Research On The Mechanism Of Neurological Impairment Caused By App's Abnormal Cleavage

Alzheimer's disease (AD), the most common progressive neurodegenerative disease, is pathologically characterized by senile plaques (SP), intracellular neurofibrillary tangles (NFT) and neuronal death. The primary protein component of SP isβ-amyloid peptide (Aβ), which is derived from the amyloid precursor protein (APP) through an initial cleavage byβ-secretase followed by an intramembranous cutting byγ-secretase.In this study, we investigated the relationship between abnormal cut of APP and neurological impairment in AD. Oxidative stress, mitochondrial dysfunction and the related apoptosis have been observed in AD brain. Thus, the study is focused on the effect of APP's cleavage on oxidative stress, mitochondrial dysfunction and apoptosis.We investigated the negative effect on oxidative stress, mitochondrial function, Ca2+ homeostasis, and apoptosis of endogenous Aβoverproduction by using N2a cell lines stably transfected with wild-type APP (N2a/APP695) or its Familial Alzheimer's Disease Swedish mutant (N2a/APPswe). It was found that N2a/APP695 and N2a/APPswe cells showed increased oxidative stress, including increased intracellular reactive (ROS), nitric oxide (NO), protein carbonyls, and malondialdehyde (MDA). Moreover, mitochondrial damage was found in N2a/APP695 and N2a/APPswe cells, embodying the reduced mitochondrial membrane potential (Δψm), COXⅣactivity, mitochondrial membrane fluidity, and ATP content. At the same time, the result showed that [Ca2+]i was significantly higher in N2a/APP695 and N2a/APPswe cells. And then, we proved that Aβlevel positively correlated with ROS levels and mitochondrial damage. In addition, N2a/APP695 and N2a/APPswe cells showed increased sensitivity of a secondary insult induce by H2O2. The ERK and JNK signal pathway as well as a significantly reduced shift in Bcl-2/Bax ratio was found involved in the apoptosis induced by H2O2 in N2a/APP695cells, especially in N2a/APPswe cells.It has been argued thatγ-secretase should be considered as a pharmacological target, as there are few mechanism-based experimental and clinical studies onγ-secretase treatment. Thus, we control the proteolytic cleavage of APP by regulatingγ-secretase activity. When the activity ofγ-secretase was inhibited by expression of the D385A PS1 variant, cells (N2a/Swe.D385A) showed reduced oxidative stress (including ROS, NO, protein carbonyls, and MDA), improved mitochondrial function (includingΔψm, COXⅣactivity, and ATP content), reduced [Ca2+]i and reduced sensitivity to apoptosis. On the contrary, when the activity ofγ-secretase was increased by expression of theΔE9 PS1 variant, cells (N2a/Swe.ΔE9) showed increased oxidative stress, impaired mitochondrial function, increased [Ca2+]i and increased sensitivity to apoptosis. Analogously, the ERK and JNK signal pathway as well as the shift in Bcl-2/Bax ratio played an important role in the process of apoptosis induced by H2O2. Thus, our data support the view thatγ-secretase is a therapeutic target for treatment of AD.Furthermore, APP has been reported to play a role in cell proliferation, adhesion, and migration. However, it is still unknown whether abnormal cleavage of APP caused by FAD-linked mutations will influence APP's natural function on cell adhesion and migration. Thus, we intend to investigate the potential effects on cell morphology, adhesion, and migration when APP was abnormally processed in FAD-linked mutants. Overexpression of wild type human APP695 (N2a/APP695) was found to stimulate the adhesion and migration of N2a cells. In the cells co-transfected by familial Alzheimer's disease (FAD)-linked Swedish mutant of APP695 gene plusΔE9 deleted presenilin1 gene (N2a/Swe.ΔE9), however, this stimulating function was impaired compared to that in the cells co-transfected by Swedish mutant of APP695 gene plus dominant negative mutant of presenilin1 D385A gene (N2a/Swe.D385A). Furthermore, it was also found that the phosphorylation of FAK Tyr-861 and GSK-3βSer-9 was reduced in N2a/Swe.ΔE9 cells, which can be possibly taken as a reasonable explanation for the underlying mechanism.