The Two Cysteines of Tau Protein Are Functionally Distinct and Contribute Differentially to Its Pathogenicity in Vivo.

Although Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease and other Tauopathies, the mechanism that initiates the aggregation of this highly soluble protein in vivo remains largely unanswered. Interestingly, in vitro Tau can be induced to form fibrillar filaments by oxidation of its two cysteine residues, generating an intermolecular disulfide bond that promotes dimerization and fibrillization. The recently solved structures of Tau filaments revealed that the two cysteine residues are not structurally equivalent since Cys-322 is incorporated into the core of the fibril, whereas Cys-291 projects away from the core to form the fuzzy coat. Here, we examined whether mutation of these cysteines to alanine affects differentially Tau mediated toxicity and dysfunction in the well-established Drosophila Tauopathy model. Experiments were conducted with both sexes, or with either sex. Each cysteine residue contributes differentially to Tau stability, phosphorylation status, aggregation propensity, resistance to stress, learning, and memory. Importantly, our work uncovers a critical role of Cys-322 in determining Tau toxicity and dysfunction.SIGNIFICANCE STATEMENT Cysteine-291 and Cysteine-322, the only two cysteine residues of Tau present in only 4-Repeat or all isoforms, respectively, have competing functions: as the key residues in the catalytic center, they enable Tau auto-acetylation; and as residues within the microtubule-binding repeat region are important not only for Tau function but also instrumental in the initiation of Tau aggregation. In this study, we present the first in vivo evidence that their substitution leads to differential consequences on Tau's physiological and pathophysiological functions. These differences raise the possibility that cysteine residues play a potential role in determining the functional diversity between isoforms.

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.

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.

Differential effects of 14-3-3 dimers on Tau phosphorylation, stability and toxicity in vivo.

Neurodegenerative dementias collectively known as Tauopathies involve aberrant phosphorylation and aggregation of the neuronal protein Tau. The largely neuronal 14-3-3 proteins are also elevated in the central nervous system (CNS) and cerebrospinal fluid of Tauopathy patients, suggesting functional linkage. We use the simplicity and genetic facility of the Drosophila system to investigate in vivo whether 14-3-3s are causal or synergistic with Tau accumulation in precipitating pathogenesis. Proteomic, biochemical and genetic evidence demonstrate that both Drosophila 14-3-3 proteins interact with human wild-type and mutant Tau on multiple sites irrespective of their phosphorylation state. 14-3-3 dimers regulate steady-state phosphorylation of both wild-type and the R406W mutant Tau, but they are not essential for toxicity of either variant. Moreover, 14-3-3 elevation itself is not pathogenic, but recruitment of dimers on accumulating wild-type Tau increases its steady-state levels ostensibly by occluding access to proteases in a phosphorylation-dependent manner. In contrast, the R406W mutant, which lacks a putative 14-3-3 binding site, responds differentially to elevation of each 14-3-3 isoform. Although excess 14-3-3ζ stabilizes the mutant protein, elevated D14-3-3ɛ has a destabilizing effect probably because of altered 14-3-3 dimer composition. Our collective data demonstrate the complexity of 14-3-3/Tau interactions in vivo and suggest that 14-3-3 attenuation is not appropriate ameliorative treatment of Tauopathies. Finally, we suggest that 'bystander' 14-3-3s are recruited by accumulating Tau with the consequences depending on the composition of available dimers within particular neurons and the Tau variant.

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.

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.

Temporally distinct phosphorylations differentiate Tau-dependent learning deficits and premature mortality in Drosophila.

Abnormally phosphorylated Tau protein, the major component of neurofibrillary tangles, is critical in the pathogenesis of Alzheimer's disease and related Tauopathies. We used Drosophila to examine the role of key disease-associated phosphorylation sites on Tau-mediated neurotoxicity. We present evidence that the late-appearing phosphorylation on Ser(238) rather than hyperphosphorylation per se is essential for Tau toxicity underlying premature mortality in adult flies. This site is also occupied at the time of neurodegeneration onset in a mouse Tauopathy model and in damaged brain areas of confirmed Tauopathy patients, suggesting a similar critical role on Tau toxicity in humans. In contrast, occupation of Ser(262) is necessary for Tau-dependent learning deficits in adult Drosophila. Significantly, occupation of Ser(262) precedes and is required for Ser(238) phosphorylation, and these temporally distinct phosphorylations likely reflect conformational changes. Because sequential occupation of Ser(262) and Ser(238) is required for the progression from Tau-mediated learning deficits to premature mortality in Drosophila, they may also play similar roles in the escalating symptom severity in Tauopathy patients, congruent with their presence in damaged regions of their brains.

