Bioorthogonal tag: A modern way to visualize polyaminated proteins in hippocampus

Although essential for protein structure, stability and function, polyamination is still poorly explored posttranslational modification. In the brain, polyamines (putrescine, spermidine and spermine), are involved in a variety of neuronal processes, including neuronal development, neurotransmission, neuroprotection and synaptic plasticity, but it remains to be identified what is the role of polyamination in these mechanisms.Hippocampal pyramidal neurons exhibit specific properties, depending on the subregion. Those found in the subregion CA1 possess plastic synapses and are very sensitive to excitotoxicity, when those in CA2 – are resistant to LTP/LTD induction and to excitotoxic cell damage. The molecular bases of these characteristics are not well known, but regional differences in protein polyamination may be a possible mechanism contributing to this heterogeneity.Here, we employed mouse hippocampus acute sections as an ex vivo model for imaging studies of polyamination profiles at regional, cellular and subcellular levels, using a novel bioorthogonal putrescine probe (TvS-Put). Following entry to the cell, TVS-PUT can be used by cellular transglutaminase 2 for protein polyamination, like endogenous, native putrescine. Then, we label TvS-Put-modified proteins in situ by attaching a fluorochrome to the probe in a specific reaction. We also show here that labeling the tissue with TVS-PUT can be combined with immunofluorescence, allowing precise localization of TVS-PUT-labeled proteins. These results show that TVS-PUT may be an efficient and versatile tool that will allow to understand better the crucial aspects of yet poorly studied process of polyamination in physiology and pathology in individual cells, regions and tissue types.