Chlorogenic Acids, Acting via Calcineurin, Are the Main Compounds in Centella asiatica Extracts That Mediate Resilience to Chronic Stress in Drosophila melanogaster.

Common symptoms of depressive disorders include anhedonia, sleep problems, and reduced physical activity. Drugs used to treat depression mostly aim to increase serotonin signaling but these can have unwanted side effects. Depression has also been treated by traditional medicine using plants like Centella asiatica (CA) and this has been found to be well tolerated. However, very few controlled studies have addressed CA's protective role in depression, nor have the active compounds or mechanisms that mediate this function been identified. To address this issue, we used Drosophila melanogaster to investigate whether CA can improve depression-associated symptoms like anhedonia and decreased climbing activity. We found that a water extract of CA provides resilience to stress induced phenotypes and that this effect is primarily due to mono-caffeoylquinic acids found in CA. Furthermore, we describe that the protective function of CA is due to a synergy between chlorogenic acid and one of its isomers also present in CA. However, increasing the concentration of chlorogenic acid can overcome the requirement for the second isomer. Lastly, we found that chlorogenic acid acts via calcineurin, a multifunctional phosphatase that can regulate synaptic transmission and plasticity and is also involved in neuronal maintenance.

Withania somnifera Extracts Promote Resilience against Age-Related and Stress-Induced Behavioral Phenotypes in Drosophila melanogaster; a Possible Role of Other Compounds besides Withanolides.

Withania somnifera (WS) extracts have been used in traditional medicine for millennia to promote healthy aging and wellbeing. WS is now also widely used in Western countries as a nutritional supplement to extend healthspan and increase resilience against age-related changes, including sleep deficits and depression. Although human trials have supported beneficial effects of WS, the study designs have varied widely. Plant material is intrinsically complex, and extracts vary widely with the origin of the plant material and the extraction method. Commercial supplements can contain various other ingredients, and the characteristics of the study population can also be varied. To perform maximally controlled experiments, we used plant extracts analyzed for their composition and stability. We then tested these extracts in an inbred Drosophila line to minimize effects of the genetic background in a controlled environment. We found that a water extract of WS (WSAq) was most potent in improving physical fitness, while an ethanol extract (WSE) improved sleep in aged flies. Both extracts provided resilience against stress-induced behavioral changes. WSE contained higher levels of withanolides, which have been proposed to be active ingredients, than WSAq. Therefore, withanolides may mediate the sleep improvement, whereas so-far-unknown ingredients enriched in WSAq likely mediate the effects on fitness and stress-related behavior.

Octopamine mediates sugar relief from a chronic-stress-induced depression-like state in Drosophila.

Chronic, uncontrollable stress can result in psychiatric syndromes, including anxiety and major depressive disorder, in humans and mammalian disease models.1,2 Similarly, several days of chronic stress can induce depression-associated behavioral alteration in Drosophila accompanied by changes in biogenic amine levels in the adult brain.3-6 In our chronic stress paradigm, flies are subjected to 3 days of repetitive phases of 300 Hz vibrations combined with overcrowding and food deprivation. This treatment reduces voluntary behavioral activity, including the motivation to climb wide gaps (risk taking) and to stop for sweets (anhedonia), suggesting a depression-like state (DLS). These behavioral changes correlate with decreased serotonin release to the mushroom body (MB), a major behavioral control center in the central brain of the fly.7,8 Stressed flies are relieved from the DLS by feeding the anti-depressant serotonin precursor 5-HTP or the selective serotonin reuptake inhibitor fluoxetine. Notably, feeding sucrose to stressed flies results in elevated serotonin levels in the brain and ameliorates the DLS.3 Here, we show that this sugar relief is mediated by the neurotransmitter octopamine signaled from ventral unpaired medial neurons located in the subesophageal ganglion. The octopamine signaling of sweet sensation is transmitted to the MB via the dopaminergic PAM neurons. In addition, neuronal-silencing experiments reveal that the serotonergic dorsal paired medial (DPM) neurons innervating the MB are essential for sugar relief. Conversely, thermogenetic or optogenetic activation of DPMs can replace sweet sensation, elucidating that serotonergic signaling from DPMs takes part in positively modulating DLS-related behavioral changes.

Drosophila Acquires a Long-Lasting Body-Size Memory from Visual Feedback.

