Synthetic Squamosamide Cyclic Derivative (FLZ)Improves Mitochondrial Function By Selectively Inhibiting γ-secretase

Amyloid precursor protein (APP) metabolism is a key factor in the pathogenesis of Alzheimer's disease (AD). Amyloid-beta (Aβ) produced by APP metabolism is proved to injury neurons and lead to the pathology of AD. But the mechanism of pathology from Aβ production to central neural system defects in AD is still not well understood. It has been recently proposed that mitochondria are involved in the biochemical pathway through which Aβ causes neuronal dysfunction. Aβ in mitochondria comes from APP mitochondrial metabolism or the uptaken Aβ from outside of mitochondria. The accumulated Aβ in mitochondria decreases the level of cytochrome c oxidase (COX Ⅳ), injuries the electron transport chain, increases the reactive oxygen species (ROS) production and attenuates the ATP production consequently. The mitochondrial dysfunction induces the abnormality in neurons related with energy supply, axonal transport and Ca2+homeostasis. The decrease of Aβ in mitochondria and the normalization of mitochondrial function may contribute to slow down the disease progression of AD.FLZ is a synthetic cyclic derivative of squamosamide from Annona glabra. The main purpose of this thesis is to investigate the neuroprotection mechanism of FLZ basing on the prophase work of our lab. The thesis is divided into3parts. Part one includes the modulation of FLZ on APP processing in mitochondria, the related improvement of mitochondrial function and the mechanism of y-secretase inhibition caused by FLZ. Part two is about the selective y-secretase inhibition of FLZ. Part three is the preliminary research of the defect BDNF axonal transport induced by APPSwe.The effect of FLZ on APP processing in mitochondria is investigated in SH-SY5Y cell overexpressing APP695(wt/Swe). FLZ treatment attenuates APP processing and decreases of Aβ production in mitochondria. The mitochondrial function is increased with the upregulation of COX IV both at protein and activity levels, and ATP production is also increased after FLZ treatment. The mechanic study showes that FLZ inhibits y-secretase activity by decreasing C-terminal fragment (CTF) protein level of presenilin (PS), the active center of y-secretase. FLZ may attenuate Aβ production in mitochondria by inhibiting y-secretase.The precise mechanism of y-secretase inhibition caused by FLZ is explored. The effect of FLZ differs from DAPT (a non-selective γ-secretase inhibitor), suggesting FLZ might be a selective y-secretase inhibitor. There are several substrates of y-secretase, among which APP and Notch are the major ones. The selective inhibition of γ-secretase is a challenge in the development of anti-AD drugs. To investigate the precise of mechanism of γ-secretase caused by FLZ, the assay system of γ-secretase activity using recombinant C terminus of APP (C-APP) and recombinant Notch intracellular domain (NICD) is constructed. In the assay system, FLZ selectively inhibits γ-secretase in the cleavage of recombinant C terminus of APP in vitro, but does not affect the processing of recombinant Notch intracellular domain (NICD). Meanwhile, DAPT inhibits the processing of C-APP and NICD respectively. These results indicate that FLZ decreases Aβ accumulation in mitochondria by selectively inhibiting γ-secretase.The previous studies of our lab indicated that FLZ upregulated BDNF signal pathway in several AD models. BDNF activates TrkB located on axon terminals and initiates the signals that culminate in axon outgrowth and neuronal survival. By formation of the ligand-receptor complex, BDNF and TrkB initiate their internalization from plasma membrane and are retrogradely transported along the microtubules in axon toward the cell body. The axonal transport is depended on the energe supply of michondria. So, the defect BDNF axonal transport induced by APPSwe is also investigated to explore the role of mitochondrial dysfunction in the pathology. In part three, APPSwe is transfected into the cultured hippocampol neurons by lentivirus and BDNF with RFP tag is produced in293T cells by recombinant expression. In the cultured hippocampol neurons overexpressing APPSwe, the amount of BDNF entrance is decreased. Meanwhile the mitochondria gather around the nuclei with decreasing mitochondrial distribution. It is indicated that APPSwe may attenuate the BDNF axonal transport by inducing abnormal mitochondrial dynamics.In summary, it is proposed that FLZ is a potential anti-AD drug candidate, and its mechanism is improving mitochondrial function by selective inhibiting γ-secretase to reduce the A(3burden in mitochondria. And the mitochondrial dysfunction may take part in the defect of BDNF axonal transport induced by APPSwe.The following are the innovation points of the thesis:1. Set up the methods for detecting Aβ in mitochondria2. Construct the assay system of γ-secretase activity in vitro3. Over-express APPSwe in cultured hippocampol neurons4. Produce BDNF which can be uptaken by neurons with RFP tag...