Isolation of highly purified and functionally active synaptic mitochondria

Abstract

Isolation of synaptic mitochondria is an important step in assessing synaptic bioenergetics. Purity and functional activity of mitochondria are important to characterize the mitochondrial proteome and determine the contribution of intrinsic factors to mitochondrial activity. We have developed and validated an isolation protocol to obtain highly purified synaptosomal mitochondria from brain tissue. The method combines Percoll step gradient centrifugation to obtain the synaptosomal fraction and nitrogen cavitation to release neuronal mitochondria and using anti-mitochondrial outer membrane protein antibodies conjugated to magnetic nanobeads to extract the mitochondria. The final products of isolation are non-synaptosomal mitochondria, which are a mixture of mitochondria isolated from different brain cells (i.e., neurons, astrocytes, oligodendrocytes, microglia) and synaptic mitochondria, which are of neuronal origin. The procedure produces mitochondria that (1) have minimal cytoplasmic contamination and (2) are functionally active based on measurements of mitochondrial matrix protein import; the process depends on the mitochondrial integrity and presence of high membrane potential. The procedure requires approximately 4 h for the isolation of synaptosomal mitochondria and can be used to isolate mitochondria from other species, including rat, monkey, and human brain tissue as well. This method allows researchers to study disease implications on highly enriched synaptosomal mitochondria without the confounding effect of cellular and organelle contaminants.