Neuronal knockdown of Cullin3 as a Drosophila model of autism spectrum disorder.

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.

Circadian autophagy drives iTRF-mediated longevity.

Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1-5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.

Vitamin preference in Drosophila.

Many animals rely on taste to identify nutritious foods and to avoid the consumption of harmful substances. The tastes of macronutrients, as well as of non-caloric micronutrients such as sodium and calcium, contribute to the regulation of ingestive behavior1,2. Whether vitamins also affect feeding behavior through taste is less clear. Here, we show that the fly Drosophila melanogaster has a strong preference for consuming a vitamin-containing diet: both sexes show a preference for folic acid, whereas only females show a preference for riboflavin. Females show a preference with vitamin concentrations as low as ∼10 nM - at least 50,000-fold lower than the concentration needed for sucrose preference. This female vitamin preference requires inputs from external and internal taste organs, suggesting that post-ingestive signals, in the absence of gustatory input, are insufficient to actuate preferential consumption of vitamin-containing diets. Our studies demonstrate that vitamin perception is an important determinant of feeding behavior.

Neurofibromin regulates metabolic rate via neuronal mechanisms in Drosophila.

Neurofibromatosis type 1 is a chronic multisystemic genetic disorder that results from loss of function in the neurofibromin protein. Neurofibromin may regulate metabolism, though the underlying mechanisms remain largely unknown. Here we show that neurofibromin regulates metabolic homeostasis in Drosophila via a discrete neuronal circuit. Loss of neurofibromin increases metabolic rate via a Ras GAP-related domain-dependent mechanism, increases feeding homeostatically, and alters lipid stores and turnover kinetics. The increase in metabolic rate is independent of locomotor activity, and maps to a sparse subset of neurons. Stimulating these neurons increases metabolic rate, linking their dynamic activity state to metabolism over short time scales. Our results indicate that neurofibromin regulates metabolic rate via neuronal mechanisms, suggest that cellular and systemic metabolic alterations may represent a pathophysiological mechanism in neurofibromatosis type 1, and provide a platform for investigating the cellular role of neurofibromin in metabolic homeostasis.

The Drosophila melanogaster Neprilysin Nepl15 is involved in lipid and carbohydrate storage.

The prototypical M13 peptidase, human Neprilysin, functions as a transmembrane "ectoenzyme" that cleaves neuropeptides that regulate e.g. glucose metabolism, and has been linked to type 2 diabetes. The M13 family has undergone a remarkable, and conserved, expansion in the Drosophila genus. Here, we describe the function of Drosophila melanogaster Neprilysin-like 15 (Nepl15). Nepl15 is likely to be a secreted protein, rather than a transmembrane protein. Nepl15 has changes in critical catalytic residues that are conserved across the Drosophila genus and likely renders the Nepl15 protein catalytically inactive. Nevertheless, a knockout of the Nepl15 gene reveals a reduction in triglyceride and glycogen storage, with the effects likely occurring during the larval feeding period. Conversely, flies overexpressing Nepl15 store more triglycerides and glycogen. Protein modeling suggests that Nepl15 is able to bind and sequester peptide targets of catalytically active Drosophila M13 family members, peptides that are conserved in humans and Drosophila, potentially providing a novel mechanism for regulating the activity of neuropeptides in the context of lipid and carbohydrate homeostasis.

Absolute ethanol intake predicts ethanol preference in Drosophilamelanogaster.

Factors that mediate ethanol preference in Drosophila melanogaster are not well understood. A major confound has been the use of diverse methods to estimate ethanol consumption. We measured fly consumptive ethanol preference on base diets varying in nutrients, taste and ethanol concentration. Both sexes showed an ethanol preference that was abolished on high nutrient concentration diets. Additionally, manipulating total food intake without altering the nutritive value of the base diet or the ethanol concentration was sufficient to evoke or eliminate ethanol preference. Absolute ethanol intake and food volume consumed were stronger predictors of ethanol preference than caloric intake or the dietary caloric content. Our findings suggest that the effect of the base diet on ethanol preference is largely mediated by total consumption associated with the delivery medium, which ultimately determines the level of ethanol intake. We speculate that a physiologically relevant threshold for ethanol intake is essential for preferential ethanol consumption.

PGC1α Controls Sucrose Taste Sensitization in Drosophila.

Perceived palatability of food controls caloric intake. Sweet taste is the primary means of detecting the carbohydrate content of food. Surprisingly, sweet taste sensitivity is responsive to extrinsic factors like diet, and this occurs by unknown mechanisms. Here, we describe an unbiased proteomic investigation into sweet taste sensitivity in the fruit fly. We identify a dopamine/cyclic AMP (cAMP)/CREB axis acting within sweet taste neurons that controls taste perception but is largely dispensable for acute taste transduction. This pathway modulates sweet taste perception in response to both sensory- and nutrient-restricted diets and converges on PGC1α, a critical regulator of metabolic health and lifespan. By electrophysiology, we found that enhanced sucrose taste sensitivity was the result of heightened sweet taste intensity and that PGC1α was both necessary and sufficient for this effect. Together, we provide the first molecular insight into how diet-induced taste perception is regulated within the sweet taste neuron.

Excreta Quantification (EX-Q) for Longitudinal Measurements of Food Intake in Drosophila.

Longitudinal measurements of food intake remain a challenge in Drosophila studies of nutrition and behavior. Here, we report an improved method for measuring fly food intake using dye-labeled food and excreta quantification (EX-Q). Reducing the surface area of the medium maximized excreta recovery and the accuracy in estimating total consumption. The EX-Q method is compatible with agar-based medium and makes it possible to measure consumption over an extended period and at multiple time points without sacrificing flies. Using EX-Q, we revealed nutrient- and age-specific features of Drosophila feeding behavior. Daily consumption of a chemically defined diet was relatively consistent over the first 25 days of adulthood. Omitting amino acids or vitamins from the diet reduced consumption in both sexes, whereas omitting sugars or cholesterol primarily affected female food intake. Our results demonstrate EX-Q as a simple, reliable, and nondestructive method for longitudinal studies of solid food intake in Drosophila.