Exploring functional links between circadian clocks, neurodegeneration, and aging in Drosophila melanogaster

Circadian clocks are endogenous molecular mechanisms that coordinate daily rhythms in gene expression, cellular activities, and physiological functions with external day/night cycles. Breakdown of circadian rhythms such as sleep/wake cycles is associated with the onset of several neurological diseases; however, it is not clear whether disruption of rhythms is a symptom or cause of neurodegeneration, or both. To address this important question, circadian rhythms were disrupted by both genetic and environmental manipulations in Drosophila mutants prone to neurodegeneration. This led to shortening of lifespan, premature accumulation of oxidative and nervous damage during aging, and overall decline in healthspan, suggesting that circadian clocks may be causally involved in neuroprotective pathways in aging Drosophila. Recent evidence suggests bidirectional relationships between circadian rhythms and aging. While disruption of the clock mechanism accelerates aging and age-related pathologies in mammals, output rhythms of sleep and hormonal fluctuations tend to deteriorate during aging in humans, rodents, and fruit flies. To understand whether this decay is caused by defects in the core transcriptional clock, or weakening of the clock output pathways, a comprehensive study on age-related changes in the behavioral and molecular circadian rhythms was conducted using the fruit fly as a model organism. Aging caused disruption of rest/activity patterns and lengthening of the free-running period of the circadian locomotor activity rhythm. Transcriptional oscillations of four genes involved in the clock mechanism, period, timeless, Par domain protein 1ε, and vrille, were significantly reduced in heads, but not in bodies of aging flies. It was further determined that reduced transcription of these genes is not caused by the deficient expression of their activators, encoded by Clock and cycle genes. Moreover, transcriptional activation by CLOCK-CYCLE complexes is impaired despite reduced levels of the PERIOD repressor protein in old flies. These data suggest that aging alters the properties of the core transcriptional clock in flies such that both the positive and the negative limbs of the clock are attenuated. In fruit flies, the protein CRYPTOCHROME (CRY) acts in a cell-autonomous manner to synchronize circadian oscillations with light-dark cycles. The oscillatory amplitude of CRY is significantly dampened in heads of old flies at both mRNA and protein levels. Rescue of CRY using the binary GAL4/UAS system in old flies significantly enhanced the dampened molecular oscillations of several clock genes, and also strengthened the locomotor activity rhythms. There was a remarkable extension of healthspan in flies with elevated CRY. Conversely, CRY deficient mutants accumulated greater oxidative damage and showed accelerated functional decline. Interestingly, rescue of CRY in central clock neurons alone was not sufficient to restore rest/activity rhythms or extend healthspan. These data suggest novel anti-aging functions of CRY and indicate that peripheral clocks play an active role in delaying behavioral and physiological aging. Taken together, research conducted for this dissertation is a first attempt to elucidate functional links between circadian clocks, neurodegeneration, and aging. While previous evidence linking these processes was of correlative nature, functional studies conducted in this dissertation demonstrate that disruption of circadian clocks causes neurodegeneration and aging. While aging disrupts circadian rhythms at the molecular and behavioral levels, restoration of these rhythms can delay aging and improve healthspan in Drosophila. Owing to the conserved nature of clocks, novel insights obtained from this research can illuminate future translational research aimed to extend human healthspan

Effects of aging on the molecular circadian oscillations in Drosophila

Circadian clocks maintain temporal homeostasis by generating daily output rhythms in molecular, cellular, and physiological functions. Output rhythms, such as sleep/wake cycles and hormonal fluctuations, tend to deteriorate during aging in humans, rodents, and fruit flies. However, it is not clear whether this decay is caused by defects in the core transcriptional clock, or weakening of the clock-output pathways, or both. The authors monitored age-related changes in behavioral and molecular rhythms in Drosophila melanogaster. Aging was associated with disrupted rest/activity patterns and lengthening of the free-running period of the circadian locomotor activity rhythm. The expression of core clock genes was measured in heads and bodies of young, middle-aged, and old flies. Transcriptional oscillations of four clock genes, period, timeless, Par domain protein 1ε, and vrille, were significantly reduced in heads, but not in bodies, of aging flies. It was determined that reduced transcription of these genes was not caused by the deficient expression of their activators, encoded by Clock and cycle genes. Interestingly, transcriptional activation by CLOCK-CYCLE complexes was impaired despite reduced levels of the PERIOD repressor protein in old flies. These data suggest that aging alters the properties of the core transcriptional clock in flies such that both the positive and the negative limbs of the clock are attenuated

