Stigmasterol activates Cdc42-Arp2 and Erk1/2-Creb pathways to enrich glutamatergic synapses in cultures of brain neurons.

The naturally occurring phytosterol stigmasterol (ST) ameliorates scopolamine-induced memory impairment and promotes neuritogenesis and synaptogenesis. Therefore, in this study, we investigated the hypothesis that ST promotes spinogenesis and the formation of glutamatergic synapses and thus improves memory in cultured rat hippocampal neurons. Immunoblots showed that ST activated extracellular signal-regulated kinase 1/2 (Erk1/2) and cAMP response element binding protein (Creb), and upregulated actin-related protein 2 (Arp2) and cell division cycle 42 (Cdc42), central components in actin organization and spine head development. Consistently, DiO staining of live neurons showed that ST increased the densities of dendritic filopodia and mushroom-type mature spines, and immunocytochemistry showed an increase in the expressions of the GluN2A and GluN2B subunits of N-methyl-D-aspartate receptors (NMDARs) at synapses. Finally, staining the recycling membranes with FM1-43 showed that ST increased the sizes of synaptic vesicle pools at presynaptic terminals. Together, our data indicate that ST upregulates Cdc42-Arp2/3 and Erk1/2-Creb signaling and thus induces the formations of mushroom-type spines and glutamatergic excitatory synapses.