| Abstract No.: | B-C2114 |
| Country: | Canada |
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| Title: | ROLE OF TAU IN THE ENDOCYTIC PATHWAY ALONG THE AXON |
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| Authors/Affiliations: | 2 Grégoire MORISSE*; 2 Johanne BERTRAND; 1 Jenny KING; 2 Roger LIPPÉ; 1 Deborah ANDERSON; 2 Nicole LECLERC;
1 Cancer Research Unit, University of Saskatchewan, Saskatoon, SK, Canada; 2 Département of Pathology and Cell Biology, University of Montréal, QC, Canada
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| Content: | Objectives: Tau is a microtubule asociated protein enriched in the axon. In Alzheimer Disease (AD), tau becomes hyperphosphorylated, detaches from microtubules and accumulates in the somato-dendritic compartment. The role of tau in the axon remains elusive. To elucidate the role of tau, we established a strategy to identify the proteins interacting with it. We found that tau can interact with Rab5a, a GTPase implicated in the fusion of early endosomes.
Materials and Methods: Tau and Rab5 proteins were fused to a GST tag, were expressed and purified from bacteria using affinity chromatography or a heat stable method for tau. The interaction between Rab5 and tau was detected by a blot-overlay assay and affinity chromatography. The Rab5 GAP activity assay was performed in the laboratory of Deborah Anderson as described previously. The presence of tau and Rab5 on the same endosomal vesicles found in a synaptosomal fraction isolated from mice adult brain was revealed by immuno-isolation experiments using an anti-tau antibody bound to magnetic beads. Primary hippocampal cultures were transfected with GFP-tau and RFP-Rab5a using lipofectamine 2000 to visualize their co-localization in vivo using fluorescence microscope or confocal microscope. The transfected neurons were stained with the endosomal markers, EEA1 and Rab7.
Results: We show that tau can interact directly with Rab5a using purified proteins. The C-terminal domain of tau (aa 205-383) is sufficient for the interaction to be detected. In hippocampal neurons overexpressing GFP-tau and RFP-Rab5a, a significant overlap between the two proteins was observed in the axon. Tau shows higher affinity towards Rab5a-GTP than Rab5a-GDP indicating that tau might be GTPase Activating Protein (GAP protein). GAP proteins inactivate Rab5 and thereby the fusion of the early endosomes. The GAP activity of tau was confirmed using an in vitro assay developed by the laboratory of Deborah Anderson. Consistent with this idea, in hippocampal neurons, the overexpression of tau resulted in a reduction of the size of Rab5-positive endosomes. This was also correlated to a decreased recruitment of EEA1 (Early Endosome Antigen 1), a Rab5 effector, to early endosomes and to an important decrease of rab7 protein levels, a marker of late endosomes.
Conclusion: Our results indicate that tau could play a central role in the endocytic pathway along the axon by negativly regulating Rab5a. This endocytic pathway is involved in the retrograde transport of neurotrophins, factors involved in neuronal survival, from the pre-synaptic terminal to the cell body. Therefore, the relocalization of tau from the axon to the somato-dendritic compartment in neurodegenerative diseases such as AD could lead to an endocytic deficit of neurotrophins and ultimately to neuronal cell death.
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