Deep Phosphoproteomic Measurements Pinpointing Drug Induced Protective Mechanisms in Neuronal Cells

Abstract

Alzheimer's disease (AD) is a progressive and irreversible neurological disorder that impairs the living quality of old population and even life spans. New compounds have shown potential inneuroprotective effects in AD, such as GFKP-19, a 2-pyrrolidone derivative which has been proved to enhance the memory of dysmnesia mouse. The molecular mechanisms remain to be established for these drug candidates. Large-scale phosphoproteomic approach has been evolved rapidly in the last several years, which holds the potential to provide a useful toolkit to understand cellular signaling underlying drug effects. To establish and test such a method, we accurately analyzed the deep quantitative phosphoproteome of the neuro-2a cells treated with and without GFKP-19 using triple SILAC labeling. A total of 14,761 Class I phosphosites were quantified between controls, damaged, and protected conditions using the high resolution mass spectrometry, with a decent inter-mass spectrometer reproducibility for even subtle regulatory events. Our data suggests that GFKP-19 can reverse Aβ25−35 induced phosphorylation change in neuro-2a cells, and might protect the neuron system in two ways: firstly, it may decrease oxidative damage and inflammation induced by NO via down regulating the phosphorylation of nitric oxide synthase NOS1 at S847; Secondly, it may decrease tau protein phosphorylation through down-regulating the phosphorylation level of MAPK14 at T180. All mass spectrometry data are available via ProteomeXchange with identifier PXD005312.

Quantitative Proteomic Study Reveals Up-Regulation of cAMP Signaling Pathway-Related Proteins in Mild Traumatic Brain Injury

“…Quantitative proteomic approaches have been used for neurological diseases in our previous studies. − Label-free quantitative proteomic strategy was employed to reveal the protein expression changes in dorsal hippocampus of heroin self-administering rats, suggesting that CDK5 and RhoB are two important molecules involved in heroin addiction. In addition, using the same approach, we performed proteomic and phosphoproteomic analysis to investigate the pharmacological mechanism of the Alzheimer’s disease-relatedneuroprotective compounds Huperzine A and GFKP-19 in the neuronal cells, demonstrating that p53 and phosphorylated Tau may be involved in the drug responses of these two compounds, respectively.…”

Commentary: Deep Phosphoproteomic Measurements Pinpointing Drug Induced Protective Mechanisms in Neuronal Cells

“…Yu and coworkers recently studied the phosphoproteomic changes in neuro-2a cells challenged by cytotoxicity from Aβ 25−35 peptide and the reversal of the changes by drug candidate- GFKP-19, a 2-pyrrolidone derivative compound (Yu et al, 2016 ). Using SILAC labels, three different treatment conditions were studied in neuro-2a cells- light (DMSO control), medium (cells with Aβ 25−35 ) and heavy (Aβ 25−35 + GFKP-19) on Q-Exactive (QE) and Q-Exactive HF (HF) mass spectrometers.…”