Tau exists as six closely related protein isoforms in the adult human brain. These are generated from alternative splicing of a single mRNA transcript and they differ in the absence or presence of two N-terminal and three or four microtubule binding domains. Typically all six isoforms have been considered functionally similar. However, their differential involvement in particular tauopathies raises the possibility that there may be isoform-specific differences in physiological function and pathological role. To explore this, we have compared the phenotypes induced by the 0N3R and 0N4R isoforms in Drosophila. Expression of the 3R isoform causes more profound axonal transport defects and locomotor impairments, culminating in a shorter lifespan than the 4R isoform. In contrast, the 4R isoform leads to greater neurodegeneration and impairments in learning and memory. Furthermore, the phosphorylation patterns of the two isoforms are distinct, as is their ability to induce oxidative stress. These differences are not consequent to different expression levels and are suggestive of bona fide physiological differences in isoform biology and pathological potential. They may therefore explain isoform-specific mechanisms of tau-toxicity and the differential susceptibility of brain regions to different tauopathies.

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.

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, Ser 238 , Thr 245 , and Ser 262 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 Ser 262 is necessary for Tau-dependent learning deficits and a "facilitatory gatekeeper" to Ser 238 occupation, which is linked to Tau toxicity. Importantly we reveal that phosphorylation at Thr 245 acts as a "suppressive gatekeeper", preventing phosphorylation of many sites including Ser 262 and consequently of Ser 238. Therefore, we elucidate novel interactions among phosphosites central to Tau mediated neuronal dysfunction and toxicity, likely driven by phosphorylation-dependent conformational plasticity.

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.

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.

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 consequent of aggregate accumulation which 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 upon suppression of new transgenic human Tau expression, but 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 hTau0N4Rexpression. 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 hTau0N4Raggregate suppression within adult mushroom body neurons, also resulted in emergence of memory deficits. Therefore, deficient PSD-M upon human Tau expression in the Drosophila CNS is not consequent 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.

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