The Function Of Autophagy Induced By Mechanical Neuronal Injury And The Regulation Of Homer1a Through PI3K/Akt Pathway

BackgroundTraumatic brain injury is the most common central nervous system disease in world,and it can cause death and disability. Traumatic brain injury can be classified to primarybrain injury and secondary brain injury. Occurrence of primary brain injury can be reducedby prevention, but brain damage induced by second brain injury must be improved bydrug intervention. At present, the main mechanism of secondary brain injury includes thegeneration of excitotoxic amino acid, release of oxygen free radicals, calcium overloadand inflammatory reaction. According to recently studies, these mechanisms are closelyrelated to the postsynaptic density (PSD) and synaptic signal transmission. Homer proteinfamily molecules-mainly exists in the PSD of mammalian neurons, interact with otherPSD proteins, and plays different functions in different brain tissue and structure.Homer1b/c-one subtypes of Homer1protein, is an important scaffold protein of PSD. Our previous studies have confirmed that the expression of Homer1b/c is stable aftermechanical neuronal injury, and downregulation of Homer1b/c significantly decreased thedamage caused by mechanical injury through regulating endoplasmic reticulum and themitochondrial pathway; Homer1a, another subtype of Homer1protein, is encoded by animmediate-early gene and is temporarily high expression after mechanical neuronal injury,Overexpression of Homer1a protectived PC12cells from the injury induced by hydrogenperoxide.Autophagy is an evolutionary conserved lysosomal degradation pathway, which canremove the large molecules or organelles of abnormal or dysfunction to maintain energybalance and homeostasis of cell. At present, the appropriate autophagy has protectiveeffects on cells, whereas less or more autophagic has the injury effects on cells. A largenumber of studies have show that, autophagy plays an important role in the centralnervous system diseases. Autophagy is significantly increased after TBI, but its functionand regulation mechanism is still unclear. Our study elucidated the occurrence andfunction of autophagy in mechanical neuronal injury, and the involvement of Homer1a inthis process.PartⅠAutophagy induced by mechanical neuronal injuryObjective: To clarify the occurrence of autophagy in mouse cortical neurons causedby mechanical injury.Methods:(1) Cell culture: cortical neurons cultured from Kunmingmouse;(2) Establishment of the traumatic brain injury model: establishment of stably andreliably mechanical injury model to study the traumatic brain injury in vitro;(3)Monitoring of Autophagy: Western blot is used to detect the expression of LC3II andBeclin-1; using the transmission electron microscope for detecting the number ofautophagosome and autolysosome in neuron; monitor the autophagy of neurons useimmunofluorescence technique. Results: At3h after mechanical neurons injury, weobserved increasing of autophagy marker LC3II expression, and Beclin-1expression, buthad no significant time trends. Immunofluorescence technique proved that injurysignificantly induced autophagy at24h. The electron microscopy observed the generation of cytoplasmic autophagosome or autophagolysosomal at24h after the mechanicalinjury.Conclusion: Mechanical injury can significantly lead to the autophagy in corticalneurons.PartⅡThe role of PI3K/Akt signaling pathway in regulation ofautophagy induced by mechanical neuronal injuryObjective: To study the role of PI3K-Akt signaling pathway to autophagy inregulation of autophagy induced by mechanical neuronal injury. Methods:(1)Inhibition ofPI3K-Akt signaling: Inhibit PI3K by LY294002in three concentrations:12.5μM,25μMand50μM, respectively; Detection of p-Akt expression by Western blot to explore thebest concentration;(2) Regulation of autophagy: Autophagy is regulated through agonist(rapamycin) or inhibitor (3-MA);(3) Monitoring of autophagy: Western blot was used todetect the expression of LC3II and Beclin-1. Results: The expression of p-Akt wasincreased at1h after mechanical neurons injury, reached the peak at3h, and thendecreased and returned to normal level at24h;25μM is the best concentration forLY294002to inhibit PI3K; p-Akt was significantly down-regulated after PI3K wasinhibited, while the expression of LC3Ⅱ and Beclin-1increased significantly.Conclusion: PI3K-Akt signaling pathway has a negative effect on the regulation ofneuronal autophagy after mechanical injury.PartⅢThe role and function of autophagy in mechanical neuronal injuryObjective: To explore the role and function of autophagy in neurons after mechanicalinjury.Methods:(1) Detection of neuron injury: LDH kit and MTT were used to measurethe damage of neurons;(2) Intervention of autophage: autophagy agonists rapamycin andautophagy inhibitor3-MA were used to regulate autophagy in further sudies;(3)Autophagy detection: LC3II and Beclin-1expression were detected by Western blot;(4)Apoptosis detection: Western blot analysis of cleaved-Caspase3and Caspase3andTUNEL staining were used to detect the apoptosis of neurons. Results: LDH values wereincreased by inhibition of autophagy at1h before or1h after injury, Inhibition beforeinjury showed higher LDH release compared to inhibition after injury. MTT values were decreased by inhibition of autophagy at1h before or1h after injury, Inhibition beforeinjury showed lower MTT value compared to inhibition after injury. The activition ofautophage at1h before injury increased LDH release, while activition of autophage1hafter injury decreased LDH release. The activition of autophage at1h before injuryincreased MTT value, whereas, activition of autophage1h after injury decreased MTTvalue. Further study found that the inhibition of autophage at1h after injury decreased thecleaved-Caspase3expression and TUNEL positive staining at24h, however, activition ofautophage1h after injury increased cleaved-Caspase3expression and TUNEL positivestaining at24h. Conclusion: Inhibition of autophagy at1h before or1h after injurydecreased neuronal viablility. Activition of autophage at1h before injury has no impact onneuron viablility, while activition of autophage at1h after injury improved neuronviablility. Inhibition of autophage at1h before injury decreased neuronal apoptosis, whileactivition of autophage at1h after injury increased neuronal apoptosis.PartⅣ The role of Homer1a in regulation of autophagy induced bymechanical neuronal injury and its possible mechanismsObjective: To explore the role of Homer1a in regulation of autophagy induced bymechanical neuronal injury and its possible mechanisms. Methods:(1) Homer1aregulation: up-regulation and down-regulation of Homer1a by transfection with lentivirusvector;(2) Regulation of autophagy: Autophagy is regulated through agonist (rapamycin)or inhibitor (3-MA);(3) The inhibittion of PI3K pathway by LY294002, and theconcentrations is25μ M;(4) Monitoring of autophagy: Western blot was used to detectthe expression of LC3II and Beclin-1. Results: Homer1a expression increased from1hafter mechanical injury to cortical neurons, reached the peak at3h, then tends to decline;immunofluorescence detection found that the expression of Homer1a in neurons wassignificantly higher than that in control group, and mainly distributed in the cytoplasm andneurites. Overexpression of Homer1a can decreased the expression of p-Akt of injuriedneurons, while expression of LC3II and Beclin-1are increased.Down regulation ofHomer1a increased the expression of p-Akt and decreased the expression of LC3Ⅱ and Beclin-1. The useage of PI3K inhibitor LY294002up-regulated expression of Homer1aafter injury while autophage inhibitor3-MA decreased Homer1a expression after injury. Inaddition, autophagy agonist rapamycin also up-regulated expression of Homer1a afterinjury. Conclusion: Homer1a can regulate autophagy induced by mechanical neuronalinjury through PI3K/Akt pathway; autophagy induced by mechanical neurons injury havea positive feedback effect on the expression of Homer1a.