Disruption of normal circadian clock function in a mouse model of tauopathy.

Disruption of normal circadian rhythm physiology is associated with neurodegenerative disease, which can lead to symptoms such as altered sleep cycles. In Alzheimer's disease (AD), circadian dysfunction has been attributed to ß-amyloidosis. However, it is unclear whether tauopathy, another AD-associated neuropathology, can disrupt the circadian clock. We have evaluated the status of the circadian clock in a mouse model of tauopathy (Tg4510). Tg4510 mice display a long free-running period at an age when tauopathy is present, and show evidence of tauopathy in the suprachiasmatic nucleus (SCN) of the hypothalamus - the site of the master circadian clock. Additionally, cyclic expression of the core clock protein PER2 is disrupted in the hypothalamus of Tg4510 mice. Finally, disruption of the cyclic expression of PER2 and BMAL1, another core circadian clock protein, is evident in the Tg4510 hippocampus. These results demonstrate that tauopathy disrupts normal circadian clock function both at the behavioral and molecular levels, which may be attributed to the tauopathy-induced neuropathology in the SCN. Furthermore, these results establish the Tg4510 mouse line as a model to study how tauopathy disrupts normal circadian rhythm biology.

Deregulated expression of LRBA facilitates cancer cell growth.

LRBA expression is induced by mitogens in lymphoid and myeloid cells. The Drosophila LRBA orthologue rugose/DAKAP550 is involved in Notch, Ras and EGFR pathways. These findings suggest that LRBA could play a role in cell types that have increased proliferative and survival capacity. Here, we show by microarray and real-time PCR analyses that LRBA is overexpressed in several different cancers relative to their normal tissue controls. We also show that LRBA promoter activity and endogenous LRBA mRNA levels are reduced by p53 and increased by E2F1, indicating that mutations in the tumor suppressors p53 and Rb could contribute to the deregulation of LRBA. Furthermore, inhibition of LRBA expression by RNA interference, or inhibition of its function by a dominant-negative mutant, leads to significant growth inhibition of cancer cells, demonstrating that deregulated expression of LRBA contributes to the altered growth properties of a cancer cell. Finally, we show that the phosphorylation of EGFR is affected by the dominant-negative mutant, suggesting LRBA plays a role in the mammalian EGFR pathway. These findings demonstrate that LRBA facilitates cancer cell growth and thus LRBA may represent a novel molecular target for cancer therapy.

Chronic shifts in the length and phase of the light cycle increase intermittent alcohol drinking in C57BL/6J mice.

INTRODUCTION: Shift workers-e.g., health care professionals, truck drivers, and factory workers-are forced to maintain daily cycles at odds with their natural circadian rhythms and as a consequence need to frequently readjust these cycles. This shift work-induced circadian desynchrony (CD) is associated with increased sleep disorders and with alcohol abuse. Nonetheless, it has proven difficult to model CD-induced changes in alcohol consumption in mouse models, which is an important step toward identifying the mechanisms by which CD increases alcohol intake. This study examined whether frequent changes in the light cycle could increase free access alcohol intake in a mouse line that readily consumes alcohol. METHODS: Free access alcohol intake, water intake, and wheel-running activity patterns of male C57BL/6J mice were measured while the mice were maintained on a normal 12HR photoperiod for baseline data for 2 weeks. The mice were then exposed to an alternating photoperiod of 12 h and 18 h, with light onset advanced 8 h during the 18HR photoperiod. The photoperiods rotated every 3 days, for 21 days total. RESULTS: The repeated pattern of phase advances and delays, with a concurrent change in the length of the photoperiod, shifted mice to a pattern of intermittent alcohol drinking without altering water intake. Wheel running activity demonstrated that mice were unable to reset their behavioral clocks during CD, showing constant, low-level activity with no peak in activity at the start of the dark phase and greater activity during the morning light phase. CONCLUSION: It is possible to model CD effects on alcohol intake in C57BL/6J mice using a pattern of phase shifts and changes in the photoperiod. Using this model, we demonstrate that mice begin intermittent drinking during CD, and this increase in alcohol intake does not correlate with an increase in overall activity or in overall fluid intake.

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

Modulation of clock gene expression by the transcriptional coregulator receptor interacting protein 140 (RIP140).

Circadian rhythms are generated in central and peripheral tissues by an intracellular oscillating timing mechanism known as the circadian clock. Several lines of evidence show a strong and bidirectional interplay between metabolism and circadian rhythms. Receptor interacting protein 140 (RIP140) is a coregulator for nuclear receptors and other transcription factors that represses catabolic pathways in metabolic tissues. Although RIP140 functions as a corepressor for most nuclear receptors, mounting evidence points to RIP140 as a dual coregulator that can repress or activate different sets of genes. Here, we demonstrate that RIP140 mRNA and protein levels are under circadian regulation and identify RIP140 as a modulator of clock gene expression, suggesting that RIP140 can participate in a feedback mechanism affecting the circadian clock. We show that the absence of RIP140 disturbs the basal levels of BMAL1 and other clock genes, reducing the amplitude of their oscillations. In addition, we demonstrate that RIP140 is recruited to retinoid-related orphan receptor (ROR) binding sites on the BMAL1 promoter, directly interacts with RORα, and increases transcription from the BMAL1 promoter in a RORα-dependent manner. These results indicate that RIP140 is not only involved in metabolic control but also acts as a coactivator for RORα, influencing clock gene expression.

Retinoic acid mediates long-paced oscillations in retinoid receptor activity: evidence for a potential role for RIP140.

BACKGROUND: Mechanisms that underlie oscillatory transcriptional activity of nuclear receptors (NRs) are incompletely understood. Evidence exists for rapid, cyclic recruitment of coregulatory complexes upon activation of nuclear receptors. RIP140 is a NR coregulator that represses the transactivation of agonist-bound nuclear receptors. Previously, we showed that RIP140 is inducible by all-trans retinoic acid (RA) and mediates limiting, negative-feedback regulation of retinoid signaling. METHODOLOGY AND FINDINGS: Here we report that in the continued presence of RA, long-paced oscillations of retinoic acid receptor (RAR) activity occur with a period ranging from 24 to 35 hours. Endogenous expression of RIP140 and other RA-target genes also oscillate in the presence of RA. Cyclic retinoid receptor transactivation is ablated by constitutive overexpression of RIP140. Further, depletion of RIP140 disrupts cyclic expression of the RA target gene HOXA5. Evidence is provided that RIP140 may limit RAR signaling in a selective, non-redundant manner in contrast to the classic NR coregulators NCoR1 and SRC1 that are not RA-inducible, do not cycle, and may be partially redundant in limiting RAR activity. Finally, evidence is provided that RIP140 can repress and be induced by other nuclear receptors in a manner that suggests potential participation in other NR oscillations. CONCLUSIONS AND SIGNIFICANCE: We provide evidence for novel, long-paced oscillatory retinoid receptor activity and hypothesize that this may be paced in part, by RIP140. Oscillatory NR activity may be involved in mediating hormone actions of physiological and pathological importance.