Choline Metabolites Reverse Differentially the Habituation Deficit and Elevated Memory of Tau Null Drosophila.

Impaired neuronal plasticity and cognitive decline are cardinal features of Alzheimer's disease and related Tauopathies. Aberrantly modified Tau protein and neurotransmitter imbalance, predominantly involving acetylcholine, have been linked to these symptoms. In Drosophila, we have shown that dTau loss specifically enhances associative long-term olfactory memory, impairs foot shock habituation, and deregulates proteins involved in the regulation of neurotransmitter levels, particularly acetylcholine. Interestingly, upon choline treatment, the habituation and memory performance of mutants are restored to that of control flies. Based on these surprising results, we decided to use our well-established genetic model to understand how habituation deficits and memory performance correlate with different aspects of choline physiology as an essential component of the neurotransmitter acetylcholine, the lipid phosphatidylcholine, and the osmoregulator betaine. The results revealed that the two observed phenotypes are reversed by different choline metabolites, implying that they are governed by different underlying mechanisms. This work can contribute to a broader knowledge about the physiologic function of Tau, which may be translated into understanding the mechanisms of Tauopathies.

Drosophila Contributions towards Understanding Neurofibromatosis 1.

Neurofibromatosis 1 (NF1) is a multisymptomatic disorder with highly variable presentations, which include short stature, susceptibility to formation of the characteristic benign tumors known as neurofibromas, intense freckling and skin discoloration, and cognitive deficits, which characterize most children with the condition. Attention deficits and Autism Spectrum manifestations augment the compromised learning presented by most patients, leading to behavioral problems and school failure, while fragmented sleep contributes to chronic fatigue and poor quality of life. Neurofibromin (Nf1) is present ubiquitously during human development and postnatally in most neuronal, oligodendrocyte, and Schwann cells. Evidence largely from animal models including Drosophila suggests that the symptomatic variability may reflect distinct cell-type-specific functions of the protein, which emerge upon its loss, or mutations affecting the different functional domains of the protein. This review summarizes the contributions of Drosophila in modeling multiple NF1 manifestations, addressing hypotheses regarding the cell-type-specific functions of the protein and exploring the molecular pathways affected upon loss of the highly conserved fly homolog dNf1. Collectively, work in this model not only has efficiently and expediently modelled multiple aspects of the condition and increased understanding of its behavioral manifestations, but also has led to pharmaceutical strategies towards their amelioration.

Human Tau Aggregates Are Permissive to Protein Synthesis-Dependent Memory in Drosophila Tauopathy Models.

Tauopathies including Alzheimer's disease, are characterized by progressive cognitive decline, neurodegeneration, and intraneuronal aggregates comprised largely of the axonal protein Tau. It has been unclear whether cognitive deficits are a consequence of aggregate accumulation thought to compromise neuronal health and eventually lead to neurodegeneration. We use the Drosophila tauopathy model and mixed-sex populations to reveal an adult onset pan-neuronal Tau accumulation-dependent decline in learning efficacy and a specific defect in protein synthesis-dependent memory (PSD-M), but not in its protein synthesis-independent variant. We demonstrate that these neuroplasticity defects are reversible on suppression of new transgenic human Tau expression and surprisingly correlate with an increase in Tau aggregates. Inhibition of aggregate formation via acute oral administration of methylene blue results in re-emergence of deficient memory in animals with suppressed human Tau (hTau)0N4R expression. Significantly, aggregate inhibition results in PSD-M deficits in hTau0N3R-expressing animals, which present elevated aggregates and normal memory if untreated with methylene blue. Moreover, methylene blue-dependent hTau0N4R aggregate suppression within adult mushroom body neurons also resulted in emergence of memory deficits. Therefore, deficient PSD-M on human Tau expression in the Drosophila CNS is not a consequence of toxicity and neuronal loss because it is reversible. Furthermore, PSD-M deficits do not result from aggregate accumulation, which appears permissive, if not protective of processes underlying this memory variant.SIGNIFICANCE STATEMENT Intraneuronal Tau aggregate accumulation has been proposed to underlie the cognitive decline and eventual neurotoxicity that characterizes the neurodegenerative dementias known as tauopathies. However, we show in three experimental settings that Tau aggregates in the Drosophila CNS do not impair but rather appear to facilitate processes underlying protein synthesis-dependent memory within affected neurons.

