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Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue.

ABSTRACT: While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

SUBMITTER: Hubbard WB

PROVIDER: S-EPMC6609636 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue.

Hubbard W Brad WB Harwood Christopher L CL Prajapati Paresh P Springer Joe E JE Saatman Kathryn E KE Sullivan Patrick G PG

Scientific reports 20190704 1

While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neu ...[more]

PMID: 31273236

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Project description:The brain requires continuously high energy production to maintain ion gradients and normal function. Mitochondria critically undergird brain energetics, and mitochondrial abnormalities feature prominently in neuropsychiatric disease. However, many unique aspects of brain mitochondria composition and function are poorly understood. Developing improved neuroprotective therapeutics thus requires more comprehensively understanding brain mitochondria, including accurately delineating protein composition and channel-transporter functional networks. However, obtaining pure mitochondria from the brain is especially challenging due to its distinctive lipid and cell structure properties. As a result, conflicting reports on protein localization to brain mitochondria abound. Here we illustrate this problem with the neuropsychiatric disease-associated L-type calcium channel Cav1.2α1 subunit previously observed in crude mitochondria. We applied a dual-process approach to obtain functionally intact versus compositionally pure brain mitochondria. One branch utilizes discontinuous density gradient centrifugation to isolate semipure mitochondria suitable for functional assays but unsuitable for protein localization because of endoplasmic reticulum (ER) contamination. The other branch utilizes self-forming density gradient ultracentrifugation to remove ER and yield ultrapure mitochondria that are suitable for investigating protein localization but functionally compromised. Through this process, we evaluated brain mitochondria protein content and observed the absence of Cav1.2α1 and other previously reported mitochondrial proteins, including the NMDA receptor, ryanodine receptor 1, monocarboxylate transporter 1, excitatory amino acid transporter 1, and glyceraldehyde 3-phosphate dehydrogenase. Conversely, we confirmed mitochondrial localization of several plasma membrane proteins previously reported to also localize to mitochondria. We expect this dual-process isolation procedure will enhance understanding of brain mitochondria in both health and disease.

Dataset Information

Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue.

Publications

Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue.

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