RNAi of Complex I and V of the electron transport chain in glutamate neurons extends life span, increases sleep, and decreases locomotor activity in Drosophila melanogaster.

RNAi targeting the electron transport chain has been proven to prolong life span in many different species, and experiments specifically with Drosophila melanogaster and Caenorhabditis elegans have shown a distinct role for neurons. To determine which subset of neurons is implicated in this life span extension, we used the GAL4/UAS system to activate RNAi against genes of Complex I and Complex V. We found life span extension of 18-24% with two glutamate neuron (D42 and VGlut) GAL4 lines. We used the GAL80 system to determine if the overlapping set of glutamate neurons in these two GAL4 lines imparts the life span extension. Limiting GAL4 activity to non-VGlut glutamate neurons in the D42 background failed to extend life span, suggesting that glutamate neurons have an important role in aging. Interestingly, RNAi of the electron transport chain in D42 glutamate neurons also caused an increase in daytime and nighttime sleep and a decrease in nighttime locomotor activity. Changes to sleep patterns and prolonged life span were not accompanied by any changes in female fertility or response to starvation. Our findings demonstrate that a small subset of neurons can control life span, and further studies can look into the contributions made by glutamate neurons.

Parkin Knockdown Modulates Dopamine Release in the Central Complex, but Not the Mushroom Body Heel, of Aging Drosophila.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons leading to reduced locomotion. Mutations of parkin gene in Drosophila produce the same phenotypes as vertebrate models, but the effect of parkin knockdown on dopamine release is not known. Here, we report age-dependent, spatial variation of dopamine release in the brain of parkin-RNAi adult Drosophila. Dopamine was repetitively stimulated by local application of acetylcholine and quantified by fast-scan cyclic voltammetry in the central complex or mushroom body heel. In the central complex, the main area controlling locomotor function, dopamine release is maintained for repeated stimulations in aged control flies, but lower concentrations of dopamine are released in the central complex of aged parkin-RNAi flies. In the mushroom body heel, the dopamine release decrease in older parkin-RNAi flies is similar to controls. There is not significant dopaminergic neuronal loss even in older parkin knockdown flies, which indicates that the changes in stimulated dopamine release are due to alterations of neuronal function. In young parkin-RNAi flies, locomotion is inhibited by 30%, while in older parkin-RNAi flies it is inhibited by 85%. Overall, stimulated dopamine release is modulated by parkin in an age and brain region dependent manner. Correlating the functional state of the dopaminergic system with behavioral phenotypes provides unique insights into the PD mechanism. Drosophila can be used to study dopamine functionality in PD, elucidate how genetics influence dopamine, and test potential therapies to maintain dopamine release.

Ring Finger Protein 11 (RNF11) Modulates Dopamine Release in Drosophila.

Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role of RNF11 in regulating dopamine neurotransmission has not been studied. In this work, we tested the effect of RNF11 RNAi knockdown or overexpression on stimulated dopamine release in the larval Drosophila central nervous system. Dopamine release was stimulated using optogenetics and monitored in real-time using fast-scan cyclic voltammetry at an electrode implanted in an isolated ventral nerve cord. RNF11 knockdown doubled dopamine release, but there was no decrease in dopamine from RNF11 overexpression. RNF11 knockdown did not significantly increase stimulated serotonin or octopamine release, indicating the effect is dopamine specific. Dopamine clearance was also changed, as RNF11 RNAi flies had a higher Vmax and RNF11 overexpressing flies had a lower Vmax than control flies. RNF11 RNAi flies had increased mRNA levels of dopamine transporter (DAT) in RNF11, confirming changes in DAT. In RNF11 RNAi flies, release was maintained better for stimulations repeated at short intervals, indicating increases in the recycled releasable pool of dopamine. Nisoxetine, a DAT inhibitor, and flupenthixol, a D2 antagonist, did not affect RNF11 RNAi or overexpressing flies differently than control. Thus, RNF11 knockdown causes early changes in dopamine neurotransmission, and this is the first work to demonstrate that RNF11 affects both dopamine release and uptake. RNF11 expression decreases in human dopaminergic neurons during PD, and that decrease may be protective by increasing dopamine neurotransmission in the surviving dopaminergic neurons.

Real-Time Measurement of Stimulated Dopamine Release in Compartments of the Adult Drosophila melanogaster Mushroom Body.

