| Parkinson’s disease(PD) is a common and adult-onset progressive neurodegenerative disorder. PD is clinically characterized by rest tremor, bradykinesia, rigidity and postural instability. Neuropathological hallmarks of PD include the degeneration and loss of dopaminergic neurons in the substantia nigra(SN) and the formation of Lewy body(LB). However, the etiology and pathogenesis of PD remain unrevealed. Genetic factors, environmental factors and aging are thought to contribute to the development of this disease. Multiple mechanisms might be involved, such as oxidative stress, inflammation, mitochondria dysfunction, apoptosis, aberrant protein aggregation and iron deposition. α-Synuclein is the main composition of Lewy body, which is closely related to both familial and sporadic PD cases. The aberrant aggregation of α-synuclein is regarded as the crucial player in PD pathogenesis. Several posttranslational modifications have already been identified in α-synuclein, including phosphorylation, ubiquitination, nitration, glycosylation and SUMO modification. Among those, phosphorylation is the most common and important protein modification for regulating protein function. While only 4% of the soluble, monomeric α-synuclein appears to be phosphorylated under physiological conditions in vivo, approximately 90% is phosphorylated in LB lesions, suggesting that the phosphorylation status of α-synuclein clearly influences its aggregation and toxicity. There is a close relationship between α-synuclein phosphorylation at S29(p S129) and its clearance mechanism. While several reports suggest that p S129 promotes inclusion formation, others suggest that phosphorylation prevents or has no effect on inclusion formation. Therefore, it is still unclear whether phosphorylation promotes or prevents expression and aggregation of α-synuclein.Post-mortem examinations reveal that individual dopaminergic neuron in substantia nigra shows raised iron levels in PD and iron levels in substantia nigra are associated with the progression of PD. Iron deposition might be a critical factor in the etiology of PD. Abnormally increased iron promotes the generation of reactive oxygen species(ROS), thus induces dopaminergic neuronal degeneration. Previous studies in our lab have demonstrated that iron could increase the expression of α-synuclein and aggravate its aggregation. However, whether the process of α-synuclein phosphorylation is involved remains unclear. Polo-like kinase 2(PLK2) comprises a family of conserved Ser/Thr protein kinases, which could mediate α-synuclein phosphorylation at S129 specifically. Casein kinase 2(CK2) is a common Ser/Thr protein kinase composed of two α or α’ catalytic subunits and β regulatory subunits. However, whether different kinases participate in iron-induced phosphorylation of α-synuclein also remains unclear. In the present study, western blot was used to detect the protein levels of α-synuclein, p S129, PLK2, CK2 and light chain 3(LC3) in SH-SY5 Y cells treated with ferric ammonium citrate(FAC). 20 S proteasome activity assay kit was used to detect the effects of FAC on intracellular proteasome activity. In addition, we investigated the effects of PLK2 inhibitor BI2536, CK2 inhibitor TBCA and antioxidant N-Acetyl-L-cysteine(NAC) on the changes mentioned above in SH-SY5 Y cells with FAC treatment. Therefore, these experiments were proposed to explore the effects of iron on phosphorylation of α-synuclein and elucidate the possible mechanisms in SH-SY5 Y cells. The results were shown as follows:1. SH-SY5 Y cells were treated with 1 mmol/L FAC for 4 h, 8 h, 12 h, 16 h, 20 h, 24 h or 48 h. α-Synuclein protein levels began to increase at 12 h(additional 81% increase compared to the non-treated group, P<0.05). Then more up-regulation was observed at 16 h(90% increase compared to the non-treated group, P<0.05). Most significant up-regulation occurred at 20 h(93% increase compared to the non-treated group, P<0.05) and then to a less extent but still much more up-regulation than controls was observed at 24 h and 48 h(66% and 51% increase compared to the non-treated group, respectively, P<0.05). The results indicated that iron could upregulate the expression of α-synuclein in a time-dependent manner.2. SH-SY5 Y cells were treated with 1 mmol/L FAC for 4 h, 8 h, 12 h, 16 h, 20 h, 24 h or 48 h. PS129 protein levels began to increase at 12 h(additional 115% increase compared to the non-treated group, P<0.05). Then more up-regulation was observed at 16 h(127% increase compared to the non-treated group, P<0.05). Most significant up-regulation occurred at 20 h(152% increase compared to the non-treated group, P<0.05) and then to a less extent but still much more up-regulation than controls was observed at 24 h and 