Differential Effects of Human Tau Isoforms to Neuronal Dysfunction and Toxicity in the Drosophila CNS.

Accumulation of highly post-translationally modified tau proteins is a hallmark of neurodegenerative disorders known as tauopathies, the most common of which is Alzheimer's disease. Although six tau isoforms are found in the human brain, the majority of animal and cellular tauopathy models utilize a representative single isoform. However, the six human tau isoforms present overlapping but distinct distributions in the brain and are differentially involved in particular tauopathies. These observations support the notion that tau isoforms possess distinct functional properties important for both physiology and pathology. To address this hypothesis, the six human brain tau isoforms were expressed singly in the Drosophila brain and their effects in an established battery of assays measuring neuronal dysfunction, vulnerability to oxidative stress and life span were systematically assessed comparatively. The results reveal isoform-specific effects clearly not attributed to differences in expression levels but correlated with the number of microtubule-binding repeats and the accumulation of a particular isoform in support of the functional differentiation of these tau isoforms. Delineation of isoform-specific effects is essential to understand the apparent differential involvement of each tau isoform in tauopathies and their contribution to neuronal dysfunction and toxicity.

Anesthesia Resistant Memories in Drosophila, a Working Perspective.

Memories are lasting representations over time of associations between stimuli or events. In general, the relatively slow consolidation of memories requires protein synthesis with a known exception being the so-called Anesthesia Resistant Memory (ARM) in Drosophila. This protein synthesis-independent memory type survives amnestic shocks after a short, sensitive window post training, and can also emerge after repeated cycles of training in a negatively reinforced olfactory conditioning task, without rest between cycles (massed conditioning-MC). We discussed operational and molecular mechanisms that mediate ARM and differentiate it from protein synthesis-dependent long-term memory (LTM) in Drosophila. Based on the notion that ARM is unlikely to specifically characterize Drosophila, we examined protein synthesis and MC-elicited memories in other species and based on intraspecies shared molecular components and proposed potential relationships of ARM with established memory types in Drosophila and vertebrates.

Loss of the Schizophrenia-linked Furin protein from Drosophila mushroom body neurons results in antipsychotic-reversible habituation deficits.

Habituation is a conserved adaptive process essential for incoming information assessment, which drives behavioral response decrement to recurrent inconsequential stimuli and does not involve sensory adaptation, or fatigue. Although the molecular mechanisms underlying the process are not well understood, habituation has been reported defective in a number of disorders including schizophrenia. We demonstrate that loss of furin1, the Drosophila homolog of a gene whose transcriptional downregulation has been linked to schizophrenia, results in defective habituation to recurrent footshocks in mixed sex populations. The deficit is reversible by transgenic expression of the Drosophila or human Furin in adult α,/ß, mushroom body neurons and by acute oral delivery of the typical antipsychotic Haloperidol and the atypical Clozapine, which are commonly used to treat schizophrenic patients. The results validate the proposed contribution of Furin downregulation in schizophrenia and suggest that defective footshock habituation is a Drosophila protophenotype of the human disorder.SIGNIFICANCE STATEMENTGenome Wide Association Studies have revealed a number of loci linked to Schizophrenia, but most have not been verified experimentally in a relevant behavioral task. Habituation deficits constitute a schizophrenia endophenotype. Drosophila with attenuated expression of the Schizophrenia-linked highly conserved Furin gene present delayed habituation reversible with acute exposure to antipsychotics This strongly suggests that footshock habituation defects constitute a Schizophrenia protophenotype in Drosophila. Furthermore, determination of the neurons whose regulated activity is required for footshock habituation provides a facile metazoan system to expediently validate putative Schizophrenia genes and variants in a well-understood simple brain.

Cold Shock Disrupts Massed Training-Elicited Memory in Drosophila.

