Behavioral neuroscience & neurodegeneration

Emi Nagoshi

Assistant Professor

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  • office 3013b (Sciences III)

Circadian locomotor behavior

Circadian rhythms

Circadian rhythms are the cyclic and persistent patterns of behavior and physiological processes exhibited by most organisms, ranging from cyanobacteria to human (Fig.1). These rhythms have a period of roughly 24 hours, matching the rotation of the Earth. Disruption of the circadian rhythms in humans causes sleep disorders and is also associated with many other health problems such as bipolar disorders and depression (1, 2).

Figure 1

At the molecular level, the core molecular clocks that present within many cells and tissues generate circadian rhythms. Accumulating evidence indicates that negative feedback loops of transcription are the design principle of the molecular clock core components in eukaryotes. Many clock genes are also conserved across a range of phylogenetic groups. These core clocks control rhythmic behavior and physiology principally by regulating rhythmic changes in downstream gene expression or protein activities (3, 4).

At the organismal level, the intrinsic periodicity is revealed as free-running rhythms when organisms are kept under constant conditions, such as constant darkness (DD). However, natural environments usually undergo daily changes such as light-dark cycles (LD) and organisms synchronize their circadian rhythms with these cycles, which is a process called “entrainment”. On the other hand, fluctuations in temperature have little impact on circadian rhythms. These seemingly opposing characteristics allow organisms to anticipate and prepare for regular and predictable environmental changes of the day, night and season. Components of the molecular clocks and the dedicated neural circuits are crucial to control both aspects of the circadian behavior.

The Drosophila circadian rhythms

Figure 2

Drosophila displays circadian rhythms in various physiological and behavioral processes (Fig.2). Locomotor activity rhythms of flies show bimodal patterns, which peak at dawn and dusk in LD cycles. These rhythms sustain endlessly in DD. Approximately 150 clock-containing neurons in the adult brain make up the circuit controlling this circadian behavior. The clock neurons are classified into 7 subgroups based on their anatomical locations and characteristics. Developing animals also have fully functional, yet simpler clock circuits, which consist of only 3 groups of clock neurons (5). It has been suggested that neurons in the lateral brain (Lateral Neurons, LNs) contain the oscillators that control morning and evening activity (M-and E- cells) and therefore serve as the central pacemakers to generate rhythms. In contrast, other clock neurons appear to mediate input of the environmental information to the central pacemakers (6, 7). Recent studies have mapped the M-cells to the small ventral Lateral Neurons (s-LNvs) and E- cells to the dorsal Lateral Neurons (LNds) and some of Dorsal Neurons 1 (DN1) (8, 9). M-cells are not only required for the morning activity in LD, but also indispensable for driving rhythms in DD. Thus, many groups now interpret the term “M-cells” as “main oscillator” (10).

Research Projects

Despite the advances in circadian rhythms research, our understanding of the circadian circuit is still limited. In particular, roles of many clock neurons, organization of the circadian circuits and neurochemical basis of the neuronal communication among clock neurons remain poorly understood.

1. Molecular mechanisms of circadian circuit organization and operation

To understand the molecular underpinning of the circadian circuit organization and functioning, we have set out the molecular characterization of clock neuron subtypes. Using a novel technique to isolate and analyze RNA expression from a small number of specific neurons, we profiled genome-wide gene expression in several key subtypes of clock neurons (11, 12). We further showed that two nuclear receptor genes, unfulfilled (unf; DHR-51) and E75 play key roles in the functioning of the M-cells (13, 14). Our continuing efforts to dissect molecular mechanisms underlying the operation of circadian circuitry include identification of UNF and E75 targets and more comprehensive transcriptome analysis of clock neuron sub clusters. 

2. Circadian neural circuit dynamics

Molecular rhythms are thought to control rhythmic neuronal activity and/or transmitter release. Conversely, there is a growing body of evidence that inter-neuronal signaling contributes to the synchronization and amplitude of the rhythms of clock neurons in both mammals and Drosophila (15, 16). Our goal is to decipher how inter-neuronal communication affects intracellular molecular clockwork, and how circuit-wide molecular and neuronal rhythms are integrated to generate rhythmic behavior. To this end, we developed fluorescence circadian reporter fly lines and live-imaging system using cultured brain and dissociated neurons. Our system allows for a spatiotemporally controlled manipulation of gene expression and neuronal activity together with the real-time recording of molecular oscillation in clock neurons.

