<em>In Vitro</em> and <em>In Vivo</em> Detection of Mitophagy in Human Cells,<em> C. Elegans</em>, and Mice

Abstract

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease

“…We established an in vivo drug screening platform using C. elegans to identify potent neuronal mitophagy inducers, using a z -score standard as defined elsewhere 22 . Two different methods were used to detect neuronal mitophagy in vivo 10,23 . First, we generated transgenic animals expressing a pan-neuronal mitochondria-targeted Rosella (mtRosella) biosensor with a GFP variant sensitive to the acidic environment of the lysosomal lumen fused to the pH-insensitive Discosoma red fluorescent protein (DsRed).…”

“…Neuronal mitophagy events were quantified using the z -scores of the colocalization between LGG-1 and DCT-1. A screening window coefficient, called Z-factor, was used for evaluation and validation; a z -score of 0.5–1.0 means that the drug screen is deemed acceptable 23 . An agent with values higher than WT ( P < 0.05) was scored as a mitophagy inducer.…”

GAK and PRKCD are positive regulators of PRKN-independent mitophagy

“…Progeny of these adults were tested on adult day 2. We performed imaging of mitophagy process in C. elegans based on the methods we had established [58][59][60] . Briefly, worms were immobilized with levamisole before mounting on 2% agarose pads for microscopic examination using EVOS Imaging System.…”

Metabolic dysfunction in human skin: Restoration of mitochondrial integrity and metabolic output by nicotinamide (niacinamide) in primary dermal fibroblasts from older aged donors

“…Primary human skin fibroblasts were grown from dermal tissue from dividing the red area by the red and green areas combined, and multiplying by 100 (Fang et al, 2017). To allow for the best visual representation of the images, the brightness and contrast of these images were altered using the best fit function in ZEN 3.1 software (Zeiss)…”