The Mechanisms Of Cell Damage In Alzheimer's Disease And The Neuroprotection Of Galantamine

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system. The main pathological change is the loss and death of cholinergic neurons in brain. Amyloidβ(Aβ) protein had been found in AD brain. As the important organelle related to cell survival and function, mitochondria have been proved to show abnormal dynamics and function in AD. However, the long-term successive dynamic study of mitochondria in AD is few. In addition, cholinergic system has been demonstrated to be abnormal in AD. Vesicular acetylcholine transporter (VAChT) is an important part of cholinergic nervous system, it has been reported that VAChT expression decreased in AD, but there was little about the dynamics and mechanisms in AD. At present, cholinesterase inhibitors have been used to treat AD in clinical, such as galantamine. Galantamine has been proved to inhibit Aβaggregation, oxidative stress, neuronal apoptosis, etc. However, the neuroprotective mechanisms of galantamine against Aβ25-35-induced apoptosis have not been completely demonstrated, need to be further investigated.In this study, we first established an AD cellular model with PC 12 cell apoptosis induced by Aβ25-35 (the active fragment of Aβ), determined 20μM Aβ25-35 incubating for 24 h as the optimum modeling condition. Then based on this model, using laser confocal microscopy, flow cytometry, fluorescence microplate reader, live cell station, fluorochrome staining, TIRFM, molecular biological technology and computer image processing, we investigated changes of mitochondrial and VAChT vesicular dynamics and fuction induced by Aβ25-35, as well as the neuroprotective mechanism of galantamine. The results are:①Aβ25-35 induced abnormal mitochondrial dynamics and function. Mitochondrial changes are reversible, depended on exposure time and drug concentration;②Normally, there were 3 main transport types of VAChT:single vesicle transported long distance along one trajectory, different vesicles transported along the same trajectory and single vesicle along one trajectory transported bidirectionally. We further demonstrated that 20μM Aβ25-35 incubating for 24 h significantly inhibited VAChT vesicle movement and protein expression;③Galantamine inhibited Aβ25-35 aggregation, calcium release from endoplasmic reticulum (ER), upregulation of ER stress-related proteins GADD153 and Grp78/94 and activation of caspase-12. In addition, galantamine could also suppress mitochondrial dysfunction, such as inhibiting mitochondrial membrane potential collapse, ROS production, cytochrome c release, Bcl-2/Bax ratio decrease and the following caspase-9 activation. Finally, galantamine inhibited caspase-3 activation and reduced cell apoptosis. These results suggest that mitochondria and VAChT play important roles in the development of AD, galantamine has antiapoptotic and neuroprotective effects through mitochondrial and ER passways, provide new sights into AD pathogenesis and new targets for drug discovery.