Parkinson’s disease

Parkinson’s disease (PD) is the movement disorder characterized by the locomotor defects such as tremor, bradykinesia, rigidity and postural instability, affecting over 1% of the global population over 60 years of age. Motor symptoms of PD primarily arise from the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN). Despite the advances in gene discovery associated with familial PD, the knowledge of the PD pathogenesis is still limited. In particular, why the degeneration is specific to DA neurons and why it is progressive remain enigmatic. Lack of animal models that show genuinely progressive DA neuron degeneration has also hindered the study on this central issue.

Overall goal of our research in this topic is to understand the mechanisms underlying selective and progressive degeneration of the DA neurons. We will address these central questions using a novel Drosophila PD model we have established, and later by generating new mouse models.

Research Projects

We have recently established a novel PD model in Drosophila that offers an unusual and exciting opportunity to address the mechanisms underlying selective and progressive degeneration of DA neurons (17). Our model flies - the Fer2 (48-related-2) gene loss-of-function mutants - show specific and progressive death of brain DA neurons, severe locomotor defects and reduced life span (Movie 1, 2). We further showed that degeneration of DA neurons in Fer2 loss-of function mutants coincides with the systemic increase in reactive oxygen species (ROS) and mitochondrial dysfunction within DA neurons. Because increased ROS production and mitochondrial dysfunctions are pathological hallmarks of PD, our results underscore that Fer2 mutants recapitulate cellular and organismal characteristics of PD (17).

Encouraged by these exciting results, we are investigating upstream and downstream pathways of Fer2 to understand molecular mechanisms contributing to the survival of DA neurons. Furthermore, based on the knowledge gained from our work using Fer2 mutant flies, we are generating novel mouse models of PD.

Video 1: Brain DA neurons in the control flies

Video 2: Brain DA neurons in the Fer21 mutant flies

References

  1. Benca R, et al. (2009) "Biological rhythms, higher brain function, and behavior: gaps, opportunities and challenges". Brain Res Rev.
  2. Stevens RG, et al. (2007) Meeting report: the role of environmental lighting and circadian disruption in cancer and other diseases. Environ Health Perspect 115(9):1357-1362.
  3. Hastings MH, Maywood ES, & O'Neill JS (2008) Cellular circadian pacemaking and the role of cytosolic rhythms. Curr Biol 18(17):R805-R815.
  4. Yu W & Hardin PE (2006) Circadian oscillators of Drosophila and mammals. J Cell Sci 119(Pt 23):4793-4795.
  5. Helfrich-Forster C (2005) Neurobiology of the fruit fly's circadian clock. Genes Brain Behav 4(2):65-76.
  6. Grima B, Chelot E, Xia R, & Rouyer F (2004) Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain. Nature 431(7010):869-873.
  7. Stoleru D, Peng Y, Agosto J, & Rosbash M (2004) Coupled oscillators control morning and evening locomotor behaviour of Drosophila. Nature 431(7010):862-868.
  8. Helfrich-Forster C, et al. (2007) The lateral and dorsal neurons of Drosophila melanogaster: new insights about their morphology and function. Cold Spring Harb Symp Quant Biol 72:517-525.
  9. Picot M, Cusumano P, Klarsfeld A, Ueda R, & Rouyer F (2007) Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock. PLoS Biol 5(11):e315.
  10. Nitabach MN & Taghert PH (2008) Organization of the Drosophila circadian control circuit. Curr Biol 18(2):R84-93.
  11. Kula-Eversole E, et al. (2010) Surprising gene expression patterns within and between PDF-containing circadian neurons in Drosophila. Proc Natl Acad Sci U S A 107(30):13497-13502.
  12. Nagoshi E, et al. (2010) Dissecting differential gene expression within the circadian neuronal circuit of Drosophila. Nat Neurosci 13(1):60-68.
  13. Beuchle D, Jaumouille E, & Nagoshi E (2012) The Nuclear Receptor unfulfilled Is Required for Free-Running Clocks in Drosophila Pacemaker Neurons. Curr Biol 22(13):1221-1227.
  14. Jaumouille E, Machado Almeida P, Stahli P, Koch R, & Nagoshi E (2015) Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr Biol 25(11):1502-1508.
  15. Freeman GM, Jr., Krock RM, Aton SJ, Thaben P, & Herzog ED (2013) GABA networks destabilize genetic oscillations in the circadian pacemaker. Neuron 78(5):799-806.
  16. Mizrak D, et al. (2012) Electrical activity can impose time of day on the circadian transcriptome of pacemaker neurons. Curr Biol 22(20):1871-1880.
  17. Bou Dib P, et al. (2014) A Conserved Role for p48 Homologs in Protecting Dopaminergic Neurons from Oxidative Stress. Plos Genet 10(10).
  • Guidelines for Genome-Scale Analysis of Biological Rhythms. J. Biol. Rhythms 2017 Nov;():748730417728663. 10.1177/0748730417728663.

