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In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called ‘master pacemakers’ because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

Inhibitors of enzymes that inactivate amine neurotransmitters (dopamine, serotonin), such as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), are thought to increase neurotransmitter levels and are widely used to treat Parkinson's disease and psychiatric disorders, yet the role of these enzymes in regulating behavior remains unclear. Here, we investigated the genetic loss of a similar enzyme in the model organism Drosophila melanogaster . Because the enzyme Ebony modifies and inactivates amine neurotransmitters, its loss is assumed to increase neurotransmitter levels, increasing behaviors such as aggression and courtship and decreasing sleep. Indeed, ebony mutants have been described since 1960 as \"aggressive mutants,\" though this behavior has not been quantified. Using automated machine learning-based analyses, we quantitatively confirmed that ebony mutants exhibited increased aggressive behaviors such as boxing but also decreased courtship behaviors and increased sleep. Through tissue-specific knockdown, we found that ebony ’s role in these behaviors was specific to glia. Unexpectedly, direct measurement of amine neurotransmitters in ebony brains revealed that their levels were not increased but reduced. Thus, increased aggression is the anomalous behavior for this neurotransmitter profile. We further found that ebony mutants exhibited increased aggression only when fighting each other, not when fighting wild-type controls. Moreover, fights between ebony mutants were less likely to end with a clear winner than fights between controls or fights between ebony mutants and controls. In ebony vs. control fights, ebony mutants were more likely to win. Together, these results suggest that ebony mutants exhibit prolonged aggressive behavior only in a specific context, with an equally dominant opponent.

Mutations in Cullin-3 ( Cul3 ), a conserved gene encoding a ubiquitin ligase, are strongly associated with autism spectrum disorder (ASD). Here, we characterize ASD-related pathologies caused by neuron-specific Cul3 knockdown in Drosophila . We confirmed that neuronal Cul3 knockdown causes short sleep, paralleling sleep disturbances in ASD. Because sleep defects and ASD are linked to metabolic dysregulation, we tested the starvation response of neuronal Cul3 knockdown flies; they starved faster and had lower triacylglyceride levels than controls, suggesting defects in metabolic homeostasis. ASD is also characterized by increased biomarkers of oxidative stress; we found that neuronal Cul3 knockdown increased sensitivity to hyperoxia, an exogenous oxidative stress. Additional hallmarks of ASD are deficits in social interactions and learning. Using a courtship suppression assay that measures social interactions and memory of prior courtship, we found that neuronal Cul3 knockdown reduced courtship and learning compared to controls. Finally, we found that neuronal Cul3 depletion alters the anatomy of the mushroom body, a brain region required for memory and sleep. Taken together, the ASD-related phenotypes of neuronal Cul3 knockdown flies establish these flies as a genetic model to study molecular and cellular mechanisms underlying ASD pathology, including metabolic and oxidative stress dysregulation and neurodevelopment.

Traumatic brain injury (TBI) affects millions annually and is associated with long-term health decline. TBI also shares molecular and cellular hallmarks with neurodegenerative diseases (NDs), typically increasing in prevalence with age, and is a major risk factor for developing neurodegeneration later in life. While our understanding of genes and pathways that underlie neurotoxicity in specific NDs has advanced, we still lack a complete understanding of early molecular and physiological changes that drive neurodegeneration, particularly as an individual ages following a TBI. Recently Drosophila has been introduced as a model organism for studying closed-head TBI. In this paper, we deliver a TBI to flies early in adult life, and then measure molecular and physiological phenotypes at short-, mid-, and long-term timepoints following the injury. We aim to identify the timing of changes that contribute to neurodegeneration. Here we confirm prior work demonstrating a TBI-induced decline in lifespan, and present evidence of a progressive decline in locomotor function, robust acute and modest chronic neuroinflammation, and a late-onset increase in protein aggregation. We also present evidence of metabolic dysfunction, in the form of starvation sensitivity and decreased lipids, that persists beyond the immediate injury response, but does not differ long-term. An intervention of dietary restriction (DR) partially ameliorates some TBI-induced phenotypes, including lifespan and locomotor function, though it does not alter the pattern of starvation sensitivity of injured flies. In the future, molecular pathways identified as altered following TBI—particularly in the short-, or mid-term—could present potential therapeutic targets.

