Analysis of synaptic Apolipoprotein E localization by immunofluorescence microscopy and flow cytometry

• Main Authors: Yi-Fong Zou, 鄒怡鳳
• Other Authors: Hwan-Ching Tai
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/40262310065761359382

碩士 === 國立臺灣大學 === 化學研究所 === 104 === Alzheimer’s disease (AD), a protein misfolding disorder of beta amyloid (Aβ) and tau, accounts for > 50% of cases of senile dementia. AD is a neurodegenerative disease characterized by the loss of neurons and synapses. Loss of synapses in the neocortex and limbic system correlates strongly with cognitive impairment, more than other known pathological markers. The ApoE ε4 allele is known to be a very strong genetic risk factor for developing AD. ApoE4 increases the level of Aβ oligomers in the brain and is thought to initiate neurotoxic events that lead to synaptic dysfunction. Direct visualization of intact synaptic terminals is one of our approaches to investigate the distribution of ApoE. We developed a novel high-throughput method for analyzes pre- and post-synaptic terminals by flow cytometry, as well as direct capturing of synaptosomes followed by fluorescence imaging. Our new strategy to capture synapses is based on electrostatic attraction. Synaptosomes surfaces are negatively charged, caused by the presence of negatively charged phospholipids, protein side chains, and glycans already present on the plasma membrane. We also modified synaptic surfaces with ethylene glycol bis(succinimidyl succinate) (EGS), a crosslinker that contains two amine-reactive NHS esters and a 12-atom spacer, which serves two important functions. EGS may crosslink two amine groups on different proteins to achieve synaptosome fixation. Alternatively, EGS may increase the net negative charge by neutralizing a single amine group on the synapse surface, and later imparting a negative charge after the free NHS ester is hydrolyzed. After EGS modification, synaptosomes thus carry high negative charge density. EGS-treated synaptosomes are easily captured via electrostatic attraction over amine-derivatized glass surfaces treated with (3-aminopropyl)triethoxysilane (APTES). Surfaced-attached synaptosomes can be immunostained under gentle conditions just like fixed cells and imaged by fluorescence microscopy or super-resolution optical microscopy. This capture strategy enables detailed analysis of brain synapses with minimal tissue samples. The negative surface charge also prevents synaptosome aggregation and centrifugation damages in flow cytometry protocols. This allows us to immunostain synaptosomes with primary and secondary antibodies just like ordinary cells before flow cytometry and sorting. Using a combination of flow cytometry and single-synapse image analysis, we determined ApoE as being localized to both pre- and post-synaptic terminals. It is a fairly abundant protein at synapses, present in over half of the synaptic terminals by either characterization method. Therefore it is very likely that the synapse is one of the critical sites of interaction between ApoE and Aβ, but the synaptic function of ApoE is still poorly understood.