Protein aggregation and therapeutic strategies in SOD1- and TDP-43- linked ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with severe socio-economic impact. A hallmark of ALS pathology is the presence of aberrant cytoplasmic inclusions composed of misfolded and aggregated proteins, including both wild-type and mutant forms. This review highlights the critical role of misfolded protein species in ALS pathogenesis, particularly focusing on Cu/Zn superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP-43), and emphasizes the urgent need for innovative therapeutic strategies targeting these misfolded proteins directly. Despite significant advancements in understanding ALS mechanisms, the disease remains incurable, with current treatments offering limited clinical benefits. Through a comprehensive analysis, the review focuses on the direct modulation of the misfolded proteins and presents recent discoveries in small molecules and peptides that inhibit SOD1 and TDP-43 aggregation, underscoring their potential as effective treatments to modify disease progression and improve clinical outcomes.

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.

Understanding the nervous system: lessons from Frontiers in Neurophotonics.

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.

IQGAP1 mediates the communication between the nucleus and the mitochondria via NDUFS4 alternative splicing.

Constant communication between mitochondria and nucleus ensures cellular homeostasis and adaptation to mitochondrial stress. Anterograde regulatory pathways involving a large number of nuclear-encoded proteins control mitochondrial biogenesis and functions. Such functions are deregulated in cancer cells, resulting in proliferative advantages, aggressive disease and therapeutic resistance. Transcriptional networks controlling the nuclear-encoded mitochondrial genes are known, however alternative splicing (AS) regulation has not been implicated in this communication. Here, we show that IQGAP1, a scaffold protein regulating AS of distinct gene subsets in gastric cancer cells, participates in AS regulation that strongly affects mitochondrial respiration. Combined proteomic and RNA-seq analyses of IQGAP1KO and parental cells show that IQGAP1KO alters an AS event of the mitochondrial respiratory chain complex I (CI) subunit NDUFS4 and downregulates a subset of CI subunits. In IQGAP1KO cells, CI intermediates accumulate, resembling assembly deficiencies observed in patients with Leigh syndrome bearing NDUFS4 mutations. Mitochondrial CI activity is significantly lower in KO compared to parental cells, while exogenous expression of IQGAP1 reverses mitochondrial defects of IQGAP1KO cells. Our work sheds light to a novel facet of IQGAP1 in mitochondrial quality control that involves fine-tuning of CI activity through AS regulation in gastric cancer cells relying highly on mitochondrial respiration.

NORMA: The Network Makeup Artist - A Web Tool for Network Annotation Visualization.

The Network Makeup Artist (NORMA) is a web tool for interactive network annotation visualization and topological analysis, able to handle multiple networks and annotations simultaneously. Precalculated annotations (e.g., Gene Ontology, Pathway enrichment, community detection, or clustering results) can be uploaded and visualized in a network, either as colored pie-chart nodes or as color-filled areas in a 2D/3D Venn-diagram-like style. In the case where no annotation exists, algorithms for automated community detection are offered. Users can adjust the network views using standard layout algorithms or allow NORMA to slightly modify them for visually better group separation. Once a network view is set, users can interactively select and highlight any group of interest in order to generate publication-ready figures. Briefly, with NORMA, users can encode three types of information simultaneously. These are 1) the network, 2) the communities or annotations of interest, and 3) node categories or expression values. Finally, NORMA offers basic topological analysis and direct topological comparison across any of the selected networks. NORMA service is available at http://norma.pavlopouloslab.info, whereas the code is available at https://github.com/PavlopoulosLab/NORMA.

Adenovirus Fibers as Ultra-Stable Vehicles for Intracellular Nanoparticle and Protein Delivery.

Protein-based carriers are promising vehicles for the intracellular delivery of therapeutics. In this study, we designed and studied adenovirus protein fiber constructs with potential applications as carriers for the delivery of protein and nanoparticle cargoes. We used as a basic structural framework the fibrous shaft segment of the adenovirus fiber protein comprising of residues 61-392, connected to the fibritin foldon trimerization motif at the C-terminal end. A fourteen-amino-acid biotinylation sequence was inserted immediately after the N-terminal, His-tagged end of the construct in order to enable the attachment of a biotin moiety in vivo. We report herein that this His-tag biotinylated construct folds into thermally and protease-stable fibrous nanorods that can be internalized into cells and are not cytotoxic. Moreover, they can bind to proteins and nanoparticles through the biotin-streptavidin interaction and mediate their delivery to cells. We demonstrate that streptavidin-conjugated gold nanoparticles can be transported into NIH3T3 fibroblast and HeLa cancer cell lines. Furthermore, two streptavidin-conjugated model proteins, alkaline phosphatase and horseradish peroxidase can be delivered into the cell cytoplasm in their enzymatically active form. This work is aimed at establishing the proof-of-principle for the rational engineering of diverse functionalities onto the initial protein structural framework and the use of adenovirus fiber-based proteins as nanorods for the delivery of nanoparticles and model proteins. These constructs could constitute a stepping stone for the development of multifunctional and modular fibrous nanorod platforms that can be tailored to applications at the sequence level.

