InteractiveFly: GeneBrief

tau: Biological Overview | Regulation | Developmental Biology | Drosophila as a Model for Human Tauopathies | References

BIOLOGICAL OVERVIEW

The mechanisms regulating synapse numbers during development and aging are essential for normal brain function and closely linked to brain disorders including dementias. Using Drosophila, this study demonstrates roles of the microtubule-associated protein Tau in regulating synapse numbers, thus unravelling an important cellular requirement of normal Tau. In this context, it was found that Tau displays a strong functional overlap with microtubule-binding spectraplakins, establishing new links between two different neurodegenerative factors. Tau and the spectraplakin Short Stop act upstream of a three-step regulatory cascade ensuring adequate delivery of synaptic proteins. This cascade involves microtubule stability as the initial trigger, JNK signalling as the central mediator, and kinesin-3 (see Drosophila Unc-103) mediated axonal transport as the key effector. This cascade acts during development (synapse formation) and aging (synapse maintenance) alike. Therefore, these findings suggest novel explanations for intellectual disability in Tau deficient individuals, as well as early synapse loss in dementias including Alzheimer's disease (Voelzmann, 2016).

The correct formation and subsequent maintenance of synapses is a key prerequisite for brain development, function and longevity. Precocious loss of synapses is observed in late onset neurodegenerative diseases including Alzheimer's disease (AD) and Frontotemporal Dementia (FTD), likely contributing to the cognitive decline and neuronal decay observed in patients. Therefore, the characterisation of mechanisms maintaining synapses during ageing would have major implications for understanding of dementias (Voelzmann, 2016).

The development of synapses and their maintenance during ageing is dependent on sustained transport of synaptic proteins from the distant soma, driven by motor proteins which trail along the bundles of microtubules in axons and dendrites. Microtubules are regulated by microtubule binding proteins which are therefore in a key position to regulate synapse formation and maintenance (Voelzmann, 2016).

Tau is a microtubule associated protein (MAP) discovered in the mid-seventies. Reduction in Tau levels has been linked to intellectual disability (Sapir, 2012) and a class of brain disorders termed 'dementias which lack distinctive histopathology' (DLDH) (Zhukareva, 2001). Tau detachment from MTs is linked to prominent neurodegenerative diseases such as Alzheimer's disease, Frontotemporal Dementia and some forms of Parkinson's disease (Kovacs, 2015). In vitro, Tau has the ability to regulate microtubule properties including stability, cross-linkage and polymerisation (Morris, 2013). Through such functions, Tau would be expected to regulate multiple aspects of neuronal cell biology, but its physiological roles are still not understood and highly debated (Morris, 2013). This might partly be due to experimental challenges posed by functional redundancy, where other MAPs are proposed to mask physiological roles of Tau (Voelzmann, 2016).

A good model in which to deal with functional redundancy is the fruit fly Drosophila melanogaster. As is ideal for studies of Tau, Drosophila neurons provide access to powerful genetics, they are readily established for research on the neuronal cytoskeleton, on neuronal transport and on synapses. Importantly, concepts and mechanisms gained from work in flies are often well conserved in higher organisms (Voelzmann, 2016).

Work in Drosophila suggested that the spectraplakin Short Stop (Shot), a large actin-MT linker molecules and potent regulators of microtubules, could display potential functional overlap with Tau during microtubule stabilisation (Alves-Silva, 2012; Prokop, 2013). This hypothesis is attractive because the well-conserved mammalian spectraplakin Dystonin is already linked to a neurodegenerative disease (type VI hereditary sensory autonomic neuropathy; OMIM #614653), and its paralogue ACF7/MACF1 plays important roles during brain development). Since ACF7 continues to be expressed in the brain, it is tempting to speculate that it might be required for neuronal maintenance (Voelzmann, 2016).

This study used Drosophila neurons, in culture and in vivo alike, to demonstrate novel roles of Tau in regulating the formation and maintenance of synapses during ageing, by coordinating the intracellular trafficking of synaptic proteins. Thus, this study shows that the role of Tau in synapse regulation occurs in functional overlap with Shot. The robust shot-tau double-mutant phenotypes enabled study of the mechanistic cascade composed of three steps: microtubule stability as the trigger, the JNK signalling pathway as the mediator and kinesin-3 mediated axonal transport of synaptic proteins as the key effector. It is propose that a new mechanism based on the loss of Tau function which could explain intellectual disability in MAPT (the human tau gene) mutant individuals and precocious synapse loss in tau-related neurodegeneration (Voelzmann, 2016).

The aim of these studies was to understand the role of endogenous Tau in neurons with particular attention to synapses. This effort was essentially aided by the finding that Tau and Shot are functionally redundant, and the subsequent incorporation of Shot into these studies. The robust phenotypes of shot-tau double-mutant neurons enabled this study to demonstrate roles of Shot-Tau during the formation and maintenance of pre-synaptic sites in axons, and unravel the underlying mechanistic cascade which involves three major steps. Firstly, the absence of Shot-Tau causes microtubule destabilisation. Secondly, this cytoskeletal stress causes aberrant JNK activity patterns with upregulation in somata and downregulation at axon tips. Thirdly, aberrant JNK activation leads to a somatic roadblock for kinesin-3 mediated transport, thus inhibiting the delivery of synaptic proteins and eventually causing synapse loss. Depending on whether the functions of Tau and/or Shot are removed during development or ageing, either the formation or the maintenance of synapses are affected, respectively (Voelzmann, 2016).

The model explaining the function of Tau and Shot in synapse establishment and maintenance by regulating intracellular transport, is supported by loss- and gain-of-function experiments, genetic interactions and cross-rescue experiments. The initial finding that shot-tau mutant neurons had reduced branch numbers, could have suggested that defects on synapse numbers is indirect. However, experiments with double knock-down in culture and in the adult brain clearly showed strong synapse reduction whilst maintaining normal branch patterns, and Unc-104 rescued synapse reduction in shot-tau mutant neurons without major increases of the branch pattern in these neurons. These results clearly demonstrate that changes in neuronal morphology are not the cause of changes in synapse number (Voelzmann, 2016).

Notably, the synaptic function of Tau described in this study for Drosophila might be conserved in higher animals or humans, since also aged Tau knock-out mice develop a reduction of synaptic proteins from the hippocampus (Ma, 2014; Voelzmann, 2016 and references therein).

These findings provide potential new mechanistic explanations for various tau related brain disorders. For example, microdeletions in the region of MAPT (the human tau gene) cause intellectual disability, and Tau's synapse-promoting roles may well contribute to this pathology. Furthermore, various tauopathies are characterised by precocious pathological loss of synapses. The currnet data suggest that loss of tau could lead to defective synapse maintenance and eventually synapse loss. For example, a prominent group of dementias which lacks distinctive histopathology (DLDH) are characterised by the loss of Tau. Further tauopathies including Alzheimer disease, typically involve hyper-phosphorylation and aggregate formation of Tau. In this scenario, there are two parallel, non-exclusive modalities through which Tau can cause pathology. Firstly, detached hyper-phosphorylated tau attains gain-of-function roles in the cytoplasm damaging neurons through a number of mechanisms. Secondly, hyper-phosphorylation of tau causes a loss-of-function condition by depleting Tau from microtubules. However, since Tau knock-out mouse models mostly failed to show significant phenotypes and the neuronal functions of endogenous tau remain little understood, the pathological importance of Tau loss from microtubules has been marginalised. The current results now re-emphasise the notion that loss of Tau from microtubules could contribute to neurodegenerative pathology and deliver mechanistic explanations (Voelzmann, 2016).

To unravel pathomechanisms caused by the loss of Tau, a combined depletion of Shot and Tau gave strong phenotypes, ideal for short-term experimental approaches. However, similar, yet milder phenotypes were found if only Tau was depleted, suggesting that the mechanisms described in this study could well contribute to slow disease progression in tauopathies. The discovery that spectraplakins are MAPs which functionally overlap with Tau, opens up new experimental avenues for Tau studies. So far, spectraplakins have been linked to the degeneration of sensory and autonomous neurons, and it remains to be elucidated whether they may have similar roles also in the brain. These results clearly hint at this possibility (Voelzmann, 2016).

The loss of Tau and/or Shot inhibits kinesin-3 mediated transport leading to accumulation of synaptic proteins in the soma of neurons. A road-block mechanism is proposed suppressing the initiation of axonal transport in somata of Shot-Tau depleted neurons, which is caused indirectly through microtubule stress and mediated by JNK (Voelzmann, 2016).

The involvement of microtubules in causing a transport block is supported by experiments using microtubule stabilising and de-stabilising drugs which rescued or mimicked the shot-tau mutant phenotypes, respectively. Similarly, axonal transport defects and cognitive deficits of PS19Tg mice (expressing the P301S mutant form of human tau) and various other mouse and fly tauopathy models were shown to be rescued by microtubule-stabilising drugs, suggesting that the mechanisms described may be conserved and relevant to disease (Voelzmann, 2016).

The somatic road-block is a novel mechanism through which the loss of Tau can interfere with the transport of synaptic proteins and provides potential explanations also for somatic accumulations of postsynaptic proteins such as PSD-95, AMPA and NMDA receptors observed in mouse tauopathy models. A likely mechanism causing a roadblock in intracellular transport could be the direct inactivation of Unc-104 or its associated adaptor proteins, for example through JNK or other kinases within its pathway. This mode of regulation has a clear precedent in kinesin-1 and its adaptor Jip which are directly phosphorylated by JNK leading to transport inhibition. Unfortunately, extensive attempts to co-immunoprecipitate JNK and Kinesin-3 were unsuccessful, leaving open for now the exact molecular mechanism (Voelzmann, 2016).

It is proposed that aberrant JNK activation downstream of microtubule destabilisation or stress is the ultimate cause for the defective delivery of synaptic proteins in Tau and/or Shot loss of function. Also in mouse, microtubule stress leads to somatic activation of the JNK pathway, suggesting this mechanism is likely to be conserved with vertebrates (Voelzmann, 2016).