Memory consolidation is a time-dependent process occurring over hours, days, or longer in different species and requires protein synthesis. An apparent exception is a memory type in Drosophila elicited by a single olfactory conditioning episode, which ostensibly consolidates quickly, rendering it resistant to disruption by cold anesthesia a few hours post-training. This anesthesia-resistant memory (ARM), is independent of protein synthesis. Protein synthesis independent memory can also be elicited in Drosophila by multiple massed cycles of olfactory conditioning, and this led to the prevailing notion that both of these operationally distinct training regimes yield ARM. Significantly, we show that, unlike bona fide ARM, massed conditioning-elicited memory remains sensitive to the amnestic treatment two hours post-training and hence it is not ARM. Therefore, there are two protein synthesis-independent memory types in Drosophila.

Bee foraging preferences, microbiota and pathogens revealed by direct shotgun metagenomics of honey.

Honeybees (Apis mellifera) continue to succumb to human and environmental pressures despite their crucial role in providing essential ecosystem services. Owing to their foraging and honey production activities, honeybees form complex relationships with species across all domains, such as plants, viruses, bacteria and other hive pests, making honey a valuable biomonitoring tool for assessing their ecological niche. Thus, the application of honey shotgun metagenomics (SM) has paved the way for a detailed description of the species honeybees interact with. Nevertheless, SM bioinformatics tools and DNA extraction methods rely on resources not necessarily optimized for honey. In this study, we compared five widely used taxonomic classifiers using simulated species communities commonly found in honey. We found that Kraken 2 with a threshold of 0.5 performs best in assessing species distribution. We also optimized a simple NaOH-based honey DNA extraction methodology (Direct-SM), which profiled species seasonal variability similarly to an established column-based DNA extraction approach (SM). Both approaches produce results consistent with melissopalinology analysis describing the botanical landscape surrounding the apiary. Interestingly, we detected a strong stability of the bacteria constituting the core and noncore gut microbiome across seasons, pointing to the potential utility of honey for noninvasive assessment of bee microbiota. Finally, the Direct-SM approach to detect Varroa correlates well with the biomonitoring of mite infestation observed in hives. These observations suggest that Direct-SM methodology has the potential to comprehensively describe honeybee ecological niches and can be tested as a building block for large-scale studies to assess bee health in the field.

Mical modulates Tau toxicity via cysteine oxidation in vivo.

Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease and other Tauopathies. However, processes leading to Tau fibrillization and reasons for its pathogenicity remain largely elusive. Mical emerged as a novel interacting protein of human Tau expressed in Drosophila brains. Mical is characterized by the presence of a flavoprotein monooxygenase domain that generates redox potential with which it can oxidize target proteins. In the well-established Drosophila Tauopathy model, we use genetic interactions to show that Mical alters Tau interactions with microtubules and the Actin cytoskeleton and greatly affects Tau aggregation propensity and Tau-associated toxicity and dysfunction. Exploration of the mechanism was pursued using a Mical inhibitor, a mutation in Mical that selectively disrupts its monooxygenase domain, Tau transgenes mutated at cysteine residues targeted by Mical and mass spectrometry analysis to quantify cysteine oxidation. The collective evidence strongly indicates that Mical's redox activity mediates the effects on Tau via oxidation of Cys322. Importantly, we also validate results from the fly model in human Tauopathy samples by showing that MICAL1 is up-regulated in patient brains and co-localizes with Tau in Pick bodies. Our work provides mechanistic insights into the role of the Tau cysteine residues as redox-switches regulating the process of Tau self-assembly into inclusions in vivo, its function as a cytoskeletal protein and its effect on neuronal toxicity and dysfunction.

Molecular Mechanisms Employed by Neurons to Receive and Transduce Signals Essential for Learning and Memory.

The ability to learn from the consequences of one's actions, extracting useful information from threatening, painful or rewarding encounters and developing the skill of predicting probable events from pre-experienced stimuli, is essential for survival and reproductive success [...].

Associative Learning Requires Neurofibromin to Modulate GABAergic Inputs to Drosophila Mushroom Bodies.

Cognitive dysfunction is among the hallmark symptoms of Neurofibromatosis 1, and accordingly, loss of the Drosophila melanogaster ortholog of Neurofibromin 1 (dNf1) precipitates associative learning deficits. However, the affected circuitry in the adult CNS remained unclear and the compromised mechanisms debatable. Although the main evolutionarily conserved function attributed to Nf1 is to inactivate Ras, decreased cAMP signaling on its loss has been thought to underlie impaired learning. Using mixed sex populations, we determine that dNf1 loss results in excess GABAergic signaling to the central for associative learning mushroom body (MB) neurons, apparently suppressing learning. dNf1 is necessary and sufficient for learning within these non-MB neurons, as a dAlk and Ras1-dependent, but PKA-independent modulator of GABAergic neurotransmission. Surprisingly, we also uncovered and discuss a postsynaptic Ras1-dependent, but dNf1-independnet signaling within the MBs that apparently responds to presynaptic GABA levels and contributes to the learning deficit of the mutants.

