| Parkinson’s disease(PD)is a common neurodegenerative disease,and genetic,environmental,and age factors may play a role in the pathogenesis of PD.The main pathological manifestations of PD include the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy Bodies(LBs)and Lewy neurites in the substantia nigra,the main components of which are α-synuclein(α-syn)in the form of aggregation.Α-syn pathological changes play a central role in the pathogenesis of PD.Recently,it has been considered that α-syn transmission between cells is an important driver of α-syn pathology and neurodegeneration,and is an important focus of research on the pathogenesis of PD.Autopsy reports confirmed the presence of high levels of iron in substantia nigra in PD patients,and iron in LBs was co-located with the aggregated form of α-syn.Studies have shown that iron deposition in neurons is closely related to α-syn aggregation.During the progression of PD disease,studies have found that α-syn can be released into the intercellular space by surviving neurons and then retaken by surrounding cells(including neurons,glial cells,etc.)so that it can be transferred between cells and between related structures.In contrast to the free form of α-syn,the intercellular transmission of α-syn in vesicular forms such as exosomes only represents a portion of its extracellular release but plays a key role in mediating the progression of PD disease due to its membranous structure such as lipids.However,whether and how intracellular iron deposition affects exosome release,especially the release of the exosome form α-syn,remains unclear.In this study,dopaminergic cell lines with wild type,WT α-syn overexpression,A53 T mutant α-syn,and Snca knockdown were constructed in PC12 cells.Construct a cell line that overexpresses cationic transporting ATPase 13A2(ATP13A2).Cells were treated with Ferric ammonium citrate(FAC),and the control group was treated with a serum-free medium for 24 h.Cells and supernatant were collected,exosomes were separated by the ultracentrifugation method,and cell number changes were measured by cell count.The shape and diameter of exosomes were determined by Transmission electron microscopy(TEM),the Intraluminal vesicles in Multivesicular bodies(MVBs),The number and density of ILVs;The protein expression levels of exosome specific markers Alix and flotillin-1 were detected by western blot,and the protein levels of ATP13A2 in PC12 cells and primary cultured Ventral Mesencephalic(VM)neurons were determined.The mRNA levels of ATP13A2 in PC12 cells and VM neurons and the mRNA levels of inflammatory cytokines TNF-α and IL-1β in primary astrocytes were detected by q PCR.ELISA was used to detect α-syn in exosomes and the proinflammatory factors TNF-α and IL-1β in astrocyte supernatant.The results are as follows:1.PC12 cells were treated with 100 μmol/L FAC or serum-free medium for 24 h,the supernatant was collected,and exosomes were extracted by ultracentrifugation.The exosomes were cupped by TEM.The average particle size of exosomes detected by nanoparticle tracking analysis(NTA)was 127.8 nm-148.2 nm.Compared with the control group,the number of cells in the FAC treatment group was increased(P < 0.001),but the expressions of exosome marker proteins Alix,flotillin-1,and CD63 were significantly down-regulated(P < 0.05,P <0.01).The results showed that iron increases the number of cells but decreases the secretion of exosomes.2.TEM results showed that the number of ILVs in MVBs was decreased in the FAC-treated group compared with the control group.3.PC12 cells were treated with serum-free medium for 12 h and 24 h,respectively,and exosomes were isolated.Western blotting showed no difference in the levels of Alix and flotillin-1 proteins in cell lysate compared with those in the 12 h group.The expressions of Alix and flotillin-1 in exosomes in the 24 h group were up-regulated(P < 0.05).The cell count results showed that compared with the 12 h group,the number of cells in the 24 h group was increased(P < 0.05).The results showed that exosome release increased during cell proliferation,suggesting that the reduction of exosome release by FAC was not related to cell proliferation.4.WT hαsyn PC12 cells(overexpressing WT human α-syn)were constructed using LV-FlagWT-SNCA,LV-Flag-A53T-SNCA,LV-Snca-RNAi and LV-vector-GFP,respectively.A53 T hαsyn PC12 cells(overexpressing A53 T human α-syn),Snca KD PC12 cells(α-syn knockdown),and vector PC12 cells.WT hα-syn PC12 cells or A53 T hα-syn PC12 cells were treated with 100 μmol/L FAC for 24 h.In both cells,the expression levels of exosome marker protein Alix and flotillin-1 in the FAC treatment group were down-regulated compared with the control group(P < 0.001,P < 0.01),the down-regulation ratio was similar to that of normal PC12 cells treated with FAC,suggesting that the overexpression of WT hαsyn or A53 T hαsyn did not affect the inhibitory effect of intracellular high iron exosome release.Snca KD PC12 cells were treated with 100 μmol/L FAC for 24 h.The expression levels of exosome marker protein Alix and flotillin-1 in the FAC treatment group were down-regulated compared with the control group(P < 0.05,P < 0.01,P < 0.001).The downregulation ratio was similar to that of normal PC12 cells treated with FAC.The results showed that the inhibitory effect of iron on exosome release was independent of α-syn expression level.5.PC12 cells and VM neurons treated with 100 μmol/L FAC for 24 h showed no significant changes in ATP13A2 mRNA and protein levels compared with the control