The JNK pathway is emerging as a central player in neurodegenerative diseases. Its activation is prompted by various neurodegeneration risk factors including oxidative stress, inflammation, and ageing. Furthermore, JNK is activated in AD patients and in several AD models where it triggers progression of the pathology. The new link between Tau/spectraplakins, JNK and synapses proposed in this study, is therefore likely to provide mechanistic explanations for synaptic pathology observed in AD and other tauopathies (Voelzmann, 2016).

This study has delivered an important conceptual advance by revealing a new mechanistic cascade which can explain synaptic decay as the consequence of Tau loss from microtubules. Furthermore, a previously unknown functional redundancy with spectraplakins was identified as a promising new avenue for research on Tau. These findings emphasize that Tau detachment from microtubules can be an important aspect contributing to the pathology of tauopathies in parallel to roles of hyper-phosphorylated Tau in the cytoplasm. Synaptic decay, axonal transport and alterations in the JNK pathway are emerging as central players in a wider range of adult-onset neurodegenerative diseases, and here this study has aligned these factors into a concrete mechanistic cascade (Voelzmann, 2016).

PAR-1 promotes microtubule breakdown during dendrite pruning in Drosophila

Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. This study shows that the kinase PAR-1 is required for pruning and dendritic microtubule breakdown. The data show that neurons lacking PAR-1 fail to break down dendritic microtubules, and PAR-1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR-1 is a known Tau kinase, and genetic interactions suggest that PAR-1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila. Finally, PAR-1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. These results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning (Herzmann, 2017).

The physiological degeneration of synapses, axons, or dendrites without loss of the parent neuron is known as pruning. Pruning is an important developmental mechanism that is used to ensure specificity of neuronal connections, and to remove developmental intermediates. While the mechanisms of neurite outgrowth and synapse formation have been studied in some detail, comparably little is known about the mechanisms underlying pruning (Herzmann, 2017).

In holometabolous insects, the nervous system is remodeled at a large scale during metamorphosis. In the peripheral nervous system (PNS) of Drosophila, several types of sensory neurons undergo either apoptosis or prune their larval processes in an ecdysone-dependent manner. The sensory class IV dendritic arborization (c4da) neurons completely and specifically prune their long and branched larval dendrites at the onset of the pupal phase, while their axons stay intact. Pruning proceeds in a stereotypical fashion: Dendrites are first severed at proximal sites close to the cell body between 5 and 10 h after puparium formation (h APF). Severed dendrites are then fragmented and phagocytosed by the epidermal cells surrounding them. First signs of dendrite pruning are visible at 2-3 h APF when dendrites start to display swellings and thinned regions in their proximal parts where they are subsequently severed. Proximal dendrites are destabilized by local disassembly of the cytoskeleton through the microtubule-severing enzyme Katanin p60-like 1 (Kat-60L1) (Lee, 2009) and possibly the actin-severing enzyme Mical. Furthermore, the plasma membrane of proximal dendrites is thinned through increased local endocytosis (Herzmann, 2017).

How proximal dendrite destabilization is orchestrated is one of the most intriguing questions in the field. Local microtubule breakdown is one of the first apparent signs of pruning before plasma membrane severing in dendrites. However, not much is known about the signals leading to microtubule breakdown. For example, Kat-60L1 is already expressed at the larval stage in c4da neurons (Stewart, 2012), opening up the question as to how it is activated temporally for dendrite pruning (Herzmann, 2017).

This study shows that the kinase PAR-1 is required for dendrite pruning and dendritic microtubule breakdown. PAR-1 is known to phosphorylate microtubule-associated proteins (MAPs) including Tau, thus leading to microtubule destabilization (Drewes, 1997). This study found that PAR-1 is required for an increase in c4da neuron microtubule dynamics during the early pupal phase. Furthermore, PAR-1 was found to interact genetically with Drosophila Tau in a manner consistent with Tau being a PAR-1 target during dendrite pruning. Tau is also known to inhibit katanin (Qiang, 2006), and this study found that PAR-1 interacts genetically with Kat-60L1. Finally, local microtubule breakdown is linked to loss of membrane stabilizing factors and dendritic membrane collapse. Thus, the results suggest a mechanism for local microtubule disassembly and the relationship between early local events during pruning (Herzmann, 2017).

The kinase PAR-1 is part of a pathway for microtubule disassembly during dendrite pruning. The data show that PAR-1 acts to enhance microtubule dynamics specifically during the early pupal phase. In the absence of PAR-1, c4da neurons accumulate stable microtubules at a time when control neurons have already degraded most of their dendritic microtubules. The genetic data suggest that Tau is a major target for PAR-1 in this process and that PAR-1 is required during pruning to remove, or inactivate Tau. It is known that Tau itself stabilizes microtubules; therefore, Tau inhibition likely serves to destabilize microtubules. Interestingly, Tau removal might also serve to activate the katanin homologue Kat-60L1 during dendrite pruning. This is an attractive possibility because Tau, but not the Futsch homolog MAP1B, has been shown to be a potent katanin inhibitor in mammalian cells (Qiang, 2006), exactly matching the observed genetic interactions with PAR-1 during dendrite pruning. Tau also becomes depleted from mammalian sensory neuron axons after trophic support withdrawal in an in vitro pruning model system (Maor-Nof, 2013). Tau depletion was not sufficient to induce pruning in mammalian sensory neurons (Maor-Nof, 2013), matching the observations in c4da neurons. However, while not sufficient, it is interesting to speculate that Tau inactivation might also be required for pruning in mammalian neurons (Herzmann, 2017).

The data suggest that PAR-1 acts specifically during the pupal phase, but PAR-1 protein levels do not seem to increase at this stage. PAR-1 can be activated through phosphorylation by upstream kinases such as LKB1. lkb1 mutants showed only mild pruning defects that likely cannot fully explain the stronger defects caused by PAR-1 downregulation. Interestingly, this study found that PAR-1 interacts genetically with ik2, another kinase required for dendrite pruning. Thus, PAR-1 activation during dendrite pruning might depend on the interplay of several kinases. Given the temporal specificity of the PAR-1 effect , it is interesting to speculate that PAR-1 might be directly activated by a ecdysone-responsive factor (Herzmann, 2017).

This study also found that loss of PAR-1 prevents several processes at the dendritic plasma membrane during the pruning process: It prevented the local loss of membrane-associated Ank2XL from proximal dendrites, abrogated Ca2+ transients, and displayed strong enhancing genetic interactions with the thinning factor Shibire. As the genetic data indicate that Tau is the primary PAR-1 target during dendrite pruning, this suggests that microtubule breakdown is required for these plasma membrane alterations. In this scenario, the data actually suggest that microtubule disruption is closely linked to plasma membrane alterations, such that it is interesting to speculate that microtubule loss might trigger local endocytosis and thinning formation during dendrite pruning. Thus, a model is proposed where PAR-1, via Tau and possibly Kat-60L1, promotes microtubule disruption. In this model, these processes are placed epistatically over plasma membrane alterations during dendrite pruning (Herzmann, 2017).

Spatial regulation of microtubule disruption during dendrite pruning in Drosophila

Large scale neurite pruning is an important specificity mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their larval dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions, but how this spatial information is encoded is not clear. Dendrite severing is preceded by local breakdown of dendritic microtubules through PAR-1-mediated inhibition of Tau. This study investigated spatial aspects of microtubule breakdown during dendrite pruning. Live imaging of fluorescently tagged tubulin shows that microtubule breakdown first occurs at proximal dendritic branchpoints, followed by breakdown at more distal branchpoints, suggesting that the process is triggered by a signal emanating from the soma. In fly dendrites, microtubules are arranged in uniformly oriented arrays where all plus ends face towards the soma. Mutants in kinesin-1 and -2, which are required for uniform microtubule orientation, cause defects in microtubule breakdown and dendrite pruning. These data suggest that the local microtubule organization at branch points determines where microtubule breakdown occurs. Local microtubule organization may therefore contribute spatial information for severing sites during dendrite pruning (Herzmann, 2018).

The physiological degeneration of synapses, axons or dendrites without loss of the parent neuron is known as pruning. Pruning is an important developmental mechanism that is used to ensure specificity of neuronal connections and to remove developmental intermediates. Whereas the mechanisms of neurite outgrowth and synapse formation have been studied in some detail, comparably little is known about the mechanisms underlying pruning (Herzmann, 2018).

In holometabolous insects, the nervous system is remodeled on a large scale during metamorphosis. In the peripheral nervous system (PNS) of Drosophila, several types of sensory neurons undergo either apoptosis or prune their larval processes in an ecdysone-dependent manner. The sensory class IV dendritic arborization (c4da) neurons completely and specifically prune their long and branched larval dendrites at the onset of the pupal phase by a degenerative mechanism, while their axons remain intact. Dendrite pruning is induced in a cell-autonomous fashion by the steroid hormone ecdysone and proceeds in a stereotypical fashion. Dendrites are first severed at proximal sites close to the cell body between 5 and 10 h after puparium formation (APF). Severed dendrites are then fragmented and phagocytosed by the epidermal cells surrounding them. First signs of dendrite pruning are visible at 2-3 h APF, when proximal dendrites take on an irregular appearance with beadings and thinnings. In these regions, microtubules are disassembled locally. Plasma membrane retrieval through increased local endocytosis also contributes to thinning of proximal dendrites. Live imaging and genetic data suggest that local microtubule breakdown precedes membrane thinning. Microtubule disassembly is therefore the earliest known local sign of dendrite pruning. Microtubule disassembly requires the kinase PAR-1, which mediates inhibition of the microtubule-associated protein Tau. Furthermore, dendrite pruning also requires the microtubule-severing enzyme Katanin p60-like 1 (Kat-60L1), possibly also downstream of PAR-1 and Tau (Herzmann, 2018).

