Nutraceutical and Probiotic Approaches to Examine Molecular Interactions of the Amyloid Precursor Protein APP in Drosophila Models of Alzheimer's Disease.

Studies using animal models have shed light into the molecular and cellular basis for the neuropathology observed in patients with Alzheimer's disease (AD). In particular, the role of the amyloid precursor protein (APP) plays a crucial role in the formation of senile plaques and aging-dependent degeneration. Here, we focus our review on recent findings using the Drosophila AD model to expand our understanding of APP molecular function and interactions, including insights gained from the fly homolog APP-like (APPL). Finally, as there is still no cure for AD, we review some approaches that have shown promising results in ameliorating AD-associated phenotypes, with special attention on the use of nutraceuticals and their molecular effects, as well as interactions with the gut microbiome. Overall, the phenomena described here are of fundamental significance for understanding network development and degeneration. Given the highly conserved nature of fundamental signaling pathways, the insight gained from animal models such as Drosophila melanogaster will likely advance the understanding of the mammalian brain, and thus be relevant to human health.

In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction.

Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low-frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic Drosophila neuromuscular junctions (NMJs), where low-frequency Ca2+ oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. We examined embryonic growth cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca2+ oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca2+ signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca2+ transients matured over several hours into regular low-frequency (0.03 Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. We provide genetic evidence suggesting a complex presynaptic Ca2+-dependent signaling network underlying refinement that involves the phosphatases calcineurin and protein phosphatase-1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts.SIGNIFICANCE STATEMENT To address the question of how synaptic connectivity is established during development, we examined the behavior of growth cone filopodia during the exploration of both correct and off-target muscle fibers in Drosophila embryos. We demonstrate that filopodia repeatedly contact off-target muscles over several hours, until they ultimately retract. We show that intracellular signals are observed in motile and stabilized "ectopic" contacts. Several genetic experiments provide insight in the molecular pathway underlying network refinement, which includes oscillatory calcium signals via voltage-gated calcium channels as a key component. Calcium orchestrates the activity of several kinases and phosphatases, which interact in a coordinated fashion to regulate chemorepulsion exerted by the muscle.

Dendrites are dispensable for basic motoneuron function but essential for fine tuning of behavior.

Dendrites are highly complex 3D structures that define neuronal morphology and connectivity and are the predominant sites for synaptic input. Defects in dendritic structure are highly consistent correlates of brain diseases. However, the precise consequences of dendritic structure defects for neuronal function and behavioral performance remain unknown. Here we probe dendritic function by using genetic tools to selectively abolish dendrites in identified Drosophila wing motoneurons without affecting other neuronal properties. We find that these motoneuron dendrites are unexpectedly dispensable for synaptic targeting, qualitatively normal neuronal activity patterns during behavior, and basic behavioral performance. However, significant performance deficits in sophisticated motor behaviors, such as flight altitude control and switching between discrete courtship song elements, scale with the degree of dendritic defect. To our knowledge, our observations provide the first direct evidence that complex dendrite architecture is critically required for fine-tuning and adaptability within robust, evolutionarily constrained behavioral programs that are vital for mating success and survival. We speculate that the observed scaling of performance deficits with the degree of structural defect is consistent with gradual increases in intellectual disability during continuously advancing structural deficiencies in progressive neurological disorders.

Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development.

Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. We combine genetic manipulation, imaging and quantitative dendritic architecture analysis in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dα7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter we demonstrate that AP-1 transcriptional activity is downstream of Dα7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. Our results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies.

Dscam1 is required for normal dendrite growth and branching but not for dendritic spacing in Drosophila motoneurons.

