Modeling Synaptic Maturation From Growth Cone to Synapse in Human Organoids.

Area of Science:

• Neuroscience
• Cell Biology
• Developmental Biology

Background:

• Human neural organoids (NOs) are valuable for studying neurodevelopment but lack methods for isolating functional synaptic structures.
• Investigating synaptic development and dysfunction requires accessible and reproducible isolation techniques.

Purpose of the Study:

• To present a novel differential centrifugation protocol for enriching growth cone particles (GCPs) and immature synaptosomes from human cerebral organoids (ALI-COs).
• To characterize these isolated structures and demonstrate their functional viability for studying synaptic development and disorders.

Main Methods:

• Differential centrifugation protocol applied to ALI-COs at Day 90 and Day 150.
• Quantitative proteomic profiling and Transmission Electron Microscopy (TEM) for structural and molecular characterization.
• Functional assessment via KCl-induced depolarization to observe protein phosphorylation changes.

Main Results:

• The protocol successfully enriched GCPs (Day 90) and immature synaptosomes (Day 150) without density gradients.
• Proteomic analysis confirmed enrichment of key growth cone and synaptosomal markers.
• TEM visualized distinct structures of GCPs and immature synaptosomes.
• Functional assays demonstrated depolarization-induced phosphorylation events in both isolated structures.

Conclusions:

• A reproducible, gradient-free centrifugation method for isolating GCPs and immature synapses from human organoids has been established.
• This protocol enables the study of synaptic development and dysfunction in scalable, patient-compatible models.
• The findings support the use of these isolated structures for investigating neurodevelopmental and neurodegenerative disorders.