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways, but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, up-regulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents over-proliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.

Understanding the biological mechanisms of aging represents an urgent biomedical challenge. AMP-activated protein kinase (AMPK) exhibits pro-longevity effects in diverse species. However, the tissue-specific mechanisms involved in AMPK regulation of aging are poorly understood. Here, we show that activation of AMPK in the adult Drosophila nervous system induces autophagy both in the brain and the intestinal epithelium. These cell autonomous and non-autonomous functions of AMPK are linked to improved intestinal homeostasis, muscle proteostasis and extended lifespan. Neuronal upregulation of the autophagy-specific protein kinase Atg1 is both necessary and sufficient to induce these inter-tissue effects during aging, resulting in prolonged lifespan. Furthermore, transgenic AMPK overexpression in neurons is sufficient to increase endogenous AMPK gene activity in distal tissues of the organism, including the intestine. In a complementary approach, transgenic upregulation of AMPK specifically in the adult intestine induces autophagy both cell autonomously and non-autonomously in the brain, exhibiting slowed systemic aging and prolonged lifespan. Additionally, we show that the organism-wide response to tissue-specific AMPK/Atg1 activation is linked to suppressed Drosophila insulin-like peptide (DILP) signaling. Together, these results reveal that localized transgenic activation of AMPK can function to relay pro-longevity signals to distal tissues. AMPK may now represent part of a novel brain-to-gut and gut-to-brain signaling axis in Drosophila.

In previous work, we found that short sleep caused sensitivity to oxidative stress; here we set out to characterize the physiological state of a diverse group of chronically short-sleeping mutants during hyperoxia as an acute oxidative stress. Using RNA-sequencing analysis, we found that short-sleeping mutants had a normal transcriptional oxidative stress response relative to controls. In both short-sleeping mutants and controls, hyperoxia led to downregulation of glycolytic genes and upregulation of genes involved in fatty acid metabolism, reminiscent of metabolic shifts during sleep. We hypothesized that short-sleeping mutants may be sensitive to hyperoxia because of defects in metabolism. Consistent with this, short-sleeping mutants were sensitive to starvation. Using metabolomics, we identified a pattern of low levels of long chain fatty acids and lysophospholipids in short-sleeping mutants relative to controls during hyperoxia, suggesting a defect in lipid metabolism. Though short-sleeping mutants did not have common defects in many aspects of lipid metabolism (basal fat stores, usage kinetics during hyperoxia, respiration rates, and cuticular hydrocarbon profiles), they were all sensitive to dehydration, suggesting a general defect in cuticular hydrocarbons, which protect against dehydration. To test the bi-directionality of sleep and lipid metabolism, we tested flies with both diet-induced obesity and genetic obesity. Flies with diet-induced obesity had no sleep or oxidative stress phenotype; in contrast, the lipid metabolic mutant, , slept significantly more than controls but was sensitive to oxidative stress. Previously, all short sleepers tested were sensitive and all long sleepers resistant to oxidative stress. mutants, the first exceptions to this rule, lack a key enzyme required to mobilize fat stores, suggesting that a defect in accessing lipid stores can cause sensitivity to oxidative stress. Taken together, we found that short-sleeping mutants have many phenotypes in common: sensitivity to oxidative stress, starvation, dehydration, and defects in lipid metabolites. These results argue against a specific role for sleep as an antioxidant and suggest the possibility that lipid metabolic defects underlie the sensitivity to oxidative stress of short-sleeping mutants.

In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these clock neurons secrete the neuropeptide Pdf and have been called \"master pacemakers\" because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It is assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient for circadian locomotor activity. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.