Milky, B. | Search bioRxiv

bioRxiv2024

Presynapses are mitophagy pit stops that prevent axon degeneration

Defects in neuronal mitophagy have been linked to neurodegenerative diseases including Parkinsons disease. However, despite the importance of mitophagy in neuronal homeostasis, the mechanistic basis for neurodegeneration when mitophagy is defective is unclear. Here, using human neurons, we discover that presynapses are mitophagy pit stops for damaged axonal mitochondria. We show that while mitochondrial damage and PINK1/Parkin activation events are distributed throughout axons, mitophagy initiation and autophagosome formation are restricted to presynapses, which we show contain the machineries required for mitophagy. Being the primary sites of axonal mitophagy, presynapses were vulnerable when PINK1/Parkin mitophagy was defective. We observed local cytochrome c release within presynapses from an accumulation of damaged mitochondria. This resulted in downstream degradative caspase activation, defining a mechanism for neurodegeneration. Pharmacological rescue of axon degeneration was achieved through synthetic upregulation of receptor mediated mitophagy with the clinically approved compound Roxadustat, revealing a potential therapeutic avenue for disease.

cell biology↗

bioRxiv2020

BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology have improved exponentially in the last ten years. At the same time, advances in cellular reprogramming and organoid engineering have quickly expanded the use of human neuronal models in vitro. Altogether this creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identified multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (e.g., phototoxicity, suboptimal fluorescence signals, and unphysiological neuronal activity). To overcome these issues, we developed a new neuromedium, "BrainPhys Imaging", in which we adjusted fluorescent and phototoxic compounds. The new medium is based on the formulation of the original BrainPhys medium, which we designed to better support the neuronal activity of human neurons in vitro 1. We tested the new imaging-optimized formulation on human neurons cultured in monolayers or organoids, and rat primary neurons. BrainPhys Imaging enhanced fluorescence signals and reduced phototoxicity throughout the entire light spectrum. Importantly, consistent with standard BrainPhys, we showed that the new imaging medium optimally supports the electrical and synaptic activity of midbrain and human cortical neurons in culture. We also benchmarked the capacity of the new medium for functional calcium imaging and optogenetic control of human neurons. Altogether, our study shows that the new BrainPhys Imaging improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting cell viability and neuronal functions.

neuroscience↗