Microtubules are polar rods with so-called 'plus' and 'minus' ends. This nomenclature reflects the fact that the rates of both growth and shrinkage are greater at the plus ends. Microtubules in larval c4da neuron dendrites are oriented uniformly with their plus ends facing the soma. This uniform orientation is particularly prevalent in the primary and secondary dendrites, while higher order dendrites can also have microtubules with mixed orientation. Uniform dendritic plus-end-in microtubule orientation is known to depend on plus-end-directed motors of the kinesin family. In c4da neurons, uniform plus-end-in orientation depends on kinesin-2, which can bind to microtubule plus ends via EB1 and thereby promotes their orientation. In C. elegans, kinesin-1 was shown to be required for plus-end-in dendritic microtubule orientation. Kinesin-1 cannot bind to plus ends, and it was suggested that it instead moved wrongly oriented microtubules out of dendrites via microtubule sliding, or that it could be anchored to the dendritic cell cortex and could thus move microtubules out of the dendrite via its motor domain (Herzmann, 2018).

Given that microtubule breakdown occurs very early during the pruning process in a spatially confined manner, it is likely that this process involves spatial cues for pruning, such as the restriction of the destructive process to proximal over distal dendrites, or the sparing of the axon. This study addressed the apparent spatial regulation of microtubule disassembly during dendrite pruning by live imaging of fluorescently marked tubulin markers as well as in genetic studies. Gaps in dendritic microtubules were found to occur first at proximal dendrite branchpoints and later at more distal ones. Kinesin-2 is required for efficient c4da neuron dendrite pruning and microtubule disassembly, and EB1 manipulation was found to modulate the phenotypes associated with other microtubule disassembly factors, indicating that the uniform plus-end-in orientation of dendritic microtubules is also required for efficient microtubule breakdown and dendrite pruning. This is further supported by the observation that mutation of kinesin-1 also affects dendritic microtubule orientation, disassembly, and dendrite pruning in c4da neurons. It is proposed that microtubule disassembly depends on a signal emanating from the soma, and that the local microtubule organization at dendrite branchpoints, including the uniform plus-end-in orientation, favors microtubule disassembly there. The data indicate that dendritic microtubule organization may represent an important spatial cue for severing site selection during dendrite pruning (Herzmann, 2018).

Microtubule disassembly is the earliest known local destabilizing process during c4da neuron dendrite pruning and therefore a likely candidate to determine proximal severing. This study has begun to address the spatial regulation of microtubule disassembly during c4da neuron dendrite pruning. Using time-lapse live imaging of fluorescently tagged tubulin, it was found that gaps in the fluorescent microtubule signal develop preferentially at branchpoints of primary dendrites, and usually extend into the smaller side branches, rather than in the primary branch. Moreover, the temporal occurrence of these gaps correlates with proximity to the soma, i.e. microtubule gaps initially develop at the first branchpoint and later at the more distal branchpoints. These observations are consistent with a microtubule-destabilizing signal emanating from the soma. Previous work has shown that microtubule disassembly is induced by PAR-1-mediated Tau inhibition (Herzmann, 2017). PAR-1 activity is often induced by phosphorylation. Previous data suggested that PAR-1 is specifically activated for microtubule disassembly at the onset of the pupal phase, possibly by ecdysone (Herzmann, 2017). It is therefore plausible that the signal emanating from the soma is activated PAR-1. In such a model, activation of PAR-1 in the soma would contribute to the preference for microtubule disruption (and later dendrite severing) in proximal dendrites (Herzmann, 2018).

This study also found that the uniform plus-end-in orientation of dendritic microtubules is required for efficient microtubule disassembly and dendrite pruning. kinesin-2 and kinesin-1, both affecting dendritic microtubule orientation, are required for dendrite pruning. Manipulation of EB1, which caused less severe dendrite pruning defects by itself, strongly modified the defects seen upon manipulation of the microtubule dynamics regulator PAR-1. Interestingly, microtubule orientation (and hence the localization of the outermost microtubule plus ends) correlates well with the sites of degeneration and the degeneration mode in several models of large-scale pruning. For example, degeneration often starts from the distal end in axons, where microtubules have uniform plus-end-out orientation. As in c4da neurons, microtubule disassembly is an early event during large-scale axon pruning and is therefore likely to carry intrinsic spatial information crucial for pruning as well. This was shown to be the case in pruning axons of Drosophila mushroom body γ neurons and for pruning motoneuron axons at the mammalian neuromuscular junction. These axons retract, i.e. they shrink from their distal ends. Intriguingly, and in an exactly opposite manner to c4da neuron dendrites, these axons degenerate from the distal tips, and degeneration stops at the next branchpoint, again implying input from neurite branchpoints in the spatial regulation of large-scale pruning (Herzmann, 2018).

What could be special about microtubules at branchpoints? Although branchpoints could be localization sites for spatial pruning regulators, it is interesting to speculate that the local microtubule organization at branchpoints might suffice. For dendrites, uniformly oriented microtubules within the microtubule array would presumably enable the formation of larger gaps if the plus ends of the array microtubules have similar positions. Alternatively, microtubules at branchpoints could be more likely to be bent, and might therefore be more susceptible to shrinkage or catastrophe. Also, the overlap between the microtubule arrays of the primary and side branches may be smaller than that within an array in a straight branch, enabling gaps to occur at these sites even with relatively little microtubule shrinkage. Of note, previous data suggest that PAR-1 acts through inhibition of Tau to promote microtubule breakdown (Herzmann, 2017), and Tau was recently shown to promote microtubule bundling. Thus, PAR-1 activation might lead to 'unbundling' of microtubules at branchpoints. Furthermore, it could also be speculated that local concentrations of activated PAR-1 might increase as the diameter of the dendrite side branch decreases, which would also favor microtubule disassembly at proximal branchpoints. In order to distinguish between these models, it will be important to better characterize the local microtubule organization at dendritic branchpoints (Herzmann, 2018).

Previously it has been proposed that microtubule disassembly acts upstream of membrane thinning. Dendrite thinning was observed to be ~55% during the time of microtubule breakdown. Importantly, dendrites can often thin out to ~20% of their original diameter before rupturing, indicating that microtubule loss correlates with the onset of dendrite thinning (Herzmann, 2018).

Taken together, these data reveal that the spatial regulation of neurite pruning depends on the local microtubule organization. The data are consistent with a model in which microtubule disassembly carries the spatial information for pruning and therefore contributes to the selection of both degeneration mode and severing sites (Herzmann, 2018).

Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, this study showed that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. This demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies (Hahn, 2021).

Axons are the enormously long cable-like cellular processes of neurons that wire nervous systems. In humans, axons of ≤15μm diameter can be up to two meters long. They are constantly exposed to mechanical challenges, yet have to survive for up to a century; ~40% of axons are lost towards high age and far more in neurodegenerative diseases (Hahn, 2021).

Their growth and maintenance absolutely require parallel bundles of microtubules (MTs) that run all along axons, providing the highways for life-sustaining transport and driving morphogenetic processes. Consequently, bundle decay through MT loss or disorganisation is a common feature in axon pathologies. Key roles must be played by MT polymerisation, which is not only essential for the de novo formation of MT bundles occurring during axon growth in development, plasticity or regeneration, but also to repair damaged and replace senescent MTs during long-term maintenance. However, the molecular mechanisms regulating MT polymerisation in axons are surprisingly little understood (Hahn, 2021).

MT polymerisation is primarily understood in vitro, where MTs can undergo polymerisation in the presence of nucleation seeds and tubulin heterodimers; the addition of catalytic factors such as CLASPs, stathmins, tau, Eb proteins or XMAP215 can enhance and refine these events. However, it is not known whether mechanisms observed in reconstitution assays are biologically relevant in the context of axons, especially when considering that none of the above-mentioned factors has genetic links to human neurological disorders on OMIM (Online Mendelian Inheritance in Man), except Tau/MAPT which features primarily with dominant mutations relating to functions less likely to represent its intrinsic MT-regulatory roles (Hahn, 2021).

To identify relevant factors regulating axonal MT polymerisation, Drosophila primary neurons were used as one consistent model, which is amenable to combinatorial genetics as a powerful strategy to decipher complex regulatory networks. Previous loss-of-function studies of 9 MT plus-end-associating factors in these Drosophila neurons (CLASP, CLIP190, dynein heavy chain, APC, p150Glued, Eb1, Short stop/Shot, doublecortin, Lis1) have taken axon length as a crude proxy readout for net polymerisation, mostly revealing relatively mild axon length phenotypes, with the exception of Eb1 and Shot, which cause severe axon shortening (Hahn, 2021).

This study has incorporated more candidate factors and additional readouts to take these analyses to the next level. Three factors, Eb1, XMAP215/Msps and Tau, share a unique combination of mutant phenotypes in culture and in vivo, including reduced axonal MT polymerisation in frog and fly neurons. These data reveal that the three factors co-operate. Eb1 and XMAP215/Msps act interdependently at MT plus-ends, whereas Tau acts through a novel mechanism: it outcompetes Eb1-binding along MT lattices, thus preventing the depletion of Eb1 pools at polymerising MT plus-ends. By upholding these Eb1 pools, the functional trio also promotes the bundle conformation of axonal MTs through a guidance mechanism mediated by the spectraplakin Shot. This work uniquely integrates molecular mechanisms into understanding of MT regulation that is biologically relevant for axon growth, maintenance and disease (Hahn, 2021).

Understanding the machinery of MT polymerisation is of utmost importance in axons where MTs form loose bundles that run along the neurite throughout its entire length; these bundles are essential for axonal morphogenesis and life-sustaining cargo transport, and must be maintained in functional state for up to a century in humans. To achieve this, MT polymerisation is required to generate MTs de novo, repair or replace them. The underpinning machinery is expected to be complex, but deciphering the involved mechanisms will pay off by delivering new strategies for tackling developmental and degenerative axon pathologies (Hahn, 2021).