Grasping an object or crossing a trench requires the integration of information on the operating distance of our limbs with precise distance estimation. The reach of our hands and step size of our legs are learned by the visual feedback we get during our actions. This implicit knowledge of our peripersonal space is first acquired during infancy but will be continuously updated throughout our whole life [1]. In contrast, body size of holometabolous insects does not change after metamorphosis; nevertheless, they do have to learn their body reaches at least once. The body size of Drosophila imagines can vary by about 15% depending on environmental factors like food quality and temperature [2]. To investigate how flies acquire knowledge about and memorize their body size, we studied their decisions to either refrain from or initiate climbing over gaps exceeding their body size [3]. Naive (dark-reared) flies overestimate their size and have to learn it from the parallax motion of the retinal images of objects in their environment while walking. Naive flies can be trained in a striped arena and manipulated to underestimate their size, but once consolidated, this memory seems to last for a lifetime. Consolidation of this memory is stress sensitive only in the first 2 h after training but cannot be retrieved for the next 12 h. We have identified a set of intrinsic, lateral neurons of the protocerebral bridge of the central complex [4, 5] that depend on dCREB2 transcriptional activity for long-term memory consolidation and maintenance.

Drosophila Full-Length Amyloid Precursor Protein Is Required for Visual Working Memory and Prevents Age-Related Memory Impairment.

The ß-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). However, its normal physiological functions are still unclear. APP is cleaved by various secretases whereby sequential processing by the ß- and γ-secretases produces the ß-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPPß fragments and the APP intracellular domain (AICD). Alternative cleavage by α-secretase results in slightly longer secreted sAPPα fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPPα fragments have been suggested to have a neurotrophic and neuroprotective role [1]. Moreover, expression of sAPPα in APP-deficient mice could rescue their deficits in learning, spatial memory, and long-term potentiation [2]. Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment [3]. We now show that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, our results show that specific memory processes require distinct APPL products.

Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment.

Major depressive disorder (MDD) affects millions of patients; however, the pathophysiology is poorly understood. Rodent models have been developed using chronic mild stress or unavoidable punishment (learned helplessness) to induce features of depression, like general inactivity and anhedonia. Here we report a three-day vibration-stress protocol for Drosophila that reduces voluntary behavioural activity. As in many MDD patients, lithium-chloride treatment can suppress this depression-like state in flies. The behavioural changes correlate with reduced serotonin (5-HT) release at the mushroom body (MB) and can be relieved by feeding the antidepressant 5-hydroxy-L-tryptophan or sucrose, which results in elevated 5-HT levels in the brain. This relief is mediated by 5-HT-1A receptors in the α-/ß-lobes of the MB, whereas 5-HT-1B receptors in the γ-lobes control behavioural inactivity. The central role of serotonin in modulating stress responses in flies and mammals indicates evolutionary conserved pathways that can provide targets for treatment and strategies to induce resilience.

Visual Working Memory Requires Permissive and Instructive NO/cGMP Signaling at Presynapses in the Drosophila Central Brain.

The gaseous second messenger nitric oxide (NO) has been shown to regulate memory formation by activating retrograde signaling cascades from post- to presynapse that involve cyclic guanosine monophosphate (cGMP) production to induce synaptic plasticity and transcriptional changes. In this study, we analyzed the role of NO in the formation of a visual working memory that lasts only a few seconds. This memory is encoded in a subset of ring neurons that form the ellipsoid body in the Drosophila brain. Using genetic and pharmacological manipulations, we show that NO signaling is required for cGMP-mediated CREB activation, leading to the expression of competence factors like the synaptic homer protein. Interestingly, this cell-autonomous function can also be fulfilled by hydrogen sulfide (H2S) through a converging pathway, revealing for the first time that endogenously produced H2S has a role in memory processes. Notably, the NO synthase is strictly localized to the axonal output branches of the ring neurons, and this localization seems to be necessary for a second, phasic role of NO signaling. We provide evidence for a model where NO modulates the opening of cGMP-regulated cation channels to encode a short-term memory trace. Local production of NO/cGMP in restricted branches of ring neurons seems to represent the engram for objects, and comparing signal levels between individual ring neurons is used to orient the fly during search behavior. Due to its short half-life, NO seems to be a uniquely suited second messenger to encode working memories that have to be restricted in their duration.

m6A modulates neuronal functions and sex determination in Drosophila.

N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. Here we carry out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. We identify the member of the split ends protein family, Spenito, as a novel bona fide subunit of the methyltransferase complex. We further demonstrate important roles of this complex in neuronal functions and sex determination, and implicate the nuclear YT521-B protein as a main m6A effector in these processes. Altogether, our work substantially extends our knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.

Serum response factor-mediated gene regulation in a Drosophila visual working memory.

