Isolation and characterization of synaptic structures from human neural organoids

Synapses are essential for neuronal function and are central to numerous neurological disorders including developmental and neurodegenerative diseases. Synapses structurally constitute a very small proportion of a neuron, and their protein content is difficult to study using whole tissue preparations. Especially studying synapses on the functional level is challenging. To overcome this limitation, synapses can be captured as synaptosomes generated through enrichment of isolated nerve terminals. Such synaptosomes have a re-sealed plasma membrane and can regenerate their membrane potential and perform physiological function, for example neurotransmitter release.

Synaptosomes are traditionally enriched from rodent or postmortem human brain tissue, but rodent models lack human-specific synaptic features, and the functionality of synaptosomes from postmortem tissues is limited by the postmortem interval and often only show disease endpoints. Furthermore, due to ethical issues and availability, only a few studies have been conducted on human samples. However, neural organoids (NOs) have emerged as a possible new source for isolation of intact and live human nerve terminals to study human-specific aspects of synaptic transmission. Further, the enrichment of synaptosomes is usually performed using density gradient centrifugation, which requires a lot of starting material. In the present study we developed a method for the enrichment of synaptic structures from human NOs applying a differential centrifugation protocol. We then used mass spectrometry-based quantitative proteomics to document the enrichment of synapse and growth cone specific proteins, and quantitative phosphoproteomics upon KCl stimulation to demonstrate viability and physiological function of the derived synaptic structures.