Postnatal Ontogenesis of the Islet Circadian Clock Plays a Contributory Role in β-Cell Maturation Process

Development of cell replacement therapies in diabetes requires understanding of the molecular underpinnings of β-cell maturation. The circadian clock regulates diverse cellular functions important for regulation of β-cell function and turnover. However, postnatal ontogenesis of the islet circadian clock and its potential role in β-cell maturation remain unknown. To address this, we studied wild-type Sprague-Dawley as well as Period1 luciferase transgenic (Per1:LUC) rats to determine circadian clock function, clock protein expression, and diurnal insulin secretion during islet development and maturation process. We additionally studied β-cell-specific Bmal1-deficient mice to elucidate a potential role of this key circadian transcription factor in β-cell functional and transcriptional maturation. We report that emergence of the islet circadian clock 1) occurs during the early postnatal period, 2) depends on the establishment of global behavioral circadian rhythms, and 3) leads to the induction of diurnal insulin secretion and gene expression. Islet cell maturation was also characterized by induction in the expression of circadian transcription factor BMAL1, deletion of which altered postnatal development of glucose-stimulated insulin secretion and the associated transcriptional network. Postnatal development of the islet circadian clock contributes to early-life β-cell maturation and should be considered for optimal design of future β-cell replacement strategies in diabetes

Impaired β-cell glucokinase as an underlying mechanism in diet-induced diabetes

High-fat diet (HFD)-fed mouse models have been widely used to study early type 2 diabetes. Decreased β-cell glucokinase (GCK) expression has been observed in HFD-induced diabetes. However, owing to its crucial roles in glucose metabolism in the liver and in islet β-cells, the contribution of decreased GCK expression to the development of HFD-induced diabetes is unclear. Here, we employed a β-cell-targeted gene transfer vector and determined the impact of β-cell-specific increase in GCK expression on β-cell function and glucose handling in vitro and in vivo. Overexpression of GCK enhanced glycolytic flux, ATP-sensitive potassium channel activation and membrane depolarization, and increased proliferation in Min6 cells. β-cell-targeted GCK transduction did not change glucose handling in chow-fed C57BL/6 mice. Although adult mice fed a HFD showed reduced islet GCK expression, impaired glucose tolerance and decreased glucose-stimulated insulin secretion (GSIS), β-cell-targeted GCK transduction improved glucose tolerance and restored GSIS. Islet perifusion experiments verified restored GSIS in isolated HFD islets by GCK transduction. Thus, our data identify impaired β-cell GCK expression as an underlying mechanism for dysregulated β-cell function and glycemic control in HFD-induced diabetes. Our data also imply an etiological role of GCK in diet-induced diabetes. This article has an associated First Person interview with the first author of the paper

Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of β cell function

Circadian rhythm disruption (CD) is associated with impaired glucose homeostasis and type 2 diabetes mellitus (T2DM). While the link between CD and T2DM remains unclear, there is accumulating evidence that disruption of fasting/feeding cycles mediates metabolic dysfunction. Here, we used an approach encompassing analysis of behavioral, physiological, transcriptomic, and epigenomic effects of CD and consequences of restoring fasting/feeding cycles through time-restricted feeding (tRF) in mice. Results show that CD perturbs glucose homeostasis through disruption of pancreatic β cell function and loss of circadian transcriptional and epigenetic identity. In contrast, restoration of fasting/feeding cycle prevented CD-mediated dysfunction by reestablishing circadian regulation of glucose tolerance, β cell function, transcriptional profile, and reestablishment of proline and acidic amino acid–rich basic leucine zipper (PAR bZIP) transcription factor DBP expression/activity. This study provides mechanistic insights into circadian regulation of β cell function and corresponding beneficial effects of tRF in prevention of T2DM