BACKGROUND: Navigation through the environment requires a working memory for the chosen target and path integration facilitating an approach when the target becomes temporarily hidden. We have previously shown that this visual orientation memory resides in the ellipsoid body, which is part of the central complex in the Drosophila brain. Former analysis of foraging and ignorant mutants have revealed that a hierarchical PKG and RSKII kinase signaling cascade in a subset of the ellipsoid-body ring neurons is required for this type of working memory in flies. RESULTS: Here we show that mutants in the ellipsoid body open (ebo) gene, which encodes the actin-binding protein Exportin 6, exhibit excessive nuclear accumulation of actin during development and in the adult brain. ebo mutants lack the orientation memory independent of the structural defect in the ellipsoid-body neuropil, and EBO activity in any type of adult ring neurons is sufficient for orientation-memory function. Moreover, genetic interaction studies revealed that nuclear actin accumulation in ebo mutants inhibits the Drosophila coactivator myocardin-related transcription factor A (dMRTF) and therefore the transcriptional activator serum response factor (dSRF). dSRF also functions in different ring neurons, suggesting that it regulates abundance of a diffusible factor that enables a working memory in ellipsoid-body ring neurons. CONCLUSIONS: To date, SRF has only been implicated in longer forms of memory formation like synaptic long-term potentiation and depression. This study provides the first evidence that SRF-mediated gene regulation is also required for a working memory that lasts only for a few seconds.

The visual orientation memory of Drosophila requires Foraging (PKG) upstream of Ignorant (RSK2) in ring neurons of the central complex.

Orientation and navigation in a complex environment requires path planning and recall to exert goal-driven behavior. Walking Drosophila flies possess a visual orientation memory for attractive targets which is localized in the central complex of the adult brain. Here we show that this type of working memory requires the cGMP-dependent protein kinase encoded by the foraging gene in just one type of ellipsoid-body ring neurons. Moreover, genetic and epistatic interaction studies provide evidence that Foraging functions upstream of the Ignorant Ribosomal-S6 Kinase 2, thus revealing a novel neuronal signaling pathway necessary for this type of memory in Drosophila.

Analysis of amyloid precursor protein function in Drosophila melanogaster.

Amyloid precursor proteins (APPs) are evolutionary conserved from nematodes to man (Jacobsen and Iverfeldt in Cell Mol Life Sci 66:2299-2318, 2009) suggesting an important physiological function of these proteins. Human APP is a key factor in the pathogenesis of Alzheimer's Disease because its proteolytic processing results in the production of the neurotoxic Aß-peptide, which accumulates in the amyloid plaques characteristic for this disease (Selkoe in Physiol Rev 81(2):741-766, 2001). However, the processing also leads to the production of several other fragments and the role of these products, as well as the function of the full-length protein is so far not well understood. The functional analysis of APP in vertebrates has been hampered by the fact that two close relatives, APLP1 and APLP2, exist and that knock-out mice for APP only show subtle defects. In contrast, invertebrates like Caenorhabditis elegans and Drosophila express only one APP-like protein but whereas a null mutation in the C. elegans APL-1 protein is lethal, flies lacking APPL (Amyloid Precursor Protein-like) are viable but show synaptic defects and behavioral abnormalities. Together with the analyses of flies that express APP proteins ectoptically or xenotopically, these studies show that APP proteins are involved in neuronal differentiation, neuritic outgrowth, and synapse formation. In addition, they play a role in long-term memory formation and maintaining brain integrity in adult flies.

Visual targeting of motor actions in climbing Drosophila.

Drosophila melanogaster flies cross surmountable gaps in their walkway of widths exceeding their body length with an astounding maneuver but avoid attempts at insurmountable gaps by visual width estimation. Different mutant lines affect specific aspects of this maneuver, indicating a high complexity and modularity of the underlying motor control. Here we report on two mutants, ocelliless(1) and tay bridge(1), that, although making a correct decision to climb, fail dramatically in aiming at the right direction. Both mutants show structural defects in the protocerebral bridge, a central complex neuropil formed like a handlebar spanning the brain hemispheres. The bridge has been implicated in step-length control in walking flies and celestial E-vector orientation in locusts. In rescue experiments using tay bridge(1) flies, the integrity of the bridge was reestablished, concomitantly leading to a significant improvement of their orientation at the gap. Although producing directional scatter, their attempts were clearly aimed at the landing site. However, this partial rescue was lost in these flies at a reduced-visibility landing site. We therefore conclude that the protocerebral bridge is an essential part of a visual targeting network that transmits directional clues to the motor output via a known projection system.

Neurotoxic effects induced by the Drosophila amyloid-beta peptide suggest a conserved toxic function.

The accumulation of amyloid-beta (Abeta) into plaques is a hallmark feature of Alzheimer's disease (AD). While amyloid precursor protein (APP)-related proteins are found in most organisms, only Abeta fragments from human APP have been shown to induce amyloid deposits and progressive neurodegeneration. Therefore, it was suggested that neurotoxic effects are a specific property of human Abeta. Here we show that Abeta fragments derived from the Drosophila orthologue APPL aggregate into intracellular fibrils, amyloid deposits, and cause age-dependent behavioral deficits and neurodegeneration. We also show that APPL can be cleaved by a novel fly beta-secretase-like enzyme. This suggests that Abeta-induced neurotoxicity is a conserved function of APP proteins whereby the lack of conservation in the primary sequence indicates that secondary structural aspects determine their pathogenesis. In addition, we found that the behavioral phenotypes precede extracellular amyloid deposit formation, supporting results that intracellular Abeta plays a key role in AD.

