| Backgroud:Methamphetamine(METH),which is a strong addictive amphetamine-typed stimulant,is commonly abused worldwide.Multiple studies,including ours,have shown that dopamine depletion,oxidative stress,mitochondrial functional impairment,endoplasmic reticulum stress and axonal transport barriers may be involved in the main mechanism of METH neurotoxicity.The major target of METH is the central nervous system,especially dopaminergic neurons.Furthermore,METH causes pathological changes similar to neurodegenerative diseases such as Parkinson's disease(PD).In the both laboratory animals and humans,METH exposure results in the high expression of alpha-Synuclein(a-Syn)in the striatum and prefrontal cortex.?-Syn,a natively unfolded neuronal protein enriched in preSynaptic terminals,has been implicated in many neurodegenerative diseases,including PD and dementia with Lewy bodies.Furthermore,a-Syn accumulates and aggregates during the pathogenic proces.Posttranslational modifications including ubiquitination,phosphorylation,and nitrosylation of a-Syn have been reported to play roles in ?-Syn accumulation and aggregation.The small ubiquitin-related modifier(SUMO)is covalently conjugated to lysine residues in a wide range of substrate proteins,modulating the functional properties of the modified protein.As with ubiquitination,the SUMOylation cycle involves a series of enzymes in a multistep process.It has been reported that the level of SUMO conjugation coincides with the expression level of UBC9.Several aggregation-prone proteins implicated in neurodegeneration like ?-Syn are found to be SUMOylated.Nevertheless,the effects of SUMOylation on ?-Syn are complex,with reports supporting negative as well as positive impacts on cells.Currently,the exact contribution of SUMOylation to the effects of METH on ?-Syn remains unclear.Objectives:The objective of this study was to investigate whether METH affects aggregation and SUMOylation of a-Syn and how the SUMOylation of a-Syn influences METH-induced neurotoxicity in dopaminergic cells.We explored that METH influences SUMOylation of ?-Syn on account of these enzymes involved in the SUMOylation cycle.Moreover,we hypothesized that METH affects SUMOylation of?-Syn,and that,in turn,SUMOylation of ?-Syn may mediate its METH-induced aggregation.In order to test this hypothesis,we measured the levels of ?-Syn SUMOylation and these enzymes involved in vitro and in vivo with METH exposure.We also determined the effect of ?-Syn SUMOylation on the aggregation of ?-Syn after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro.Then,we made mutations in the major SUMOylation acceptor sites of ?-Syn in vitro and in vivo,and measured the UPS and ALP-related markers.The results showed that METH exposure decreases the SUMOylation level of a-Syn with the reduction of UBC9 level.The change of a-Syn SUMOylation regulates a-Syn aggregation induced by METH.Furthermore,mutations in the major SUMOylation acceptor sites of a-Syn aggravate a-Syn aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo.This study also provides a potential target for gene therapy for neurodegenerative diseases,even those caused by METH abuse.Method:1 To analyze whether METH affects SUMO-1 and a-Syn protein expression,we treated SH-SY5Y cells and primary culture neurons with a range of METH doses for 24 h or treated with 2.0 mM or 1.0mM METH for 2-24 h.Western blot and immunofluorescence results also show that SUMO-1 and a-Syn protein expression were increased in the METH-treated SH-SY5Y cells and primary culture neurons.We also determined SUMO-1 and a-Syn protein expression in the prefrontal cortex and striatum of mice following subacute METH exposure.Western blot and immunohistochemistry was used to examine the levels of SUMO-1 and a-Syn in the mouse striatum following subacute METH exposure.2 Co-immunoprecipitation results showed that the SUMOylation level of a-Syn was decreased by