Analysis of synaptic tau protein localization by immunofluorescence microscopy and flow cytometry
Date Issued
2014
Date
2014
Author(s)
Wang, Boyao
Abstract
The accumulation of neurofibrillary tangles in Alzheimer disease (AD) propagates with characteristic spatiotemporal patterns following brain network connections, which may imply trans-synaptic transmission of tauopathy. A prerequisite of this transmission theory would require misfolded tau to accumulate at synapses—i.e. that it be present in both pre- and post-synaptic locations. However, tau is thought to normally be primarily an axonal protein, and it is widely hypothesized that tau is mislocalized or mistrafficked to the neuronal somatodendritic compartment in AD. We isolated intact, bipartite synapses from cortical tissues of AD subjects and detected misfolded tau by immunofluorescence microscope with a distribution ratio of 15.4%:16.4%:2.9% (presynaptic-only/postsynaptic-only/both), while hyperphosphorylated tau exhibited a ratio of 23.1%:26.9%:3.8%; total tau (any form) exhibited a ratio of 34.6%:47.1%:3.8%. Non-demented controls showed total tau distribution similar to that of AD subjects, but with little phosphorylation or misfolding. In AD-affected synapses, we observed tau misfolded into SDS-resistant oligomers. Thus tau appears not to be mislocalized, but instead adopts misfolded, oligomeric, and phosphorylated forms within both pre- and post-synaptic sites. Based on these observations, we propose two models for the transmission of misfolded tau at synapses. We are also develop a high throughput method to analyze tau protein localization in synaptic terminals. We analyzed synaptosomes of mouse brains and immunostaining for pre- and post-synaptic terminals with synaptophysin and PSD-95 antibody. We found 35.2% and 33.5% of total particles to be pre- and post-synaptic terminals, respectively consistent with immunofluorescence counting under the microscope.
Subjects
神經突觸
tau蛋白
螢光顯微鏡
流式細胞術
阿茲海默症
Type
thesis
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