48 h(106% and 68% increase compared to the non-treated group, respectively, P<0.05). The p S129 expression was in parallel with that of α-synuclein. The phosphorylation level of α-synuclein began to increase at 16 h. After treatment with 1 mmol/L FAC for 16~48 h, the phosphorylation level of α-synuclein was increased by 42%, 42%, 47% or 51%, compared with control(P<0.05). The results indicated that iron could upregulate the phosphorylation level of α-synuclein in a time-dependent manner.3. SH-SY5 Y cells were treated with 1 mmol/L FAC for 4 h, 8 h, 12 h, 16 h, 20 h, 24 h or 48 h. PLK2 protein levels began to increase at 12 h(additional 33% increase compared to the non-treated group, P<0.05). Most significant up-regulation occurred at 20 h(53% increase compared to the non-treated group, P<0.05) and then to a less extent but still much more up-regulation than controls was observed at 24 h and 48 h(44% and 35% increase compared to the non-treated group, respectively, P<0.05). CK2 protein levels began to increase at 24 h. After treatment with 1 mmol/L FAC for 24 h or 48 h, CK2 expression was increased by 46% or 48%, compared with control(P<0.05). The results indicated that iron could upregulate the expression of PLK2 and CK2 in a time-dependent manner. There was a much earlier response of PLK2 up-regulation compared with CK2 up-regulation.4. SH-SY5 Y cells were pretreated with 20 μmol/L TBCA, 1 μmol/L BI2536 or 20 μmol/L TBCA and 1 μmol/L BI2536 for 30 min and then co-incubated with 1 mmol/L FAC for 24 h. α-Synuclein expression was decreased by 43%, 44% or 41%, compared with FAC group(P<0.01). The phosphorylation level of α-synuclein was decreased by 27%, 31% or 32%, compared with FAC group(P<0.01). The results indicated that inhibitors of PLK2 or CK2 could block the upregulation of α-synuclein or p S129 induced by iron overload.5. 20 μmol/L TBCA treatment increased the protein levels of PLK2 by 41%, compared with control(P<0.05). There was no obvious alteration of CK2 protein levels in 1 μmol/L BI2536 treated group compared with control. The results indicated that the change of CK2 activity in SH-SY5 Y cells could influence the expression of PLK2. There might be mutual influences among different kinases.6. SH-SY5 Y cells were pretreated with 0.5 mmol/L NAC for 30 min and then co-incubated with 1 mmol/L FAC for 24 h. The expression of PLK2, CK2 and the phosphorylation level of α-synuclein were brought back to the normal levels. However, the protein levels of α-synuclein were still increased by 32%, compared with control(P<0.05). The results indicated that NAC could completely block the upregulation of PLK2, CK2 or the phosphorylation level of α-synuclein induced by iron overload. Iron-induced upregulation of α-synuclein could only be partially blocked by NAC pretreatment.7. Twenty-four-hour treatment of SH-SY5 Y cells with 1 mmol/L FAC led to both a LC3Ⅱ/LC3Ⅰ ratio elevation and increased intracellular proteasome activity(147% or 12%, respectively) compared with control(P<0.01). LC3 Ⅱ /LC3 Ⅰ ratio was decreased by 22% after pretreatment with 0.5 mmol/L NAC, compared with FAC group(P<0.05). Intracellular proteasome activity was decreased by 5% after pretreatment with 0.5 mmol/L NAC, compared with FAC group. The results indicated that iron overload could enhance intracellular autophagy and proteasome activity, which could be partially blocked by NAC pretreatment. This was in parallel with that of α-synuclein.The above results suggest iron could upregulate the expression of α-synuclein, which could be partially blocked by antioxidant NAC. That hinted that oxidative stress partially participated in iron-induced upregulation of α-synuclein. Iron overload increased the protein levels of p S129 via upregulating the expression of PLK2 or CK2 that could phosphorylate α-synuclein at S129. PLK2 or CK2 inhibitors could block the upregulation of α-synuclein and p S129 induced by iron overload. Compared to CK2, PLK2 might contribute more to the phosphorylation of α-synuclein induced by iron. The upregulation of PLK2 or CK2 protein levels and α-synuclein phosphorylation induced by iron could be blocked by NAC, indicating that oxidative stress played a critical role in iron-induced phosphorylation of α-synuclein. Iron overload could incease intracellular autophagy and proteasome activity, which could be partially blocked by NAC pretreatment. The trend was in aparallel with that of α-synuclein, indicating that the activation of the two systems seemed to act as potential compensatory responses to enhance intracellular clearance of α-synuclein. These results provide new experimental basis for further exploring the mechanisms of interaction between iron and α-synuclein and intervention strategies in PD. |
PDF Full Text Request