Role Of Alpha-Synucle In Oligomerization In DEHP-Induced Neurotoxicity In Quail Brain

Di-（2-ethylhexyl）phthalate（DEHP）has gained increasing concern in recent years due to being commonly used as a plasticizer to add elasticity to many products,including toys,food wrapping,and medical devices and lubricants to render them.DEHP is a lipophilic compound and not chemically bound to the polymer chains,which means that it can easily migrate with the material and be released into the environment,food and water.Recent studies have shown that DEHP exposure can cause damage to the nervous system.Neurons,astrocytes,and microglia play a key role in maintaining the nervous system.However,the impact of DEHP on the nervous system is still unclear.To explore the mechanism of neurotoxicity caused by DEHP,30 quails were randomly divided into 5 groups（6 quails per group）in this experiment:Control group（Con group）,vehicle control group（Vcon group）,250mg/kg DEHP group（D250 group）,500 mg/kg DEHP group（D500 group）,and 750 mg/kg DEHP group（D750 group）were fed continuously for 45 days.We recorded the mental state of quails in each group,observed the histopathology and ultrastructure changes.We detected the changes of autophagy-related indicators and alpha-Synuclein.Meanwhile,the expression of related neurotransmitter receptor proteins and changes of ion content and inflammation related protein content were detected.Molecular dynamics was used to simulate the structural changes of alpha-Synuclein under DEHP exposure.Immunofluorescence detection alpha-Synuclein changes among neurons,astrocytes,and microglia.The detection results showed that:（1）The quails showed good mental state and normal feeding habits in the Con and Vcon groups.There were no deaths,mental depression,and decreased food intake in the DEHP treatment group.Pathological structure shows that dendrites was shorten or even disappear,the number of neurons decreased,swells,and deforms,and vacuolar degeneration occured in the cytoplasm.Nissl staining became shallow and dissolved.LFB staining showed varying shades of myelin staining,loss of refractive power,and decreased density.Ultrastructural observation showed that the morphology of the myelin sheath was disordered,with obvious swelling,expansion of layer space,and myelin sheath rupture.In addition,the presence of typical autophages was observed in the DEHP treatment groups.These results suggested that DEHP can damage neurons and induce brain demyelination and autophagy,leading to neurotoxicity.（2）DEHP exposure increased the expression of autophagy related factors（Beclin1,LC3B）via the AKT/PI3K/m TOR pathway,promoting the formation of autophagic bodies.However,the content of p62 increased and autophagy bodies dissolved by lysosomesobserved were not observed under electron microscopy.These results indicated that DEHP activated the formation of autophagosomes but inhibit autophagic flux.When the neuronal dependent autophagy lysosomal pathway was blocked,various proteins were misfolded.（3）DEHP exposure led to protein misfolding and structural dynamic changes.The proportion of dimers was higher than that of monomers and tetramers in the DEHP treatment groups.Molecular docking experiments have found that DEHP mainly combined with dimers through the formation of hydrogen bonds and hydrophobic forces.Molecular dynamics simulation experiments found that the presence of DEHP during the formation of dimers increased the electrostatic force and hydrogen bonds between the two chains of the dimer.These results suggested that nuclear protein dimers were the key structures responsible for DEHP neurotoxicity in quails.（4）Exposure to DEHP resulted in increased overlap between alpha-Synuclein and astrocytes.DEHP upregulated the content of ionic glutamate receptors NMDAR and increased the glutamate（Glu）content,and significantly increased the Ca²⁺and K⁺content.The expression of"A1"markers（H2-T23,Ggta1,and C3）in astrocytes was significantly upregulated.In addition,DEHP exposure led to alpha-Synuclein overlaps with microglia and significantly upregulated the inflammatory factor TNF-αAnd IL-1βand inhibited the expression of the anti-inflammatory factor IL-10.These results indicated that DEHP transfered nuclear proteins from neurons to astrocytes and microglia,leading to convert microglia to the pro-inflammatory M1 subtype and to convert astrocytes to the A1 subtype via Notch signals.In summary,DEHP caused neuronal damage,demyelination,activated the formation of autophagic body,and inhibited of autophagic flux,leding to the formation of alpha-Synuclein dimer.The dimer was transferred from neurons to astrocytes and microglia,triggering microglia into the pro-inflammatory M1 subtype,which in turn activated neurotoxic A1 type astrocytes through the Notch pathway,ultimately causing damage to the nervous system.