This study has made important advances to this end. Having screened through 13 candidates, it was found the three factors Eb1, Msps and Tau stand out by expressing the same combination of phenotypes, and by displaying functional interaction in both Drosophila and Xenopus neurons. It was found that their functions are not only important to maintain MT polymerisation, but also to align MTs into parallel arrangements, thus contributing in two ways to MT bundle formation and maintenance, both in culture and in vivo. The observed impact on MT organisation is also consistent with roles of XMAP215 during MT guidance in growth cones of frog neurons (Hahn, 2021).

The data reveal that various mechanisms observed in vitro or in non-neuronal cells, apply in the biological context of axons, which was unpredictable for two reasons: Firstly, of the three proteins only the human tau homologue has OMIM-listed links to human axonopathies, and these do not necessarily relate to MT polymerisation. Absence of such disease links might well be due to the fact that these proteins are functionally too important, causing embryonic lethality when dysfunctional. Secondly, axons and non-neuronal cells can display significant mechanistic deviations as shown for CLIP170/190 and for the MT localisation of Msps that is facilitated by Sentin or dTACC in non-neuronal cells, dendrites and in vitro but seemingly not in axons. However, other mechanisms observed in axons matched previous reports: (1) the complementary binding preferences of Eb1 and Tau for GTP-/GDP-tubulin; (2) the mutual enhancement of Eb1 and XMAP215/Msps; (3) the correlation of GTP cap size with comet velocity. Furthermore, it was observed that depletion of α1-tubulin in neurons mutant for αtub84B or stathmin (a promoter of tubulin availability) affects comet numbers but not Eb1 amounts: this is consistent with observations that MT nucleation in vitro is far more sensitive to tubulin levels than polymerisation (Hahn, 2021).

Apart from demonstrating the relevance of various molecular mechanisms in the context of axonal MT regulation, this work provides key insights as to how they integrate into one consistent mechanistic model of biological function (see A mechanistic model consistent with all reported data. The TOG-domain protein XMAP215/Msps is relevant for neuronal morphogenesis in fly and Xenopus, likely through its expected function as a MT polymerase. In contrast, Drosophila and vertebrate Eb proteins are only moderate promoters of MT polymerisation in vitro, but rather act as scaffolds. Conserved binding partners of Eb proteins are the spectraplakins, which can guide extending MT plus-ends along actin networks in axons and non-neuronal cells (Hahn, 2021).

It is proposed therefore that Eb1 is the key mediator of MT guidance into bundles (as supported by data throughout this work), and Msps is the key promoter of MT polymerisation. To execute these functions, both proteins depend on each other: MT plus-end localisation of Msps is reduced upon loss of Eb1 and vice versa. This mutual dependency is unlikely to involve their physical interaction, since MT plus-end localisation of Eb1 is known to occur tens of nanometres behind XMAP215, as seems to be the case also for axonal MTs. Furthermore, the data do not support an obvious role of the Sentin or dTACC adaptors in mediating Eb1-XMAP215 interactions (Hahn, 2021).

Potential indirect mechanisms explaining this co-dependency are provided by the promotion of MT polymerisation through XMAP215/Msps, which maintains a prominent GTP-cap which, in turn, mediates Eb1 binding. Restricted GTP-cap formation as a limiting factor for Eb1 binding would also explain why Eb1 over-expression fails to improve Msps-deficient phenotypes. Vice versa, Eb proteins promote lateral protofilament contacts which could assist in sheet formation at the very plus tip, thus facilitating the binding of XMAP215/Msps (Hahn, 2021).

Tau and Map1b/Futsch are known to bind along MT lattices, to promote MT polymerisation in vitro, and to enhance axon growth in mouse and fly neurons through mechanisms that remain unclear (Hahn, 2021).

In the cellular model used in this study, loss of the Map1b homologue Futsch has no obvious effects, whereas Tau shares all assessed loss-of-function mutant phenotypes with Msps and Eb1, although with weaker expression. Of these phenotypes, reduced MT plus-end localisation of Eb proteins upon loss of Tau function was likewise reported for frog neurons, N1E-115 mouse neuroblastoma cells and primary mouse cortical neurons (Hahn, 2021).

One proposed mechanism involves direct interaction where Tau recruits Eb proteins at MT plus ends, consistent with other reports that Tau can bind Eb1. However, further reports argue against overlap of Tau and Eb1 and rather show that Eb1 and Tau have complementary preferences for GTP- and GDP-tubulin, respectively; this is also consistent with the current data. Furthermore, a recruitment model is put in question by the finding that Eb1 lattice localisation increases rather than decreases upon loss of Tau, as similarly observed for mammalian tau and Eb1 in vitro (Hahn, 2021).

Therefore, a different mechanism based on competitive binding is proposed where Tau's preferred binding to GDP-tubulin along the lattice prevents Eb1 localisation, comparable to Tau's role in preventing other proteins including MAP6 and MAP7 from binding in certain regions of the MT lattice. Given the high density of MTs especially in small-diameter axons, lattice binding could generate a sink large enough to reduce Eb1 levels at MT plus-ends, and the experiments with Eb1::GFP overexpression strongly support this notion. In this way, loss of Tau generates a condition comparable to a mild Eb1 loss-of-function mutant phenotype, thus explaining why Tau shares its repertoire of loss-of-function phenotypes with Msps and Eb1, but with more moderate presentation. This competition mechanism might apply in axons with high MT density, for example explain the reduction in axonal MT numbers upon Tau deficiency in C. elegans. It might be less relevant in larger diameter axons of vertebrates where MT densities are low (Hahn, 2021).

This study has used a standardised Drosophila neuron system amenable to combinatorial genetics to gain understanding of MT regulation at the cellular level in axons. A consistent mechanistic model is proposed that can integrate all the data, mechanisms reported in the literature, and previous mechanistic model explaining Eb1/Shot-mediated MT guidance. This understanding offers new opportunities to investigate the mechanisms behind other important observations (Hahn, 2021).

For example, the presence of an axonal sleeve of cortical actin/spectrin networks was shown to be important to maintain MT polymerisation, likely relevant in certain axonopathies; the underlying mechanisms are now far easier to dissect. As another example, it was found that loss of either Eb1, XMAP215/Msps or Tau all caused a reduction in comet numbers, consistent with reports of nucleation-promoting roles of XMAP215 in non-neuronal contexts or reactivating neuronal stem cells. This might offer opportunities to investigate how axonal MT numbers can be determined through the regulation of local acentrosomal nucleation in reproducible, neuron/axon-specific ways, thus addressing a fundamental aspect of axon morphology (Hahn, 2021).

By gradually assembling molecular mechanisms into regulatory networks that can explain axonal MT regulation at the cellular level, i.e., the level at which diseases become manifest, these studies come closer to explaining axonal pathologies which can then form the basis for the development of remedial strategies (Hahn, 2021).

Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease

Common neurodegenerative proteinopathies, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by the misfolding and aggregation of toxic protein species, including the amyloid β (Aβ) peptide, microtubule-associated protein Tau (Tau), and alpha-synuclein (αSyn) protein. These factors also show toxicity in Drosophila. Using standardized conditions and medium-throughput assays, this study expressed human Tau, Aβ or αSyn selectively in neurons of the adult Drosophila retina and monitored age-dependent changes in both structure and function, based on tissue histology and recordings of the electroretinogram (ERG), respectively. Each protein was found to cause a unique profile of neurodegenerative pathology. Strikingly, expression of Tau leads to progressive loss of ERG responses whereas retinal architecture and neuronal numbers are largely preserved. By contrast, Aβ induces modest, age-dependent neuronal loss without degrading the retinal ERG. αSyn expression is characterized by marked retinal vacuolar change, progress photoreceptor cell death, and delayed-onset but modest ERG changes. Surprisingly, Tau and αSyn each cause prominent but distinct synaptotoxic profiles, including disorganization or enlargement of photoreceptor terminals, respectively. These findings suggest that Drosophila may be useful for revealing determinants of neuronal dysfunction that precede cell loss, including synaptic changes, in the adult nervous system (Chouhan, 2016).

Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila.

Accumulations of Tau, a microtubule-associated protein (MAP), into neurofibrillary tangles is a hallmark of Alzheimer's disease and other tauopathies. However, the mechanisms leading to this pathology are still unclear: the aggregates themselves could be toxic or the sequestration of Tau into tangles might prevent Tau from fulfilling its normal functions, thereby inducing a loss of function defect. Surprisingly, the consequences of losing normal Tau expression in vivo are still not well understood, in part due to the fact that Tau knockout mice show only subtle phenotypes, presumably due to the fact that mammals express several MAPs with partially overlapping functions. In contrast, flies express fewer MAP, with Tau being the only member of the Tau/MAP2/MAP4 family. Therefore, this study used Drosophila to address the physiological consequences caused by the loss of Tau. Reducing the levels of fly Tau (dTau) ubiquitously resulted in developmental lethality, whereas deleting Tau specifically in neurons or the eye caused progressive neurodegeneration. Similarly, chromosomal mutations affecting dTau also caused progressive degeneration in both the eye and brain. Although photoreceptor cells initially developed normally in dTau knockdown animals, they subsequently degenerated during late pupal stages whereas weaker dTau alleles caused an age-dependent defect in rhabdomere structure. Expression of wild type human Tau partially rescued the neurodegenerative phenotype caused by the loss of endogenous dTau, suggesting that the functions of Tau proteins are functionally conserved from flies to humans (Bolkan, 2014).

Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer's disease-related tau phosphorylation via PAR-1

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. This study investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, it was found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and this study found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, this study found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. These results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD (Iijima-Ando, 2012).

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 two novel putative phosphorylation sites, Ser(238) and Thr(245), on WT hTau were identified that are 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 (Kosmidis, 2010).

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin-dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. This study reports age-dependent constitutive activation of glycogen synthase kinase 3beta, protein kinase B, mitogen-activated protein kinase, and c-Jun-N-terminal kinase in adult p62(-/-) mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62(-/-) brain led to recovery of aggregated K63-ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain-derived neurotrophic factor levels. These findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer-like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid-beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity (Ramesh Babu, 2008).