Functional Interactions of Tau Phosphorylation Sites That Mediate Toxicity and Deficient Learning in Drosophila melanogaster.

Hyperphosphorylated Tau protein is the main component of the neurofibrillary tangles, characterizing degenerating neurons in Alzheimer's disease and other Tauopathies. Expression of human Tau protein in Drosophila CNS results in increased toxicity, premature mortality and learning and memory deficits. Herein we use novel transgenic lines to investigate the contribution of specific phosphorylation sites previously implicated in Tau toxicity. These three different sites, Ser238, Thr245, and Ser262 were tested either by blocking their phosphorylation, by Ser/Thr to Ala substitution, or pseudophosphorylation, by changing Ser/Thr to Glu. We validate the hypothesis that phosphorylation at Ser262 is necessary for Tau-dependent learning deficits and a "facilitatory gatekeeper" to Ser238 occupation, which is linked to Tau toxicity. Importantly we reveal that phosphorylation at Thr245 acts as a "suppressive gatekeeper", preventing phosphorylation of many sites including Ser262 and consequently of Ser238. Therefore, we elucidate novel interactions among phosphosites central to Tau mediated neuronal dysfunction and toxicity, likely driven by phosphorylation-dependent conformational plasticity.

Altered Proteostasis in Neurodegenerative Tauopathies.

Tauopathies are a heterogeneous group of neurodegenerative dementias involving perturbations in the levels, phosphorylation or mutations of the neuronal microtubule-binding protein Tau. Tauopathies are characterized by accumulation of hyperphosphorylated Tau leading to formation of a range of aggregates including macromolecular ensembles such as Paired Helical filaments and Neurofibrilary Tangles whose morphology characterizes and differentiates these disease states. Why nonphysiological Tau proteins elude the surveillance normal proteostatic mechanisms and eventually form these macromolecular assemblies is a central mostly unresolved question of cardinal importance for diagnoses and potential therapeutic interventions. We discuss the response of the Ubiquitin-Proteasome system, autophagy and the Endoplasmic Reticulum-Unfolded Protein response in Tauopathy models and patients, revealing interactions of components of these systems with Tau, but also of the effects of pathological Tau on these systems which eventually lead to Tau aggregation and accumulation. These interactions point to potential disease biomarkers and future potential therapeutic targets.

Drosophila Bruton's Tyrosine Kinase Regulates Habituation Latency and Facilitation in Distinct Mushroom Body Neurons.

Habituation is the adaptive behavioral outcome of processes engaged in timely devaluation of non-reinforced repetitive stimuli, but the neuronal circuits and molecular mechanisms that underlie them are not well understood. To gain insights into these processes we developed and characterized a habituation assay to repetitive footshocks in mixed sex Drosophila groups and demonstrated that acute neurotransmission from adult α/ß mushroom body (MB) neurons prevents premature stimulus devaluation. Herein we demonstrate that activity of the non-receptor tyrosine kinase dBtk protein is required within these neurons to prevent premature habituation. Significantly, we also demonstrate that the complementary process of timely habituation to the repetitive stimulation is facilitated by α'/ß' MB neurons and also requires dBtk activity. Hence our results provide initial insights into molecular mechanisms engaged in footshock habituation within distinct MB neurons. Importantly, dBtk attenuation specifically within α'/ß' neurons leads to defective habituation, which is readily reversible by administration of the antipsychotics clozapine and risperidone suggesting that the loss of the kinase may dysregulate monoamine receptors within these neurons, whose activity underlies the failure to habituate.SIGNIFICANCE STATEMENT Habituation refers to processes underlying decisions to attend or ignore stimuli, which are pivotal to brain function as they underlie selective attention and learning, but the circuits involved and the molecular mechanisms engaged by the process therein are poorly understood. We demonstrate that habituation to repetitive footshock involves two phases mediated by distinct neurons of the Drosophila mushroom bodies and require the function of the dBtk non-receptor tyrosine kinase. Moreover, habituation failure upon dBtk abrogation in neurons where it is required to facilitate the process is readily reversible by antipsychotics, providing conceptual links to particular symptoms of schizophrenia in humans, also characterized by habituation defects and ameliorated by these pharmaceuticals.