group.The results showed that the ATP13A2 expression was independent of iron accumulation.6.Vector-PC12 cells and ATP13A2 PC12 cells overexpressing ATP13A2 were constructed using Lv-Flag-vector and Lv-Flag-ATP13A2 viruses,respectively.When vector-PC12 cells and ATP13A2 PC12 cells were treated with serum-free medium for 24 h,the expression levels of exosome labeled protein Alix and flotillin-1 in ATP13A2 PC12 cells were increased compared with those in Vector-PC12 cells(P < 0.001,P < 0.01),indicating that the overexpression of ATP13A2 can promote the release of exosomes.vector-PC12 cells and ATP13A2 PC12 cells were treated with 100 μmol/L FAC for 24 h.flotillin-1 protein expression levels were also increased compared with vector-PC12 cells(P < 0.001,P < 0.01),and the proportion of increased Flotillin-1 protein expression was similar to that of serum-free medium.The results showed that intracellular high iron did not affect the expression of ATP13A2,and its overexpression could promote the release of exosomes.The inhibitory effect of FAC on the release of exosomes was independent of ATP13A2.7.PC12 cells were treated with 100 μmol/L FAC or serum-free medium for 24 h,and then 200ng/mL epidermal growth factor(EGF)was used to treat cells for 0 h,1 h,2 h,3 h and 4 h,respectively.Protein expression levels of the serum-free medium group epidermal growth factor receptor(EGFR)were reduced to 81.28 %,64.80 %,56.20 %,and 45.92 %,respectively,as EGF treatment time was prolonged.EGFR expression level in the FAC treatment group decreased to 39.28 % of that in the serum-free medium treatment group at 0 h after EGF treatment and no longer showed a time-dependent decrease(P < 0.05,P < 0.01,P < 0.001).The results suggest that FAC induces impaired endocytosis and inhibits exosome release.8.Exosomes derived by PC12 cells for 24 h were collected from 100 μmol/L FAC or serumfree medium,and primary astrocytes were treated with the above two groups of exosomes for12 h.mRNA levels of IL-1β and TNF-α were detected from cells and supernatant,respectively.Compared with the non-exosome-treated group,IL-1β mRNA level was significantly increased in both groups of exosome-treated primary astrocytes(P < 0.001),while TNF-αmRNA level was not significantly changed.The levels of IL-1β and TNF-α in the supernatant of primary astrocytes treated with exosome were significantly increased between the two groups(P < 0.001),but there was no difference in the levels and levels of the mRNA levels of proinflammatory cytokines induced by exosome from PC12 cells treated with FAC or in serum-free medium.Primary astrocytes pretreated with 0.2 μg/mL LPS for 12 h could induce the increase of IL-1β mRNA level in astrocytes(P < 0.001),and the release of IL-1β and TNF-α in the supernatant increased(P < 0.01,P < 0.001),suggesting the activation of astrocytes.After 12 h treatment of primary astrocytes activated by the exosome,IL-1β mRNA level in primary astrocytes activated by exosome did not further increase,nor did IL-1β and TNF-αrelease in cell supernatant.The results suggest that both exosomes and LPS released by PC12 cells can induce astrocyte proinflammatory response,but there is no synergistic effect between the two.Whether exosomes are derived from PC12 cells with high iron content in the cell has no effect on the astrocyte proinflammatory response induced by the exosomes.9.The supernatant of PC12 cells was collected using flotillin-1 antibody for immunoprecipitation,and the exosome-expressed specific markers Alix and CD63 were isolated.After exosome separation,the content of α-syn in supernatant decreased from 2999pg/mL ± 193.0 pg/mL to 2276 pg/mL ± 81.81 pg/mL(P < 0.01),and the concentration decreased by 28.28 % ± 7.271 %.Exosomes produced by WT Hα-SYN PC12 cells or A53 T Hα-SYN PC12 cells treated with 100 μmol/L FAC for 24 h were collected and the α-syn levels in the exosomes were detected by ELISA.In the exosomes produced by these two cells,compared with the Control group,α-syn levels in exosomes in FAC treatment groups were increased(P < 0.01,P < 0.001).These results suggest that FAC can increase the level of α-syn in PC12 exosomes.High iron in PC12 cells can inhibit the release of exosomes and reduce the number of ILVs in MVB.The regulatory effect of iron on exosome release did not change in PC12 cells with either overexpression or α-syn knockout,suggesting that the inhibitory effect of high iron on exosome release in cells was independent of α-syn expression level.There was no change in ATP13A2 mRNA and protein levels in PC12 cells and VM neurons,and ATP13A2 overexpression promoted exosome release regardless of intracellular high iron,suggesting that intracellular ATP13A2 did not participate in the inhibition of high iron exosome release.High iron in PC12 cells reduced EGFR protein level and no longer showed a time-dependent decrease after EGF treatment,suggesting that the inhibitory mechanism of high iron exosome release in PC12 cells may be related to impaired endocytosis.Whether exosomes are derived from PC12 cells with high iron content in the cell has no effect on the astrocyte proinflammatory response induced by the exosomes.Intracellular iron can increase the level of exosome α-syn.In this study,through the establishment of an intracellular high iron model,the changes of exosome release in the process of PD when neurons are high iron and the mechanism is preliminarily discussed.The research results provide new research ideas for the pathogenesis of PD. |
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