Journal article
Dual-process brain mitochondria isolation preserves function and clarifies protein composition
Proceedings of the National Academy of Sciences - PNAS, Vol.118(11), e2019046118
03/16/2021
DOI: 10.1073/pnas.2019046118
PMCID: PMC7980376
PMID: 33836587
Abstract
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.
Details
- Title: Subtitle
- Dual-process brain mitochondria isolation preserves function and clarifies protein composition
- Creators
- Maria F Noterman - Interdisciplinary Graduate Program in Neuroscience, University of Iowa, Iowa City, IA 52242Kalyani Chaubey - Department of Psychiatry, Case Western Reserve University, Cleveland, OH 44106Kristi Lin-Rahardja - Department of Systems Biology and Bioinformatics, Case Western Reserve University, Cleveland, OH 44106Anjali M Rajadhyaksha - Pediatric Neurology, Pediatrics, Weill Cornell Medicine of Cornell University, New York, NY 10065Andrew A Pieper - Department of Neuroscience, School of Medicine, Case Western Reserve University, Cleveland, OH 44106Eric B Taylor - Iowa Neuroscience Institute, University of Iowa, Iowa City, IA 52242
- Resource Type
- Journal article
- Publication Details
- Proceedings of the National Academy of Sciences - PNAS, Vol.118(11), e2019046118
- DOI
- 10.1073/pnas.2019046118
- PMID
- 33836587
- PMCID
- PMC7980376
- NLM abbreviation
- Proc Natl Acad Sci U S A
- ISSN
- 0027-8424
- eISSN
- 1091-6490
- Publisher
- National Academy of Sciences; United States
- Grant note
- F31 NS106773 / NINDS NIH HHS R01 DK104998 / NIDDK NIH HHS R01 NS084190 / NINDS NIH HHS P30 CA086862 / NCI NIH HHS R01 DA029122 / NIDA NIH HHS
- Language
- English
- Date published
- 03/16/2021
- Academic Unit
- Molecular Physiology and Biophysics; Psychiatry; Fraternal Order of Eagles Diabetes Research Center
- Record Identifier
- 9984071794902771
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