The network makeup artist (NORMA-2.0): distinguishing annotated groups in a network using innovative layout strategies.

Motivation: Network biology is a dominant player in today's multi-omics era. Therefore, the need for visualization tools which can efficiently cope with intra-network heterogeneity emerges. Results: NORMA-2.0 is a web application which uses efficient layouts to group together areas of interest in a network. In this version, NORMA-2.0 utilizes three different strategies to make such groupings as distinct as possible while it preserves all of the properties from its first version where one can handle multiple networks and annotation files simultaneously. Availability and implementation: The web resource is available at http://norma.pavlopouloslab.info/. The source code is freely available at https://github.com/PavlopoulosLab/NORMA.

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.

Differential effects of Tau on the integrity and function of neurons essential for learning in Drosophila.

Tauopathies are a heterogeneous group of neurodegenerative dementias involving perturbations in the levels, phosphorylation, or mutations of the microtubule-binding protein Tau. The heterogeneous pathology in humans and model organisms suggests differential susceptibility of neuronal types to wild-type (WT) and mutant Tau. WT and mutant human Tau-encoding transgenes expressed pan-neuronally in the Drosophila CNS yielded specific and differential toxicity in the embryonic neuroblasts that generate the mushroom body (MB) neurons, suggesting cell type-specific effects of Tau in the CNS. Frontotemporal dementia with parkinsonism-17-linked mutant isoforms were significantly less toxic in MB development. Tau hyperphosphorylation was essential for these MB aberrations, and we identified two novel putative phosphorylation sites, Ser(238) and Thr(245), on WT hTau essential for its toxic effects on MB integrity. Significantly, blocking putative Ser(238) and Thr(245) phosphorylation yielded animals with apparently structurally normal but profoundly dysfunctional MBs, because animals accumulating this mutant protein exhibited strongly impaired associative learning. Interestingly, the mutant protein was hyperphosphorylated at epitopes typically associated with toxicity and neurodegeneration, such as AT8, AT100, and the Par-1 targets Ser(262) and Ser(356), suggesting that these sites in the context of adult intact MBs mediate dysfunction and occupation of these sites may precede the toxicity-associated Ser(238) and Thr(245) phosphorylation. The data support the notion that phosphorylation at particular sites rather than hyperphosphorylation per se mediates toxicity or dysfunction in a cell type-specific manner.

Phosphorylation differentiates tau-dependent neuronal toxicity and dysfunction.

The heterogeneous pathology of tauopathies and the differential susceptibility of different neuronal types to WT (wild-type) and mutant tau suggest that phosphorylation at particular sites rather than hyperphosphorylation mediates toxicity or dysfunction in a cell-type-specific manner. Pan-neuronal accumulation of tau in the Drosophila CNS (central nervous system) specifically affected the MBs (mushroom body neurons), consistent with neuronal type-specific effects. The MB aberrations depended, at least in part, on occupation of two novel phosphorylation sites: Ser(238) and Thr(245). The degree of isoform-specific MB aberrations was paralleled by defects in associative learning, as blocking putative Ser(238) and Thr(245) phosphorylation yielded structurally normal, but profoundly dysfunctional, MBs, as animals accumulating the mutant protein exhibited strongly impaired associative learning. Similarly dysfunctional MBs were obtained by temporally restricting tau accumulation to the adult CNS, which also altered the tau phosphorylation pattern. Our data clearly distinguish tau-dependent neuronal degeneration and dysfunction and suggest that temporal differences in occupation of the same phosphorylation sites are likely to mediate these distinct effects of tau.

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.

An assessment of the translational relevance of Drosophila in drug discovery.

INTRODUCTION: Drosophila melanogaster offers a powerful expedient and economical system with facile genetics. Because of the high sequence and functional conservation with human disease-associated genes, it has been cardinal in deciphering disease mechanisms at the genetic and molecular level. Drosophila are amenable to and respond well to pharmaceutical treatment which coupled to their genetic tractability has led to discovery, repositioning, and validation of a number of compounds. Areas covered: This review summarizes the generation of fly models of human diseases, their advantages and use in elucidation of human disease mechanisms. Representative studies provide examples of the utility of this system in modeling diseases and the discovery, repositioning and testing on pharmaceuticals to ameliorate them. Expert opinion: Drosophila offers a facile and economical whole animal system with many homologous organs to humans, high functional conservation and established methods of generating and validating human disease models. Nevertheless, it remains relatively underused as a drug discovery tool probably because its relevance to mammalian systems remains under question. However, recent exciting success stories using Drosophila disease models for drug screening, repositioning and validation strongly suggest that fly models should figure prominently in the drug discovery pipeline from bench to bedside.