Mild traumatic brain injury in Drosophila melanogaster alters reactive oxygen and nitrogen species in a sex-dependent manner.

Traumatic brain injury (TBI) is an outside force causing a modification in brain function and/or structural brain pathology that upregulates brain inducible nitric oxide synthase (iNOS), instigating increased levels of nitric oxide activity which is implicated in secondary pathology leading to behavioral deficits (Hall et al., 2012; Garry et al., 2015; Kozlov et al., 2017). In mammals, TBI-induced NO production activates an immune response and potentiates metabolic crisis through mitochondrial dysfunction coupled with vascular dysregulation; however, the direct influence on pathology is complicated by the activation of numerous secondary cascades and activation of other reactive oxygen species. Drosophila TBI models have demonstrated key features of mammalian TBI, including temporary incapacitation, disorientation, motor deficits, activation of innate immunity (inflammation), and autophagy responses observed immediately after injury (Katzenberger et al., 2013; Barekat et al., 2016; Simon et al., 2017; Anderson et al., 2018; Buhlman et al., 2021b). We hypothesized that acute behavioral phenotypes would be associated with deficits in climbing behavior and increased oxidative stress. Because flies lack mammalian-like cardiovascular and adaptive immune systems, we were able to make our observations in the absence of vascular disruption and adaptive immune system interference in a system where highly targeted interventions can be rapidly evaluated. To demonstrate the induction of injury, ten-day-old transgenic flies received an injury of increasing angles from a modified high impact trauma (HIT) device where angle-dependent increases occurred for acute neurological behavior assessments and twenty-four-hour mortality, and survival was significantly decreased. Injury caused sex-dependent effects on climbing activity and measures of oxidative stress. Specifically, after a single 60-degree HIT, female flies exhibited significant impairments in climbing activity beyond that observed in male flies. We also found that several measures of oxidative stress, including Drosophila NOS (dNOS) expression, protein nitration, and hydrogen peroxide production were significantly decreased in female flies. Interestingly, protein nitration was also decreased in males, but surpassed sham levels with a more severe injury. We also observed decreased autophagy demand in vulnerable dopaminergic neurons in female, but not male flies. In addition, mitophagy initiation was decreased in females. Collectively, our data suggest that TBI in flies induces acute behavioral phenotypes and climbing deficits that are analogous to mammalian TBI. We also observed that various indices of oxidative stress, including dNOS expression, protein tyrosine nitration, and hydrogen peroxide levels, as well as basal levels of autophagy, are altered in response to injury, an effect that is more pronounced in female flies.

Folic Acid Improves Parkin-Null Drosophila Phenotypes and Transiently Reduces Vulnerable Dopaminergic Neuron Mitochondrial Hydrogen Peroxide Levels and Glutathione Redox Equilibrium.

Loss-of-function parkin mutations cause oxidative stress and degeneration of dopaminergic neurons in the substantia nigra. Several consequences of parkin mutations have been described; to what degree they contribute to selective neurodegeneration remains unclear. Specific factors initiating excessive reactive oxygen species production, inefficient antioxidant capacity, or a combination are elusive. Identifying key oxidative stress contributors could inform targeted therapy. The absence of Drosophila parkin causes selective degeneration of a dopaminergic neuron cluster that is functionally homologous to the substantia nigra. By comparing observations in these to similar non-degenerating neurons, we may begin to understand mechanisms by which parkin loss of function causes selective degeneration. Using mitochondrially targeted redox-sensitive GFP2 fused with redox enzymes, we observed a sustained increased mitochondrial hydrogen peroxide levels in vulnerable dopaminergic neurons of parkin-null flies. Only transient increases in hydrogen peroxide were observed in similar but non-degenerating neurons. Glutathione redox equilibrium is preferentially dysregulated in vulnerable neuron mitochondria. To shed light on whether dysregulated glutathione redox equilibrium primarily contributes to oxidative stress, we supplemented food with folic acid, which can increase cysteine and glutathione levels. Folic acid improved survival, climbing, and transiently decreased hydrogen peroxide and glutathione redox equilibrium but did not mitigate whole-brain oxidative stress.