    abstract

    Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

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  • Transforming Growth Factor β/Activin signaling in neurons increases susceptibility to starvation. PLoS ONE 2017 ;12(10):e0187054. 10.1371/journal.pone.0187054. PONE-D-17-08341.

    abstract

    Animals rely on complex signaling network to mobilize its energy stores during starvation. We have previously shown that the sugar-responsive TGFβ/Activin pathway, activated through the TGFβ ligand Dawdle, plays a central role in shaping the post-prandial digestive competence in the Drosophila midgut. Nevertheless, little is known about the TGFβ/Activin signaling in sugar metabolism beyond the midgut. Here, we address the importance of Dawdle (Daw) after carbohydrate ingestion. We found that Daw expression is coupled to dietary glucose through the evolutionarily conserved Mio-Mlx transcriptional complex. In addition, Daw activates the TGFβ/Activin signaling in neuronal populations to regulate triglyceride and glycogen catabolism and energy homeostasis. Loss of those neurons depleted metabolic reserves and rendered flies susceptible to starvation.

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  • Evaluating the Autonomy of the Drosophila Circadian Clock in Dissociated Neuronal Culture. Front Cell Neurosci 2017 ;11():317. 10.3389/fncel.2017.00317. PMC5643464.

    abstract

    Circadian behavioral rhythms offer an excellent model to study intricate interactions between the molecular and neuronal mechanisms of behavior. In mammals, pacemaker neurons in the suprachiasmatic nucleus (SCN) generate rhythms cell-autonomously, which are synchronized by the network interactions within the circadian circuit to drive behavioral rhythms. However, whether this principle is universal to circadian systems in animals remains unanswered. Here, we examined the autonomy of the Drosophila circadian clock by monitoring transcriptional and post-transcriptional rhythms of individual clock neurons in dispersed culture with time-lapse microscopy. Expression patterns of the transcriptional reporter show that CLOCK/CYCLE (CLK/CYC)-mediated transcription is constantly active in dissociated clock neurons. In contrast, the expression profile of the post-transcriptional reporter indicates that PERIOD (PER) protein levels fluctuate and ~10% of cells display rhythms in PER levels with periods in the circadian range. Nevertheless, PER and TIM are enriched in the cytoplasm and no periodic PER nuclear accumulation was observed. These results suggest that repression of CLK/CYC-mediated transcription by nuclear PER is impaired, and thus the negative feedback loop of the molecular clock is incomplete in isolated clock neurons. We further demonstrate that, by pharmacological assays using the non-amidated form of neuropeptide pigment-dispersing factor (PDF), which could be specifically secreted from larval LNvs and adult s-LNvs, downstream events of the PDF signaling are partly impaired in dissociated larval clock neurons. Although non-amidated PDF is likely to be less active than the amidated one, these results point out the possibility that alteration in PDF downstream signaling may play a role in dampening of molecular rhythms in isolated clock neurons. Taken together, our results suggest that Drosophila clocks are weak oscillators that need to be in the intact circadian circuit to generate robust 24-h rhythms.

