Mechanism Of Ferroptosis In Dopaminergic Neurons:α-synuclein-induced Phospholipid Peroxidation

Research background:Parkinson’s disease（PD）is a chronic neurodegeneration disease characterized by the loss of dopaminergic neurons in the SNc of midbrain and the appearance of Lewy bodies（the main component isα-synuclein（α-Syn））in neurons,but its pathogenesis is not completely clear.The oligomerization ofα-Syn is an early pathological phenomenon of Parkinson’s disease,and lipid peroxidation of midbrain neurons is also an important pathological indicator of Parkinson’s disease.However,there is no definite conclusion in the current research as to whether the two are etiological or pathological results,and whether there is any correlation between the two.This study proposes a scientific assumption:peroxidation of membrane phospholipid polyunsaturated fatty acids（PL-PUFA）induced byα-Syn aggregates may be the potential cause of death of dopaminergic neurons and also an important pathogenesis of Parkinson’s disease.Lipid peroxidation damage is involved in various disease mechanisms.The large accumulation of phospholipid peroxidation products can cause a new type of cell death-ferroptosis,which is mainly characterized by increased iron ion load and accumulation of large amounts of lipid peroxides,which are highly consistent with the molecular biological characteristics of the brain in patients clinically with Parkinson’s disease.Therefore,ferroptosis of dopaminergic neurons caused by membrane phospholipid peroxidation may be an important pathological mechanism of Parkinson’s diseas.Among the major pathways of ferroptosis reported so far,glutathione peroxidase GPX4,as the only enzyme that can remove lipid peroxides on biofilms,plays a key role in the regulation of ferroptosis.Accordingly,in this study,the in vivo and in vitro model of mutantα-Syn^A53Toverexpression were adopted to explore the important role ofα-Syn-induced membrane phospholipid peroxidation in the pathogenesis of Parkinson’s disease,and mice model with midbrain-specific Gpx4 knockout was used to further confirmed the molecular mechanism of membrane phospholipid peroxidation-mediated ferroptosis of dopaminergic neurons,and LC-MS/MS oxidative lipidomics analysis was administrated to detect the content and typing of oxidized phospholipids.This study provides a new exploration perspective for the etiology ofα-Syn in Parkinson’s disease,with a view to providing new research ideas for the development of related prevention and treatment measures.Research method:First,we used PC12 cells that conditionally overexpressα-Syn^A53T and transgenic mice that overexpressα-Syn^A53T to explore the relationship betweenα-Syn and lipid peroxidation-mediated ferroptosis.In vitro,the expression ofα-Syn was induced by doxycycline hydrochloride（Dox）with different time,and then the level of lipid peroxidation and the expression of ferroptosis pathway-related proteins were detected by flow cytometry and Western blot.For transgenic mice with natural disease（15 months old）,we used the pole test,rotarod test and catwalk test to detect the movement and balance and gait analysis in mice,immunofluorescence was utilized to detect the expression of tyrosine hydroxylase TH,a marker of dopaminergic neurons,and the kit was utilized to detect the content of MDA in midbrain,one of a lipid peroxidation product.Western blot was applied to detect the expression ofα-Syn and ferroptosis pathway-related proteins in midbrain tissue,q-PCR was applied to detect the expression of ferroptosis pathway-related genes,and LC-MS/MS oxidative lipidomics analysis was administrated to detect the content and typing of oxidized phospholipids.Secondly,in order to further explore whetherα-Syn has any influence on the sensitivity of dopaminergic neurons to lipid peroxidation-mediated ferroptosis,we respectively gave ferroptosis inducers to the above-mentioned in vitro and in vivo models.RSL3（GPX4 inhibitor）and erastin（System x_c^-inhibitor）were given to PC12 cells that conditionally overexpressα-Syn^A53T,and sorafenib（System x_c^-inhibitor）was given to transgenic mice that overexpressα-Syn^A53T.Then,the accumulation of lipid peroxides,the damage of cell membrane,the expression ofα-Syn and ferroptosis pathway-related genes and proteins,the Parkinson’s disease related behavior and dopaminergic neuron indexes of mice were detected,and the content and typing of oxidized phospholipids were detected by LC-MS/MS oxidative lipidomics analysis.Finally,we used Cre-loaded adeno-associated virus（AAV）to inject GPX4^flox/flox mice in brain location to specifically knock out midbrain Gpx4,so as to explore the damage of lipid peroxidation on dopaminergic neurons induced by midbrain Gpx4 deficiency.The behavior ability of mice was tested by pole test,rotarod test and catwalk test.The number of dopaminergic neurons in the SNc of midbrain was detected by immunohistochemistry,the content of MDA in midbrain was detected by detection kit,and the expressions ofα-Syn and ferroptosis