Fluorescence Imaging Of The Synergistic Changes On Lysosomal And Mitochondrial Hydrogen Peroxide In The Brain Of Depression Mice

Depression is a common mental disease,mainly characterized by significant and persistent down in spirits,which seriously affects people’s cognitive function,quality of life and overall health.However,the etiology and pathogenesis of depression have not been fully elucidated,which greatly hinders the development of effective anti-depression methods.In recent years,studies have shown that oxidative stress might play a vital role.Mitochondria are the main place for cells to carry out aerobic respiration and are the organelles that produce the most reactive oxygen species.The oxidative stress can cause a large accumulation of ROS in the mitochondria,which can lead to neuronal metabolism disorders and cause depression and other mental diseases.The lysosome is the main digestive chamber in the cell.It contains a variety of hydrolytic enzymes,which can decompose endogenous or exogenous substances.Its function is closely related to the activity of the enzyme.The increase of ROS in the lysosome can cause the inactivation of a variety of lysosomal enzymes,thereby affecting the function of neurons.Hydrogen peroxide（H₂O₂）,as the most abundant ROS in the cell,can be converted into other ROS,and its concentration directly determines the degree of oxidative stress in the cell.Therefore,it is of great significance to explore the relationship of the changes in hydrogen peroxide concentration between in mitochondria and lysosomes to the pathogenesis,diagnosis and treatment of depression.Fluorescence imaging technology,with its advantages of simple operation,real-time imaging and high accuracy,has become a powerful tool for studying biologically active molecules at the cellular and living level,and has played an important role in the study of the pathogenesis of depression.However,most of the design ideas of fluorescent probes are more biased towards the detection of biologically active molecules in a single organelle,rather than studying the synergistic changes between biomolecules in multiple organelles at the same time.Therefore,fluorescent probes that can locate different organelles are urgently needed to explore the relationship between active molecules in different organelles,so as to elucidate the etiology and pathogenesis of depression as soon as possible.In order to solve the problem of simultaneous visualization of H₂O₂ in mouse brain mitochondria and lysosomes,this thesis has carried out the following two aspects of work:（1）Fluorescence imaging of H₂O₂ in lysosomes and mitochondria in the brains of depression mice.Two fluorescent probes were designed and synthesized to specifically image brain lysosomes and mitochondria H₂O₂ in depressed mice.LY-H₂O₂ use phenylborate as the recognition group of H₂O₂,coumarin as the chromophore fluorophore,and morpholine as the lysosomal targeting group.MI-H₂O₂use phenylborate as the recognition group for H₂O₂,naphthalene fluorescein as the chromophore,and triphenylphosphine as the mitochondrial targeting group.The spectral resolubility of MI-H₂O₂ and LY-H₂O₂ enables the imaging of H₂O₂ in both cells and in vivo.By using LY-H₂O₂and MI-H₂O₂,it was found that under oxidative stress,the concentration of hydrogen peroxide in both mitochondria and lysosomes of PC12 cells is increased.Two-photon imaging showed that the concentration of H₂O₂ in the brain lysosomes and mitochondria of depressed mice was higher than that of normal mice.This work provides an important tool for exploring the close link between depression and oxidative stress.（2）Fluorescence imaging was used to analyze the relationship between the concentration of H₂O₂ in the mitochondria and lysosome in the brain of depressed miceOn the basis of the above work,the relationship between the changes of H₂O₂ concentration in mitochondria and lysosomes and the relationship between the concentration of H₂O₂ inlysosomes and the activity of hydrolase in lysosomes under oxidative stress in cells and in vivo was further explored.Through the cell and the vivo fluorescence imaging,it was found that H₂O₂ in mitochondria was produced earlier than lysosomes in the process of oxidative stress induced by excessive glutamate in PC12 cells,and H₂O₂ in brain mitochondria was also produced earlier than lysosomes in the process of depression in mice.Further experiments revealed that H₂O₂ in the brain lysosomes of mice decreased the activity of the GCase enzyme,aggravated thesymptoms of depression.This work will promote the discovery of potential antidepressant targets.