Down syndrome cell adhesion molecule, Dscam, serves diverse neurodevelopmental functions, including axon guidance and synaptic adhesion, as well as self-recognition and self-avoidance, depending on the neuron type, brain region, or species under investigation. In Drosophila, the extensive molecular diversity that results from alternative splicing of Dscam1 into >38,000 isoforms provides neurons with a unique molecular code for self-recognition in the nervous system. Each neuron produces only a small subset of Dscam1 isoforms, and distinct Dscam1 isoforms mediate homophilic interactions, which in turn, result in repulsion and even spacing of self-processes, while allowing contact with neighboring cells. While these mechanisms have been shown to underlie mushroom body development and spacing of mechanosensory neuron dendrites, here we report that Dscam1 plays no role in adult Drosophila motoneuron dendrite spacing, but is required for motoneuron dendritic growth. Targeted expression of Dscam-RNAi in an identified flight motoneuron did not impact dendrite spacing, but instead produced overgrowth. Increasing the knockdown strength severely reduced dendritic growth and branching. Similarly, Dscam mutant motoneurons in an otherwise control background (MARCM) were completely devoid of mature dendrites. These data suggest that Dscam1 is required cell autonomously for normal adult motoneuron dendrite growth in Drosophila. This demonstrates a previously unreported role of Drosophila Dscam1 in central neuron development, and expands the current understanding that Dscam1 operates as a cell adhesion molecule that mediates homophilic repulsion.

Maximizing Access to Cell Biology for PEERS: Retracting the term minority in favor of a more inclusive lexicon.

The word minority, when used incorrectly, is a condescending term that segregates, inaccurately represents groups as being smaller or less important, and fuels microaggressions. Scientific societies and other institutions have normalized using the word minority, or the "M word," to refer to members of underrepresented groups in Science, Technology, Engineering, and Mathematics (STEM). The message put forth using the term minority often directly conflicts with the inclusive agenda these societies seek to enact. More inclusive acronyms such as PEER (Persons Excluded because of their Ethnicity or Race) have been created to more accurately reflect the active process of exclusion by institutions. Here, we detail the rationale behind the decision to eradicate the word minority from the name of a prominent committee within the American Society for Cell Biology (ASCB). The ASCB Minority Affairs Committee changed its name to the Maximizing Access to Cell Biology for PEERS Committee. Herein, we emphasize the basis for the name change and highlight the contradictions intrinsic to the word minority in this context. We highlight why swift action is required for this rewording within the context of a committee dedicated to supporting the inclusion of PEERs in the scientific community.

Dual treatment with kynurenine pathway inhibitors and NAD+ precursors synergistically extends life span in Drosophila.

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD+. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD+, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD+ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD+ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD+ precursors, or a combination of KP inhibitors with NAD+ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD+ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD+ metabolism can synergistically increase life span.

Food supplementation with wheat gluten leads to climbing performance decline in Drosophila melanogaster.

Gluten sensitivity is associated with digestive and neurological disorders, correlating with abnormal amino acid levels, innate immune responses, gut dysbiosis and movement incoordination. However, the molecular mechanisms linking dietary gluten and brain function remain incompletely understood. We used Drosophila melanogaster to test the effects of gluten ingestion in locomotion performance. Whereas flies on control food showed decreased climbing performance after five weeks, flies exposed to food supplemented with different gluten concentrations showed a significant locomotion decline after three weeks of treatment. Future studies will determine the mechanisms underlying the observed gluten-dependent phenotypes to establish Drosophila models for gluten sensitivity.

Corazonin Neurons Contribute to Dimorphic Ethanol Sedation Sensitivity in Drosophila melanogaster.

Exposure to alcohol has multiple effects on nervous system function, and organisms have evolved mechanisms to optimally respond to the presence of ethanol. Sex differences in ethanol-induced behaviors have been observed in several organisms, ranging from humans to invertebrates. However, the molecular mechanisms underlying the dimorphic regulation of ethanol-induced behaviors remain incompletely understood. Here, we observed sex differences in ethanol sedation sensitivity in Drosophila Genome Reference Panel (DGRP) lines of Drosophila melanogaster compared to the absence of dimorphism in standard laboratory wildtype and control lines. However, in dose response experiments, we were able to unmask dimorphic responses for the control mutant line w 1118 by lowering the testing ethanol concentration. Notably, feminization of the small population of Corazonin (Crz) neurons in males was sufficient to induce female-like sedation sensitivity. We also tested the role of the transcription factor apontic (apt) based on its known expression in Crz neurons and its regulation of sedation responses. Interestingly, loss of function apt mutations increased sedation times in both males and females as compared to controls. No significant difference between male and female apt mutants was observed, suggesting a possible role of apt in the regulation of dimorphic ethanol-induced responses. Thus, our results shed light into the mechanisms regulating sex-differences in ethanol-induced behaviors at the cellular and molecular level, suggesting that the genetic sex in a small neuronal population plays an important role in modulating sex differences in behavioral responses to ethanol.

