An improved procedure for isolation of functional synaptosomes for the transient generation of cybrids from frozen human brain

Abstract

Cybrids are cell lines derived from the fusion of mtDNA‐depleted cells with cytoplasts or enucleated cells. To date, there are no reports of cybrid fusions from frozen human tissue. In this study, we developed a new method of synaptosome isolation, the “Frozen Brain Synaptosomes” or FBS method. Synaptosomes were isolated from fresh mouse, frozen mouse and frozen human brain by centrifugation in an iodixanol gradient. The effectiveness of our FBS method was compared with current synaptosome isolation procedures that have been utilized to make cybrid cell lines from fresh tissue. The FBS method yielded the most structurally intact synaptosome preparation from frozen tissue as judged by electron microscopy. We assessed cytochrome c oxidase and citrate synthase activity, phospho‐synapsin‐1 and total ATP levels. The FBS method yielded the greatest enrichment of intact synaptosomes, high protein, mitochondrial enzyme activity and ATP levels, and functional synaptosomes as defined by their indirect response to a phosphatase inhibitor okadaic acid (OKA). We expected that the synaptosomes isolated from frozen postmortem brain would be sufficient in quantity and quality to generate cybrid cells. From ten fusion experiments, we concluded that two putative cybrid cell lines contained the exogenous synaptosomal mitochondria based upon mtDNA sequence analysis. Research support was from the Virginia Center on Aging.