Drosophila melanogaster, a fruit fly, is an exquisite model organism to understand neurotransmission. Dopaminergic signaling in the Drosophila mushroom body (MB) is involved in olfactory learning and memory, with different compartments controlling aversive learning (heel) vs. appetitive learning (medial tip). Here, the goal was to develop techniques to measure endogenous dopamine in compartments of the MB for the first time. We compared three stimulation methods: acetylcholine (natural stimulus), P2X2 (chemogenetics), and CsChrimson (optogenetics). Evoked dopamine release was measured with fast-scan cyclic voltammetry in isolated adult Drosophila brains. Acetylcholine stimulated the largest dopamine release (0.40 µM) followed by P2X2 (0.14 µM) and CsChrimson (0.07 µM). With the larger acetylcholine and P2X2 stimulations, there were no regional or sex differences in dopamine release. However, with CsChrimson, dopamine release was significantly higher in the heel than the medial tip, and females had more dopamine than males. Michaelis-Menten modeling of the single-light pulse revealed no significant regional differences in Km, but the heel had a significantly lower Vmax (0.12 µM/s vs. 0.19 µM/s) and higher dopamine release (0.05 µM vs. 0.03 µM). Optogenetic experiments are challenging because CsChrimson is also sensitive to blue light used to activate green fluorescent protein, and thus, light exposure during brain dissection must be minimized. These experiments expand the toolkit for measuring endogenous dopamine release in Drosophila, introducing chemogenetic and optogenetic experiments for the first time. With a variety of stimulations, different experiments will help improve our understanding of neurochemical signaling in Drosophila.

Drosophila as a Model System for Neurotransmitter Measurements.

Drosophila melanogaster, the fruit fly, is an important, simple model organism for studying the effects of genetic mutations on neuronal activity and behavior. Biologists use Drosophila for neuroscience studies because of its genetic tractability, complex behaviors, well-known and simple neuroanatomy, and many orthologues to human genes. Neurochemical measurements in Drosophila are challenging due to the small size of the central nervous system. Recently, methods have been developed to measure real-time neurotransmitter release and clearance in both larvae and adults using electrochemistry. These studies have characterized dopamine, serotonin, and octopamine release in both wild type and genetic mutant flies. Tissue content measurements are also important, and separations are predominantly used. Capillary electrophoresis, with either electrochemical, laser-induced fluorescence, or mass spectrometry detection, facilitates tissue content measurements from single, isolated Drosophila brains or small samples of hemolymph. Neurochemical studies in Drosophila have revealed that flies have functioning transporters and autoreceptors, that their metabolism is different than in mammals, and that flies have regional, life stage, and sex differences in neurotransmission. Future studies will develop smaller electrodes, expand optical imaging techniques, explore physiological stimulations, and use advanced genetics to target single neuron release or study neurochemical changes in models of human diseases.

Extension of Drosophila life span by RNAi of the mitochondrial respiratory chain.

BACKGROUND: Mitochondria have long been proposed to play an important role in the aging process. In the nematode Caenorhabditis elegans, genes important for mitochondrial electron transport chain (ETC) function stand out as a principal group of genes affecting life span. However, it has been suggested that this may be a peculiarity of nematode biology. In the present study, we have used an in vivo RNA interference (RNAi) strategy to inactivate ETC genes in Drosophila melanogaster and examine the impact on longevity. RESULTS: Here, we report that RNAi of five genes encoding components of mitochondrial respiratory complexes I, III, IV, and V leads to increased life span in flies. Long-lived flies with reduced expression of ETC genes do not consistently show reduced assembly of respiratory complexes or reduced ATP levels. In addition, extended longevity is not consistently correlated with reduced fertility or increased resistance to the free-radical generator paraquat. Targeted RNAi of two complex I genes in adult tissues or in neurons alone is sufficient to extend life span. CONCLUSIONS: Our data suggest that the role of mitochondrial ETC function in modulating animal aging is evolutionarily conserved and might also operate in humans. Furthermore, our findings suggest that the longer life span of flies with reduced ETC gene expression cannot simply be attributed to reduced energy production leading to decreased "rate of living."

echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases.

BACKGROUND: Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. RESULTS: We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. CONCLUSION: The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions suggests that echinus acts at a novel point(s) to regulate interommatidial cell sorting and/or cell death in the fly eye.