relatively higher doses of METH in vitro and invivo.3 To identify the mechanisms underlying regulation of SUMOylation a-Syn induced by METH,we measured the levels of enzymes involved in the SUMOylation cycle.4 To test how cellular SUMOylation of a-Syn relies on the catalytic activity of UBC9,we effectively overexpressed the UBC9 gene using a plasmid in SH-SY5Y cells.To verify the results in SH-SY5Y cells,we also overexpressed the expression of the UBC9 gene using LV-UBC9 in primary culture neurons.5 To further assess the role of SUMO-1 in METH-induced a-Syn aggregation,we investigated whether SUMO-1 knockdown affects a-Syn aggregation triggered by METH in vitro.To verify the results in SH-SY5Y cells,we also infected primary culture neurons with LV-SUMO-1-RNAi or control LV-GFP followed by METH exposure.6 To further assess the role of a-Syn SUMOylation in METH-induced neurotoxicity,we compared the aggregation propensity and neurotoxicity of ?-Syn with K96R and K102R mutations and WT ?-Syn.For this purpose,we screened for SH-SY5Y cells stably expressing WT ?-Syn,?-Syn-2KR or GFP and treated those cells with METH.We effectively overexpressed the UBC9 gene in SH-SY5Y cells stably expressing WT a-Syn or a-Syn-2KR.We also tested the expression of UbE1 and ubiquitin,which are involved in the UPS,and LC3-?,P-62 and LAMP2A,which are involved in the ALP.7 To further testify whether mutations of lysine 96 and lysine 102 exacerbate METH-induced a-Syn aggregation in vivo,AAV2-EGFP,AAV2-WT ?-Syn and AAV2-a-Syn-2KR were injected into the right striatum of mice using a standard stereotaxic positioning system and then mice were treated with or without METH.We also tested the expression of UbEl and ubiquitin,which are involved in the UPS,and LC3-II,P-62 and LAMP2A,which are involved in the ALP.Results:1 METH influences SUMO-1 and a-Syn protein expression in vitro and in vivo.We treated SH-SY5Y cells and primary culture neurons with a range of METH doses for 24 h or treated with 2.0 mM or 1.0mM METH for 2-24 h.Western blot results showed that METH increased SUMO-1 and ?-Syn protein expression in a dose-dependent and time-dependent manner.For example,after 24 h of exposure,the SUMO-1 protein expression was significantly increased by 1.77-fold in the METH-treated(2.5mM)SH-SY5Y cells(p<0.05).Similarly,?-Syn protein expression was significantly increased by 1.73-fold in the 24-h METH-treated(2.5 mM)SH-SY5Y cells(p<0.05).Aggregated a-Syn was also increased by 1.76-fold in a dose-dependent manner upon METH exposure(2.5mM,24h)(p<0.05).In addition,after 24 h of exposure,the SUMO-1 protein expression was significantly increased by 2.23-fold in the METH-treated(1.0 mM)primary culture neurons(p<0.05).Similarly,a-Syn protein expression was observably increased by 2.71-fold in the 24-h METH-treated(1.0 mM)primary culture neurons(p<0.05).We determined SUMO-1 and a-Syn protein expression in the prefrontal cortex and striatum of mice following subacute METH exposure.Western blot results showed that,in the prefrontal cortex,the SUMO-1 protein level was 1.69-fold higher in the subacute exposure groups than in the control group(p<0.05).This ?-Syn protein level was increased 2.06-fold in the subacute exposure groups compared with that in the control group(p<0.05).Similarly,subacute METH exposure increased SUMO-1 and a-Syn protein expression(2.14-fold and 1.56-fold,respectively)in the striatum(p<0.05).2 METH decreases the SUMOylation level of ?-Syn.Co-immunoprecipitation results showed that the SUMOylation level of ?-Syn was decreased by relatively higher doses of METH.The SUMOylation level of ?