Circadian clock disruption promotes the degeneration of dopaminergic neurons in male Drosophila.

Sleep and circadian rhythm disruptions are frequent comorbidities of Parkinson's disease (PD), a disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. However, the causal role of circadian clocks in the degenerative process remains uncertain. We demonstrated here that circadian clocks regulate the rhythmicity and magnitude of the vulnerability of DA neurons to oxidative stress in male Drosophila. Circadian pacemaker neurons are presynaptic to a subset of DA neurons and rhythmically modulate their susceptibility to degeneration. The arrhythmic period (per) gene null mutation exacerbates the age-dependent loss of DA neurons and, in combination with brief oxidative stress, causes premature animal death. These findings suggest that circadian clock disruption promotes dopaminergic neurodegeneration.

The utility and caveat of split-GAL4s in the study of neurodegeneration.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, afflicting over 1% of the population of age 60 y and above. The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) is the primary cause of its characteristic motor symptoms. Studies using Drosophila melanogaster and other model systems have provided much insight into the pathogenesis of PD. However, little is known why certain cell types are selectively susceptible to degeneration in PD. Here, we describe an approach to identify vulnerable subpopulations of neurons in the genetic background linked to PD in Drosophila, using the split-GAL4 drivers that enable genetic manipulation of a small number of defined cell populations. We identify split-GAL4 lines that target neurons selectively vulnerable in a model of leucine-rich repeat kinase 2 (LRRK2)-linked familial PD, demonstrating the utility of this approach. We also show an unexpected caveat of the split-GAL4 system in ageing-related research: an age-dependent increase in the number of GAL4-labelled cells.

Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor.

Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.

Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP-PKA signaling.

Various behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila melanogaster, in which only ~0.1% of the brain's neurons contain circadian clocks. Clock neurons transmit the timing information to a plethora of non-clock neurons via poorly understood mechanisms. Here, we address the molecular underpinning of this phenomenon by profiling circadian gene expression in non-clock neurons that constitute the mushroom body, the center of associative learning and sleep regulation. We show that circadian clocks drive rhythmic expression of hundreds of genes in mushroom body neurons, including the Neurofibromin 1 (Nf1) tumor suppressor gene and Pka-C1. Circadian clocks also drive calcium rhythms in mushroom body neurons via NF1-cAMP/PKA-C1 signaling, eliciting higher mushroom body activity during the day than at night, thereby promoting daytime wakefulness. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep.

Uncovering the Roles of Clocks and Neural Transmission in the Resilience of Drosophila Circadian Network.

Studies of circadian locomotor rhythms in Drosophila melanogaster gave evidence to the preceding theoretical predictions on circadian rhythms. The molecular oscillator in flies, as in virtually all organisms, operates using transcriptional-translational feedback loops together with intricate post-transcriptional processes. Approximately150 pacemaker neurons, each equipped with a molecular oscillator, form a circuit that functions as the central pacemaker for locomotor rhythms. Input and output pathways to and from the pacemaker circuit are dissected to the level of individual neurons. Pacemaker neurons consist of functionally diverse subclasses, including those designated as the Morning/Master (M)-oscillator essential for driving free-running locomotor rhythms in constant darkness and the Evening (E)-oscillator that drives evening activity. However, accumulating evidence challenges this dual-oscillator model for the circadian circuit organization and propose the view that multiple oscillators are coordinated through network interactions. Here we attempt to provide further evidence to the revised model of the circadian network. We demonstrate that the disruption of molecular clocks or neural output of the M-oscillator during adulthood dampens free-running behavior surprisingly slowly, whereas the disruption of both functions results in an immediate arrhythmia. Therefore, clocks and neural communication of the M-oscillator act additively to sustain rhythmic locomotor output. This phenomenon also suggests that M-oscillator can be a pacemaker or a downstream path that passively receives rhythmic inputs from another pacemaker and convey output signals. Our results support the distributed network model and highlight the remarkable resilience of the Drosophila circadian pacemaker circuit, which can alter its topology to maintain locomotor rhythms.

Identification of a micropeptide and multiple secondary cell genes that modulate Drosophila male reproductive success.

