The Role Of Pink1/Parkin-mediated Mitophagy In Insulin Resistance-induced Neuronal Damage

Aims:Studies have shown that insulin signaling pathway is involved in the regulation of cognitive function.Abnormal insulin signaling pathway,which is also named as insulin resistance,can significantly increase the incidence of age-related neurodegenerative diseases.However,it is still unclear how insulin resistance leads to the damage of neuronal structure and function.Therefore,further investigations are needed.Cognitive function relies on the communication between neuronal synapses.The release of neurotransmitters needs to be triggered by the influx of calcium ions and is supported by a lot of energy.Mitochondria,which serve as the "powerhouse" and calcium ion buffer in eukaryotic cells,play an important role in regulating the electrophysiological activities of neurons.Therefore,maintaining the homeostasis of mitochondrial function is vital to neuronal function.The Pink1/Parkin-mediated mitophagy is a key pathway in the mitochondrial quality control system.However,under insulin resistant condition,changes of this pathway in neurons and their effects on neuronal function have not been well studied.Based on the above,we aim to establish an insulin resistant model in the neuron-astrocyte coculture system,to discover the impacts of central insulin resistance on neuronal morphology and functions,and to explore the role of Pink1/Parkin-mediated mitophagy in insulin resistance-induced neuronal dysfunction.After that,we will use GLP-1 receptor agonist,a kind of hypoglycemic agent,to treat neurons with insulin resistance and explore the effects of this drug on neuronal and mitochondrial functions,as well as mitophagy.Methods:(1)Establishment of insulin resistance model: A neuron-astroglia coculture system was established using an improved "sandwich" coculture method,which can better mimic the microenvironment between neural cells in brain;0.1m M palmitic acid was used to induce insulin resistance.The expression of proteins related to the insulin signaling pathway were detected by Western blot to evaluate whether the insulin resistance model was successfully established.(2)Detection of neuronal morphology and function: After the establishment of the insulin resistance model,we examined the changes of neuronal morphology by HE staining;detected neuronal apoptosis by flow cytometry and Western blot;evaluated the expression of synaptic plasticity-related proteins by Western blot.(3)Mitochondria related detection: We used fluorescent probes and flow cytometry to detect mitochondrial membrane potential and ROS generation;assay kits were used to detect the mitochondrial oxidation respiratory chain complex IV activity and ATP generation;Western blot was used to detect the expression of mitochondrial dynamics,mitophagy and mitochondrial biosynthesis-related proteins;immunofluorescence staining was used to detect mitochondrial morphology and mitophagy.(4)Detection the effects of hypoglycemic drugs: GLP-1 receptor agonist,exenatide,was used to detect its improvements in neuronal morphology,apoptosis,synaptic plasticity and mitochondrial functions.The expression of proteins related to mitochondrial dynamics,mitophagy and mitochondrial biosynthesis were also examined after the hypoglycemic agent intervention.(5)Neuronal function detection after interference and overexpression of neuronal Pink1:we used si RNA or adenovirus to knock down or induce the overexpression of neuronal Pink1,respectively.Then,we examined their effects on mitochondrial function and synaptic plasticity.Results:(1)When 0.1 m M palmitic acid was administered,insulin resistance was found to occur in neurons after 12 h of administration,as evidenced by the significant decrease of IRS1 and AKT phosphorylation in the PA group.(2)Insulin resistance can impair neuronal morphology,synaptic plasticity,promote neuronal apoptosis and accelerate neuronal aging.(3)Insulin resistance can disrupt mitochondrial function,as evidenced by decreased mitochondrial membrane potential,oxidative respiratory chain complex IV activity,ATP production and increased ROS production.Mitochondrial fusion was diminished and the fission was enhanced,which leading to the mitochondrial fragmentation.Moreover,mitophagy and mitochondrial biogenesis were also inhibited.(4)GLP-1 receptor agonist treatment can improve neuronal morphology,synaptic plasticity and apoptosis.Meanwhile,it can also ameliorate mitochondrial dysfunction,promote mitochondrial fusion,mitophagy and mitochondrial biogenesis.(5)Pink1 protein can protect mitochondrial and synaptic function under insulin resistance condition.Conclusions:We used a modified "sandwich" coculture method to establish a neuronal-astrocyte coculture system.The following conclusions can be drawn:(1)Central insulin resistance can impair neuronal morphology,synaptic function,accelerate neuronal apoptosis.(2)Insulin resistance can impair mitochondrial function,promote mitochondrial fragmentation,inhibit mitochondrial biogenesis and autophagy.The accumulation of these dysfunctional mitochondria plays an important role in insulin resistance-induced neuronal damage.(3)The GLP-1 receptor agonist can significantly improve the neuronal function,ameliorate mitochondrial dysfunction,promote mitochondrial fusion,mitophagy and mitochondrial biogenesis.(4)Pink1/Parkin pathway-mediated mitophagy protects both neuronal function and mitochondrial function under insulin resistance conditions.Therefore,Pink1/Parkin pathway may be a potential therapeutic target for insulin resistance-induced cognitive dysfunction.