Surface charge manipulation and electrostatic immobilization of synaptosomes for super-resolution imaging: a study on tau compartmentalization

Abstract

Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

Hyperphosphorylated Human Tau Accumulates at the Synapse, Localizing on Synaptic Mitochondrial Outer Membranes and Disrupting Respiration in a Mouse Model of Tauopathy

“…Our findings lend support to the role of non-mutant human tau in synaptic dysfunction in AD. However, a recent report using super-resolution microscopy of fixed synaptosomes from WT (C57BL/6) mice observed significant colocalization of non-mutant murine tau with synaptophysin (marking pre-synaptic terminals) ( Bhattacharya et al, 2021 ). Interestingly, the authors noted tau also colocalized with the post-synaptic terminal marker protein post-synaptic density protein 95 (PSD-95), suggesting tau accumulates in both compartments of the synapse ( Bhattacharya et al, 2021 ).…”

“…However, a recent report using super-resolution microscopy of fixed synaptosomes from WT (C57BL/6) mice observed significant colocalization of non-mutant murine tau with synaptophysin (marking pre-synaptic terminals) ( Bhattacharya et al, 2021 ). Interestingly, the authors noted tau also colocalized with the post-synaptic terminal marker protein post-synaptic density protein 95 (PSD-95), suggesting tau accumulates in both compartments of the synapse ( Bhattacharya et al, 2021 ). Subcellular fractionation analysis from mutant tau P 301 L mice showed that both murine tau and the human tau P 301 L were present in the synaptosomal fraction; however, tau P 301 L was preferentially distributed in the synaptosomal fraction while murine endogenous tau was more distributed in the cytosolic fraction ( Sahara et al, 2014 ).…”