Nicotine Has a Therapeutic Window of Effectiveness in a Drosophila melanogaster Model of Parkinson's Disease.

Strong epidemiological evidence and studies in models of Parkinson's disease (PD) suggest that nicotine may be therapeutically beneficial in PD patients. However, a number of clinical trials utilizing nicotine in PD patients have had mixed results, indicating that either nicotine is not beneficial in PD patients, or an important aspect of nicotine therapy was absent. We hypothesized that nicotine must be administered early in the adult fly life in order to have beneficial effects. We show that continuous early nicotine administration improves both climbing and flight deficiencies present in homozygous park 25 mutant PD model Drosophila melanogaster. Using a new climbing assay, we identify several climbing deficiencies in this PD model that are improved or rescued by continuous nicotine treatment. Amongst these benefits, it appears that nicotine improves the ability of the park 25 flies to descend the climbing vial by being able to climb down more. In support of our hypothesis, we show that in order for nicotine benefits on climbing and flight to happen, nicotine administration must occur in a discrete time frame following adult fly eclosure: within one day for climbing or five days for flight. This therapeutic window of nicotine administration in this PD model fly may help to explain the lack of efficacy of nicotine in human clinical trials.

Drosophila as a model to explore secondary injury cascades after traumatic brain injury.

Drosophilae are emerging as a valuable model to study traumatic brain injury (TBI)-induced secondary injury cascades that drive persisting neuroinflammation and neurodegenerative pathology that imposes significant risk for long-term neurological deficits. As in mammals, TBI in Drosophila triggers axonal injury, metabolic crisis, oxidative stress, and a robust innate immune response. Subsequent neurodegeneration stresses quality control systems and perpetuates an environment for neuroprotection, regeneration, and delayed cell death via highly conserved cell signaling pathways. Fly injury models continue to be developed and validated for both whole-body and head-specific injury to isolate, evaluate, and modulate these parallel pathways. In conjunction with powerful genetic tools, the ability for longitudinal evaluation, and associated neurological deficits that can be tested with established behavioral tasks, Drosophilae are an attractive model to explore secondary injury cascades and therapeutic intervention after TBI. Here, we review similarities and differences between mammalian and fly pathophysiology and highlight strategies for their use in translational neurotrauma research.

Measuring Mitochondrial Hydrogen Peroxide Levels and Glutathione Redox Equilibrium in Drosophila Neuron Subtypes Using Redox-Sensitive Fluorophores and 3D Imaging.

Disruptions in mitochondrial redox activity are implicated in maladies ranging from those in which cells degenerate to those in which cell division is unregulated. This is not surprising given the pivotal role of mitochondria as ATP producers, reactive oxygen species (ROS) generators, and gatekeepers of apoptosis. While increased ROS are implicated in such a wide variety of disorders, pinpointing the cause of their hyperproduction is challenging. Elevated levels of ROS can result from increases in their production and/or decreases in their turnover. Disruptions in and/or hyperactivity of NADH-ubiquinone oxidoreductase or ubiquinone-cytochrome c oxidoreductase can cause excessive ROS generation. Alternatively, if respiration is functioning in a homeostatic manner, decreases in levels or activity of antioxidants like glutathione, CuZn- and Mn-superoxide dismutase, and catalase could result in excessive ROS. Because of the diversity of disorders in which oxidative damage occurs, the most effective therapeutic strategies may be those that address the putatively diverse causes of increased ROS. Strategies for determining antioxidant activity typically involve semiquantitative measurement of relative protein levels using immunochemistry and mass spectrometry. These methods can be applied to a variety of samples, but they do not lend themselves to detection of cell-specific analyses within tissue like brain.Because we are interested in elucidating the cause of oxidative stress in selectively vulnerable brain neurons, we have taken advantage of the easily manipulatable genetics and high fecundity of the fly. Using a cell type-targeting approach, we have driven redox sensitive green fluorescent proteins (roGFP2 ) into the mitochondria of tyrosine hydroxylase-producing (dopaminergic) neurons. In oxidizing conditions, the fluorophore's maximal excitation wavelength reversibly shifts. Therefore, the relative amount of mitochondrial protein oxidation can be determined by taking the ratio of fluorescence excited with two different lasers. In addition, these GFPs have been independently fused to human glutaredoxin-1 (mito-roGFP2-Grx1) and yeast oxidant receptor peroxidase (mito-roGFP2-Orp1), facilitating measurements of relative mitochondrial glutathione redox potential and H2O2 levels, respectively. In order to obtain a more comprehensive observation of redox states, we capture 3D images of roGFP2 excited by two different lasers. Mito- and cytoplasmic-roGFP2 -Grx1 and -Orp1 expression can be driven by hundreds of genetic drivers in Drosophila , facilitating fixed or living whole organism or tissue- and cell-specific redox measurements.

