Proteomic Analysis of Huperzine A-Treated N2a Cells

Huperzine A is a Chinese drug derived from the Chinese herb Huperzia serrata and is commonly used in China for the treatment of Alzheimer’s disease (AD). With the same targets of FDA-approved AD treatments in the U.S., Huperzine A is an AChE inhibitor and is known to effectively penetrate the blood-brain barrier. Its effects also have a longer duration than traditional medications prescribed for AD.¹ Clinical trials involving Huperzine A have demonstrated increased memory function and cognitive abilities in Alzheimer’s patients.²

To better understand Huperzine targets in AD, Tao et al.³ performed a label-free quantitative proteomic analysis of mouse neuroblastoma N2a cells treated and untreated with Huperzine A. In their experiments, proteins were processed using a centrifugal proteomic reactor developed by Zhou et al.⁴ This reactor was used for processing three experimental groups. The control group, known as the Con group, included proteins from untreated N2a cells. The HupA group contained cells preincubated with Huperzine A followed by Aβ (1–42) oligomers. Lastly, the Aβ group contained cells only treated with Aβ (1–42) oligomers.

Proteins were purified and trypsin digests were performed in preparation for LC-MS/MS on a Easy nano-UPLC 1000 (Thermo Scientific) coupled to a nanoESI Velos Pro Orbitrap Elite mass spectrometer (Thermo Scientific). The resulting MS/MS spectra were searched against version 3.87 of the decoyed mouse International Protein Index protein sequence database, containing 59,534 protein sequences.

From these data, 2860 proteins were identified, and 198 proteins were significantly changed (with p value < 0.05) when the HupA and Aβ groups were compared. These proteins were mapped to the KEGG pathway database and found to be implicated in eight pathways in the AD, RNA transport, ribosome, spliceosome, oxidative phosphorylation, protein processing in endoplasmic reticulum, endocytosis, and Huntington’s disease pathways. HupA and Aβ groups did not show significant change (p value = 0.653), which showed that the Huperzine A incubation did not change the quantity of Aβ cells entering N2a cells. In addition, Tao et al.³ further hypothesize that Huperzine A may protect N2a cells from Aβ-induced cell death through downregulation of p53 levels.

References

1. Wang, R., et al. (2006) ‘Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine‘, Acta Pharmacologica Sinica, 27 (1) (pp. 1–26)

2. Zhang, H.Y. (2012) ‘New insights into huperzine A for the treatment of Alzheimer’s disease‘, Acta Pharmacologica Sinica, 33 (9), (pp. 1170–1175)

3. Tao, Y., et al. (2013) ‘Quantitative proteomic analysis reveals the neuroprotective effects of huperzine A for amyloid beta treated neuroblastoma N2a cells‘, Proteomics, published online February 19, 2013. doi: /10.1002/pmic.201200437

4. Zhou, H., et al. (2011) ‘Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor‘, Molecular and Cellular Proteomics, 10 (10), O111.008425.