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  • A screening of UNF targets identifies Rnb, a novel regulator of Drosophila circadian rhythms. J. Neurosci. 2017 Jun;():. JNEUROSCI.3286-16.2017. 10.1523/JNEUROSCI.3286-16.2017.

    abstract

    Behavioral circadian rhythms are controlled by multi-oscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly Drosophila melanogaster are regulated differently in clock neuron subclasses to support their specific functions (Lee et al., 2016; Top et al., 2016). The nuclear receptor unfulfilled (unf) represents a regulatory node that provides the small ventral Lateral Neurons (s-LNvs) unique characteristics as the master pacemaker (Beuchle et al., 2012). We previously showed that UNF interacts with the s-LNv molecular clocks by regulating transcription of the core clock gene period (per) (Jaumouille et al., 2015). To gain more insight into the mechanisms by which UNF contributes to the functioning of the circadian master pacemaker, we identified UNF target genes using chromatin immunoprecipitation. Our data demonstrate that a previously uncharacterized gene CG7837, which we termed R and B (Rnb), acts downstream of UNF to regulate the function of s-LNvs as the master circadian pacemaker. Mutations and LNv-targeted adult-restricted knockdown of Rnb impair locomotor rhythms. RNB localizes to the nucleus and its loss-of-function blunts the molecular rhythms and output rhythms of the s-LNvs, particularly the circadian rhythms in PDF accumulation and axonal arbor remodeling. These results establish a second pathway by which UNF interacts with the molecular clocks in the s-LNvs and highlight the mechanistic differences in the molecular clockwork within the pacemaker circuit.SIGNIFICANCE STATEMENTCircadian behavior is generated by a pacemaker circuit comprising diverse classes of pacemaker neurons, each of which contains a molecular clock. In addition to the anatomical and functional diversity, recent studies have shown the mechanistic differences in the molecular clockwork among the pacemaker neurons in Drosophila Here, we identified the molecular characteristics distinguishing the s-LNvs, the master pacemaker of the locomotor rhythms, from other clock neuron subtypes. We demonstrated that a newly identified gene Rnb is a s-LNv-specific regulator of the molecular clock and essential for the generation of circadian locomotor behavior. Our results provide additional evidence to the emerging view that the differential regulation of the molecular clocks underlies the functional differences among the pacemaker neuron subgroups.

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  • Fluorescence circadian imaging reveals a PDF-dependent transcriptional regulation of the Drosophila molecular clock. Sci Rep 2017 Jan;7():41560. srep41560. 10.1038/srep41560. PMC5278502.

    abstract

    Circadian locomotor behaviour is controlled by a pacemaker circuit composed of clock-containing neurons. To interrogate the mechanistic relationship between the molecular clockwork and network communication critical to the operation of the Drosophila circadian pacemaker circuit, we established new fluorescent circadian reporters that permit single-cell recording of transcriptional and post-transcriptional rhythms in brain explants and cultured neurons. Live-imaging experiments combined with pharmacological and genetic manipulations demonstrate that the neuropeptide pigment-dispersing factor (PDF) amplifies the molecular rhythms via time-of-day- and activity-dependent upregulation of transcription from E-box-containing clock gene promoters within key pacemaker neurons. The effect of PDF on clock gene transcription and the known role of PDF in enhancing PER/TIM stability occur via independent pathways downstream of the PDF receptor, the former through a cAMP-independent mechanism and the latter through a cAMP-PKA dependent mechanism. These results confirm and extend the mechanistic understanding of the role of PDF in controlling the synchrony of the pacemaker neurons. More broadly, our results establish the utility of the new live-imaging tools for the study of molecular-neural interactions important for the operation of the circadian pacemaker circuit.

