Role Of Mitophagy Regulated By VPS34 For Learn And Memory Damaged Induced By AlNPs On Zebrafish

Objective:Aluminum oxide nanoparticles(Al NPs)have a wide range of applications,but autophagy abnormalities and mitochondrial morphological deformation can lead to impairment of learning and memory abilities.The Vps34(vacuum protein supporting 34)gene plays a key regulatory role in the excessive autophagy induced by nano alumina,and plays an important role in the normal functioning of the nervous system.The purpose of this study was to investigate the role of Vps34 in regulating mitochondrial autophagy in the learning and memory impairment of zebrafish induced by nano-alumina.By knocking down the expression of Vps34 gene during the embryonic stage,we observed the changes in the spatial learning and memory abilities of adult fish,and explored the potential protective mechanism of Vps34 regulating mitochondrial autophagy in the damage to the nervous system caused by nano-alumina.Methods:The study Embryos from zebrafish within 1 hour after fertilization were collected and microinjected with 1 mmol/L antisense morpholino oligonucleotide(MO)to reduce the expression of Vps34.Embryos from 6 hpf(hours post fertilization,hpf)were randomly divided into 5 groups exposed to 144 hpf: normal control group,negative control group,knockdown Vps34 group(Vps34-),Al NPs group(100 mg/L),and Al NPs+knockdown Vps34 group(Al NPs+Vps34-).The general toxicity(mortality,hatching rate,and deformity rate)of nano alumina and the behavioral changes(speed,moving distance,and cumulative time of outer circle)of juvenile fish were detected at the age of 3 months.The m RNA expression levels of Vps34,VDAC1(Voltage dependent region channel 1),and PARK2(parking RBR E3 ubiquitin protein ligase)were also detected;The expression levels of mitochondrial outer membrane protein TOMM20 and mitochondrial protein P62 in fish brain were detected by ELISA.The levels of oxidative stress,including lactate dehydrogenase(LDH),reactive oxygen species(ROS),and superoxide dismutase(SOD),were measured in adult fish;Observe and analyze the exploration ability and use T-maze experiment to detect the learning and memory ability of zebrafish.Next,use flow cytometry to detect the level of mitochondrial membrane potential;The ATPase activity and the m RNA expression levels of mitochondrial autophagy genes PINK(PTEN-induced putative kinase 1),FUNDC1(FUNDC1),and BNIP3L(BNIP3-like)were measured;Western blot was used to detect the expression levels of TOMM20 and TIM23(the translation of the inner member)proteins in each group.The pathological changes of the fish brain were analyzed by Nissl staining,and the ultrastructural changes of mitochondrial autophagy in the fish brain were observed by electron microscopy.Results:(1)General toxicity: Juvenile zebrafish fish exhibit varying degrees of malformation at different periods of time,with no significant difference in overall development.At 24 hpf for zebrafish,the development of the nano-alumina group was delayed,while knockdown of the Vps34 gene and exposure to nano-alumina significantly improved,showing significant tail extension.Some embryos may exhibit abnormalities during hatching,including tail abnormalities,yolk sac edema,and spinal curvature.The results of incubation rate,mortality,and deformity rate among the groups showed no statistical difference(P>0.05).(2)Spontaneous movement changes: During the period of 7 days to 3 months,the average speed,movement distance,and tactile ability of the nano alumina group decreased(P<0.05).When young fish were exposed to nano-alumina after knocking down Vps34,it was observed that the average speed of young fish increased,their tactile ability increased,their movement distance increased,and their movement trajectory was relatively regular,significantly improving the movement behavior of young fish.Knocking down Vps34 reduced the neurotoxicity of nano-alumina on the movement behavior of young zebrafish.(3)Mitochondrial autophagy gene level in three-month-old zebrafish: The expression level of Vps34 m RNA is still at a level of about 40% lower(P<0.001).Exposure to nano-alumina resulted in increased gene m RNA expression levels of Vps34,VDAC1,and PARK2 in zebrafish(P<0.001),and increased mitochondrial autophagy levels.In addition,the Vps34 knockdown group decreased the expression of VDAC1 and PARK2 genes(P<0.001,P<0.05).The expression levels of Vps34,VDAC1,and PARK2 genes in zebrafish exposed to nano-alumina after knocking down Vps34 decreased(P<0.05),indicating that Vps34 interferes with the process of promoting mitochondrial autophagy by nano-alumina,reducing mitochondrial autophagy levels,and having a protective effect on maintaining the normal structure and function of mitochondria.(4)The level of mitochondrial autophagy protein in three-month-old zebrafish: The expression of P62 in the nano-alumina group decreased(P.The protein expression in the nano alumina+knockdown Vps34 group was higher than that in the nano alumina group(P<0.05).In addition,compared with the control group,the expression of mitochondrial outer membrane protein TOMM20 in zebrafish induced by nano-alumina showed a significant downward trend,with no statistical significance(P>0.05).However,the knockdown Vps34 gene was close to normal level,and ultimately increased expression under the combined action of nano-alumina and knockdown Vps34,protecting the gene expression level of mitochondrial outer membrane,thereby maintaining the normal function of mitochondrial autophagy.