Protocols for assessing mitophagy in neuronal cell lines and primary neurons

Mitochondria are organelles that regulate essential eukaryotic functions including generating energy, sequestering excess calcium, and modulating cell survival. In order for neurons to thrive, mitochondria have to be continuously replenished by maintaining autophagic-lysosomal mediated degradation of mitochondria (mitophagy) and mitochondrial biogenesis. While a plethora of image- and biochemical-based techniques have been developed for measuring autophagy (macroautophagy) in eukaryotic cells, the molecular toolbox for quantifying and assessing mitophagy in neurons continues to evolve. Compared to proliferating cells, quantifying mitophagy in neurons poses a technical challenge given that mitochondria are predominantly present in neurites (axons and dendrites) and are highly dynamic. In this chapter, we provide a brief overview on mitophagy and provide a list of validated fluorescence- and biochemistry-based techniques used for assessing mitophagy in neuronal cells and primary neurons. Secondly, we provide comprehensive guidelines for interpreting steady-state levels of mitophagy and mitophagic flux in neurons using modern fluorescence- and biochemistry-based techniques. Finally, we provide a comprehensive list of common pitfalls to avoid when assessing mitophagy and offer practical solutions to overcome technical issues.