JNK and PI3K signaling pathways mediate synapse formation and network spontaneous activities in primary neurons

Abstract

Background: Cellular signals orchestrating synapse formation and neuronal network function remain poorly understood. To explore the critical signaling pathways in neurons and their influence on network development, pharmacological assays were employed to inhibit multiple signaling pathways in cultured neurons. Methods: Immunofluorescence and western blotting are applied to identify the expression of synapse-related proteins within neurons. micro-electrode arrays (MEAs) are employed to study the developmental characteristics of neuronal networks. RNA sequencing is utilized to determine the gene expression profiles pertaining to multiple signaling pathways. Results: Canonical c-jun N-terminal kinases (JNK) pathway is necessary for pre- and post-synaptic specializations, while phosphatidylinositide3-kinases (PI3K) is a key to postsynaptic specialization and affects the puncta sizes of presynaptic marker. Unexpectedly, pharmacological inhibition of JNK pathway significantly suppressed the mean firing rate (MFR), network burst frequency (NBF) and regularity of network firing after 4 weeks, but did not alter the synchrony of the network. During network development, PI3K pathway regulates the longer burst duration and lower network synchrony. Gene sets associated with neurodevelopmental processes and myelination was disturbed during restraining these signal pathways. Furthermore, inhibition of the PI3K signaling pathway obviously transformed voltage-gated ion channel activity, synaptic transmission and synaptic plasticity of neurons. Conclusion: This study reveals that JNK and PI3K signaling pathways play different roles during synapse formation, and these signaling pathways have a lasting impact on the development of neuronal networks. Thus, this study provides further insights into the intracellular signaling pathways associated with synapse formation in the development of neuronal networks.