(5)The exploratory ability of 3-month-old zebrafish: the average velocity,the percentage of cumulative time of outer zone movement and the distance of movement of nano-alumina and Vps34 fish were decreased(P<0.001).In the adult fish,nano-alumina + Vps34 knockdown group had higher mean velocity,cumulative time percentage of outer zone movement,and movement distance(P<0.05),indicating that the damage to the exploratory ability of adult fish was reduced and the exploratory behavior of adult fish was improved under the condition of combined effects.(6)Observation on the number of nerves in zebrafish at the age of six months: Nissl staining results showed that the number of brain nerves in zebrafish in the nano-alumina group significantly decreased(P<0.05),while the number of brain nerves in the knockdown Vps34 group and the knockdown Vps34+nano-alumina group increased(P<0.05).(7)Observation on the ultrastructure of mitochondrial autophagy in 6-month-old zebrafish: The results of transmission electron microscopy showed that the mitochondria cristae in the brain of adult fish exposed to nano-alumina were ruptured and partially degraded into vacuoles,and more and more structures of damaged mitochondria were enveloped by the bilayer membrane,forming a mitochondrial autophagy structure.The mitochondria in the knockdown Vps34 group had slight deformation,while the mitochondria in the knockdown Vps34+nano-alumina group showed a vague structure wrapped in the autophagy body,It may be due to the degradation of the contents of autophagic lysozyme,resulting in the degradation of most of the damaged organelles.(8)Changes in oxidative stress of zebrafish at the age of six months: ROS levels in the nano alumina group increased significantly,SOD activity decreased significantly,and LDH levels increased significantly(P<0.05).In the nano alumina+knockdown Vps34 group,the ROS activity was significantly decreased,the SOD level was significantly increased,and the LDH level was significantly decreased(P<0.05).(9)Results of learning and memory abilities of zebrafish aged six months: The incubation period of the 13 nm Al NPs group increased the most(P<0.001),and the cumulative residence time in the outer zone decreased significantly(P<0.001),indicating that nano alumina affects the nervous system in adult fish,resulting in delayed response and low learning ability.The incubation period of adult fish in the knockdown Vps34 group and the knockdown Vps34+nano-alumina group decreased(P<0.001),and the cumulative residence time in the outer zone increased significantly(P<0.05),indicating that knockdown Vps34 made adult fish more sensitive to learning and memory after exposure to nano-alumina.(10)Mitochondrial membrane potential level of 6-month-old zebrafish: Compared with the control group,the average fluorescence intensity of the group exposed to nano-alumina significantly decreased(P<0.001),and the mitochondrial membrane potential decreased(P<0.001).Under the common condition of knocking down Vps34+nano-alumina,the average fluorescence intensity of the group compared with nano-alumina showed an upward trend(P<0.001),indicating that Vps34 participated in protecting the ionic activity of the inner and outer membranes of mitochondria and maintaining the potential balance of the inner and outer membranes of mitochondria.(11)Changes in enzyme activity of zebrafish at the age of six months: Nano alumina caused a decrease in mitochondrial enzyme activity of Na+-K+ATPase and Ca2+-Mg2+ATPase in adult fish(P<0.05),while the expression of mitochondrial enzyme activity of knockdown Vps34 and nano alumina+knockdown Vps34 increased(P<0.05).knockdown Vps34 protected mitochondrial normal respiratory function.(12)Changes in mitochondrial autophagy genes in zebrafish at the age of six months:Nano-alumina significantly increased the expression level of mitochondrial autophagy gene PINK in adult fish(P<0.05),decreased the m RNA expression levels of FUNDC1 and BNIP3L(P<0.05),significantly decreased the expression level of PINK in the knockdown Vps34 group and knockdown Vps34+nano-alumina group(P<0.05),and significantly increased the expression level of FUNDC1 and BNIP3L(P<0.05).After knockdown Vps34,damaged mitochondria may be degraded and cleared through the PINK pathway.(13)Changes in mitochondrial autophagy proteins in zebrafish at the age of six months: nano-alumina resulted in a decrease in the expression of mitochondrial outer membrane protein TOMM20(P<0.05),an increase in the expression of mitochondrial inner membrane protein TIM23(P<0.05),a significant increase in the expression level of TOMM20 in the knockdown Vps34 group,a decrease in the expression level of TIM23 in the knockdown Vps34+nano-alumina group,and an increase in the permeability of mitochondrial inner and outer membranes,resulting in excessive autophagy,After degradation of outer membrane proteins,inner membrane proteins leak out into the cytoplasm,causing mitochondrial dysfunction,while knocking down Vps34 protects the normal transport function of the inner and outer membranes of mitochondria.Conclusions:(1)Nano alumina can cause behavioral damage and increase the expression of Vps34 in young zebrafish fish;At the same time,the mitochondrial membrane potential decreases and the level of mitochondrial autophagy increases,ultimately leading to a decrease in the learning and memory abilities of adult zebrafish.(2)Vps34 regulates nano-alumina induced mitophagy to protect learning and memory ability caused by nano-alumina.