Khattar Nicolas K, Yablonska Svitlana, Baranov Sergei V, Baranova Oxana V, Kretz Eric S, Larkin Timothy M, Carlisle Diane L, Richardson R Mark, Friedlander Robert M
Neuroapoptosis and Translational Therapeutics Laboratory, Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, PA, United States.
Brain Modulation Laboratory, Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, PA, United States; University of Pittsburgh Medical Center, Pittsburgh, PA, United States.
J Neurosci Methods. 2016 Apr 1;263:1-6. doi: 10.1016/j.jneumeth.2016.01.017. Epub 2016 Jan 22.
Functional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available.
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.
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.
This method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants.
线粒体的功能和结构特性高度依赖于组织和细胞，但目前尚无法分离出高度纯化的人神经元线粒体。
我们开发并验证了一种从脑组织中分离纯化神经元线粒体的方法。该方法将使用Percoll梯度离心法获得突触体组分与通过氮空化介导的突触体破坏以及使用与磁珠偶联的抗线粒体外膜蛋白抗体提取线粒体相结合。分离的最终产物是非突触体线粒体，它是从不同脑细胞（即神经元、星形胶质细胞、少突胶质细胞、小胶质细胞）分离的线粒体的混合物，以及源自神经元的突触线粒体。该方法非常适合从手术提取的人皮质组织中制备功能性线粒体。
该方法产生的线粒体具有最小的细胞质污染，基于线粒体呼吸以及线粒体蛋白导入的测量，这些线粒体具有功能活性。分离人神经元线粒体的过程大约需要四个小时，也可用于从小鼠/大鼠/猴脑中分离线粒体。
该方法将使研究人员能够研究高度富集的神经元线粒体，而不受细胞和细胞器污染物的混杂影响。
