Introduction of macromolecules into synaptosomes using electroporation

Synaptic terminals are sites of high metabolic activity and thus are partic ularly vulnerable to oxidative stress. Oxidative damage to proteins can be toxic to neurons and may cause irreversible cell damage and neurodegenerati on. A neuroprotective mechanism used by cells to combat oxidative damage is to selectively degrade damaged proteins. Therefore, it is of interest to s tudy the mechanism of degradation of oxidatively damaged proteins in synapt osomes. One way of oxidizing: synaptosomal proteins in vitro is by incubati ng intact synaptosomes in the presence of an oxidizing agent. A problem wit h this approach is that it may also cause oxidative damage to the machinery required to recognize and degrade oxidized proteins. We have, therefore, i ntroduced a fluorescent macromolecule into synaptosomes to assess the feasi bility of using this technique to study how oxidized proteins are degraded and removed from synaptic terminals. Synaptosomes were subjected to electro poration in the presence of FITC labelled-dextran with an average molecular weight of 70 000 (FD-70) and non-specific binding was determined by runnin g parallel experiments in lysed synaptosomes. Following extensive washing, synaptosomes were assayed for the presence of intra-synaptosomal FD-70 by m easuring fluorescence in a microplate fluorescence reader. Significant diff erences in fluorescence were found between intact and lysed synaptosomes wi th maximal uptake at 100 V/ 1500 mu F (approx. 36 pmol/mg protein). To dete rmine if membrane transport was compromised by electroporation, uptake of H -3-arginine was compared in control and electroporated synaptosomes. While untreated electroporated synaptosomes showed a loss of 22% in the ability t o transport arginine, preincubation in the presence of 1 mM ATP resulted in a complete restoration of arginine transport. These results show that elec troporation is a potentially useful technique for introducing a specific ox idized protein, into synaptic terminals so its metabolic fate can be examin ed. (C) 2000 Elsevier Science B.V. All rights reserved.