Drosophila Tau Negatively Regulates Translation and Olfactory Long-Term Memory, But Facilitates Footshock Habituation and Cytoskeletal Homeostasis.

Although the involvement of pathological tau in neurodegenerative dementias is indisputable, its physiological roles have remained elusive in part because its abrogation has been reported without overt phenotypes in mice and Drosophila This was addressed using the recently described Drosophila tauKO and Mi{MIC} mutants and focused on molecular and behavioral analyses. Initially, we show that Drosophila tau (dTau) loss precipitates dynamic cytoskeletal changes in the adult Drosophila CNS and translation upregulation. Significantly, we demonstrate for the first time distinct roles for dTau in adult mushroom body (MB)-dependent neuroplasticity as its downregulation within α'ß'neurons impairs habituation. In accord with its negative regulation of translation, dTau loss specifically enhances protein synthesis-dependent long-term memory (PSD-LTM), but not anesthesia-resistant memory. In contrast, elevation of the protein in the MBs yielded premature habituation and depressed PSD-LTM. Therefore, tau loss in Drosophila dynamically alters brain cytoskeletal dynamics and profoundly affects neuronal proteostasis and plasticity.SIGNIFICANCE STATEMENT We demonstrate that despite modest sequence divergence, the Drosophila tau (dTau) is a true vertebrate tau ortholog as it interacts with the neuronal microtubule and actin cytoskeleton. Novel physiological roles for dTau in regulation of translation, long-term memory, and footshock habituation are also revealed. These emerging insights on tau physiological functions are invaluable for understanding the molecular pathways and processes perturbed in tauopathies.

Human Tau isoform-specific presynaptic deficits in a Drosophila Central Nervous System circuit.

Accumulation of normal or mutant human Tau isoforms in Central Nervous System (CNS) neurons of vertebrate and invertebrate models underlies pathologies ranging from behavioral deficits to neurodegeneration that broadly recapitulate human Tauopathies. Although some functional differences have begun to emerge, it is still largely unclear whether normal and mutant Tau isoforms induce differential effects on the synaptic physiology of CNS neurons. We use the oligosynaptic Giant Fiber System in the adult Drosophila CNS to address this question and reveal that 3R and 4R isoforms affect distinct synaptic parameters. Whereas 0N3R increased failure rate upon high frequency stimulation, 0N4R compromised stimulus conduction and response speed at a specific cholinergic synapse in an age-dependent manner. In contrast, accumulation of the R406W mutant of 0N4R induced mild, age-dependent conduction velocity defects. Because 0N4R and its mutant isoform are expressed equivalently, this demonstrates that the defects are not merely consequent of exogenous human Tau accumulation and suggests distinct functional properties of 3R and 4R isoforms in cholinergic presynapses.

Accessing Olfactory Habituation in Drosophila melanogaster with a T-maze Paradigm.

Habituation is the process whereby perceptual changes alter the value of environmental stimuli, enabling salience filtering. This behavioral response decrement is a form of non-associative learning, where the subject learns about the stimulus and does not involve sensory adaptation, sensory or motor fatigue. The range of behavioral responses in D. melanogaster led to the development of a number of habituation paradigms addressing various sensory modalities. Habituation of osmotactic responses has previously been measured with the Y-maze test and required 30 min of odor exposure. Here, we describe an olfactory habituation assay utilizing the widely used in associative learning paradigms T-maze. Continuous or repetitive odor exposure for 4 min is adequate to attenuate osmotactic responses both to attractive and aversive odors. Importantly, the decreased response conforms to habitation parameters, presenting dishabituation and spontaneous recovery. This assay allows the study of habituation after brief odor exposure, but also discriminates between the two distinct phases of the response, an initial habituation latency period followed by habituation. In addition, the characterization of the neuronal circuits implicated in each phase facilitates further study of the molecular components underlying this process.