Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue
Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue
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DOI：
10.1038/s41598-019-45568-3
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发表时间：
2019-07-04
影响因子：
4.6
通讯作者：
Sullivan, Patrick G.
中科院分区：
文献类型：
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作者：
Hubbard, W. Brad;Harwood, Christopher L.;Sullivan, Patrick G.
虽然线粒体维持着重要的细胞功能，例如能量产生、钙稳态和调节程序性细胞死亡，但它们也在许多神经系统疾病的病理生理学中发挥着重要作用。此外，一些神经退行性疾病与突触损伤和突触线粒体功能障碍密切相关。不幸的是，在神经退行性疾病和中枢神经系统损伤的实验模型中评估线粒体功能障碍和线粒体靶向治疗的功效的能力受到当前线粒体分离技术的限制。密度梯度超速离心 (UC) 是目前唯一可以分离突触和非突触线粒体亚群的技术，但由于线粒体产量低而无法分析小脑区域。为了解决这一限制，我们使用分级线粒体磁分离 (FMMS)，采用磁性抗 Tom22 抗体，开发了一种新策略，在不使用 UC 的情况下从小鼠皮层和海马中分离功能性突触和非突触线粒体。我们将使用 FMMS 获得的线粒体与通过 UC 获得的线粒体的产量和功能进行了比较。与 UC 相比，在相同数量的小鼠海马组织中，FMMS 产生的突触线粒体蛋白产量高出 3 倍。与 UC 分离相比，FMMS 测量线粒体呼吸减少的灵敏度也有所提高，这在轻度闭合性头部损伤的范例中得到了证明。总而言之，FMMS 能够提高脑源性线粒体产量，用于线粒体评估，并更好地检测中枢神经系统损伤和神经退行性疾病中的线粒体损伤。
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.