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  • USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level. PLoS ONE 2016 ;11(1):e0145155. 10.1371/journal.pone.0145155. PONE-D-15-15381. PMC4710524.

    abstract

    The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd), predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

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  • Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr. Biol. 2015 Jun;25(11):1502-8. S0960-9822(15)00430-3. 10.1016/j.cub.2015.04.017. PMC4454776.

    abstract

    Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and CLOCK/CYCLE (CLK/CYC) (in Drosophila) drive the expression of the period (per) family genes. The PER-containing complexes inhibit the activity of CLOCK/BMAL1 or CLK/CYC, thereby forming a negative feedback loop [1]. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation [2]. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. We show here that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor UNF are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and UNF bind to per regulatory sequences and act together to enhance the CLK/CYC-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. Our results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks.

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  • RNA-seq profiling of small numbers of Drosophila neurons. Meth. Enzymol. 2015 ;551():369-86. S0076-6879(14)00026-3. 10.1016/bs.mie.2014.10.025.

    abstract

    Drosophila melanogaster has a robust circadian clock, which drives a rhythmic behavior pattern: locomotor activity increases in the morning shortly before lights on (M peak) and in the evening shortly before lights off (E peak). This pattern is controlled by ~75 pairs of circadian neurons in the Drosophila brain. One key group of neurons is the M-cells (PDF(+) large and small LNvs), which control the M peak. A second key group is the E-cells, consisting of four LNds and the fifth small LNv, which control the E peak. Recent studies show that the M-cells have a second role in addition to controlling the M peak; they communicate with the E-cells (as well as DN1s) to affect their timing, probably as a function of environmental conditions (Guo, Cerullo, Chen, & Rosbash, 2014). To learn about molecules within the M-cells important for their functional roles, we have adapted methods to manually sort fluorescent protein-expressing neurons of interest from dissociated Drosophila brains. We isolated mRNA and miRNA from sorted M-cells and amplified the resulting DNAs to create deep-sequencing libraries. Visual inspection of the libraries illustrates that they are specific to a particular neuronal subgroup; M-cell libraries contain timeless and dopaminergic cell libraries contain ple/TH. Using these data, it is possible to identify cycling transcripts as well as many mRNAs and miRNAs specific to or enriched in particular groups of neurons.

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  • A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress. PLoS Genet. 2014 Oct;10(10):e1004718. 10.1371/journal.pgen.1004718. PGENETICS-D-14-00993. PMC4207665.

    abstract

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.

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  • The nuclear receptor unfulfilled is required for free-running clocks in Drosophila pacemaker neurons. Curr. Biol. 2012 Jul;22(13):1221-7. S0960-9822(12)00472-1. 10.1016/j.cub.2012.04.052.

    abstract

    An intricate neural circuit composed of multiple classes of clock neurons controls circadian locomotor rhythms in Drosophila. Evidence indicates that the small ventral lateral neurons (s-LNvs, M cells) are the dominant pacemaker neurons that synchronize the clocks throughout the circuit and drive free-running locomotor rhythms. Little is known, however, about the molecular underpinning of this unique function of the s-LNvs. Here, we show that the nuclear receptor gene unfulfilled (unf; DHR51) is required for the function of the s-LNvs. UNFULFILLED (UNF) is rhythmically expressed in the s-LNvs, and unf mutant flies are behaviorally arrhythmic. Knockdown of unf in developing LNvs irreversibly destroys the ability of adult s-LNvs to generate free-running rhythms, whereas depletion of UNF from adult LNvs dampens the rhythms of the s-LNvs only in constant darkness. These temporally controlled LNv-targeted unf knockdowns desynchronize circuit-wide molecular rhythms and disrupt behavioral rhythms. Therefore, UNF is a prerequisite for free-running clocks in the s-LNvs and for the function of the entire circadian circuit.

