DHA elongates the neuronal primary cilium through GPR40-mediated signaling

Docosahexaenoic acid (DHA), the predominant n-3 polyunsaturated fatty acid (PUFA) in the brain, promotes neuronal differentiation, neurite outgrowth, and synapse formation. However, the subcellular mechanisms underlying these effects remain unclear. The primary cilium functions as a signaling antenna, with its length influencing signaling capacity; however, the physiological factors regulating cilium length in neurons are largely unknown. This study investigated whether DHA promotes primary cilium elongation in neurons and examined the underlying signaling pathways.

Primary cilium length was measured in cultured mouse hippocampal neurons immunostained for adenylyl cyclase 3, and in the embryonic cerebral cortex immunostained for ARL13B after maternal dietary or pharmacological manipulation of DHA and GPR40 signaling. The pathway was dissected using selective receptor agonists and antagonists, RNAi-mediated knockdown, live-cell calcium imaging, RT-qPCR, Western blotting, and ELISA.

DHA increased cilium length in a concentration- and time-dependent manner, with the increase observable within 24 h and sustained thereafter in cultured neurons. Consistently, maternal dietary n-3 fatty acid intake modulated cilium length in the developing fetal brain, with high n-3 diet leading to elongation and low n-3 diet resulting in shortening. DHA-induced ciliary elongation was mediated by GPR40, as this effect was abolished by a selective GPR40 antagonist in vitro and recapitulated by selective GPR40 agonists both in vitro and in vivo. DHA rapidly increased intracellular Ca²⁺ levels, and pharmacological inhibition of calcineurin-NFAT signaling suppressed DHA-induced ciliary elongation. DHA selectively induced cyclooxygenase-2 (COX-2) at both the mRNA and protein levels, and COX-2 activity was essential for elongation. Finally, DHA elevated prostaglandin E2 (PGE2) levels, and pharmacological blockade of EP4 suppressed DHA-induced ciliary elongation, indicating that PGE2-EP4 signaling contributes to this process.

By identifying the regulation of cilium length as a previously unrecognized action of DHA, this study uncovers a subcellular mechanism by which dietary n-3 fatty acids may influence neuronal primary cilia.