Mechanism Of Mitophagy In Alzheimer’s Disease Cell Model

Background:Alzheimer’s disease (AD), is a chronic degenerative disease of the central nervous system, cognitive function decline and personality change as the main clinical manifestation, Pathological changes are mainly concentrated in the temporal, prefrontal and parietal lobe, and the most obvious change is in the hippocampus. Neurofibrillary tangles are formed with hyperphosphorylated tau protein, and senile plaques(NPS) are formed with β-amyloid protein(Aβ) abnormal deposit. NPS, Ap and the selectivity of neurons and synapses loss are the major pathological features of AD. amyloid-β protein precursor (APP) and presenilin (PS1) 1 and presenilin 2 (PS2) genes mutations are involved in early-onset familial of AD. However, the molecular mechanism of the pathogenesis of AD is unclear, prior to the formation of neurofibrillary tangles cell energy metabolism and ROS generation increased has occurred, indicating oxidative stress and mitochondrial dysfunction is an early event in AD. Currently several hypotheses are focus on the early events in the development of AD research, the purpose is to provide effective clinical intervention for an early disease.Autophagy is a catabolic process to eliminate damaged organelles and protein, and through this process helps the cell adapt to their metabolism needs and achieve cell organelles updates. Autophagy includes macroautophagy, microautophagy and chaperon-mediated autophagy three forms and according to the selective autophagy also divided into selective and non-selective autophagy. Mitochondria are the energy conversion sites in eukaryotic cells, can produce reactive oxygen species (ROS) at the same time, excessive ROS can cause the protein, nucleic acid and fatty acid peroxidation, cause cell damage, therefore removal of damaged mitochondria in time plays an important role in maintaining the normal physiological functions of cells. When the cells under starvation, ROS, aging and other circumstances, can make the mitochondria to the depolarization, depolarization of mitochondria are selectively packaged into autophagosomes and combined with lysosomes, so that the process to clear the damage mitochondria known as mitophagy. Mitophagy are selective autophagy. Studies have shown that, in AD patients brain mitochondrial dysfunction, cristae damaged, and the number of mitochondrial decreased. Aβ can inhibit cell respiratory chain and the key enzymes of the tricarboxylic acid cycle, and lead to mitochondrial dysfunction, decrease the mitochondrial membrane potential, mitochondrial swelling, change the granular characters, decrease the production of ATP, ROS increase, causing cell injury.Mitochondrial targeting serine/threonine kinase PTEN inducible kinase 1 (PINK1) and E3 ubiquitin ligase Parkin by regulating the membrane potential, cristae structure, calcium homeostasis, mitochondrial DNA integrity, respiratory activities and a number of physiological process to maintain mitochondrial integrity. In normal mitochondria, PINK1 is degraded rapidly, when the mitochondrial membrane potential depolarizing, PINK1 with a voltage dependent manner stable expression in the mitochondrial outer membrane. The PINK1/Parkin pathway is the key of autophagy related to remove abnormal mitochondrial.Based on the above background, this research mainly to investigate whether cell model of AD have abnormal mitophagy, what is the mechanism of mitophagy, and whether PINK1 and Parkin is involved in the regulation of mitophagy. The purpose of this study is to research on the early AD events -- mitophagy, provides the new ideas and methods for early clinical intervention and prevention of AD. Objective:This study is to investigate whether cell model of AD have abnormal mitophagy, what is the mechanism of mitophagy, and whether PINK1 and Parkin is involved in the regulation of mitophagy. The purpose of this study is to provides the new ideas and methods for early clinical intervention of AD.Methods:1. We used the 20E2 cells (HEK293 cells stably expressing Swedish mutant APP) as the experimental group, HEK293 cells were used as the control group, and they cultured under the same culture conditions.2. To determine whether the 20E2 cells model was successfully established, we detected the extracellular concentration of Aβ1-40 by ELISA kit, and the expression of APP protein level was detected by Western blotting.3. Observation of mitochondria in cells under electron microscopy.4. The mitochondrial membrane potential of both cells were detected by fluorescence probe JC-1.5. Detection of both cells apoptosis by flow cytometry.6. The expression levels of LC3-II, PINK1 and Parkin were detected by Western blotting.7. The expression of Parkin and it’s mitochondrial localization observed by Immunofluorescence.8. Cells treated with PINK1 siRNA, western blotting detected the expression levels of autophagy related protein LC3-II, PINK1. Results:1. Compared with HEK293 cells, the expression leves of APP protein and Aβ1-40 in 20E2 cells increased(P<0.05).2. Compared with HEK293 cells, mitochondrial swollen, cristae disappeared and vacuolization were obviously observed in 20E2, mitochondrial membrane potential decrease.3. Compared with HEK293 cells, the apoptosis rate showed no significant difference in 20E2 cell (P>0.05).4. Compared with HEK293 cells, the expression levels of LC3-Ⅱ, PINK1 and Parkin increased in 20E2.5. Compared with HEK293 cells, Immunofluorescence showed the expression of Parkin increased and mitochondrial localization was obvious in 20E26. The expression of LC3-Ⅱ reduced after PINK1 siRNA interference in 20E2 cell, indicating that PINK 1/Parkin pathway is involved in the regulation of mitophagy. Conclusion:In Alzheimer’s disease cell model 20E2, the mitochondrial morphology changed obviously and membrane potential of mitochondria declined. Mitophagy increased, the expression levels of PINK1, Parkin increased, autophagy reduced after siRNA treatment, and these changes may cause the increase of mitochondrial autophagy through PINK1 and Parkin pathway.