-Syn was decreased by 59.6%and 64.9%in METH-treated SH-SY5Y cells(2.0mM,24h)and primary culture neurons(1.0mM,24h)(p<0.05).The SUMOylation level of a-Syn was also reduced by 60.7%and 54.6%in the prefrontal cortex and striatum of mice,respectively,following subacute METH exposure(p<0.05).3 METH decreases the specific E2-conjugating enzyme,UBC9.Compared with the control group,UBC9 protein expression was lessened in SH-SY5Y cells and primary culture neurons that were treated with a range of METH doses for 24 h(p<0.05),while two SUMOylation factors the El ligase SAE1 and the E3 ligase PIAS1 showed no obvious change.4 The SUMOylation level of a-Syn in METH-treated cells is increased when UBC9 is overexpressed.We effectively overexpressed the UBC9 gene using a plasmid in SH-SY5Y cells.UBC9 protein expression level was significantly increased by 1.80-fold compared with the level in the negative control group(p<0.05).The SUMOylation level of a-Syn was increased by 1.49-fold in cells transfected with the UBC9 plasmid compared with the level in negative control group(p<0.05),and the SUMOylation level of ?-Syn was increased by 1.85-fold in METH-treated cells transfected with the UBC9 plasmid compared with the level in METH-treated group(p<0.05).More importantly,METH exposure significantly increased the expression of a-Syn protein;this increase was mitigated by 27.2%after transfection with the UBC9 plasmid(p<0.05).The aggregation of a-Syn was also mitigated by 33.1%compared with that of the group treated only with METH(p<0.05).To verify the results in SH-SY5Y cells,we also overexpressed the expression of the UBC9 gene using LV-UBC9 in primary culture neurons.With UBC9 protein expression increased by 1.44-fold,the SUMOylation level of a-Syn was increased by 1.57-fold(p<0.05).In the same manner,the increase in a-Syn protein expression in response to METH was diminished by 37.4%after exposure to LV-UBC9(p<0.05),the SUMOylation level of a-Syn in response to METH was increased by 1.69-fold after exposure to LV-UBC9(p<0.05).5 Silencing of SUMO-1 expression exacerbates a-Syn aggregation inMETH-treated cells.We respectively transfected each of the two siRNA sequences(siSUMO-1#1 and#2)or negative control siRNA(siNC)into SH-SY5Y cells.The expression of SUMO-1 was markedly reduced by 65.2%and 42.5%transfected with siSUMO-1#1 and#2(p<0.05).Subsequently,we selected siSUMO-1#1 as the siSUMO-1 for the next experiment.Western blot analyses showed that silencing of SUMO-1 expression decreased the level of a-Syn SUMOylation by 38.8%(p<0.05).In addition,METH exposure increased the expression and protein aggregation of a-Syn,and this effect was significantly exacerbated by 1.70-fold(p<0.05)and 1.50-fold(p<0.05)with silencing of SUMO-1 expression.To verify the results in SH-SY5Y cells,we also infected primary culture neurons with LV-SUMO-1-RNAi or control LV-GFP followed by METH exposure.We observed that the level of a-Syn SUMOylation was decreased by 70.1%in primary culture neurons infected with lentivirus LV-SUMO-1-RNAi(p<0.05).Notably,a-Syn expression was increased by 1.23-fold after co-exposure to METH and LV-SUMO-1-RNAi compared with the level in the METH group(p<0.05).6 Impaired SUMOylation of ?-Syn accelerates a-Syn aggregation induced by METH in SH-SY5Y cells.We screened for SH-SY5Y cells stably expressing WT ?-Syn,?-Syn-2KR or GFP and treated those cells with METH.Western blot analyses showed that the a-Syn expression levels of SH-SY5Y cells stably expressing WT ?-Syn(1.74-fold)and?-Syn-2KR(1.5 7-fold)were apparently higher than that in negative control group(p<0.05).SUMOylation of a-Syn was impaired by 61.6%in SH-SY5Y cells stably expressing a-Syn-2KR compared with that in SH-SY5Y cells stably expressing WT?-Syn(p<0.05).Furthermore,METH exposure significantly increased the expression and aggregation of ?-Syn,and these increases were accelerated by 1.21-fold and 1.25-fold,respectively,with impaired SUMOylation of ?-Syn(p<0.05).The expression of UBC9 was increased by 1.30-fold and 1.61-fold in SH-SY5Y cells stably expressing WT ?