Abstract
Background: The trans-neuronal spread of protein aggregation in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson’s disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. Indeed, in vivo models of aSyn prion-like propagation yield results whose interpretation is hindered by the complexity of brain networks, and in vitro models have for now failed to properly model trans-neuronal propagation in synaptically connected neural networks.Results: To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic neuronal culture system. This system is the first to guide in vitro the development of fully oriented, synaptically connected and fluidically isolated neural networks. It thus uniquely permitted us to model the trans-neuronal propagation of aggregation: we selectively exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size (Fibrils and Oligomers), and quantitatively followed their dissemination to postsynaptic neurons. Both aggregates were anterogradely transferred to through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates, and found that while endogenous aSyn expression level controlled the seeding of aSyn aggregation, it did not affect the transfer rate of exogenous aggregates. Conclusion: Overall, we describe here the first culture system for reconstructing in vitro fully oriented and synaptically connected neural networks, and demonstrate it is uniquely suitable to quantitatively interrogate original aspects of the trans-neuronal propagation of prion-like aggregates.