Over-expression of tau results in defective synaptic transmission in Drosophila neuromuscular junctions
Introduction
Tauopathies are a group of neurodegenerative diseases that include Alzheimer's disease (AD) and the fronto-temporal dementias (FTD). These diseases are characterised by aggregation of the microtubule associated protein tau into neurofibrillary tangles, which are the pathological hallmarks of tauopathies (Goedert et al., 1997 and for a review Lee et al., 2001). Although it is now well established that abnormalities in tau play a central role in tauopathies, the mechanisms underlying tau-mediated neuronal dysfunction are unclear. Drosophila has become an established model system for analysing the cellular and molecular mechanisms that underlie a variety of neurodegenerative diseases. In particular, Drosophila has made significant contributions to our understanding of the pathogenic events that underlie tau-associated neurodegeneration (Lee et al., 2005). Williams et al. (2000) showed that the over-expression of tau in Drosophila sensory neurons produces progressive neurodegeneration in the absence of filamentous aggregates. This work was confirmed and extended by the demonstration that over-expression of mutant tau also induces neurodegeneration in Drosophila without the formation of neurofibrillary aggregates (Wittmann et al., 2001) and that hyperphosphorylation of tau not only exacerbates the neurodegeneration caused by tau but also stimulates filament and tangle formation in Drosophila (Jackson et al., 2002). These findings demonstrate that although hyperphosphorylation, and possibly filament formation can exacerbate tau-mediated neurodegeneration, over-expression of tau alone is sufficient to cause neurodegeneration.
To examine the mechanisms of tau-mediated neurodegeneration in a defined neuronal population, we used a GAL4 line (Brand and Perrimon, 1993) to target human tau expression in motor neurons in Drosophila. This provides a powerful system for analysing the consequences of tau over-expression on neuronal morphology, physiology and biochemistry at the level of single identified neurons. Using this system, we have shown that over-expression of tau disrupts axonal transport causing transport vesicles to aggregate and produces a decline of locomotor function (Mudher et al., 2004). We also showed that co-expression of a constitutively active form of the tau-kinase glycogen synthase kinase 3 beta (GSK-3β) enhances, and that inhibitors of GSK-3β, ameliorate the development of the tau phenotype. This suggests that the detrimental effects of tau are phosphorylation dependent. Furthermore, these detrimental effects were seen in the absence of neuronal death indicating that abnormalities in tau are sufficient to cause neuronal dysfunction. We have used our Drosophila model to dissect the mechanism of tau mediated neuronal dysfunction, which is not well understood. The impaired locomotion caused by over-expression of tau in motor neurons (Mudher et al., 2004) suggested that a likely cause of this deficit could be due to abnormal functioning of the neuromuscular junction (NMJ). To test this, we have investigated the effects of over-expression of tau on NMJ function and examined the effects of tau on the morphology and physiology of this synapse.
We now report that over-expression of tau induces a dramatic defect in synaptic transmission at the Drosophila NMJ, which was seen in the absence of filament formation or synaptic loss. These results begin to elucidate the mechanisms by which tau abnormalities disrupt neuronal function in tauopathies.
Section snippets
Culture and fly stocks
Flies were raised at 23°C on Bloomington agar based media. All experiments were done using the GAL4D42 line (Yeh et al., 1995) to drive expression of GAL4 responder lines in motor neurons (Duffy, 2002). Three UAS responder lines were used: (1) A UAS-GFP labelled neuropeptide Y fusion protein recombined onto the same chromosome as the GAL4D42 activator (Mudher et al., 2004). This line was called vGFP (vesicular GFP). (2) UAS human 0N3R-tau responder line (Williams et al., 2000). (3) A UAS-GFP
The morphology of neuromuscular junctions over-expressing tau are abnormal
Confocal examination of the NMJ on muscle 4 using anti HRP revealed that all the NMJs present in the wild type larvae were also present in the tau over-expressing larvae indicating the over-expression of tau did not cause neuronal loss. However, the NMJs of tau over-expressing motor neurons are abnormal (Fig. 1). First, the tau over-expressing NMJ appeared smaller than wild type (Figs. 1A and B). Quantification of NMJ size showed that NMJs over-expressing tau were significantly (P < 0.01)
Discussion
Abnormalities in tau protein play a central role in neurodegenerative diseases such as AD and FTD. The mechanism by which tau mediates neuronal dysfunction in these diseases is unclear. We present here direct evidence that over-expression of human tau in Drosophila motor neurons has a deleterious effect on the function of the synapse in vivo. Over-expression of the human 0N3R tau isoform disrupts: NMJ morphology, synaptic vesicle recycling, synaptotagmin distribution, electrophysiology of
Conclusion
It is now well established that tau plays a central role in neuronal dysfunction in neurodegenerative diseases but the mechanism by which it does is still not well understood. To explore the mechanism of tau mediated dysfunction, we simulated over-expression of tau in Drosophila and we have previously reported that this leads to disruption of axonal transport and behavioural defects (Mudher et al., 2004). We now report that these transgenic flies also exhibit a dramatic defect in synaptic
Acknowledgments
Supported by the Welcome Trust. We thank H. Perry and P. Newland for critical comments on the manuscript; and I. Robinson and J. Drummond for supplying the UAS-vGFP line, H. Bellen and K. Schulze at the Howard Hughes Medical Institute, Baylor College of Medicine for supplying the anti-synaptotagmin I (DSYT2), E. Buchner at Biozentrum for supplying the nc82.
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