Calotropis giganteaPromotes Neuritogenesis and Synaptogenesis through Activation of NGF-TrkA-Erk1/2 Signaling in Rat Hippocampal Neurons

Calotropis gigantea (L.) R. Br (Apocynaceae) (commonly known as milkweed or crown flower) is a large shrub native to temperate regions of Asia, including China, Bangladesh and India and has a long history of use in traditional medicines. In this study, we investigated the neuromodulatory effects of the ethanol extracts of C. gigantea leaves (CGE) during synaptogenesis in the late stage of neuronal development and during early stage neuritogenesis in cultured rat hippocampal neurons. Maximum neuritogenic activity was achieved at a CGE concentration of 7.5[Formula: see text][Formula: see text]g/ml. At this concentration, CGE facilitated the early development of cytoarchitecture, as evidenced by increases in morphometric parameters, such as, the numbers, lengths, and number of branches of initial neurites, axon and dendrites. During the synaptogenic stage (DIV 14), immunocytochemistry (ICC) showed that CGE upregulated synaptic vesicle 2 (SV2, a marker of axon terminals) and postsynaptic density-95 (PSD-95, a postsynaptic marker) and their colocalization. CGE upregulated nerve growth factor (NGF) and activated extracellular signal-regulated kinase 1/2 (Erk1/2), which is blocked by a TrkA-specific inhibitor suggesting the neuritogenic and synaptogenic potential of CGE was due to the activation of NGF-TrkA-Erk1/2 signaling. Moreover, UPLC of CGE did not detect stigmasterol, an active component of C. gigantea. However, the chloroform-methanol and ethyl acetate subfractions of CGE exhibited initial neuritogenic activity, suggesting that multiple active components were responsible for the neurotrophic-mimetic properties of CGE. Our data prove the neuromodulatory ability of CGE and provide a means of identifying new active phytochemicals with potential nootropic, preventative or therapeutic effects on the human brain.