Dalton Cardiovascular Research Center, University of Missouri-Columbia.
Dalton Cardiovascular Research Center, University of Missouri-Columbia; Department of Biomedical, Biological and Chemical Engineering, University of Missouri-Columbia.
J Vis Exp. 2022 Jan 22(179). doi: 10.3791/62917.
Mitochondrial Ca plays a critical role in controlling cytosolic Ca buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca signaling, as well as astrocyte-neuron interactions.
线粒体钙在控制细胞溶质钙缓冲、能量代谢和细胞信号转导中起着关键作用。线粒体钙超载导致各种病理状况，包括神经退行性疾病中的神经变性和凋亡性细胞死亡。在这里，我们提出了一种细胞类型特异性和线粒体靶向的分子方法，用于在体外和体内研究星形胶质细胞和神经元中的线粒体钙成像。我们构建了编码线粒体靶向基因编码钙指示剂（GECI）GCaMP5G 或 GCaMP6s（GCaMP5G/6s）的 DNA 质粒，这些质粒带有星形胶质细胞和神经元特异性启动子 gfaABC1D 和 CaMKII 以及线粒体靶向序列（mito-）。对于体外线粒体钙成像，将质粒转染到培养的星形胶质细胞和神经元中以表达 GCaMP5G/6s。对于体内线粒体钙成像，制备腺相关病毒载体（AAV）并注射到小鼠大脑中，以在星形胶质细胞和神经元中的线粒体中表达 GCaMP5G/6s。我们的方法提供了一种有用的手段，可以对星形胶质细胞和神经元中的线粒体钙动态进行成像，以研究细胞溶质和线粒体钙信号之间的关系，以及星形胶质细胞-神经元相互作用。