The Distinctive Role of Tau and Amyloid beta in Mitochondrial Dysfunction through Alteration in Mfn2 and Drp1 mRNA Levels: A Comparative Study in Drosophila melanogaster

Alzheimer's disease (AD) is one of the most common forms of neurodegenerative diseases. Aggregation of Aβ42 and hyperphosphorylated tau are two major hallmarks of AD. Whether different forms of tau (soluble or hyperphosphorylated) or Aβ are the main culprit in the events observed in AD is still under investigation. This study examined the effect of wild-type, prone to hyperphosphorylation and hyperphosphorylated tau, and also Aβ42 peptide on the brain antioxidant defense system and two mitochondrial genes, Marf (homologous to human MFN2) and Drp1 involved in mitochondrial dynamics in transgenic Drosophila melanogaster. AD is an age associated disease. Therefore, the activity of 3 antioxidant agents, CAT, SOD, and GSH levels and the mRNA levels of Marf and Drp1 was assessed in different time points of flies' life cycle. Reduction in cognitive function and antioxidant activity was observed in all lines and time points. The most and the least effect on the eye phenotype was exerted by hyperphosphorylated tau and Aβ42, respectively. In addition, the most remarkable alteration in Marf and Drp1 mRNA level was observed in transgenic flies expressing hyperphosphorylated tau when pan neuronal expression of transgenes was applied. However, when the disease causing gene expression was confined to the mushroom body, Marf and Drp1 mRNA level alteration was more prominent in tau(WT) and tau(E14) transgenic flies, respectively. This may suggest a role for propagation of tau(WT) compared to hyperphosphorylated tau or Aβ42. In conclusion, in spite of antioxidant deficiency caused by different types of tau and Aβ42, it seems that tau exerts more toxic effect on the eye phenotype and mitochondrial genes regulation (Marf and Drp1). Moreover, different mechanisms seem to be involved in mitochondrial gene dysregulation when various forms of tau are expressed (Abtahi, 2020).

Role of Tau protein in remodeling of circadian neuronal circuits and sleep

Multiple neurological, physiological, and behavioral functions are synchronized by circadian clocks into daily rhythms. Neurodegenerative diseases such as Alzheimer's disease and related tauopathies are associated with a decay of circadian rhythms, disruption of sleep patterns, and impaired cognitive function but the mechanisms underlying these alterations are still unclear. Traditional approaches in neurodegeneration research have focused on understanding how pathology impinges on circadian function. Since in Alzheimer's disease and related tauopathies tau proteostasis is compromised, this study sought to understand the role of tau protein in neuronal circadian biology and related behavior. Considering molecular mechanisms underlying circadian rhythms are conserved from Drosophila to humans, advantage was taken of a recently developed tau-deficient Drosophila line to show that loss of tau promotes dysregulation of daily circadian rhythms and sleep patterns. Strikingly, tau deficiency dysregulates the structural plasticity of the small ventral lateral circadian pacemaker neurons by disrupting the temporal cytoskeletal remodeling of its dorsal axonal projections and by inducing a slight increase in the cytoplasmic accumulation of core clock proteins. Taken together, these results suggest that loss of tau function participates in the regulation of circadian rhythms by modulating the correct operation and connectivity of core circadian networks and related behavior (Arnes, 2019).

Vesicular axonal transport is modified in vivo by Tau deletion or overexpression in Drosophila

Structural microtubule associated protein Tau is found in high amount in axons and is involved in several neurodegenerative diseases. Although many studies have highlighted the toxicity of an excess of Tau in neurons, the in vivo understanding of the endogenous role of Tau in axon morphology and physiology is poor. Indeed, knock-out mice display no strong cytoskeleton or axonal transport phenotype, probably because of some important functional redundancy with other microtubule-associated proteins (MAPs). Here, advantage was taken of the model organism Drosophila, which has only one homologue of the Tau/MAP2/MAP4 family, to decipher (endogenous) Tau functions. Tau depletion was found to lead to a decrease in microtubule number and microtubule density within axons, while Tau excess leads to the opposite phenotypes. Analysis of vesicular transport in tau mutants showed altered mobility of vesicles, but no change in the total amount of putatively mobile vesicles, whereas both aspects were affected when Tau was overexpressed. In conclusion, this study shows that loss of Tau in tau mutants not only leads to a decrease in axonal microtubule density, but also impairs axonal vesicular transport, albeit to a lesser extent compared to the effects of an excess of Tau (Talmat-Amar, 2018).

Deletion of endogenous Tau proteins is not detrimental in Drosophila

Human Tau (hTau) is a highly soluble and natively unfolded protein that binds to microtubules within neurons. Its dysfunction and aggregation into insoluble paired helical filaments is involved in the pathogenesis of Alzheimer's disease (AD), constituting, together with accumulated β-amyloid (Aβ) peptides, a hallmark of the disease. Deciphering both the loss-of-function and toxic gain-of-function of hTau proteins is crucial to further understand the mechanisms leading to neurodegeneration in AD. As the fruit fly Drosophila melanogaster expresses Tau proteins (dTau) that are homologous to hTau, this study aimed to better comprehend dTau functions by generating a specific tau knock-out (KO) fly line using homologous recombination. It was observed that the specific removal of endogenous dTau proteins does not lead to overt, macroscopic phenotypes in flies. Indeed, survival, climbing ability and neuronal function are unchanged in tau KO flies. In addition, any overt positive or negative effect of dTau removal on human Aβ-induced toxicity were not found. Altogether, these results indicate that the absence of dTau proteins has no major functional impact on flies, and suggest that the tau KO strain is a relevant model to further investigate the role of dTau proteins in vivo, thereby giving additional insights into hTau functions (Burnouf, 2016).

Identification and characterization of the Drosophila tau homolog

A pathological hallmark of neurodegenerative tauopathies, including Alzheimer's disease and a group of clinically heterogeneous frontotemporal dementias, is the presence of intracellular neurofibrillary protein lesions. The principal component of these structures is the microtubule-associated protein tau. Although tau is normally a highly soluble protein enriched in axons, in these deposits, it is abnormally hyperphosphorylated, insoluble, and redistributed to the somatodendritic compartments of neurons. Through ultrastructual analyses, it has been determined that the tau protein in these lesions is filamentous and organized into paired-helical filaments, straight filaments, or ribbon-like filaments. By the dynamic binding of microtubules, tau is thought to promote the structural stability of axons, but whether tau aggregates contribute to neurodegeneration through a direct toxicity on normal cellular functions such as organelle transport or an indirect effect on microtubule stability, is currently unknown. The identification of mutations in the tau locus in patients with familial frontotemporal dementia and Parkinsonism linked to chromosome 17 has demonstrated that mutations in tau are sufficient to cause neurodegenerative disease). To elucidate the mechanisms by which tau dysfunction contributes to neuronal loss, this study sought to model human tauopathies in a genetically tractable organism. This study describes the isolation of a Drosophila tau cDNA, the production of antibodies that recognize the encoded protein, and their use in determining the expression and subcellular localization of the fly tau protein (Heidary, 2001).

Pragati, S. S. (2021). Shaggy functions downstream of dMyc and their concurrent downregulation confers additive rescue against tau toxicity in Drosophila. Biofactors. PubMed ID: 33651466

Shaggy functions downstream of dMyc and their concurrent downregulation confers additive rescue against tau toxicity in Drosophila

Neurodegenerative tauopathies such as Alzheimer's and Parkinson's diseases are characterized by hyperphosphorylation of tau protein and their subsequent aggregation in the forms of paired helical filaments and/or neurofibrillary tangles in specific areas of the brain. Despite several attempts, it remains a challenge to develop reliable biomarkers or effective drugs against tauopathies. It is increasingly evident now that due to the involvement of multiple cellular cascades affected by the pathogenic tau molecules, a single genetic modifier or a molecule is unlikely to be efficient enough to provide an inclusive rescue. Hence, multitargets based combinatorial approach(s) have been suggested to provide an efficient rescue against tauopathies. It has been reported that targeted downregulation of dmyc (a Drosophila homolog of human cmyc proto-oncogene) restricts tau etiology by limiting tau hyperphosphorylation and heterochromatin loss. Although, dmyc generates a significant rescue; however, it is not proficient enough to provide a complete alleviation against tauopathies. This study reports that tissue-specific concurrent downregulation of dmyc and gsk3β conveys a near-complete rescue against tau toxicity in Drosophila. It is noted that combinatorial downregulation of dmyc and gsk3β reduces tau hyperphosphorylation, restricts the formation of neurofibrillary tangles, and restores heterochromatin loss to the physiological level. Subsequent investigations revealed that dmyc regulates gsk3β via protein phosphatase 2A (dPP2A) in a dose-dependent manner to regulate tau pathogenesis. It is proposed that dmyc and gsk3β candidates can be utilized in a synergistic manner for the development of an efficient combinatorial therapeutic approach against the devastating human tauopathies (Pragati, 2021).

Suppression of tau-induced phenotypes by vitamin E demonstrates the dissociation of oxidative stress and phosphorylation in mechanisms of tau toxicity

Various lines of evidence implicate oxidative stress in the pathogenic mechanism(s) underpinning tauopathies. Consequently, antioxidant therapies have been considered in clinical practice for the treatment of tauopathies such as Alzheimer's disease (AD), but with mixed results. Previous studies have reported increased protein oxidation upon expression of both human 0N3R (hTau(0N3R)) and 0N4R (hTau(0N4R)) tau (see Drosophila Tau) in vivo. Building on these studies, this study demonstrates here the suppression of hTau(0N3R) associated phenotypes in Drosophila melanogaster after treatment with vitamin C or vitamin E. Curiously the rescue of phenotype was seen without alteration in total tau level or alteration in phosphorylation at a number of disease-associated sites. Moreover, treatment with paraquat, a pro-oxidant drug, did not exacerbate the hTau(0N3R) phenotypes. This result following paraquat treatment is reminiscent of previous findings with hTau(0N4R) which also causes greater oxidative stress when compared to hTau(0N3R) but has a milder phenotype. Collectively these data imply that the role of oxidative stress in tau-mediated toxicity is not straight forward and there may be isoform-specific effects as well as contribution of other factors. This may explain the ambiguous effects of anti-oxidant treatments on clinical outcome in dementia patients (Cowan, 2020).