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  • Surprising gene expression patterns within and between PDF-containing circadian neurons in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 2010 Jul;107(30):13497-502. 1002081107. 10.1073/pnas.1002081107. PMC2922133.

    abstract

    To compare circadian gene expression within highly discrete neuronal populations, we separately purified and characterized two adjacent but distinct groups of Drosophila adult circadian neurons: the 8 small and 10 large PDF-expressing ventral lateral neurons (s-LNvs and l-LNvs, respectively). The s-LNvs are the principal circadian pacemaker cells, whereas recent evidence indicates that the l-LNvs are involved in sleep and light-mediated arousal. Although half of the l-LNv-enriched mRNA population, including core clock mRNAs, is shared between the l-LNvs and s-LNvs, the other half is l-LNv- and s-LNv-specific. The distribution of four specific mRNAs is consistent with prior characterization of the four encoded proteins, and therefore indicates successful purification of the two neuronal types. Moreover, an octopamine receptor mRNA is selectively enriched in l-LNvs, and only these neurons respond to in vitro application of octopamine. Dissection and purification of l-LNvs from flies collected at different times indicate that these neurons contain cycling clock mRNAs with higher circadian amplitudes as well as at least a 10-fold higher fraction of oscillating mRNAs than all previous analyses of head RNA. Many of these cycling l-LNv mRNAs are well expressed but do not cycle or cycle much less well elsewhere in heads. The results suggest that RNA cycling is much more prominent in circadian neurons than elsewhere in heads and may be particularly important for the functioning of these neurons.

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  • Dissecting differential gene expression within the circadian neuronal circuit of Drosophila. Nat. Neurosci. 2010 Jan;13(1):60-8. nn.2451. 10.1038/nn.2451. PMC3878269. HHMIMS467346.

    abstract

    Behavioral circadian rhythms are controlled by a neuronal circuit consisting of diverse neuronal subgroups. To understand the molecular mechanisms underlying the roles of neuronal subgroups within the Drosophila circadian circuit, we used cell-type specific gene-expression profiling and identified a large number of genes specifically expressed in all clock neurons or in two important subgroups. Moreover, we identified and characterized two circadian genes, which are expressed specifically in subsets of clock cells and affect different aspects of rhythms. The transcription factor Fer2 is expressed in ventral lateral neurons; it is required for the specification of lateral neurons and therefore their ability to drive locomotor rhythms. The Drosophila melanogaster homolog of the vertebrate circadian gene nocturnin is expressed in a subset of dorsal neurons and mediates the circadian light response. The approach should also enable the molecular dissection of many different Drosophila neuronal circuits.

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  • The period length of fibroblast circadian gene expression varies widely among human individuals. PLoS Biol. 2005 Oct;3(10):e338. 04-PLBI-RA-0698R2. 10.1371/journal.pbio.0030338. PMC1233413.

    abstract

    Mammalian circadian behavior is governed by a central clock in the suprachiasmatic nucleus of the brain hypothalamus, and its intrinsic period length is believed to affect the phase of daily activities. Measurement of this period length, normally accomplished by prolonged subject observation, is difficult and costly in humans. Because a circadian clock similar to that of the suprachiasmatic nucleus is present in most cell types, we were able to engineer a lentiviral circadian reporter that permits characterization of circadian rhythms in single skin biopsies. Using it, we have determined the period lengths of 19 human individuals. The average value from all subjects, 24.5 h, closely matches average values for human circadian physiology obtained in studies in which circadian period was assessed in the absence of the confounding effects of light input and sleep-wake cycle feedback. Nevertheless, the distribution of period lengths measured from biopsies from different individuals was wider than those reported for circadian physiology. A similar trend was observed when comparing wheel-running behavior with fibroblast period length in mouse strains containing circadian gene disruptions. In mice, inter-individual differences in fibroblast period length correlated with the period of running-wheel activity; in humans, fibroblasts from different individuals showed widely variant circadian periods. Given its robustness, the presented procedure should permit quantitative trait mapping of human period length.

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  • Circadian gene expression in cultured cells. Meth. Enzymol. 2005 ;393():543-57. S0076687905930280. 10.1016/S0076-6879(05)93028-0.

    abstract

    In mammals, circadian oscillators not only exist in specialized neurons of the suprachiasmatic nucleus, but in almost all peripheral cell types. These oscillators are operative even in established fibroblast cell lines, such as Rat-1 cells or NIH3T3 cells, and in primary fibroblasts from mouse embryos or adult animals. This can be demonstrated by treating such cells for a short time period with high concentrations of serum or chemicals that activate a large number of known signaling pathways. The possibility of studying circadian rhythms in cultured cells should facilitate the biochemical and genetic dissection of the circadian clockwork and should promote the discovery of new clock components.