Drosophila Corazonin Neurons as a Hub for Regulating Growth, Stress Responses, Ethanol-Related Behaviors, Copulation Persistence and Sexually Dimorphic Reward Pathways.

The neuronal mechanisms by which complex behaviors are coordinated and timed often involve neuropeptidergic regulation of stress and reward pathways. Recent studies of the neuropeptide Corazonin (Crz), a homolog of the mammalian Gonadotrophin Releasing Hormone (GnRH), have suggested its crucial role in the regulation of growth, internal states and behavioral decision making. We focus this review on Crz neurons with the goal to (1) highlight the diverse roles of Crz neuron function, including mechanisms that may be independent of the Crz peptide, (2) emphasize current gaps in knowledge about Crz neuron functions, and (3) propose exciting ideas of novel research directions involving the use of Crz neurons. We describe the different developmental fates of distinct subsets of Crz neurons, including recent findings elucidating the molecular regulation of apoptosis. Crz regulates systemic growth, food intake, stress responses and homeostasis by interacting with the short Neuropeptide F (sNPF) and the steroid hormone ecdysone. Additionally, activation of Crz neurons is shown to be pleasurable by interacting with the Neuropeptide F (NPF) and regulates reward processes such as ejaculation and ethanol-related behaviors in a sexually dimorphic manner. Crz neurons are proposed to be a motivational switch regulating copulation duration using a CaMKII-dependent mechanism described as the first neuronal interval timer lasting longer than a few seconds. Lastly, we propose ideas to use Crz neuron-induced ejaculation to study the effects of fictive mating and sex addiction in flies, as well as to elucidate dimorphic molecular mechanisms underlying reward behaviors and feeding disorders.

Activity-Dependent Synaptic Refinement: New Insights from Drosophila.

During development, neurons establish inappropriate connections as they seek out their synaptic partners, resulting in supernumerary synapses that must be pruned away. The removal of miswired synapses usually involves electrical activity, often through a Hebbian spike-timing mechanism. A novel form of activity-dependent refinement is used by Drosophila that may be non-Hebbian, and is critical for generating the precise connectivity observed in that system. In Drosophila, motoneurons use both glutamate and the biogenic amine octopamine for neurotransmission, and the muscle fibers receive multiple synaptic inputs. Motoneuron growth cones respond in a time-regulated fashion to multiple chemotropic signals arising from their postsynaptic partners. Central to this mechanism is a very low frequency (<0.03 Hz) oscillation of presynaptic cytoplasmic calcium, that regulates and coordinates the action of multiple downstream effectors involved in the withdrawal from off-target contacts. Low frequency calcium oscillations are widely observed in developing neural circuits in mammals, and have been shown to be critical for normal connectivity in a variety of neural systems. In Drosophila these mechanisms allow the growth cone to sample widely among possible synaptic partners, evaluate opponent chemotropic signals, and withdraw from off-target contacts. It is possible that the underlying molecular mechanisms are conserved widely among invertebrates and vertebrates.

Cyclic nucleotide signaling is required during synaptic refinement at the Drosophila neuromuscular junction.