3D quantification of autophagy activation and autophagosome-to-mitochondria recruitment in a Drosophila model of Parkinson's disease.

Here, we describe a protocol for comprehensive quantification of autophagosome recruitment to mitochondria as an early step in mitophagy. Data collected using this protocol can be useful in the study of neurodegenerative disease, cancer, and metabolism-related disorders using models in which co-expression of mito-GFP and mCherry-Atg8a is feasible. This protocol has the advantage of assessment in an in vivo model organism (Drosophila melanogaster), where tissue-specific mitophagy can be investigated. For complete details on the use and execution of this protocol, please refer to (Cackovic et al., 2018).

Vulnerable Parkin Loss-of-Function Drosophila Dopaminergic Neurons Have Advanced Mitochondrial Aging, Mitochondrial Network Loss and Transiently Reduced Autophagosome Recruitment.

Selective degeneration of substantia nigra dopaminergic (DA) neurons is a hallmark pathology of familial Parkinson's disease (PD). While the mechanism of degeneration is elusive, abnormalities in mitochondrial function and turnover are strongly implicated. An Autosomal Recessive-Juvenile Parkinsonism (AR-JP) Drosophila melanogaster model exhibits DA neurodegeneration as well as aberrant mitochondrial dynamics and function. Disruptions in mitophagy have been observed in parkin loss-of-function models, and changes in mitochondrial respiration have been reported in patient fibroblasts. Whether loss of parkin causes selective DA neurodegeneration in vivo as a result of lost or decreased mitophagy is unknown. This study employs the use of fluorescent constructs expressed in Drosophila DA neurons that are functionally homologous to those of the mammalian substantia nigra. We provide evidence that degenerating DA neurons in parkin loss-of-function mutant flies have advanced mitochondrial aging, and that mitochondrial networks are fragmented and contain swollen organelles. We also found that mitophagy initiation is decreased in park (Drosophila parkin/PARK2 ortholog) homozygous mutants, but autophagosome formation is unaffected, and mitochondrial network volumes are decreased. As the fly ages, autophagosome recruitment becomes similar to control, while mitochondria continue to show signs of damage, and climbing deficits persist. Interestingly, aberrant mitochondrial morphology, aging and mitophagy initiation were not observed in DA neurons that do not degenerate. Our results suggest that parkin is important for mitochondrial homeostasis in vulnerable Drosophila DA neurons, and that loss of parkin-mediated mitophagy may play a role in degeneration of relevant DA neurons or motor deficits in this model.

α6 subunit-containing nicotinic receptors mediate low-dose ethanol effects on ventral tegmental area neurons and ethanol reward.