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 (AD) 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. This study 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, this work uncovers a critical role of Cys-322 in determining Tau toxicity and dysfunction (Prifti, 2020).

Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS

Expansions of trinucleotide or hexanucleotide repeats lead to several neurodegenerative disorders, including Huntington disease [caused by expanded CAG repeats (CAGr) in the HTT gene], and amyotrophic lateral sclerosis [ALS, possibly caused by expanded GGGGCC repeats (G4C2r) in the C9ORF72 gene], of which the molecular mechanisms remain unclear. This study demonstrated that lowering the Drosophila homologue of tau protein (dtau) significantly rescued in vivo neurodegeneration, motor performance impairments, and the shortened life-span in Drosophila expressing expanded CAGr or expanded G4C2r. Expression of human tau (htau4R) restored the disease-related phenotypes that had been mitigated by the loss of dtau, suggesting an evolutionarily-conserved role of tau in neurodegeneration. This study further revealed that G4C2r expression increased tau accumulation by inhibiting autophagosome-lysosome fusion, possibly due to lowering the level of BAG3, a regulator of autophagy and tau. Taken together, these results reveal a novel mechanism by which expanded G4C2r causes neurodegeneration via an evolutionarily-conserved mechanism. These findings provide novel autophagy-related mechanistic insights into C9ORF72-ALS and possible entry points to disease treatment (Wen, 2020).

Moderate Overexpression of Tau in Drosophila Exacerbates Amyloid-beta-Induced Neuronal Phenotypes and Correlates with Tau Oligomerization

Alzheimer's disease (AD) is neuropathologically defined by two key hallmarks: extracellular senile plaques composed primarily of amyloid-beta (Abeta) peptide and intraneuronal neurofibrillary tangles, containing abnormally hyperphosphorylated tau protein. The tau protein is encoded by the MAPT gene. Recently, the H1 and H2 haplotypes of the MAPT gene were associated with AD risk. The minor MAPT H2 haplotype has been linked with a decreased risk of developing late-onset AD (LOAD). MAPT haplotypes show different levels of MAPT/Tau expression with H1 being approximately 1.5-fold more expressed than H2, suggesting that MAPT expression level could be related to LOAD risk. This study investigated whether this moderate difference in MAPT/Tau expression could influence Abeta-induced toxicity in vivo. It was shown that modest overexpression of tau protein in Drosophila exacerbates neuronal phenotypes in AbetaPP/BACE1 (amyloid-beta protein precursor/Beta-Secretase 1) flies. The exacerbation of neuronal defects correlates with the accumulation of insoluble dTau oligomers, suggesting that the moderate difference in level of tau expression observed between H1 and H2 haplotypes could influence Abeta toxicity through the production of oligomeric Tau insoluble species (Miguel, 2020).

Developmental expression of human tau in Drosophila melanogaster glial cells induces motor deficits and disrupts maintenance of PNS axonal integrity, without affecting synapse formation

Tauopathies are a class of neurodegenerative diseases characterized by the abnormal phosphorylation and accumulation of the microtubule-associated protein, tau (see Drosophila Tau), in both neuronal and glial cells. Though tau pathology in glial cells is a prominent feature of many of these disorders, the pathological contribution of these lesions to tauopathy pathogenesis remains largely unknown. Moreover, while tau pathology is predominantly found in the central nervous system, a role for tau in the cells of the peripheral nervous system has been described, though not well characterized. To investigate the effects of glial tau expression on the development and maintenance of the peripheral nervous system, a Drosophila melanogaster model of tauopathy was used that expresses human wild-type tau in glial cells during development. Glial tau expression during development was found to result in larval locomotor deficits and organismal lethality at the pupal stage, without affecting larval neuromuscular junction synapse development or post-synaptic amplitude. There was, however, a significant decrease in the decay time of synaptic potentials upon repeated stimulation of the motoneuron. Behavioral abnormalities were accompanied by glial cell death, disrupted maintenance of glial-axonal integrity, and the abnormal accumulation of the presynaptic protein, Bruchpilot, in peripheral nerve axons. Together, these data demonstrate that human tau expression in Drosophila glial cells does not affect neuromuscular junction synapse formation during development, but is deleterious to the maintenance of glial-axonal interactions in the peripheral nervous system (Scarpelli, 2019).

Small-molecule drug screening identifies drug Ro 31-8220 that reduces toxic phosphorylated tau in Drosophila melanogaster

The intraneuronal aggregates of hyperphosphorylated and misfolded tau (neurofibrillary tangles, NFTs) cause a stereotypical spatiotemporal Alzheimer's disease (AD) progression. Hyperactivation of kinases including the conventional protein kinase C (PKC) is a defective molecular event accompanying associative memory loss, tau phosphorylation, and progression of AD. This study investigated the ability of small therapeutic compounds (a custom library) to improve tau-induced rough-eye phenotype in a Drosophila melanogaster model of frontotemporal dementia. Tau phosphorylation in vivo and selected hit compounds was also assessed. Among the potential hits, Ro 31-8220, described earlier as a potent PKCalpha inhibitor, was tested. Ro 31-8220 robustly improved the rough-eye phenotype, reduced phosphorylated tau species in vitro and in vivo, reversed tau-induced memory impairment, and improved the fly motor functions. In a human neuroblastoma cell line, Ro 31-8220 reduced the PKC activity and the tau phosphorylation pattern, bthe compound's wide range of biological activity is also acknowledged. Nevertheless, Ro 31-8220 is a novel therapeutic mitigator of tau-induced neurotoxocity (Shim, 2019).

Inhibition of GSK-3 ameliorates Aβ pathology in an adult-onset Drosophila model of Alzheimer's disease

Aβ peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimer's disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. This study developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Aβ42 specifically in adult neurons, to avoid developmental effects. Aβ42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Aβ42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Aβ42 toxicity. Aβ42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Aβ42. The GSK-3-mediated effects on Aβ42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Aβ42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Aβ42 peptide level, in the absence of APP processing. This study points to the need both to identify the mechanisms by which GSK-3 modulates Aβ42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD (Sofola, 2010).

Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death

In neurodegenerative diseases like Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown in particular with respect to the microtubule-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of patients. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for microtubules, there has been no indication that an excess of this protein could affect mitosis. The effect of an excess of human Tau (hTau) protein on cell mitosis was studied in vivo. Using the Drosophila developing wing disc epithelium as a model, this study shows that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. The mechanism of action of hTau was studied and it was found that the MT-binding domain of hTau is responsible for these defects. hTau effects occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). This deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc) as well as in human Hela cells.By demonstrating that microtubule-bound Tau inhibits the Eg5/KIF11 kinesin and cell mitosis, this work provides a new framework to consider the role of Tau in neurodegenerative diseases (Bouge, 2016).

Developmental Expression of 4-Repeat-Tau Induces Neuronal Aneuploidy in Drosophila Tauopathy Models

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. This study used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. The results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, a mitotic delay was observed in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, a high level of aneuploidy was found in post-mitotic differentiated tissue. Finally, it was shown that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, these results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development (Malmanche, 2017).

Tau-induced nuclear envelope invagination causes a toxic accumulation of mRNA in Drosophila

The nucleus is a spherical dual-membrane bound organelle that encapsulates genomic DNA. In eukaryotes, messenger RNAs (mRNA) are transcribed in the nucleus and transported through nuclear pores into the cytoplasm for translation into protein. In certain cell types and pathological conditions, nuclei harbor tubular invaginations of the nuclear envelope known as the "nucleoplasmic reticulum." Nucleoplasmic reticulum expansion has recently been established as a mediator of neurodegeneration in tauopathies, including Alzheimer's disease. While the presence of pore-lined, cytoplasm-filled, nuclear envelope invaginations has been proposed to facilitate the rapid export of RNAs from the nucleus to the cytoplasm, the functional significance of nuclear envelope invaginations in regard to RNA export in any disorder is currently unknown. This study reports that polyadenylated RNAs accumulate within and adjacent to tau-induced nuclear envelope invaginations in a Drosophila model of tauopathy. Genetic or pharmacologic inhibition of RNA export machinery reduces accumulation of polyadenylated RNA within and adjacent to nuclear envelope invaginations and reduces tau-induced neuronal death. These data are the first to point toward a possible role for RNA export through nuclear envelope invaginations in the pathogenesis of a neurodegenerative disorder and suggest that nucleocytoplasmic transport machinery may serve as a possible novel class of therapeutic targets for the treatment of tauopathies (Cornelison, 2018).

A conserved cytoskeletal signaling cascade mediates neurotoxicity of FTDP-17 tau mutations in vivo

The microtubule binding protein tau is strongly implicated in multiple neurodegenerative disorders, including frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), which is caused by mutations in tau. In vitro, FTDP-17 mutant versions of tau can reduce microtubule binding and increase aggregation of tau, but the mechanism by which these mutations promote disease in vivo is not clear. This study took a combined biochemical and in vivo modeling approach to define functional properties of tau driving neurotoxicity in vivo. Wild type human tau and five FTDP-17 mutant forms of tau were expressed in Drosophila using a site-directed insertion strategy to ensure equivalent levels of expression. Multiple markers of neurodegeneration and neurotoxicity were analyzed in transgenic animals, including analysis of both males and females. FTDP-17 mutations act to enhance phosphorylation of tau and thus promote neurotoxicity in an in vivo setting. Further, it was demonstrated that phosphorylation-dependent excess stabilization of the actin cytoskeleton is a key phosphorylation-dependent mediator of the toxicity of wild type tau, and of all the FTDP-17 mutants tested. Finally, it was shown that important downstream pathways, including autophagy and the unfolded protein response, are co-regulated with neurotoxicity and actin cytoskeletal stabilization in brains of flies expressing wild type human and various FTDP-17 tau mutants, supporting a conserved mechanism of neurotoxicity of wild type tau and FTDP-17 mutant tau in disease pathogenesis (Bardai, 2017).