Analysis of a spatial orientation memory in Drosophila.

Flexible goal-driven orientation requires that the position of a target be stored, especially in case the target moves out of sight. The capability to retain, recall and integrate such positional information into guiding behaviour has been summarized under the term spatial working memory. This kind of memory contains specific details of the presence that are not necessarily part of a long-term memory. Neurophysiological studies in primates indicate that sustained activity of neurons encodes the sensory information even though the object is no longer present. Furthermore they suggest that dopamine transmits the respective input to the prefrontal cortex, and simultaneous suppression by GABA spatially restricts this neuronal activity. Here we show that Drosophila melanogaster possesses a similar spatial memory during locomotion. Using a new detour setup, we show that flies can remember the position of an object for several seconds after it has been removed from their environment. In this setup, flies are temporarily lured away from the direction towards their hidden target, yet they are thereafter able to aim for their former target. Furthermore, we find that the GABAergic (stainable with antibodies against GABA) ring neurons of the ellipsoid body in the central brain are necessary and their plasticity is sufficient for a functional spatial orientation memory in flies. We also find that the protein kinase S6KII (ignorant) is required in a distinct subset of ring neurons to display this memory. Conditional expression of S6KII in these neurons only in adults can restore the loss of the orientation memory of the ignorant mutant. The S6KII signalling pathway therefore seems to be acutely required in the ring neurons for spatial orientation memory in flies.

Locomotor control by the central complex in Drosophila-An analysis of the tay bridge mutant.

Several aspects of locomotor control have been ascribed to the central complex of the insect brain; however, the role of distinct substructures of this complex is not well known. The tay bridge1 (tay1) mutant of Drosophila melanogaster was originally isolated on the basis of reduced walking speed and activity. In addition, tay1 is defective in the compensation of rotatory stimuli during walking and histologically, tay1 causes a mid-sagittal constriction of the protocerebral bridge, a constituent of the central complex. Cloning of the tay gene revealed that it encodes a novel protein with no significant homology to any known protein. To associate the behavioral phenotypes with the anatomical defect in the protocerebral bridge, we used different driver lines to express the tay cDNA in various neuronal subpopulations of the central brain in tay1-mutant flies. These experiments showed an association of the aberrant walking speed and activity with the structural defect in the protocerebral bridge. In contrast, the compensation of rotatory stimuli during walking was rescued without a restoration of the protocerebral bridge. The results of our differential rescue approach are supported by neuronal silencing experiments using conditional tetanus toxin expression in the same subset of neurons. These findings show for the first time that the walking speed and activity is controlled by different substructures of the central brain than the compensatory locomotion for rotatory stimuli.

Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila.

Patients with polyglutamine expansion diseases, like Huntington's disease or several spinocerebellar ataxias, first present with neurological symptoms that can occur in the absence of neurodegeneration. Behavioral symptoms thus appear to be caused by neuronal dysfunction, rather than cell death. Pathogenesis in polyglutamine expansion diseases is largely viewed as a cell-autonomous process in neurons. It is likely, however, that this process is influenced by changes in glial physiology and, at least in the case of DRPLA glial inclusions and glial cell death, seems to be an important part in the pathogenesis. To investigate these aspects in a Drosophila model system, we expressed polyglutamine proteins in the adult nervous system. Glial-specific expression of a polyglutamine (Q)-expanded (n=78) and also a nonexpanded (n=27) truncated version of human ataxin-3 led to the formation of protein aggregates and glial cell death. Behavioral changes were observed prior to cell death. This reveals that glia is susceptible to the toxic action of polyglutamine proteins. Neuronal expression of the same constructs resulted in behavioral changes similar to those resulting from glial expression but did not cause neurodegeneration. Behavioral deficits were selective and affected two analyzed fly behaviors differently. Both glial and neuronal aggregates of Q78 and Q27 appeared early in pathogenesis and, at the electron microscopic resolution, had a fibrillary substructure. This shows that a nonexpanded stretch can cause similar histological and behavioral symptoms as the expanded stretch, however, with a significant delay.

Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye form connections in different optic ganglia. The R1-R6 subclass connects to the first optic ganglion, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development including migration of glial cells into the target region. Here, we show that the JAB1/CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and through the analysis of a unique set of missense mutations, is required in R cells to induce lamina glial cell migration. In these CSN5 alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles further revealed that the COP9 signalosome is required at an earlier stage of development for R cell differentiation.