Effects Of Lanthanum Chloride On Mitochondrial Fission/Fusion And Mitophagy In Primary Astrocytes

Objective:Rare earth elements（REE）include 15 lanthanides and yttrium and thorium in the same family.Among them,lanthanum（La）,a representative of light rare earth elements,is widely used,has a high content in environmental media and foods,and has very active chemical properties and strong biological activity.With the extensive application of REE in various fields,it has more and more entered the production and living environment of human beings,and can be accumulated in the body by means of enrichment of the respiratory tract,skin and food chain.The environmental residual and cumulative effects caused by REE,especially the impact on human health,have become a public health issue worthy of attention.Epidemiological survey results show that children living in rare earth mining areas have significantly lower IQ,learning and memory abilities,attention,and reasoning skills than children in the control area,and their IQ scores are related to the distance from their residences to the rare earth mining areas and whether rare earths are stacked in their homes.Animal experiments have also confirmed that REEs can cause neurobehavioral changes,learning and memory disorders,central nervous system developmental defects,and neuronal morphology and structural abnormalities.Based on the results of these studies,it can be determined that REEs are a class of environmental factors that are neurotoxic and can affect cognitive function.Astrocyte（AC）is one of the main types of glial cells,accounting for about 50%of neurons in the brain.AC needs a lot of energy to perform physiological functions,and these energy mainly come from mitochondria.Mitochondria can regulate cell growth,mitosis,signal transduction,intracellular calcium,iron ions,electrolyte homeostasis,and reactive oxygen species（ROS）production.In recent years,AC’s important role in maintaining and regulating brain function has received widespread attention.Studies have confirmed that dynammol/Lin-related protein 1（Drp1）,is a core regulator of mitochondrial division.Drp1 can sensitively sense the changes of calcium ions in the mitochondria.When the calcium ions are abnormally increased,the S616site of Drp1 is phosphorylated,and is recruited to the outer mitochondrial membrane.Among them,the progeny mitochondria of normal membrane potential are fused by the mitochondrial fusion protein and return to the mitochondrial network.Mitochondria with lower membrane potential are specifically cleared by mitochondrial autophagy.It is known that the PINK1/Parkin signaling pathway is most closely related to mitochondrial autophagy induced by damage stress.PINK1 is a mitochondrial outer membrane protein.Under normal circumstances,PINK1 enters the mitochondrial inner membrane in a potential-dependent manner and is rapidly degraded with a low expression level.But when the mitochondria are damaged,the depolarization of the mitochondrial membrane prevents PINK1 from entering the inner membrane of the mitochondria,causing it to accumulate in the outer membrane of the mitochondria.It is recruited to the mitochondria by phosphorylating Parkin in the cytoplasm,and then mediates mitochondrial outer membrane protein ubiquitination.The autophagy substrate p62 recognizes ubiquitinated proteins and acts as an adapter.By binding to p62,LC3 is localized on depolarized mitochondria,which guides the membrane to specifically recognize and envelop mitochondria,thereby initiating mitochondrial autophagy.The purpose of this project is to explore the effects of lanthanum on mitochondrial division/fusion and mitochondrial autophagy in AC through in vitro cell tests.The key role played by the Drp1 and PINK1/Parkin signaling pathways is clarified,which provides a new theoretical basis for further explaining the molecular mechanism of toxic effects of the central nervous system caused by lanthanum.Methods:Wistar pups within 24 hours of birth were used for primary culture of AC.AC was identified using glial fibrillary acidic protein（GFAP）.LDH method was used to detect cell viability and determine the concentration of LaCl3 treated AC;After treating AC with 0.025,0.05 and 0.1mmol/L LaCl₃,Co-localization of mitochondrial membrane potential,mitochondrial ROS,Mito Traker Green with Drp1,LC3B,and p62,respectively,by immunofluorescence;Real-time PCR detection of mRNA levels of Drp1,Mfn1,Mfn2,PINK1,Parkin,p62,LC3B;Western blot was used to determine the protein expression levels of Drp1,p-Drp1S616,Mfn1,Mfn2,PINK1,Parkin,p62,and LC3B in AC;Transmission electron microscopy was used to detect and observe AC mitochondrial damage and autophagosome formation.After intervention with mitochondrial division inhibitor Mdivi-1,mitochondrial division/fusion and mitochondrial damage and autophagy-related indicators were again tested.Result:1.La Cl₃ exposure leads to a decrease in AC mitochondrial membrane potential and an increase in ROS production.With the increase of the concentration of exposure,ROS levels continue to increase,and membrane potential damage increases.2.La Cl₃ up-regulates the m RNA transcription and protein expression levels of AC mitochondrial division proteins Drp1,p-Drp1S616,down-regulates mRNA transcription and protein expression levels of mitochondrial fusion proteins Mfn1,Mfn2,and promotes the translocation of activated Drp1 to mitochondria.3.LaCl₃up-regulates PINK1,Parkin,LC3B mRNA transcription levels and PINK1 protein expression levels,increases mitochondrial recruitment of Parkin and LC3B-II/LC3B-I protein expression ratio,down-regulates p62 mRNA transcription and protein expression levels,increases LC3B,and decreases p62.4.Mitochondria inhibitor Mdivi-1 can antagonize the aforementioned effects of La Cl3,reduce ROS generation,restore mitochondrial membrane potential,inhibit Drp1 activation and translocation,down-regulate the PINK1/Parkin signaling pathway,and reduce the LC3B-II/LC3B-I protein expression ratio Up-regulates p62 mRNA transcription and protein expression levels,reduces LC3B,and increases co-localization of p62 and mitochondria.5.The ultrastructure of AC was observed under an electron microscope.In the 0.1 mmol/L LaCl₃ treatment group,mitochondria were swollen,ridges disappeared,and more autophagosomes with double-layer membrane structures appeared.The number of autophagosomes was significantly reduced after Mdivi-1intervention.Conclusion:1.LaCl3 can increase the generation of mitochondrial ROS in the cortex of the offspring rats,reduce the membrane potential,and cause mitochondrial damage.2.LaCl3 can up-regulate the expression levels of AC Drp1,p-Drp1S616,translocate activated Drp1 to mitochondria,and down-regulate the expression levels of Mfn1 and Mfn2,thereby promoting mitochondrial division and inhibiting mitochondrial fusion.3.LaCl3 can activate the AC PINK1/Parkin signaling pathway,down-regulate the expression level of p62,increase the proportion of LC3BⅡ/LC3BⅠprotein expression,and cause a large number of autophagosomes in AC.4.Mdivi-1 can antagonize the effects of LaCl₃ on promoting mitochondrial division,inducing excessive mitochondrial autophagy,and reducing mitochondrial damage.