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  • Importin alpha transports CaMKIV to the nucleus without utilizing importin beta. EMBO J. 2005 Mar;24(5):942-51. 7600587. 10.1038/sj.emboj.7600587. PMC554133.

    abstract

    Ca(2+)/calmodulin-dependent protein kinase type IV (CaMKIV) plays an essential role in the transcriptional activation of cAMP response element-binding protein-mediated signaling pathways. Although CaMKIV is localized predominantly in the nucleus, the molecular mechanism of the nuclear import of CaMKIV has not been elucidated. We report here that importin alpha is able to carry CaMKIV into the nucleus without the need for importin beta or any other soluble proteins in digitonin-permeabilized cells. An importin beta binding-deficient mutant (DeltaIBB) of importin alpha also carried CaMKIV into the nucleus, which strongly suggests that CaMKIV is transported in an importin beta-independent manner. While CaMKIV directly interacted with the C-terminal region of importin alpha, the CaMKIV/importin alpha complex did not form a ternary complex with importin beta, which explains the nonrequirement of importin beta for the nuclear transport of CaMKIV. The cytoplasmic microinjection of importin alpha-DeltaIBB enhanced the rate of nuclear translocation of CaMKIV in vivo. This is the first report to demonstrate definitely that mammalian importin alpha solely carries a cargo protein into the nucleus without utilizing the classical importin beta-dependent transport system.

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  • Importin alpha/beta mediates nuclear transport of a mammalian circadian clock component, mCRY2, together with mPER2, through a bipartite nuclear localization signal. J. Biol. Chem. 2005 Apr;280(14):13272-8. M413236200. 10.1074/jbc.M413236200.

    abstract

    Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin alpha/beta system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin alphas, alpha1 (Rch1), alpha3 (Qip-1), and alpha7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin alpha/beta system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.

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  • Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells. Cell 2004 Nov;119(5):693-705. S0092867404010542. 10.1016/j.cell.2004.11.015.

    abstract

    The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus (SCN) of the brain and subsidiary oscillators in most peripheral cell types. While oscillators in SCN neurons are known to function in a self-sustained fashion, peripheral oscillators have been thought to damp rapidly when disconnected from the control exerted by the SCN. Using two reporter systems, we monitored circadian gene expression in NIH3T3 mouse fibroblasts in real time and in individual cells. In conjunction with mathematical modeling and cell co-culture experiments, these data demonstrated that in vitro cultured fibroblasts harbor self-sustained and cell-autonomous circadian clocks similar to those operative in SCN neurons. Circadian gene expression in fibroblasts continues during cell division, and our experiments unveiled unexpected interactions between the circadian clock and the cell division clock. Specifically, the circadian oscillator gates cytokinesis to defined time windows, and mitosis elicits phase shifts in circadian cycles.

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  • The mammalian circadian timing system: from gene expression to physiology. Chromosoma 2004 Sep;113(3):103-12. 10.1007/s00412-004-0296-2.

    abstract

    Many physiological processes in organisms from bacteria to man are rhythmic, and some of these are controlled by self-sustained oscillators that persist in the absence of external time cues. Circadian clocks are perhaps the best characterized biological oscillators and they exist in virtually all light-sensitive organisms. In mammals, they influence nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrinology, body temperature, renal activity, physiology of the gastro-intestinal tract, and hepatic metabolism. The master pacemaker is located in the suprachiasmatic nuclei, two small groups of neurons in the ventral part of the hypothalamus. However, most peripheral body cells contain self-sustained circadian oscillators with a molecular makeup similar to that of SCN (suprachiasmatic nucleus) neurons. This organization implies that the SCN must synchronize countless subsidiary oscillators in peripheral tissues, in order to coordinate cyclic physiology. In this review, we will discuss some recent studies on the structure and putative functions of the mammalian circadian timing system, but we will also point out some apparent inconsistencies in the currently publicized model for rhythm generation.

