Isolation of functionally active and highly purified neuronal mitochondria from human cortex.
BACKGROUND
Functional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available.


NEW METHOD
We 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.


RESULTS
The 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 CONCLUSIONS
This method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants.
['Khattar|Nicolas K|NK|', 'Yablonska|Svitlana|S|', 'Baranov|Sergei V|SV|', 'Baranova|Oxana V|OV|', 'Kretz|Eric S|ES|', 'Larkin|Timothy M|TM|', 'Carlisle|Diane L|DL|', 'Richardson|R Mark|RM|', 'Friedlander|Robert M|RM|']
D000906:Antibodies D002458:Cell Fractionation D002540:Cerebral Cortex D006680:HLA Antigens D006801:Humans D053078:Membrane Potential, Mitochondrial D008928:Mitochondria D033681:Mitochondrial Membrane Transport Proteins D000090622:Mitochondrial Precursor Protein Import Complex Proteins D024101:Mitochondrial Proteins D009474:Neurons D013574:Synaptosomes
Isolation Functional Purified Neurons Mitochondria Human cortex Percoll Synaptosome Non-synaptosomal mitochondria Differential centrifugation