Dopamine (DA) neuron excitability is regulated by inhibitory GABAergic synaptic transmission and modulated by nicotinic acetylcholine receptors (nAChRs). The aim of this study was to evaluate the role of α6 subunit-containing nAChRs (α6*-nAChRs) in acute ethanol effects on ventral tegmental area (VTA) GABA and DA neurons. α6*-nAChRs were visualized on GABA terminals on VTA GABA neurons, and α6*-nAChR transcripts were expressed in most DA neurons, but only a minority of VTA GABA neurons from GAD67 GFP mice. Low concentrations of ethanol (1-10 mM) enhanced GABAA receptor (GABAA R)-mediated spontaneous and evoked inhibition with blockade by selective α6*-nAChR antagonist α-conotoxins (α-Ctxs) and lowered sensitivity in α6 knock-out (KO) mice. Ethanol suppression of VTA GABA neuron firing rate in wild-type mice in vivo was significantly reduced in α6 KO mice. Ethanol (5-100 mM) had no effect on optically evoked GABAA R-mediated inhibition of DA neurons, and ethanol enhancement of VTA DA neuron firing rate at high concentrations was not affected by α-Ctxs. Ethanol conditioned place preference was reduced in α6 KO mice compared with wild-type controls. Taken together, these studies indicate that relatively low concentrations of ethanol act through α6*-nAChRs on GABA terminals to enhance GABA release onto VTA GABA neurons, in turn to reduce GABA neuron firing, which may lead to VTA DA neuron disinhibition, suggesting a possible mechanism of action of alcohol and nicotine co-abuse.

Parkin loss-of-function pathology: Premature neuronal senescence induced by high levels of reactive oxygen species?

Parkinson's and Alzheimer's diseases (PD and AD, respectively) are considered to be diseases of advanced brain ageing, which seems to involve high levels of reactive oxygen species (ROS). AD neurodegeneration is initially apparent in the hippocampus; as AD progresses, many more brain regions are affected. PD-associated neurodegeneration is relatively limited to dopaminergic neurons of the substantia nigra pars compacta (SNpc), especially in cases in which patients inherit particular disease-causing mutations. Thus, the task of elucidating mechanisms by which loss of function of one particular protein triggers death of a subset of neurons may be more approachable. Understanding the mechanisms of neurodegeneration in these forms of PD may not only shed light on avenues leading toward therapeutic strategies in PD and other neurodegenerative diseases, but also on those leading toward understanding natural ageing. Neurodegeneration in PD patients harboring homozygous loss-of-function mutations in the PARK2 gene may result from unbalanced levels of ROS, which are mostly produced in mitochondria and can irreparably damage macromolecules and trigger apoptosis. This review discusses mitochondrial sources of ROS, how ROS can trigger apoptosis, mechanisms by which Parkin loss-of-function may cause neurodegeneration by increasing ROS levels, and concludes with hypotheses regarding selective SNpc dopaminergic neuron vulnerability.

Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance.

Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified. We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.

Nicotine increases lifespan and rescues olfactory and motor deficits in a Drosophila model of Parkinson's disease.

Drosophila melanogaster is an attractive model of familial Parkinson's disease, as flies with loss-of-function mutations of the parkin gene exhibit many pathologies observed in PD patients. Progressive motor deficits found in homozygous parkin mutants seem to result from mitochondrial pathology that causes indirect flight muscle and dopaminergic neuronal degeneration [1,2]. We have found that heterozygous parkin mutants have decreased lifespan, generally progressive motor dysfunction and olfactory deficits compared to control flies, suggesting that mutation of this gene produces a dominant phenotype. Tobacco smokers are dose-dependently less likely to develop PD [3,4]; subsequent in vitro and in vivo studies show that nicotine is protective in models of sporadic PD [6]. Literature addressing the potential protection by nicotine in Parkin loss-of-function models spans limited concentrations and selected time points in the organism's lifespan. We have found that parkin heterozygotes have late-onset climbing and flying deficits as well as decreased viability and olfactory deficits that precede motor defects. While chronic nicotine exposure decreases lifespan and climbing and flying abilities in control flies, it can improve viability and flying capability as well as rescue climbing and olfactory deficits in parkin heterozygotes. Dopaminergic neurons are spared in the parkin heterozygote, perhaps because this phenotype is less severe than in the homozygous parkin mutants. Nicotine pretreatment may be protective in sporadic PD patients and models; however, timely diagnosis remains to be an obstacle. Our results suggest that nicotine also may be protective in familial PD patients, who can be easily identified before motor symptoms occur.

