Proteomic identification of calcium-binding chaperone calreticulin as a potential mediator for the neuroprotective and neuritogenic activities of botanical drug amygdalin

Abstract

Amygdalin is a potent botanical glycoside with multiple bioactivities, such as anti-cancers, hypertension and chronic inflammation. Little is known about the effect of amygdalin on neurogenesis and neuroprotection. In the present study, we initially demonstrated that amygdalin profoundly enhanced nerve growth factor (NGF)-induced neuritogenesis and attentuated the cytotoxicity of neurotoxin 6-hydroxydopamine (6-OHDA) in rat dopaminergic PC12 cells. To define the molecular mechanisms underlying the neuroprotective and neuritogenic activities of amygdalin, we employed a proteomics approach to profile the cellular responses to the treatment with amygdalin in PC12 cells. A total of 11 mostly regulated protein spots were selected from 2-D SDS-PAGE gels and subsequently identified by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and six representative proteins including Hsp90β, calreticulin, 14-3-3ζ/δ, 14-3-3η, Grp94 and rab GDP dissociation inhibitor-α were verified by Western blot analysis using specific antibodies. Based on the strong relevance to the survival and differentiation of neurons, we hypothesized that the calcium-binding chaperone calreticulin might play a role in the neuroprotective and neuritogenic activities of amygdalin. By silencing calreticulin expression with specific siRNA, we found that amygdalin did no longer enhance NGF-induced neuritogenesis, whereas no change was observed by introducing negative control siRNA. Amygdalin could also protect PC12 cells against the toxicity of 6-OHDA via increasing calreticulin expression. Moreover, amygdalin elevated the intracellular calcium concentration in a timing dependent manner, possibly when calreticulin was well induced. Taken together, our results suggest that amygdalin may potentiate NGF-induced neuritogenesis in PC12 cells via up-regulating calreticulin expression. We anticipate that amygdalin may serve as a new drug lead for the treatment of Parkinson’s disease.