The application of amine-reactive crosslinking reagents in synaptosome research

• Main Authors: Jia-Fong Jhou, 周家鋒
• Other Authors: Hwan-Ching Tai
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/qf6372

碩士 === 國立臺灣大學 === 化學研究所 === 105 === The synapse plays important roles in the pathogenesis and pathology of many neurological diseases, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, etc. In recent years, there is growing interest in immunocytochemistry studies of isolated synaptosomes, because it provides the ability to observe the subtle biochemical changes by eliminating signals from the shaft of dendrites and axons. To use immunofluorescence to study synaptosomes, chemical crosslinking of synaptic proteins is necessary. Commonly, formaldehyde has been used as the fixative to for crosslinking, but it has the apparent drawback of causing synaptosome aggregation. Here, we used amine-reactive crosslinking agents instead of formaldehyde, thereby improving the results in immunofluorescence microscopy and flow cytometry. Furthermore, we are able to develop a new strategy for studying synaptic transcriptomics. We accidentally discovered that crude synaptosomes adhered to positively-charged glass surfaces without any additional manipulation, due to electrostatic interaction. Acidic glycans (e.g. sialic acids), acidic protein side chains (Asp and Glu), and negatively charged phospholipids give synaptic surface a negative Zeta potential which is -15.0 ± 0.4 mV. We used ethylene glycol bis(succinimidyl succinate) (EGS) as an alternative to formaldehyde, which has two N-hydroxysuccinimidyl (NHS) esters that react with lysines and promote protein crosslinking. EGS can increase the negative charge of synaptic surfaces when combined with glycine quenching, thereby reducing synaptosomal aggregation and enhancing attraction between the synaptosomes and the glass surface. Based on these principles, we devised a very simple and effective method for fixing and capturing synaptosomes for immunofluorescence studies. We can carry out three-channel super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to simultaneously image tau, synaptophysin (pre-synapse) and PSD-95 (post-synapse). In studying the transcriptome of synaptosomes, we chose dithiobis(succinimidyl propionate) (DSP) as the fixative due to its reversible nature. DSP also enhances negative surface charges of synaptosomes to prevent synaptosome aggregation in the flow cytometry and sorting protocol. DSP has a cleavable disulfide bond in its spacer arm, which makes fixation reversible. Thus, DSP fixation way allow mRNAs to be isolated from sorted synaptosomes after reducing disulfide bonds.