pathway-related proteins and genes in midbrain were detected by Western blot and q-PCR.Research results:Dox can induce PC12^A53T cells to overexpressα-Syn,resulting in increased expression of4-HNE,one of a lipid peroxide,decreased expression of antioxidant enzymes:GPX4 and i PLA2β,and increased expression of transferrin receptor 1（TFR1）.Compared with WT mice of the same age,amongα-Syn^A53T transgenic mice with natural disease（15-month-old）,the duration time in the pole test was significantly longer,the elapse time in the rotarod test was shorter.Step cycle and walking cadence and other indexes in catwalk test show that the motor and coordination ability was seriously impaired.In addition,the expression of TH in the SNc of midbrain was decreased.In terms of ferroptosis-related indicators,transgenic mice were compared with WT mice,the content of MDA in midbrain of mice increased.Western blot and q PCR results all showed that the expression of GPX4 and i PLA2βwas decreased and the expression of TFR1 was increased.The results of LC-MS/MS oxidative lipidomics analysis showed that the midbrain tissue of transgenic mice had obvious phospholipid peroxidation,and the content of oxidized phosphatidylcholine（PC-ox）increased most significantly.The above experimental results indicated that overexpression ofα-Syn^A53T could induce lipid peroxidation damage and ferroptosis in dopaminergic neurons.We gave RSL3 to PC12^A53T cells.PC12 cells overexpressingα-Syn were more sensitive to RSL3,which showed more severe cell membrane damage,more accumulation of lipid peroxides,and more obvious decrease in GPX4 and i PLA2βprotein expression.At the same time,the q-PCR results also showed similar results,indicating thatα-Syn induced PC12 cells to produce lipid peroxidation,which made PC12 cells more sensitive to ferroptosis.In addition,we examined transgenic mice that had not yet developed the disease（8 months old）,it was found that the mice without disease had no changes related to exercise injury and ferroptosis.However,the administration of sorafenib to these non-diseased transgenic mice,could obviously prolong the duration time in the pole test and shorten the elapse time in the rotarod test of these non-diseased transgenic mice,catwalk test results also showed significant impairment of gait,these showed that sorafenib could accelerate the occurrence of the motor and coordination dysfunction in transgenic mice.In addition,for transgenic mice without disease,administration of sorafenib can significantly increase MDA in midbrain and reduce the expression of TH in SNc.In terms of ferroptosis-related indicators,administration of sorafenib accelerated the reduction of the expression of GPX4 and i PLA2βin non-diseased transgenic mice,and the results of m RNA detection showed similar effects.In addition,the results of LC-MS/MS oxidative lipidomics analysis showed that sorafenib significantly increased the content of PC-ox in the midbrain of transgenic mice.The experimental results showed that there was a synergistic effect betweenα-Syn and sorafenib（ferroptosis inducer）,sorafenib could increase the level of phospholipid peroxidation in transgenic mice without disease,and accelerate the occurrence of ferroptosis in dopaminergic neurons.The above in vitro and in vivo experimental results indicated that phospholipid peroxidation increases the sensitivity ofα-Syn overexpressed dopaminergic neurons to ferroptosis.Finally,we performed midbrain specific Gpx4 knockout on GPX4^flox/floxmice,and found that compared with the control group,the weight of the midbrain Gpx4 knockout mice was not affected,but its the duration time in the pole test was significantly prolonged,the elapse time in the rotarod test was significantly shortened,catwalk test results also showed impaired gait,which showed that their ability of movement and coordination had declined.At the same time,knocking out Gpx4 in midbrain leaded to the obvious reduction of TH expression in the SNc of midbrain.In addition,the content of MDA increased,the expressions of GPX4 and i PLA2βdecreased,the expressions of TFR1 increased in midbrain of Gpx4^-/-mice.The results of LC-MS/MS oxidative lipidomics analysis also showed that the increase of PC-ox in the midbrain tissues of Gpx4 knockout mice was the most obvious.All of which showed the participation of ferroptosis,it is suggested that phospholipid peroxidation caused by midbrain Gpx4 deficiency can lead to ferroptosis of dopaminergic neurons and then lead to motor dysfunction in mice.Research conclusions:In this subject,the molecular mechanism of ferroptosis in dopaminergic neurons was detected by in vivo and in vitro Parkinson’s disease models overexpressingα-Syn^A53T and mice with midbrain specific lipid peroxidation models.It was suggested that phospholipid peroxidation caused by accumulation ofα-Syn is closely related to its neurotoxicity,and the intervention of meditated ferroptosis in dopaminergic neurons byα-Syn might provide a new strategy for the treatment of Parkinson’s disease.