Effects Of α-synuclein On DMT1 Protein Stability And Its Underlying Mechanisms In Parkinson’s Disease

Parkinson’s disease（PD）,a common neurodegenerative disease,is pathologically characterized by selective loss of dopaminergic neurons in the substantia nigra（SN）and the presence of a-synuclein（a-Syn）aggragates in intracellular inclusions known as Lewy bodies（LB s）.Amounting evidence demonstrate that dysfunction of iron metabolism plays a key role in the pathogenesis of PD.There exists large amounts of iron deposition in the SN of PD patients and animal models.The iron content in the remaining dopaminergic neurons is also remarkably increased.Although the cause of selective iron deposition in PD has not been fully elucidated,our lab previously demonstrated that abnormal expressions of iron transporters divalent metal transporter 1（DMT1）was involved in the nigral iron accumulation in PD models.Iron is also included in the LBs,a characteristic pathological marker of PD.As a major component of LBs,abnormal aggregation of a-Syn further aggravates the increased intracellular iron levels,and a-Syn has been confirmed to be an iron reductase,which can reduce Fe3+ to Fe²⁺.Thus,a-Syn accumulation will inevitably affect iron metabolism,which provides great possibility for a-Syn to participate in iron deposition in the SN of PD.How a-Syn affects iron homeostasis in the brain and its role in the nigral iron deposition are still unclear.We found that overexpression of wild-type a-synuclein（WT a-Syn）and A53T mutant a-synuclein（A53T a-Syn）led to the increased DMT1 protein levels,but its mRNA levels had no obvious change.The molecular mechanism that a-Syn increased the protein levels of DMT1 was unknown.Therefore,understanding the effects of a-Syn on DMT1 protein stability is of great importance for elucidating the mechanism of iron accumulation in the SN of PD.Ubiquitin proteasome system（UPS）is considered as an important way to maintain DMT1 protein stability.As we known,UPS is the main pathway responsible for the intracellular protein degradation.Two basic processes are included:ubiquitylation of target proteins and proteasomal degradation.Parkin is a specific E3 ligase responsible for the ubiquitylation of DMT1,which can protect cells from iron toxicity.However,the post-translational modification of parkin,such as S-nitrosylation and phosphorylation,can inhibit its E3 activity and substrate degradation,which further accelerate the neurodegeneration.26S proteasome is a multi-subunit complex and can be divided into regulatory particle and core protease.The 19S regulatory particle imparts regulation on the proteasome by recognizing,unfolding and translocating polyubiquitylated proteins to the 20S for degradatio.β1,β2 and β5 contain the proteolytic active sites,which present caspase-like,trypsin-like and chymotrypsin-like activities,respectively.There exists another form of proteasome in eukaryotic cells,that is immunoproteasome.β1,β2 and β5 are replaced by β1i,β2i,and β5i,respectively,and assembled into the core protease,which play an important role in antigen presentation and protein degradation.It has been reported that protein degradation via UPS is impaired in PD.Furthermore,abnormal aggregation of a-Syn further inhibits the activity and function of proteasome.Proteasome inhibitor could induce the increased expression of DMT1.Thus,we speculated that whether a-Syn induced the up-regulation of DMT 1 by inhibiting its ubiquitylation and proteasomal degradation,which further resulted in the elevated intracellular iron levels in dopaminergic neurons.In the present study,we aimed to investigate the effects of a-Syn on DMT1 protein stability and its underlying mechanism.The ubiquitylation and proteasomal degradation of DMT1 were studied in a-Syn transfected SH-SY5Y cells and human A53T mutant a-Syn transgenic mice.Firstly,the effects of a-Syn on DMT1 expression were detected and we further observed the ferrous iron uptake and changes in mitochondrial