Intercellular mitochondrial transfer in neural cells

Intercellular mitochondrial transfer is the acquisition of exogenous mitochondria by one cell from another cell. The transfer event is generally not stochastic but directed. It largely results in a benefit for the recipient cell. In the brain, the support structure astrocytes provide to neurons establishes a framework to investigate mitochondrial transfer between neural cells. During neurodegeneration or brain development, mitochondrial transfer between neural cells may alleviate disease phenotypes or help form the early cytoarchitecture and connectome of the brain. To begin to investigate intercellular mitochondrial transfer in neural cells, co-culture experiments were performed, between two independent, fluorescently labelled cell populations, either in a monolayer or in three-dimensional spheroid culture. Initially, fluorescently labelled mitochondria were co-cultured with neonatal neurons. Although punctate mitochondrial fluorescence was found in neurons, the conventional tools and methods used to label mitochondria and assess their location were found to have limitations, and new methods were developed. Transfer is most often seen in response to injury. Stable mtDNA-depleted (ρ⁰) astrocytes were generated as a mitochondrial specific injury. These astrocytes cannot respire, have impaired biosynthesis and ATP production, fractured mitochondrial networks with swollen mitochondria, and require pyruvate and uridine to meet their bioenergetic demands. When ρ⁰ astrocytes were co-cultured with the appropriate cell type in the absence of pyruvate and uridine, mitochondrial transfer was observed, leading to restoration of an entire mitochondrial network. The mechanism of mitochondrial transfer into ρ⁰ astrocytes was highly suggestive of cell fusion and was donor cell-type specific. This went against the convention in the literature that tunneling nanotubes (TNTs) or extracellular vesicles (EVs) discretely transfer clusters of mitochondria to damaged recipient cells. This PhD dissertation challenges the conventional tools to produce microscopic images proving mitochondrial transfer and suggests cell fusion as the mechanism by which non-native mitochondria exist inside a cell