| Abstract No.: | C-B3041 |
| Country: | Canada |
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| Title: | INTERSECTIN REGULATES SOMATODENDRITIC BUT NOT SYNAPTIC VESICLE ENDOCYTOSIS IN HIPPOCAMPAL NEURONS |
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| Authors/Affiliations: | 1 Sébastien Thomas, Ph.D.*; 1 Brigitte Ritter; 1 Claire Sanson; 1 Claire Sanson; 1 Lyne Bourbonnière; 1 Peter S. McPherson; 1 Peter S. McPherson
1 Montreal Neurological Institute, QC, Canada
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| Content: | Objective: Intersectin (ITSN) is a multi-modular protein that binds numerous endocytic accessory proteins, including dynamin and synaptojanin. ITSN has been shown to regulate constitutive and regulated forms of endocytosis in non-neuronal cells and synaptic vesicle recycling at the Drosophila neuromuscular junction. In mammals, ITSN is alternatively spliced to produce either ITSN short (equivalent to the Drosophila ortholog) or ITSN long. Whereas ITSN short is ubiquitously expressed, ITSN long is exclusively expressed in neurons. This specific tissue distribution argues for a role of ITSN in synaptic vesicle recycling in mammalian neurons but this has never been directly assessed. We thus sought to investigate the role of ITSN in mammalian neurons.
Materials and Methods: Experiments were performed on neurons from primary hippocampal cultures. To knock-down the expression of ITSN, we designed miRNAs and delivered them into neurons through standard transfection or viral-mediated transduction. To determine the efficiency of the miRNAs, we used immunofluorescence and western blot analysis of cell lysates from infected cultures.
The cellular localization of ITSN was studied using confocal imaging and immunofluorescence. Synaptic vesicle recycling was measured using activity-dependent staining of synaptic vesicle membrane using FM4-64, a fluorescent membrane marker. Transferrin endocytosis was monitored using Alexa Fluor-546 transferrin conjugate and live-cell, time-lapse confocal imaging.
Results: Immunofluorescence revealed that ITSN was predominantly distributed in the somatodendritic regions of neurons. More detailed analysis using endocytic and synaptic markers indicated that ITSN colocalized with AP-2 and chlatrin heavy chain at cell bodies and dendrites. ITSN was also present near dendritic spines and occasionnaly colocalized with PSD-95. ITSN was undetectable at synaptic terminals labeled by synaptophysin. Moreover, ITSN knock-down did not affect synaptic vesicle recycling. Instead, ITSN regulated somato-dendritic uptake of transferrin in neurons: ITSN knock-down greatly reduced transferrin uptake rate.
Conclusion: In conclusion, we show that, in mammalian neurons, ITSN regulates constitutive endocytosis in the somato-dendritic region of neurons and plays no role in the regulated endocytosis taking place during synaptic vesicle recycling in axon terminals. |
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