Proteomic Profile of Carbonylated Proteins Screen the Regulation of Calmodulin-Dependent Protein Kinases-AMPK-Beclin1 in Aerobic Exercise-Induced Autophagy in Middle-Aged Rat Hippocampus.

BACKGROUND

Carbonylation is an oxidative modification of the proteins and a marker of oxidative stress. The accumulation of toxic carbonylated proteins might be one of the onsets of pathogenesis in hippocampal aging or neurodegeneration. Enormous evidence indicates that regular aerobic exercise might alleviate the dysfunction of carbonylated proteins, but the adaptational mechanisms in response to exercise are unclear.

OBJECTIVE

This study explored the carbonyl stress mechanism in the hippocampus using proteomics and the role of calmodulin-dependent protein kinases (CAMK)-AMP-activated protein kinase (AMPK)-Beclin1 signaling pathways in alleviating aging or improving function with regular aerobic exercise.

METHODS

Twenty-four healthy 13-month-old male Sprague-Dawley rats (average 693.21 ± 68.85 g) were randomly divided into middle-aged sedentary control group (M-SED, n = 12) and middle-aged aerobic exercise runner group (M-EX, n = 12). The M-EX group participated in regular aerobic exercise - treadmill running - with exercise intensity increasing gradually from 50-55% to 65-70% of maximum oxygen consumption (V˙O2max) over 10 weeks. The targeted proteins of oxidative modification were profiled by avidin magnetic beads and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Western blots were used to test for molecular targets.

RESULTS

Regular aerobic exercise restores the intersessional habituation and rescues the hippocampus morphological structure in middle-aged rats. -ESI-Q-TOF-MS screened 56 carbonylated proteins only found in M-SED and 16 carbonylated proteins only found in M-EX, indicating aerobic exercise decreased carbonyl stress. Intriguingly, Ca2+/CAMK II alpha (CAMKIIα) was carbonylated only in the M-SED group at the oxidative modification site of 4-hydroxynonenal adducts, while regular aerobic exercise alleviated CAMKIIα carbonylation. Regular aerobic exercise significantly increased the expression and phosphorylated, active levels of CAMKIIα and AMPKα1. It also upregulated the expression of Beclin1 and microtubule-associated protein1-light chain 3 in the hippocampus.

CONCLUSION

Quantification of CAMKIIα carbonylation may be a potential biomarker of the hippocampal senescence. Additionally, regular aerobic exercise-induced autophagy via the activation of CAMK-AMPK-Beclin1 signaling pathway may mitigate the hippocampal neurodegeneration or pathological changes by alleviating protein carbonylation (carbonyl stress).