transmembrane potential（ΔΨm）and reactive oxygen species（ROS）to reflect the function of up-regulated DMT1.Secondly,the ubiquitylation of DMT1 was detected by ubiquitylation assay.The phosphorylation of DMT1 specific E3 ubiquitin ligase parkin and its effects on DMT1 ubiquitylation were assessed by western blot and ubiquitylation assay,respectively.At last,transcriptional sequencing of SH-SY5Y cells with a-Syn overexpression was performed to acquire differential expressed genes and enriched signaling pathways.The change of DMT1 protein levels was detected with proteasome inhibitor MG 132 and lysosomal inhibitor chloroquine treatment.The activity of the proteasome was examined using specific fluorescent peptide substrates.We detected the protein levels of DMT1 when treated with rolipram to enhance proteasomal activity.The expression of proteasome subunits were measured by real-time PCR and western blot.The results were as follows:1.α-Syn prevented the ubiquitylation of DMT1 in PD（1）The mRNA levels of DMT1 had no obvious change in WT α-Syn and A53T α-Syn transfected SH-SY5Y cells（P>0.05）.However,DMT 1 protein levels were significantly increased by 59.1%and 66.9%,respectively,compared with the control（P<0.01）.In addition,the nigral DMT1 protein levels were significantly increased by 36.2%and 40.9%at the age of twelve and fifteen months,respectively,compared with their WT littermates（P<0.05）.（2）Ferrous iron uptake was significantly enhanced in WT α-Syn and A53T α-Syn transfected SH-SY5Y cells.Moreover,WT α-Syn and A53T α-Syn overexpression significantly aggravated the decrease in ΔΨm induced by ferrous iron,which showed a 2.36-fold and 2.52-fold decrease,respectively,when compared with solely Fe²⁺ group.Intracellular ROS generation was further increased in SH-SY5Y cells transfected with WT a-Syn and A53T α-Syn,which was 46.6%and 50.4%higher,respectively,than that of Fe²⁺ group.（3）Flag empty vector/HA empty vector/Myc-DMT 1,Flag empty vector/HA-Ub/Myc-DMT1,Flag-WT α-Syn/HA-Ub/Myc-DMT1 and Flag-A53Tα-Syn/HA-Ub/Myc-DMT1 were transfected into HEK293T cells,respectively.Compared with Flag empty vector/HA-Ub/Myc-DMT1 group,the ubiquitylation of DMT1 was significantly decreased in Flag-WT α-Syn/HA-Ub/Myc-DMT1 group and Flag-A53T α-Syn/HA-Ub/Myc-DMT1 group.（4）The phosphorylation of parkin at Ser131 in WT α-Syn and A53T α-Syn transfected SH-SY5Y cells was increased by 42.6%and 43.9%,respectively,compared with the control（P<0.05）.When transfected with S131A parkin,phosphorylation of parkin at Ser131 induced by α-Syn was eliminated.Furthermore,the phosphorylation of parkin at Ser131 in the SN of nine and twelve-monthα-SynA53T+/+ mice was increased by 32.2%and 30.9%,respectively,compared with their WT littermates（P<0.05）.（5）EGFP empty vector/HA empty vector/Flag-S131A parkin/Myc-DMT1,EGFP empty vector/HA-Ub/Flag-S 131A parkin/Myc-DMT1,EGFP-WT α-Syn/HA-Ub/Flag-S 131A parkin/Myc-DMT1 and EGFP-A53T α-Syn/HA-Ub/Flag-S131A parkin/Myc-DMT1 were transfected into HEK293T cells,respectively.Compared with EGFP empty vector/HA-Ub/Flag-S 131A parkin/Myc-DMT1 group,the ubiquitylation of DMT1 was significantly increased in EGFP-WT α-Syn/HA-Ub/Flag-S 131A parkin/Myc-DMT1 group and EGFP-A53T α-Syn/HA-Ub/Flag-S131A parkin/Myc-DMT1 group.（6）The phosphorylation of p38 MAPK in WT α-Syn and A53T α-Syn transfected SH-SY5Y cells was increased by 65.6%and 73.8%,respectively,compared with the control（P<0.05）.After treatment with p38 MAPK inhibitor SB203580,the activation of p38 MAPK was significantly inhibited.Compared with WT α-Syn group and A53T α-Syn group,the phosphorylation of parkin at Ser131 was decreased by 25.6%and 27.9%,recpectively（P<0.01）.In the SN of a-SynA53T+/+mice at the age of nine and twelve months,p38 MAPK phosphorylation was also increased by 32.2%and 35.1%,respectively,compared with their WT littermates（P<0.05）.2.a-Syn inhibited the proteasomal degradation of DMT 1 in PD（1）Transcriptome sequencing was performed in SH-SY5Y cells transfected with WTα-Syn and A53T α-Syn.The raw reads were assessed and filtered to clean reads.The minimum mapping rate was 94.93%and the maximum rate was 95.55%.And the ratio of exon is the highest.By calculating the fold change and significance of differential expressed genes,a total of 1047 differential genes were obtained.Genes related to ubiquitin proteasome,such as PSMB8,PSMB9,PSME1,PSME2,UBC,UBA7 and UBE2L6,were down-regulated in α-Syn group.Furthermore,differential genes enriched signaling pathway was also associated with proteasome.