Functional nicotinic acetylcholine receptors containing α6 subunits are on GABAergic neuronal boutons adherent to ventral tegmental area dopamine neurons.

Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR α6 subunit message is abundant in the VTA, but α6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for α6ß2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. α-Conotoxin (α-Ctx) MII labeling coupled with immunocytochemical staining localizes putative α6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABA(A) receptors. These increases are abolished by α6*-nAChR-selective α-Ctx MII or α-Ctx PIA (1 nm) but not by α7 (10 nm methyllycaconitine) or α4* (1 µm dihydro-ß-erythroidine)-nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR ß2 knock-out mice. Moreover, ACh induces an α-Ctx PIA-sensitive elevation in intraterminal Ca(2+) in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that α6ß2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic α6ß2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.

Heterologous expression of human {alpha}6{beta}4{beta}3{alpha}5 nicotinic acetylcholine receptors: binding properties consistent with their natural expression require quaternary subunit assembly including the {alpha}5 subunit.

Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits (alpha6(*) nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human alpha6beta2, alpha6beta4, alpha6beta2beta3, alpha6beta4beta3, or alpha6beta4beta3alpha5 nAChR. Cells expressing subunits needed to form alpha6beta4beta3alpha5 nAChR exhibited saturable [(3)H]epibatidine binding (K(d) = 95.9 +/- 8.3 pM and B(max) = 84.5 +/- 1.6 fmol/mg of protein). The rank order of binding competition potency (K(i)) for prototypical nicotinic compounds was alpha-conotoxin MII (6 nM) > nicotine (156 nM) approximately methyllycaconitine (200 nM) > alpha-bungarotoxin (>10 microM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that beta3 and alpha5 subunits are likely partners of the alpha6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that alpha6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human alpha5 subunit makes a critical contribution to alpha6beta4beta3alpha5 nAChR assembly into a ligand-binding form with native alpha6(*)-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.

Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells.

Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). Immunofluorescence studies show alpha4 and beta2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of 125I-labeled iodo-deschloroepibatidine binding sites in most cells. H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.

Post-hatching hormonal modulation of a sexually dimorphic neuromuscular system controlling song in zebra finches.

Sexual dimorphisms are present throughout the zebra finch song system, from forebrain centers to the tracheosyringeal portion of the hypoglossal nucleus (nXIIts) to the muscles of the syrinx (vocal organ). In females, gonadal steroids administered during development can partially masculinize the telencephalic areas, and in adulthood can increase the size of syrinx muscles. In the present study, two experiments were designed to investigate the role of early androgen and estrogen in the development of nXIIts and the ventralis and dorsalis muscles of the syrinx. In experiment one, males and females were treated with testosterone, estradiol, dihydrotestosterone, the anti-androgen flutamide, or a vehicle control for 21 days after hatching. At day 60, nXIIts volume, motoneuron soma size and number were assessed, as well as syrinx weight and the size of ventralis and dorsalis fibers. In experiment two, animals were administered either the estrogen synthesis inhibitor, fadrozole, or vehicle, and the syrinx measurements were taken at day 60. Male-biased sex differences were detected on all measures in both experiments, and several right-side biases were detected. In females, dihydrotestosterone masculinized soma size in nXIIts and testosterone slightly increased syrinx weight. E2 feminized the syrinx of males. However, flutamide did not prevent masculine development of either structure, and fadrozole did not inhibit feminine syrinx development. These results are consistent with the idea that, as in the forebrain, steroid hormones can stimulate aspects of sexual differentiation, but they may not be direct triggers for the process.