Beta-sheet assembly of Tau and neurodegeneration in Drosophila melanogaster

The assembly of Tau into abundant beta-sheet-rich filaments characterizes human tauopathies. A pathological pathway leading from monomeric to filamentous Tau is believed to be at the heart of these diseases. However, in Drosophila models of Tauopathy, neurodegeneration has been observed in the absence of abundant Tau filaments. This study investigated the role of Tau assembly into beta-sheets by expressing wild-type and Delta306-311 human Tau-383 in the retina and brain of Drosophila. Both lines were examined for eye abnormalities, brain vacuolization, Tau phosphorylation and assembly, as well as climbing activity and survival. Flies expressing wild-type Tau-383 showed MC-1 staining, Tau hyperphosphorylation, and neurodegeneration. By contrast, flies expressing Delta306-311 Tau-383 had less MC-1 staining, reduced Tau hyperphosphorylation, and no detectable neurodegeneration. Their climbing ability and lifespan were similar to those of nontransgenic flies. Fluorescence spectroscopy after addition of Thioflavin T, a dye that interacts with beta-sheets, showed no signal when Delta306-311 Tau-383 was incubated with heparin. These findings demonstrate that the assembly of Tau into beta-sheets is necessary for neurodegeneration (Passarella, 2018).

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. This study used 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 (Kadas, 2019).

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

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. This study used the Drosophila system to investigate in vivo whether 14-3-3s are causal or synergistic with Tau accumulation in precipitating pathogenesis. 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 14-3-3zeta stabilizes the mutant protein, elevated D14-3-3epsilon has a destabilizing effect probably because of altered 14-3-3 dimer composition. These 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, it is suggested 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 (Papanikolopoulou, 2018).

Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila

The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., beta-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. In an analysis of available expression data sets this study indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled prediction of the occurrence of AD with remarkable accuracy in a test cohort and identification of a set of key genes whose expression determines this classification. Then the effect of reducing expression of homologs of 6 of these genes in was studied in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. These results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology (Frenkel-Pinter, 2017).

An AMPK-dependent regulatory pathway in tau-mediated toxicity

Neurodegenerative tauopathies are characterized by accumulation of hyperphosphorylated tau aggregates primarily degraded by autophagy. The 5'AMP-activated protein kinase (AMPK) is expressed in most cells, including neurons. Alongside its metabolic functions, it is also known to be activated in Alzheimer's brains, phosphorylate tau, and be a critical autophagy activator. While stress conditions can result in AMPK activation enhancing tau-mediated toxicity, AMPK activation is not always concomitant with autophagic induction. Using a Drosophila in vivo quantitative approach, this study has analysed the impact of AMPK and autophagy on tau-mediated toxicity, recapitulating the AMPK-mediated tauopathy condition: increased tau phosphorylation, without corresponding autophagy activation. It was demonstrated that AMPK, binding to and phosphorylating tau at Ser-262, a site reported to facilitate soluble tau accumulation, affects its degradation. This phosphorylation results in exacerbation of tau toxicity and is ameliorated via rapamycin-induced autophagy stimulation. These findings support the development of combinatorial therapies effective at reducing tau toxicity targeting tau phosphorylation and AMPK-independent autophagic induction. The proposed in vivo tool represents an ideal readout to perform preliminary screening for drugs promoting this process (Galasso, 2017).

Astrocyte transport of glutamate and neuronal activity reciprocally modulate tau pathology in Drosophila

Abnormal buildup of the microtubule associated protein tau is a major pathological hallmark of Alzheimer's disease (AD) and various tauopathies. The mechanisms by which pathological tau accumulates and spreads throughout the brain remain largely unknown. It is known that a restoration of the major astrocytic glutamate transporter, GLT1, ameliorates a buildup of tau pathology and rescues cognition in a mouse model of AD. In this study, it was hypothesized that aberrant extracellular glutamate and abnormal neuronal excitatory activities promote tau pathology. Consequently, the genetic interactions between tau and the GLT1 homolog dEaat1 were investigated in Drosophila melanogaster. Neuronal-specific overexpression of human wildtype tau markedly shortens lifespan and impairs motor behavior. RNAi depletion of dEaat1 in astrocytes worsens these phenotypes, whereas overexpression of dEaat1 improves them. However, the synaptic neuropil appears unaffected, and there is no major neuronal loss with tau overexpression in combination with dEaat1 depletion. To mimic glutamate-induced aberrant excitatory input in neurons, repeated depolarization of neurons via transgenic TrpA1 was applied to the adult Drosophila optic nerves, and the change of tau deposits was examined. Repeated depolarization significantly increases the accumulation of tau in these neurons. The study propose that increased neuronal excitatory activity exacerbates tau-mediated neuronal toxicity and behavioral deficits (Kilian, 2017).

Salidroside reduces tau hyperphosphorylation via up-regulating GSK-3β phosphorylation in a tau transgenic Drosophila model of Alzheimer's disease

Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease that causes substantial public health care burdens. Intensive efforts have been made to find effective and safe treatment against AD. The plant product Salidroside (Sal) is the main effective component of Rhodiola rosea L., which has several pharmacological activities. The objective of this study was to investigate the efficacy of Sal in the treatment of AD transgenic Drosophila and the associated mechanisms. Microtubule associated protein tau transgenic Drosophila line (TAU) was used in which tau protein is expressed in the central nervous system and eyes by the Gal4/UAS system. After feeding flies with Sal, the lifespan and locomotor activity were recorded. The appearance of vacuoles in the mushroom body was examined using immunohistochemistry, and the levels of total glycogen synthase kinase 3β (t-GSK-3β), phosphorylated GSK-3β (p-GSK-3β), t-tau and p-tau was detected in the brain by western blot analysis. The results showed that the longevity was improved in salidroside-fed Drosophila groups as well as the locomotor activity. Less vacuoles in the mushroom body, upregulated level of p-GSK-3β and downregulated p-tau were detected following Sal treatment. These data presented the evidence that Sal was capable of reducing the neurodegeneration in tau transgenic Drosophila and inhibiting neuronal loss. The neuroprotective effects of Sal were associated with its up-regulation of the p-GSK-3β and down-regulation of the p-tau (Zhang, 2016).

Stabilization of microtubule-unbound Tau via Tau phosphorylation at Ser262/356 by Par-1/MARK contributes to augmentation of AD-related phosphorylation and Aβ42-induced Tau toxicity

To prevent the cascade of events leading to neurodegeneration in Alzheimer's disease (AD), it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. In this study, using transgenic Drosophila co-expressing human tau and Aβ, tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau was found in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD (Ando, 2016).

Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo

Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer's disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. This study generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. It was observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, these results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD (Gorsky, 2016).

Search PubMed for articles about Drosophila Tau

Abtahi, S. L., Masoudi, R. and Haddadi, M. (2020). The Distinctive Role of Tau and Amyloid beta in Mitochondrial Dysfunction through Alteration in Mfn2 and Drp1 mRNA Levels: A Comparative Study in Drosophila melanogaster. Gene: 144854. PubMed ID: 32525045

Alves-Silva, J., Sanchez-Soriano, N., Beaven, R., Klein, M., Parkin, J., Millard, T. H., Bellen, H. J., Venken, K. J., Ballestrem, C., Kammerer, R. A. and Prokop, A. (2012). Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins). J Neurosci 32(27): 9143-9158. PubMed ID: 22764224

Ando, K., Maruko-Otake, A., Ohtake, Y., Hayashishita, M., Sekiya, M. and Iijima, K. M. (2016). Stabilization of microtubule-unbound Tau via Tau phosphorylation at Ser262/356 by Par-1/MARK contributes to augmentation of AD-related phosphorylation and Aβ42-induced Tau toxicity. PLoS Genet 12: e1005917. PubMed ID: 27023670

Arnes, M., Alaniz, M. E., Karam, C. S., Cho, J. D., Lopez, G., Javitch, J. A. and Santa-Maria, I. (2019). Role of Tau protein in remodeling of circadian neuronal circuits and sleep. Front Aging Neurosci 11: 320. PubMed ID: 31824299

Bardai, F. H., Wang, L., Mutreja, Y., Yenjerla, M., Gamblin, T. C. and Feany, M. B. (2017). A conserved cytoskeletal signaling cascade mediates neurotoxicity of FTDP-17 tau mutations in vivo. J Neurosci. PubMed ID: 29138281

Bolkan, B. J. and Kretzschmar, D. (2014). Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila. Dev Neurobiol 74(12): 1210-1225. PubMed ID: 24909306

Bouge, A. L. and Parmentier, M. L. (2016). Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death. Dis Model Mech [Epub ahead of print]. PubMed ID: 26822478

Burnouf, S., Grönke, S., Augustin, H., Dols, J., Gorsky, M.K., Werner, J., Kerr, F., Alic, N., Martinez, P. and Partridge, L. (2016). Deletion of endogenous Tau proteins is not detrimental in Drosophila. Sci Rep 6: 23102. PubMed ID: 26976084

Chouhan, A.K., Guo, C., Hsieh, Y.C., Ye, H., Senturk, M., Zuo, Z., Li, Y., Chatterjee, S., Botas, J., Jackson, G.R., Bellen, H.J. and Shulman, J.M. (2016). Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease. Acta Neuropathol Commun 4: 62. PubMed ID: 27338814

Cowan, C. M., Sealey, M. A. and Mudher, A. (2020). Suppression of tau-induced phenotypes by vitamin E demonstrates the dissociation of oxidative stress and phosphorylation in mechanisms of tau toxicity. J Neurochem. PubMed ID: 33251603

Cornelison, G. L., Levy, S. A., Jenson, T. and Frost, B. (2018). Tau-induced nuclear envelope invagination causes a toxic accumulation of mRNA in Drosophila. Aging Cell: e12847. PubMed ID: 30411463