-Syn(p<0.05)and a-Syn-2KR(p<0.05)with UBC9 plasmid.As expected,compared with the level in SH-SY5Y cells stably expressing WT a-Syn followed with METH,a-Syn expression level was reduced by 22.7%and the SUMOylation level of a-Syn was increased by 1.71-fold in cells transfected with the UBC9 plasmid(p<0.05).On the contrary,compared with the level in SH-SY5Y cells stably expressing a-Syn-2KR with METH exposure,a-Syn expression level and the SUMOylation level of a-Syn had no significant difference in cells transfected with the UBC9 plasmid(p>0.05).Western blot results showed that METH increased Ub,LC3-?,P-62 and LAMP2A protein expression and decreased UbE1 protein expression in a dose-dependent in SH-SY5Y cells(p<0.05).The METH-induced decrease in UbEl protein expression and the UbEl/Ub protein expression ratio in SH-SY5Y cells stably expressing a-Syn-2KR were aggravated by 45.3%and 74.5%,respectively,compared with the values in SH-SY5Y cells stably expressing WT ?-Syn(p<0.05).In addition,the METH-induced increases in LC3-?,P-62 and LAMP2A protein expression in SH-SY5Y cells stably expressing a-Syn-2KR were also aggravated by 2.01-fold,1.62-fold and 2.53-fold,respectively,compared with the values in similarly METH-treated SH-SY5Y cells stably expressing WT ?-Syn(p<0.05).7 Mutations in the major SUMOylation acceptor sites of ?-Syn exacerbate METH-induced ?-Syn aggregation in dopaminergic neurons in vivo.Western blot results manifested that striatal ?-Syn protein expression in mice treated with AAV2-WT-?-Syn and AAV2-?-Syn-2KR was observably increased by 2.02-fold and 2.81-fold,respectively,compared with the expression in the negative control(p<0.05).The striatal SUMOylation level of ?-Syn was impaired by 44.2%for AAV2-?-Syn-2KR compared with AAV2-WT-?-Syn treatment(p<0.05),and the striatal SUMOylation level of ?-Syn was impaired by 62.8%for AAV2-?-Syn-2KR with METH exposure compared with AAV2-WT-?-Syn with METH exposure(P<0.05).Consistent with results in vitro,METH exposure significantly increased the expression of a-Syn,and this increase was intensified by 1.79-fold with mutations of lysine 96 and lysine 102 of ?-Syn(p<0.05).Subacute METH exposures decreased UbE1 protein expression and increased Ub,LC3-?,P-62 and LAMP2A protein expression in the striatum of mice(p<0.05).The METH-induced decreases in UbEl and UbEl/Ub protein expression in mice injected with AAV2-?-Syn-2KR were aggravated by 55.0%and 71.7%,respectively,compared with those in mice injected with AAV2-WT-?-Syn(p<0.05).In addition,METH exposure also aggravated the increases in LC3-?,P-62 and LAMP2A protein expression in mice injected with AAV2-?-Syn-2KR by 2.22-fold,1.40-fold and 1.30-fold,respectively compared with the values in mice injected with AAV2-WT-?-Syn(p<0.05).ConclusionIn the present study,we report that SUMO-1 and ?-Syn expression are increased after high-dose METH exposure in vitro and in vivo.However,SUMOylation level of?-Syn is decreased by METH in vitro and in vivo.A blockade of ?-Syn SUMOylation may contribute to METH-induced neurotoxicity.One possible reason is that UBC9,a key factor that regulates the SUMOylation level of a-Syn,is reduced in METH-induced cells.If UBC9 is overexpressed,the SUMOylation level of ?-Syn is increased remarkably,and the increase in ?-Syn SUMOylation plays a critical role in relieving ?-Syn aggregation induced by METH.Conversely,the SUMOylation level of ?Syn is decreased when SUMO-1 expression is blocked.?-Syn aggregation induced by METH is aggravated by the reduction of ?-Syn SUMOylation.Furthermore,mutations in the major SUMOylation acceptor sites of a-Syn block SUMOylation of ?-Syn and exacerbate METH-induced ?-Syn aggregation and neurotoxicity in dopaminergic neurons in vitro and in vivo.Additionally,impaired SUMOylation of a-Syn may act through the UPS and the ALP to influence a-Syn aggregation and degradation.These findings together with previous studies indicate that ?-Syn SUMOylation has a protective effective not only against several neurodegenerative diseases,but also against neurotoxicity induced by METH. |
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