Even in well-characterized genomes, many transcripts are considered noncoding RNAs (ncRNAs) simply due to the absence of large open reading frames (ORFs). However, it is now becoming clear that many small ORFs (smORFs) produce peptides with important biological functions. In the process of characterizing the ribosome-bound transcriptome of an important cell type of the seminal fluid-producing accessory gland of Drosophila melanogaster, we detected an RNA, previously thought to be noncoding, called male-specific abdominal (msa). Notably, msa is nested in the HOX gene cluster of the Bithorax complex and is known to contain a micro-RNA within one of its introns. We find that this RNA encodes a "micropeptide" (9 or 20 amino acids, MSAmiP) that is expressed exclusively in the secondary cells of the male accessory gland, where it seems to accumulate in nuclei. Importantly, loss of function of this micropeptide causes defects in sperm competition. In addition to bringing insights into the biology of a rare cell type, this work underlines the importance of small peptides, a class of molecules that is now emerging as important actors in complex biological processes.

Fluorescence Live Imaging of Drosophila Circadian Pacemaker Neurons.

Live imaging of the molecular clockwork within the circadian pacemaker neurons offers the unique possibility to study complex interactions between the molecular clock and neuronal communication within individual neurons and throughout the entire circadian circuitry. Here we describe how to establish brain explants and dissociated neuron culture from Drosophila larvae, guidelines for time-lapse fluorescence microscopy, and the method of image analysis. This approach enables the long-term monitoring of fluorescence signals of circadian reporters at single-cell resolution and can be also applicable to analyze real-time expression of other fluorescent probes in Drosophila neurons.

Nitric oxide mediates neuro-glial interaction that shapes Drosophila circadian behavior.

Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. Here we demonstrate that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. We show that mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, we also demonstrate that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, we identify the perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output.

Decoding Drosophila circadian pacemaker circuit.

Drosophila circadian circuit is one of the best described neural circuits but is complex enough to obscure our understanding of how it actually works. Animals' rhythmic behavior, the seemingly simple outcome of their internal clocks, relies on the interaction of heterogeneous clock neurons that are spread across the brain. Direct observations of their coordinated network interactions can bring us forward in understanding the circuit. The current challenge is to observe activity of each of these neurons over a long span of time - hours to days - in live animals. Here we review the progress in circadian circuit interrogation powered by in vivo calcium imaging.

Drosophila Models of Sporadic Parkinson's Disease.

Parkinson's disease (PD) is the most common cause of movement disorders and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. It is increasingly recognized as a complex group of disorders presenting widely heterogeneous symptoms and pathology. With the exception of the rare monogenic forms, the majority of PD cases result from an interaction between multiple genetic and environmental risk factors. The search for these risk factors and the development of preclinical animal models are in progress, aiming to provide mechanistic insights into the pathogenesis of PD. This review summarizes the studies that capitalize on modeling sporadic (i.e., nonfamilial) PD using Drosophila melanogaster and discusses their methodologies, new findings, and future perspectives.

Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons.

Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. Here, we investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. We show that dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. We further show that dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture.

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Evaluating the Autonomy of the Drosophila Circadian Clock in Dissociated Neuronal Culture.

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.

Transforming Growth Factor ß/Activin signaling in neurons increases susceptibility to starvation.

Animals rely on complex signaling network to mobilize its energy stores during starvation. We have previously shown that the sugar-responsive TGFß/Activin pathway, activated through the TGFß ligand Dawdle, plays a central role in shaping the post-prandial digestive competence in the Drosophila midgut. Nevertheless, little is known about the TGFß/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 TGFß/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.

A Screening of UNF Targets Identifies Rnb, a Novel Regulator of Drosophila Circadian Rhythms.

Behavioral circadian rhythms are controlled by multioscillator 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) (Jaumouillé 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 the 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 STATEMENT Circadian 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 an 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.

Fluorescence circadian imaging reveals a PDF-dependent transcriptional regulation of the Drosophila molecular clock.

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.

USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

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+.

Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock.

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 ß, 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.

RNA-seq profiling of small numbers of Drosophila neurons.

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.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

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.