Drewes, G., Ebneth, A., Preuss, U., Mandelkow, E. M. and Mandelkow, E. (1997). MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89(2): 297-308. PubMed ID: 9108484

Frenkel-Pinter, M., Stempler, S., Tal-Mazaki, S., Losev, Y., Singh-Anand, A., Escobar-Alvarez, D., Lezmy, J., Gazit, E., Ruppin, E. and Segal, D. S. Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila. Neurobiol Aging. PubMed ID: 28552182

Galasso, A., Cameron, C. S., Frenguelli, B. G. and Moffat, K. G. (2017). An AMPK-dependent regulatory pathway in tau-mediated toxicity. Biol Open [Epub ahead of print]. PubMed ID: 28808138

Gorsky, M. K., Burnouf, S., Dols, J., Mandelkow, E. and Partridge, L. (2016). Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo. Sci Rep 6: 22685. PubMed ID: 26940749

Hahn, I., Voelzmann, A., Parkin, J., Fulle, J. B., Slater, P. G., Lowery, L. A., Sanchez-Soriano, N. and Prokop, A. (2021). Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons. PLoS Genet 17(7): e1009647. PubMed ID: 34228717

Heidary, G. and Fortini, M. E. (2001). Identification and characterization of the Drosophila tau homolog. Mech Dev 108(1-2): 171-178. PubMed ID: 11578871

Herzmann, S., Krumkamp, R., Rode, S., Kintrup, C. and Rumpf, S. (2017). PAR-1 promotes microtubule breakdown during dendrite pruning in Drosophila. EMBO J 36(13): 1981-1991. PubMed ID: 28554895

Herzmann, S., Gotzelmann, I., Reekers, L. F. and Rumpf, S. (2018). Spatial regulation of microtubule disruption during dendrite pruning in Drosophila. Development [Epub ahead of print]. PubMed ID: 29712642

Iijima-Ando, K., Sekiya, M., Maruko-Otake, A., Ohtake, Y., Suzuki, E., Lu, B. and Iijima, K. M. (2012). Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer's disease-related tau phosphorylation via PAR-1. PLoS Genet 8(8): e1002918. PubMed ID: 22952452

Kadas, D., Papanikolopoulou, K., Xirou, S., Consoulas, C. and Skoulakis, E. M. C. (2019). Human Tau isoform-specific presynaptic deficits in a Drosophila central nervous system circuit. Neurobiol Dis 124: 311-321. PubMed ID: 30529489

Kilian, J.G., Hsu, H.W., Mata, K., Wolf, F.W. and Kitazawa, M. (2017). Astrocyte transport of glutamate and neuronal activity reciprocally modulate tau pathology in Drosophila. Neuroscience [Epub ahead of print]. PubMed ID: 28215745

Kosmidis, S., Grammenoudi, S., Papanikolopoulou, K. and Skoulakis, E. M. (2010). Differential effects of Tau on the integrity and function of neurons essential for learning in Drosophila. J Neurosci 30(2): 464-477. PubMed ID: 20071510

Kovacs, G. G. (2015). Invited review: Neuropathology of tauopathies: principles and practice. Neuropathol Appl Neurobiol 41(1): 3-23. PubMed ID: 25495175

Lee, H. H., Jan, L. Y. and Jan, Y. N. (2009). Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis. Proc Natl Acad Sci U S A 106(15): 6363-6368. PubMed ID: 19329489

Ma, Q. L., Zuo, X., Yang, F., Ubeda, O. J., Gant, D. J., Alaverdyan, M., Kiosea, N. C., Nazari, S., Chen, P. P., Nothias, F., Chan, P., Teng, E., Frautschy, S. A. and Cole, G. M. (2014). Loss of MAP function leads to hippocampal synapse loss and deficits in the Morris Water Maze with aging. J Neurosci 34(21): 7124-7136. PubMed ID: 24849348

Malmanche, N., Dourlen, P., Gistelinck, M., Demiautte, F., Link, N., Dupont, C., Vanden Broeck, L., Werkmeister, E., Amouyel, P., Bongiovanni, A., Bauderlique, H., Moechars, D., Royou, A., Bellen, H. J., Lafont, F., Callaerts, P., Lambert, J. C. and Dermaut, B. (2017). Developmental Expression of 4-Repeat-Tau Induces Neuronal Aneuploidy in Drosophila Tauopathy Models. Sci Rep 7: 40764. PubMed ID: 28112163

Maor-Nof, M., Homma, N., Raanan, C., Nof, A., Hirokawa, N. and Yaron, A. (2013). Axonal pruning is actively regulated by the microtubule-destabilizing protein kinesin superfamily protein 2A. Cell Rep 3(4): 971-977. PubMed ID: 23562155

Miguel, L., Frebourg, T., Campion, D. and Lecourtois, M. (2020). Moderate Overexpression of Tau in Drosophila Exacerbates Amyloid-beta-Induced Neuronal Phenotypes and Correlates with Tau Oligomerization. J Alzheimers Dis. PubMed ID: 32065789

Morris, M., Hamto, P., Adame, A., Devidze, N., Masliah, E. and Mucke, L. (2013). Age-appropriate cognition and subtle dopamine-independent motor deficits in aged tau knockout mice. Neurobiol Aging 34(6): 1523-1529. PubMed ID: 23332171

Papanikolopoulou, K., Grammenoudi, S., Samiotaki, M. and Skoulakis, E. M. C. (2018). Differential effects of 14-3-3 dimers on Tau phosphorylation, stability and toxicity in vivo. Hum Mol Genet. PubMed ID: 29659825

Passarella, D. and Goedert, M. (2018). Beta-sheet assembly of Tau and neurodegeneration in Drosophila melanogaster. Neurobiol Aging 72: 98-105. PubMed ID: 30240946

Prifti, E., Tsakiri, E. N., Vourkou, E., Stamatakis, G., Samiotaki, M. and Papanikolopoulou, K. (2020). The two Cysteines of Tau protein are functionally distinct and contribute differentially to its pathogenicity in vivo. J Neurosci. PubMed ID: 33334867

Prokop, A. (2013). The intricate relationship between microtubules and their associated motor proteins during axon growth and maintenance. Neural Dev 8: 17. PubMed ID: 24010872

Qiang, L., Yu, W., Andreadis, A., Luo, M. and Baas, P. W. (2006). Tau protects microtubules in the axon from severing by katanin. J Neurosci 26(12): 3120-3129. PubMed ID: 16554463

Ramesh Babu, J., Lamar Seibenhener, M., Peng, J., Strom, A. L., Kemppainen, R., Cox, N., Zhu, H., Wooten, M. C., Diaz-Meco, M. T., Moscat, J. and Wooten, M. W. (2008). Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration. J Neurochem 106(1): 107-120. PubMed ID: 18346206

Sapir, T., Frotscher, M., Levy, T., Mandelkow, E. M. and Reiner, O. (2012). Tau's role in the developing brain: implications for intellectual disability. Hum Mol Genet 21(8): 1681-1692. PubMed ID: 22194194

Scarpelli, E. M., Trinh, V. Y., Tashnim, Z., Krans, J. L., Keller, L. C. and Colodner, K. J. (2019). Developmental expression of human tau in Drosophila melanogaster glial cells induces motor deficits and disrupts maintenance of PNS axonal integrity, without affecting synapse formation. PLoS One 14(12): e0226380. PubMed ID: 31821364

Shim, K. H., Kim, S. H., Hur, J., Kim, D. H., Demirev, A. V. and Yoon, S. Y. (2019). Small-molecule drug screening identifies drug Ro 31-8220 that reduces toxic phosphorylated tau in Drosophila melanogaster. Neurobiol Dis: 104519. PubMed ID: 31233882

Sofola, O., Kerr, F., Rogers, I., Killick, R., Augustin, H., Gandy, C., Allen, M. J., Hardy, J., Lovestone, S. and Partridge, L. (2010). Inhibition of GSK-3 ameliorates Aβ pathology in an adult-onset Drosophila model of Alzheimer's disease. PLoS Genet 6(9): e1001087. PubMed ID: 20824130

Stewart, A., Tsubouchi, A., Rolls, M. M., Tracey, W. D. and Sherwood, N. T. (2012). Katanin p60-like1 promotes microtubule growth and terminal dendrite stability in the larval class IV sensory neurons of Drosophila. J Neurosci 32(34): 11631-11642. PubMed ID: 22915107

Talmat-Amar, Y., Arribat, Y. and Parmentier, M. L. (2018). Vesicular Axonal Transport is Modified In Vivo by Tau Deletion or Overexpression in Drosophila. Int J Mol Sci 19(3). PubMed ID: 29509687

Voelzmann, A., Okenve-Ramos, P., Qu, Y., Chojnowska-Monga, M., Del Caño-Espinel, M., Prokop, A. and Sanchez-Soriano, N. (2016). Tau and spectraplakins promote synapse formation and maintenance through Jun kinase and neuronal trafficking. Elife 5. PubMed ID: 27501441

Wen, X., An, P., Li, H., Zhou, Z., Sun, Y., Wang, J., Ma, L. and Lu, B. (2020). Tau Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS. Neurosci Bull. PubMed ID: 32500377

Zhang, B., Li, Q., Chu, X., Sun, S. and Chen, S. (2016). Salidroside reduces tau hyperphosphorylation via up-regulating GSK-3β phosphorylation in a tau transgenic Drosophila model of Alzheimer's disease. Transl Neurodegener 5: 21. PubMed ID: 27933142

Zhukareva, V., Vogelsberg-Ragaglia, V., Van Deerlin, V. M., Bruce, J., Shuck, T., Grossman, M., Clark, C. M., Arnold, S. E., Masliah, E., Galasko, D., Trojanowski, J. Q. and Lee, V. M. (2001). Loss of brain tau defines novel sporadic and familial tauopathies with frontotemporal dementia. Ann Neurol 49(2): 165-175. PubMed ID: 11220736

date revised: 20 September 2025

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