（2）In SH-SY5Y cells transfected with WT α-Syn and A53T α-Syn,200 nM MG132 treatment for 24 hrs significantly aggravated the increase of DMT1 protein levels induced by α-Syn.Compared with WT α-Syn group and A53T α-Syn group,DMT1 protein levels increased by 27.6%and 28.1%,respectively（P<0.05）.There was no significant change in DMT 1 protein levels with 30 μM chloroquine treatment when compared with WT α-Syn group and A53T α-Syn group（P>0.05）.（3）The chymotrypsin-like,trypsin-like and caspase-like activity of proteasome were significantly decreased in WT α-Syn and A53T α-Syn transfected SH-SY5Y cells and twelve-month α-SynA53T+/+mice when compared with the control（P<0.01）.（4）In SH-SY5Y cells transfected with WT α-Syn and A53T α-Syn,10 μM rolipram treatment significantly enhanced the chymotrypsin-like,trypsin-like and caspase-like activity of proteasome（P<0.01）.Moreover,rolipram significantly antagonized the up-regulation of DMT1 protein levels induced by a-Syn,which was 30.3%lower than that of the control,38.6%lower than that of WT a-Syn group and 40.1%lower than that of A53T a-Syn group（P<0.01）.Likewise,the proteasomal chymotrypsin-like,trypsin-like and caspase-like activity in the SN of twelve-month a-SynA53T+/+mice were enhanced with 0.05 mg/kg rolipram intraperitoneal injection for 21 d（P<0.05）.DMT1 protein levels were reduced by 51.9%with rolipram treatment when compared with the vehicle group（/P<0.05）.（5）In SH-SY5Y cells transfected with WT a-Syn,the mRNA levels of β1i,β5i,PA28α,PA28β were decreased by 34.7%,43.8%,30.2%,28.9%,respectively,and their protein levels were decreased by 46.1%,47.4%,30.1%,37.5%,respectively（P<0.05）.In SH-SY5Y cells transfected with A53T a-Syn,the mRNA levels of β1i,β5i,PA28α,PA28β were decreased by 59.2%,40.8%,37.5%,38.6%,respectively,and their protein levels were decreased by 62.5%,53.1%,39.3%,34.6%,respectively（P<0.05）.Moreover,in the SN of α-SynA53T+/+mice at the age of nine and twelve months,the protein levels of β1i decreased by 25.2%and 34.9%,respectively,and the protein levels of PA28β decreased by 31.6%and 37.6%,respectively（P<0.05）.（6）In SH-SY5Y cells transfected with WT α-Syn and A53T α-Syn,the mRNA levels of the 26S proteasome regulatory particle subunits Rpn11,Rpn10,Rpn13,Rpt1-6 and core protease subunits β1,β2,β5 had no obvious change when compared with the control（P>0.05）.In conclusion,a-Syn overexpression led to the up-regulation of DMT1 protein levels in PD models.On the one hand,α-Syn induced the activation of p38 MAPK,which led to the phosphorylation of parkin at Ser131,thus inhibiting its E3 ubiquitin ligase function,and reducing the ubiquitylation of DMT1.On the other hand,α-Syn inhibited the proteasomal degradation of DMT1 by inhibiting the activity of proteasome and the expression of immunoproteasome subunits.In addition,α-Syn overexpression could significantly enhance cellular ferrous iron influx via the up-regulation of DMT1,which further decreased ΔΨm and increased ROS generation leading to aggravated ferrous iron-induced oxidative stress in dopaminergic neurons.The present study elucidated the regulation of α-Syn on DMT1 protein stability through ubiquitin-proteasome pathway in PD,indicating that aggregated α-Syn enhanced iron uptake and subsequent aggravated oxidative stress in dopaminergic neurons by up-regulating DMT1 expression.Our study might provide new insight into the involvement of α-Syn in nigral iron deposition and iron metabolism dysfunction in PD.