Fluorescent pulse-chase labeling to monitor long-term mitochondrial degradation in primary hippocampal neurons

• Published in: STAR protocols 2022-12, Vol.3 (4), p.101822-101822, Article 101822
• Main Authors: Schneider, Jordan R., Evans, Chantell S.
• Format: Article
• Language: English
• Subjects: Animals; Cell Biology; Cell culture; Cell-based Assays; Coloring Agents - metabolism; Hippocampus - metabolism; Metabolism; Microscopy; Mitochondria - metabolism; Mitophagy; Molecular Biology; Molecular/Chemical Probes; Neurons - metabolism; Neuroscience; Protocol; Rats
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101822

The accumulation of dysfunctional mitochondria is a hallmark of neurodegenerative diseases, yet the dynamics of mitochondrial turnover in neurons are unclear. Here, we describe a protocol to monitor the degradation of spectrally distinct, “aged” mitochondrial populations. We describe the preparation and transfection of primary rat hippocampal neuron cultures. We detail a mitochondrial-damaging assay, a SNAP pulse-chase labeling paradigm, and live imaging to visualize the mitochondrial network. Finally, we provide steps to quantify mitochondrial turnover via lysosomal fusion. For complete details on the use and execution of this protocol, please refer to Evans and Holzbaur (2020a). [Display omitted] •Primary hippocampal cultures to monitor long-term neuronal mitochondrial degradation•Inducing mitochondrial damage through global antioxidant deprivation•A fluorescent pulse-chase protocol to visualize “aged” mitochondrial populations•Live imaging and quantitative analysis of mitochondrial turnover via lysosomal fusion Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The accumulation of dysfunctional mitochondria is a hallmark of neurodegenerative diseases, yet the dynamics of mitochondrial turnover in neurons are unclear. Here, we describe a protocol to monitor the degradation of spectrally distinct, “aged” mitochondrial populations. We describe the preparation and transfection of primary rat hippocampal neuron cultures. We detail a mitochondrial-damaging assay, a SNAP pulse-chase labeling paradigm, and live imaging to visualize the mitochondrial network. Finally, we provide steps to quantify mitochondrial turnover via lysosomal fusion.