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  • The structure of importin-beta bound to SREBP-2: nuclear import of a transcription factor. Science 2003 Nov;302(5650):1571-5. 10.1126/science.1088372. 302/5650/1571.

    abstract

    The sterol regulatory element-binding protein 2 (SREBP-2), a nuclear transcription factor that is essential for cholesterol metabolism, enters the nucleus through a direct interaction of its helix-loop-helix leucine zipper domain with importin-beta. We show the crystal structure of importin-beta complexed with the active form of SREBP-2. Importin-beta uses characteristic long helices like a pair of chopsticks to interact with an SREBP-2 dimer. Importin-beta changes its conformation to reveal a pseudo-twofold symmetry on its surface structure so that it can accommodate a symmetric dimer molecule. Importin-beta may use a similar strategy to recognize other dimeric cargoes.

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  • Crystallization and preliminary crystallographic analysis of the importin-beta-SREBP-2 complex. Acta Crystallogr. D Biol. Crystallogr. 2003 Oct;59(Pt 10):1866-8. S0907444903018328.

    abstract

    The nuclear-transport protein importin-beta mediates the nuclear import of the transcription factor SREBP-2 without requiring adaptor proteins such as importin-alpha. An importin-beta-SREBP-2 HLHZ domain complex was purified and crystallized. The crystals belong to space group P2(1)2(1)2(1) and show diffraction to at least 3.0 A resolution. The unit-cell parameters are a = 101.0, b = 113.2, c = 240.0 A. Structure determination using the MAD or SAD method is under way.

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  • Basic peptides as functional components of non-viral gene transfer vehicles. Curr. Protein Pept. Sci. 2003 Apr;4(2):141-50.

    abstract

    Improving the performance of non-viral gene-delivery vehicles that consist of synthetic compounds and nucleic acids is a key to successful gene therapy. Supplementing synthetic vehicles with various biological functions by using natural or artificial peptides is a promising approach with which to achieve this goal. One of the obstacles hindering this effort is that some of the potentially useful peptides, especially those with many basic amino acid residues, interfere with the formation of the complex owing to strong electrostatic interactions with the nucleic acid. In this review, we describe our recent work in examining the potential of these peptides in gene delivery, using a recombinant lambda phage particle as the model for the gene-delivery complex. Lambda phage encapsulates large duplex DNA in a rigid polyplex-like shell with a diameter of 55 nm, and can display various peptides on this capsid, independently of particle formation. By examining the expression of marker genes encapsulated in the phage capsid, we have demonstrated that the protein transduction domain of HIV Tat protein and the nuclear localization signal derived from SV40 T antigen can remarkably facilitate the delivery of these marker genes across the two major barriers, the cell membrane and the nuclear membrane, respectively. Our results indicate that these basic peptides can constitute effective components of synthetic gene-transfer complexes, as long as sufficient copies are displayed on the outer surface of the complex.

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  • Enhancement of phage-mediated gene transfer by nuclear localization signal. Biochem. Biophys. Res. Commun. 2002 Oct;297(4):779-86. S0006291X02022829.

    abstract

    The cell membrane and the nuclear membrane are two major barriers hindering the free movement of various macromolecules through animal cells. Nevertheless, some proteins can actively bypass these barriers by dint of intrinsic peptidic signals, so incorporation of these signals might improve the efficacy of artificial gene delivery vehicles. We examined the role of the nuclear localization signal (NLS) in gene transfer, using recombinant lambda phage as a model of the polymer/DNA complexes. We prepared a lambda phage displaying a 32-mer NLS of SV40 T antigen on its surface (NLS phage), and found that this NLS phage, delivered into the cytoplasm by appropriate devices, has higher affinity for the nucleus and induces the expression of encapsulated marker genes more efficiently than does the wild-type phage. This suggests that the 32-mer NLS peptide will become a practical tool for artificial gene delivery vehicles with enhanced nuclear targeting activity.

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