Primary cortical neurons precipitate and extrude large mitochondria-associated calcium-phosphate sheets with a bone-precursor-like ultrastructure

Calcium-phosphate (CaP) is a ubiquitous inorganic compound that plays an important structural role in healthy bone and teeth formation, but its pathologic buildup can occur in dyshomeostatic calcium disorders like Alzheimer's disease and Leigh syndrome. The nexus of pathologic extracellular CaP in the nervous system is not well understood, but prior evidence suggests mitochondria could be a source. We have observed mitochondria-sized sheet-like CaP aggregates within functional wild type cortical neuron cultures at 1 and 20 days in vitro. Neurons were extracted from embryonic day 18 (E18) rat embryos following standard protocols to study neuronal structure and function. We have used a combination of cryo-ET, cryo-CLEM, and LDSAED to demonstrate that these aggregates are octacalcium phosphate-like, are associated with mitochondria, and that at least a portion are extruded via migrasomes. Visually similar aggregates were previously observed in Huntington's disease model neurons, but in that study they were not observed in WT controls. These findings show that this CaP aggregation process occurs routinely in WT neurons and may reveal an important link for how mitochondria may participate in calcification, highlighting them as potential therapeutic targets in neurological disorders characterized by pathological calcification, such as Alzheimer's disease.