The removal of miswired synapses is a fundamental prerequisite for normal circuit development, leading to clinical problems when aberrant. However, the underlying activity-dependent molecular mechanisms involved in synaptic pruning remain incompletely resolved. Here the dynamic properties of intracellular calcium oscillations and a role for cAMP signaling during synaptic refinement in intact Drosophila embryos were examined using optogenetic tools. We provide In vivo evidence at the single gene level that the calcium-dependent adenylyl cyclase rutabaga, the phosphodiesterase dunce, the kinase PKA, and Protein Phosphatase 1 (PP1) all operate within a functional signaling pathway to modulate Sema2a-dependent chemorepulsion. It was found that presynaptic cAMP levels were required to be dynamically maintained at an optimal level to suppress connectivity defects. It was also proposed that PP1 may serve as a molecular link between cAMP signaling and CaMKII in the pathway underlying refinement. The results introduced an in vivo model where presynaptic cAMP levels, downstream of electrical activity and calcium influx, act via PKA and PP1 to modulate the neuron's response to chemorepulsion involved in the withdrawal of off-target synaptic contacts. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 39-60, 2017.

Intra-neuronal Competition for Synaptic Partners Conserves the Amount of Dendritic Building Material.

Brain development requires correct targeting of multiple thousand synaptic terminals onto staggeringly complex dendritic arbors. The mechanisms by which input synapse numbers are matched to dendrite size, and by which synaptic inputs from different transmitter systems are correctly partitioned onto a postsynaptic arbor, are incompletely understood. By combining quantitative neuroanatomy with targeted genetic manipulation of synaptic input to an identified Drosophila neuron, we show that synaptic inputs of two different transmitter classes locally direct dendrite growth in a competitive manner. During development, the relative amounts of GABAergic and cholinergic synaptic drive shift dendrites between different input domains of one postsynaptic neuron without affecting total arbor size. Therefore, synaptic input locally directs dendrite growth, but intra-neuronal dendrite redistributions limit morphological variability, a phenomenon also described for cortical neurons. Mechanistically, this requires local dendritic Ca2+ influx through Dα7nAChRs or through LVA channels following GABAAR-mediated depolarizations. VIDEO ABSTRACT.

Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

Mutations in KATNB1 cause complex cerebral malformations by disrupting asymmetrically dividing neural progenitors.

Exome sequencing analysis of over 2,000 children with complex malformations of cortical development identified five independent (four homozygous and one compound heterozygous) deleterious mutations in KATNB1, encoding the regulatory subunit of the microtubule-severing enzyme Katanin. Mitotic spindle formation is defective in patient-derived fibroblasts, a consequence of disrupted interactions of mutant KATNB1 with KATNA1, the catalytic subunit of Katanin, and other microtubule-associated proteins. Loss of KATNB1 orthologs in zebrafish (katnb1) and flies (kat80) results in microcephaly, recapitulating the human phenotype. In the developing Drosophila optic lobe, kat80 loss specifically affects the asymmetrically dividing neuroblasts, which display supernumerary centrosomes and spindle abnormalities during mitosis, leading to cell cycle progression delays and reduced cell numbers. Furthermore, kat80 depletion results in dendritic arborization defects in sensory and motor neurons, affecting neural architecture. Taken together, we provide insight into the mechanisms by which KATNB1 mutations cause human cerebral cortical malformations, demonstrating its fundamental role during brain development.

Drosophila as a model for MECP2 gain of function in neurons.

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans loss of MECP2 function causes classic Rett syndrome, but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. We show that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, our data show that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model.

Dendrite elongation and dendritic branching are affected separately by different forms of intrinsic motoneuron excitability.

Dendrites are the fundamental determinant of neuronal wiring. Consequently dendritic defects are associated with numerous neurological diseases and mental retardation. Neuronal activity can have profound effects on dendritic structure, but the mechanisms controlling distinct aspects of dendritic architecture are not fully understood. We use the Drosophila genetic model system to test the effects of altered intrinsic excitability on postembryonic dendritic architecture development. Targeted dominant negative knock-downs of potassium channel subunits allow for selectively increasing the intrinsic excitability of a selected subset of motoneurons, whereas targeted expression of a genetically modified noninactivating potassium channel decrease intrinsic excitability in vivo. Both manipulations cause significant dendritic overgrowth, but by different mechanisms. Increased excitability causes increased dendritic branch formation, whereas decreased excitability causes increased dendritic branch elongation. Therefore dendritic branching and branch elongation are controlled by separate mechanisms that can be addressed selectively in vivo by different manipulations of neuronal intrinsic excitability.