Isolation of functionally active and highly purified neuronal mitochondria from human cortex

• Combined Percoll gradient centrifugation and anti-TOM22 magnetic bead extraction is used to isolate mitochondria.
• The mitochondria obtained are neuronal (from synaptosome disruption).
• The mitochondria obtained have minimal cytoplasmic contaminants (plasma membrane, peroxisomes, lysosomes, synaptosomes, endoplasmic reticulum).
• The mitochondria are functionally active based on measurements of respiration and protein import.

BackgroundFunctional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available.New methodWe developed and validated a procedure to isolate purified neuronal mitochondria from brain tissue. The method combines Percoll gradient centrifugation to obtain synaptosomal fraction with nitrogen cavitation mediated synaptosome disruption and extraction of mitochondria using anti mitochondrial outer membrane protein antibodies conjugated to magnetic beads. 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. This method is well suited for preparing functional mitochondria from human cortex tissue that is surgically extracted.ResultsThe procedure produces mitochondria with minimal cytoplasmic contaminations that are functionally active based on measurements of mitochondrial respiration as well as mitochondrial protein import. The procedure requires approximately four hours for the isolation of human neuronal mitochondria and can also be used to isolate mitochondria from mouse/rat/monkey brains.Comparison with existing methods and conclusionsThis method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants.