Yeast Prion Protein Sup35 Initiates α-Synuclein Pathology in Parkinson's Disease.

Sinus infection of Saccharomyces cerevisiae accelerates the aggregation of α-synuclein (α-syn) in A53T mice, which was caused by prion protein Sup35. Sup35 promotes α-syn aggregation in vitro and in vivo and leads to Parkinson's disease (PD)-like motor impairment in wildtype mice, suggesting that the yeast Sup35 triggers α-syn pathology in PD.

Extracellular α-Synuclein Modulates Iron Metabolism Related Proteins via Endoplasmic Reticulum Stress in MES23.5 Dopaminergic Cells.

Alpha-synuclein plays a vital role in the pathology of Parkinson's disease (PD). Spreading of α-synuclein in neighboring cells was believed to contribute to progression in PD. How α-synuclein transmission affects adjacent cells is not full elucidated. Here, we used recombinant α-synuclein to mimic intercellular transmitted α-synuclein in MES23.5 dopaminergic cells, to investigate whether and how it could modulate iron metabolism. The results showed that α-synuclein treatment up-regulated divalent metal transporter 1 (DMT1) and down-regulated iron transporter (FPN), also up-regulated iron regulatory protein 1 (IRP1) protein levels and hepcidin mRNA levels. Endocytosis inhibitor dynasore pretreatment completely abolished and even reversed the upregulation of DMT1 and IRP1 induced by α-synuclein, however, FPN down-regulation was partially blocked by dynasore. Autophagy-inducing agent rapamycin reversed DMT1 up-regulation and FPN down-regulation, and fully blocked the upregulation of IRP1. Elevated hepcidin levels induced by α-synuclein was fully blocked by dynasore pretreatment, however, even higher with rapamycin pretreatment. Alpha-synuclein treatment triggered endoplasmic reticulum (ER) stress. ER stress inducer thapsigargin induced similar responses elicited by α-synuclein. ER stress inhibitor salubrinal blocked the up-regulation of IRP1 and hepcidin, as well as DMT1 up-regulation and FPN down-regulation, also dramatically abolished cAMP-response elements binding protein phosphorylation induced by α-synuclein. Taken together, these finding indicated that extracellular α-synuclein could regulate cellular iron metabolism, probably mediated by ER stress. It provides novel evidence to elucidate the relationships between transmitted α-synuclein and iron metabolism disturbance in PD.

Apelin-13 Protects Dopaminergic Neurons against Rotenone-Induced Neurotoxicity through the AMPK/mTOR/ULK-1 Mediated Autophagy Activation.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Several brain-gut peptides are able to exert neuroprotective effects on the nigrostriatal dopaminergic system. Apelin-13 is a neuropeptide, conveying potential neuroprotective activities. However, whether, and how, apelin-13 could antagonize rotenone-induced neurotoxicity has not yet been elucidated. In the present study, rotenone-treated SH-SY5Y cells and rats were used to clarify whether apelin-13 has protective effects on dopaminergic neurons, both in vivo and in vitro. The results showed that apelin-13 could protect SH-SY5Y cells from rotenone-induced injury and apoptosis. Apelin-13 was able to activate autophagy, and restore rotenone induced autophagy impairment in SH-SY5Y cells, which could be blocked by the autophagy inhibitor 3-Methyladenine. Apelin-13 activated AMPK/mTOR/ULK-1 signaling, AMPKα inhibitor compound C, as well as apelin receptor blockage via siRNA, which could block apelin-13-induced signaling activation, autophagy activation, and protective effects, in rotenone-treated SH-SY5Y cells. These results indicated that apelin-13 exerted neuroprotective properties against rotenone by stimulating AMPK/mTOR/ULK-1 signaling-mediated autophagy via the apelin receptor. We also observed that intracerebroventricular injection of apelin-13 could alleviate nigrostriatal dopaminergic neuron degeneration in rotenone-treated rats. Our findings provide new insights into the mechanism by which apelin-13 might attenuate neurotoxicity in PD.

miR-151-5p modulates APH1a expression to participate in contextual fear memory formation.

Long-term memory formation requires gene expression and new protein synthesis. MicroRNAs (miRNAs), a family of small non-coding RNAs that inhibit target gene mRNA expression, are involved in new memory formation. In this study, elevated miR-151-5p (miR-151) levels were found to be responsible for hippocampal contextual fear memory formation. Using a luciferase reporter assay, we demonstrated that miR-151 targets APH1a, a protein that has been identified as a key factor in γ-secretase activity, namely APH1a. Blocking miR-151 can upregulate APH1a protein levels and subsequently impair hippocampal fear memory formation. These results indicate that miR-151 is involved in hippocampal contextual fear memory by inhibiting APH1a protein expression. This work provides novel evidence for the role of miRNAs in memory formation and demonstrates the implication of APH1a protein in miRNA processing in the adult brain.

IL-35 Decelerates the Inflammatory Process by Regulating Inflammatory Cytokine Secretion and M1/M2 Macrophage Ratio in Psoriasis.

IL-35 downregulates Th17 cell development and suppresses certain types of autoimmune inflammation such as collagen-induced arthritis and experimental autoimmune uveitis. Psoriasis is thought to be initiated by abnormal interactions between cutaneous keratinocytes and systemic immune cells. However, the role of IL-35 in psoriasis remains unclear. In this study, we assessed IL-35 in three well-known psoriasis models: a human keratinocyte cell line (HaCaT), a keratin 14 (K14)-vascular endothelial growth factor A (VEGF-A)-transgenic (Tg) mouse model, and an imiquimod-induced psoriasis mouse model. First, we found that IL-35 suppressed the expression of IL-6, CXCL8, and S100A7, which are highly upregulated by a mixture of five proinflammatory cytokines in HaCaT. Second, a plasmid coding for the human IL-35 sequence coated with cationic liposomes showed potent immunosuppressive effects on K14-VEGF-A-Tg and imiquimod-induced psoriasis mouse models. In the K14-VEGF-A-Tg model, our results showed that several types of proinflammatory cytokines were significantly reduced, whereas IL-10 was remarkably induced by IL-35. Compared with pcDNA3.1, there was a small number of CD4(+)IL-17(+) T cells and a large number of CD4(+)IL-10(+) and CD4(+)CD25(+)Foxp3(+) T cells in the IL-35 group. Most importantly, we found that IL-35 decreased the total number of macrophages and ratio of M1/M2 macrophages, which has not been reported previously. In addition, compared with dexamethasone, IL-35 showed long-term therapeutic efficacy. In summary, our results strongly indicate that IL-35 plays a potent immunosuppressive role in psoriasis. Thus, IL-35 has potential for development as a new therapeutic strategy for patients with chronic psoriasis and other cutaneous inflammatory diseases.

Effects of Alpha-Synuclein on Primary Spinal Cord Neurons Associated with Apoptosis and CNTF Expression.

Spinal cord injury (SCI) often causes neurological deficits with poor recovery; the treatment, however, is far from satisfaction, and the mechanisms remain unclear. Using immunohistochemistry and western blotting analysis, we found α-synuclein (SNCA) was significantly up-regulated in the spinal caudal segment of rats subjected to spinal cord transection at 3 days post-operation. Moreover, the role of SNCA on neuronal growth and apoptosis in vitro was determined by using overexpressing and interfering SNCA recombined plasmid vectors, and the underlying mechanism was detected by QRT-PCR and western blotting. Spinal neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival, while it results in cell apoptosis in SNCA-ORF group. In molecular level, SNCA silence induced the up-regulation of CNTF and down-regulation of Caspase7/9. Together, endogenous SNCA plays a crucial role in spinal neuronal survival, in which the underlying mechanism may be linked to the regulation both apoptotic genes (Caspase7/9) and CNTF. The present findings therefore provide novel insights into the role of SNCA in spinal cord and associated mechanism, which may provide novel cue for the treatment of SCI in future clinic trials.

Knockdown of α-synuclein in cerebral cortex improves neural behavior associated with apoptotic inhibition and neurotrophin expression in spinal cord transected rats.

Spinal cord injury (SCI) often causes severe functional impairment with poor recovery. The treatment, however, is far from satisfaction, and the mechanisms remain unclear. By using proteomics and western blot, we found spinal cord transection (SCT) resulted in a significant down-regulation of α-synuclein (SNCA) in the motor cortex of SCT rats at 3 days post-operation. In order to detect the role of SNCA, we used SNCA-ORF/shRNA lentivirus to upregulate or knockdown SNCA expression. In vivo, SNCA-shRNA lentivirus injection into the cerebral cortex motor area not only inhibited SNCA expression, but also significantly enhanced neurons' survival, and attenuated neuronal apoptosis, as well as promoted motor and sensory function recovery in hind limbs. While, overexpression SNCA exhibited the opposite effects. In vitro, cortical neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival and neurite outgrowth, while it was accompanied by reverse efficiency in SNCA-ORF group. In molecular level, SNCA silence induced the upregulation of Bcl-2 and the downregulation of Bax, and the expression of NGF, BDNF and NT3 was substantially upregulated in cortical neurons. Together, endogenous SNCA play a crucial role in motor and sensory function regulation, in which, the underlying mechanism may be linked to the regulation of apoptosis associated with apoptotic gene (Bax, Bcl2) and neurotrophic factors expression (NGF, BDNF and NT3). These finds provide novel insights to understand the role of SNCA in cerebral cortex after SCT, and it may be as a novel treatment target for SCI repair in future clinic trials.

Bone Marrow Stromal Cells Promote Neuronal Restoration in Rats with Traumatic Brain Injury: Involvement of GDNF Regulating BAD and BAX Signaling.

BACKGROUND/AIMS: To investigate the effects of bone marrow stromal cells (BMSCs) and underlying mechanisms in traumatic brain injury (TBI). METHODS: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43) and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. RESULTS: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF). The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. CONCLUSION: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

Transplantation of olfactory ensheathing cells promotes the recovery of neurological functions in rats with traumatic brain injury associated with downregulation of Bad.

BACKGROUND AIMS: The neuroprotective effects of olfactory ensheathing cells (OECs) after transplantation have largely been known in the injured nervous system. However, the underlying mechanisms still must be further elucidated. We explored the effects of OEC transplantation on the recovery of neurophysiologic function and the related anti-apoptosis mechanism in acute traumatic brain injury. METHODS: The OECs from neonatal Sprague-Dawley rats were isolated, identified and labeled and then were immediately transplanted into the regions surrounding the injured brain site that is resulted from free-weight drop injury. RESULTS: Nerve growth factor and it's recepor, p75 was expressed in cultured OECs. Transplanted OECs survived, migrated around the injury site and significantly improved the neurological severe scores compared with the control group (P < 0.05). OEC transplantation significantly increased the number of GAP-43-immunopositive fibers and synaptophysin-positive vesicles (P < 0.05) but significantly decreased the number of apoptotic cells (P < 0.05). On the molecular level, the expression of Bad in the OEC transplantation group was significantly downregulated (P < 0.05). CONCLUSIONS: OEC transplantation could effectively improve neurological deficits in TBI rats; the underlying mechanism may be related with their effects on neuroprotection and regeneration induction, which is associated with the downregulation of the apoptotic molecule Bad.

Potential convergence of olfactory dysfunction in Parkinson's disease and COVID-19: The role of neuroinflammation.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder that affects 7-10 million individuals worldwide. A common early symptom of PD is olfactory dysfunction (OD), and more than 90% of PD patients suffer from OD. Recent studies have highlighted a high incidence of OD in patients with SARS-CoV-2 infection. This review investigates the potential convergence of OD in PD and COVID-19, particularly focusing on the mechanisms by which neuroinflammation contributes to OD and neurological events. Starting from our fundamental understanding of the olfactory bulb, we summarize the clinical features of OD and pathological features of the olfactory bulb from clinical cases and autopsy reports in PD patients. We then examine SARS-CoV-2-induced olfactory bulb neuropathology and OD and emphasize the SARS-CoV-2-induced neuroinflammatory cascades potentially leading to PD manifestations. By activating microglia and astrocytes, as well as facilitating the aggregation of α-synuclein, SARS-CoV-2 could contribute to the onset or exacerbation of PD. We also discuss the possible contributions of NF-κB, the NLRP3 inflammasome, and the JAK/STAT, p38 MAPK, TLR4, IL-6/JAK2/STAT3 and cGAS-STING signaling pathways. Although olfactory dysfunction in patients with COVID-19 may be reversible, it is challenging to restore OD in patients with PD. With the emergence of new SARS-CoV-2 variants and the recurrence of infections, we call for continued attention to the intersection between PD and SARS-CoV-2 infection, especially from the perspective of OD.

Neural stem cells grafts decrease neural apoptosis associated with caspase-7 downregulation and BDNF upregulation in rats following spinal cord hemisection.

Transplantation of neural stem cells (NSCs) into lesioned spinal cord demonstrated a beneficial effect for neural repair, the underlying mechanism, however, remains to be elusive. Here, we showed that NSCs, possessing the capacity to differentiate toward into neurons and astrocytes, exhibit a neuroprotective effect by anti-apoptosis mechanism in spinal cord hemi-transected rats despite it did not improve behavior. Intravenous NSCs injection substantially upregulated the level of BDNF mRNA but not its receptor TrkB in hemisected spinal cord, while caspase-7, a downstream apoptosis gene of caspase-3, has been largely down-regulated. TUNEL staining showed that the number of apoptosis cells in injured spinal cord decreased significantly, compared with seen in rats with no NSCs administration. The present finding therefore provided crucial evidence to explain neuroprotective effect of NSCs grafts in hemisected spinal cord, which is associated with BDNF upregulation and caspase-7 downregulation.

Dopaminergic neurodegeneration in the substantia nigra is associated with olfactory dysfunction in mice models of Parkinson's disease.

Olfactory dysfunction represents a prodromal stage in Parkinson's disease (PD). However, the mechanisms underlying hyposmia are not specified yet. In this study, we first observed an early olfactory dysfunction in mice with intragastric rotenone administration, consistent with dopaminergic neurons loss and α-synuclein pathology in the olfactory bulb. However, a much severer olfactory dysfunction was observed without severer pathology in olfactory bulb when the loss of dopaminergic neurons in the substantia nigra occurred. Then, we established the mice models by intrastriatal α-synuclein preformed fibrils injection and demonstrated the performance in the olfactory discrimination test was correlated to the loss of dopaminergic neurons in the substantia nigra, without any changes in the olfactory bulb analyzed by RNA-sequence. In mice with intranasal ferric ammonium citrate administration, we observed olfactory dysfunction when dopaminergic neurodegeneration in substantia nigra occurred and was restored when dopaminergic neurons were rescued. Finally we demonstrated that chemogenetic inhibition of dopaminergic neurons in the substantia nigra was sufficient to cause hyposmia and motor incoordination. Taken together, this study shows a direct relationship between nigral dopaminergic neurodegeneration and olfactory dysfunction in PD models and put forward the understandings that olfactory dysfunction represents the early stage of neurodegeneration in PD progression.

Corrigendum: MicroRNA339 Targeting PDXK Improves Motor Dysfunction and Promotes Neurite Growth in the Remote Cortex Subjected to Spinal Cord Transection.

[This corrects the article DOI: 10.3389/fcell.2020.00577.].

In vivo Direct Conversion of Astrocytes to Neurons Maybe a Potential Alternative Strategy for Neurodegenerative Diseases.

Partly because of extensions in lifespan, the incidence of neurodegenerative diseases is increasing, while there is no effective approach to slow or prevent neuronal degeneration. As we all know, neurons cannot self-regenerate and may not be replaced once being damaged or degenerated in human brain. Astrocytes are widely distributed in the central nervous system (CNS) and proliferate once CNS injury or neurodegeneration occur. Actually, direct reprogramming astrocytes into functional neurons has been attracting more and more attention in recent years. Human astrocytes can be successfully converted into neurons in vitro. Notably, in vivo direct reprogramming of astrocytes into functional neurons were achieved in the adult mouse and non-human primate brains. In this review, we briefly summarized in vivo direct reprogramming of astrocytes into functional neurons as regenerative strategies for CNS diseases, mainly focusing on neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD). We highlight and outline the advantages and challenges of direct neuronal reprogramming from astrocytes in vivo for future neuroregenerative medicine.

LRRK2-NFATc2 Pathway Associated with Neuroinflammation May Be a Potential Therapeutic Target for Parkinson's Disease.

Neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). However, the molecular mechanisms involved in extracellular α­synuclein-induced proinflammatory microglial responses through Toll-like receptor 2 (TLR2) are unclear. Leucine-rich repeat kinase 2 (LRRK2) is a serine/threonine kinase, and its mutations are closely related to autosomal dominant PD. Recently, Masliah et al characterized a novel-specific neuroinflammation cascade dependent on LRRK2-NFATc2 in microglia activated by neuron-released α-synuclein. LRRK2 selectively phosphorylated and induced nuclear translocation of NFATc2 to activate a neuroinflammation cascade. In this cascade, LRRK2 kinase was activated by neuron-released α-synuclein in microglia via TLR2. Further, NFATc2, as a kinase substrate for LRRK2, was directly phosphorylated, which accelerated nuclear translocation of NFATc2, where cytokine/chemokine gene expression including TNF-α and IL-6 is regulated by NFATc2 transcriptional activity, resulting in a neurotoxic inflammatory environment. Moreover, an abnormal increase of NFATc2 in nuclear was observed in the brains of patients and a mouse model of PD. Additionally, the administration of an LRRK2 inhibitor could ameliorate neuroinflammation, prevent neuronal loss, and improve motor function. Therefore, modulation of LRKK2-NFATc2 signaling cascade might be a potential therapeutic target for the treatment of PD.

Corynoxine Protects Dopaminergic Neurons Through Inducing Autophagy and Diminishing Neuroinflammation in Rotenone-Induced Animal Models of Parkinson's Disease.

Recent studies have shown that impairment of autophagy is related to the pathogenesis of Parkinson's disease (PD), and small molecular autophagy enhancers are suggested to be potential drug candidates against PD. Previous studies identified corynoxine (Cory), an oxindole alkaloid isolated from the Chinese herbal medicine Uncaria rhynchophylla (Miq.) Jacks, as a new autophagy enhancer that promoted the degradation of α-synuclein in a PD cell model. In this study, two different rotenone-induced animal models of PD, one involving the systemic administration of rotenone at a low dosage in mice and the other involving the infusion of rotenone stereotaxically into the substantia nigra pars compacta (SNpc) of rats, were employed to evaluate the neuroprotective effects of Cory. Cory was shown to exhibit neuroprotective effects in the two rotenone-induced models of PD by improving motor dysfunction, preventing tyrosine hydroxylase (TH)-positive neuronal loss, decreasing α-synuclein aggregates through the mechanistic target of the rapamycin (mTOR) pathway, and diminishing neuroinflammation. These results provide preclinical experimental evidence supporting the development of Cory into a potential delivery system for the treatment of PD.

DPYSL2 is a novel regulator for neural stem cell differentiation in rats: revealed by Panax notoginseng saponin administration.

BACKGROUND: The limited neuronal differentiation of the endogenous or grafted neural stem cells (NSCs) after brain injury hampers the clinic usage of NSCs. Panax notoginseng saponins (PNS) were extensively used for their clinical value, such as in controlling blood pressure, blood glucose, and inhibiting neuronal apoptosis and enhancing neuronal protection, but whether or not it exerts an effect in promoting neuronal differentiation of the endogenous NSCs is completely unclear and the potential underlying mechanism requires further exploration. METHODS: Firstly, we determined whether PNS could successfully induce NSCs to differentiate to neurons under the serum condition. Mass spectrometry and quantitative polymerase chain reaction (Q-PCR) were then performed to screen the differentially expressed proteins (genes) between the PNS + serum and serum control group, upon which dihydropyrimidinase-like 2 (DPYSL2), a possible candidate, was then selected for the subsequent research. To further investigate the actual role of DPYSL2 in the NSC differentiation, DPYSL2-expressing lentivirus was employed to obtain DPYSL2 overexpression in NSCs. DPYSL2-knockout rats were constructed to study its effects on hippocampal neural stem cells. Immunofluorescent staining was performed to identify the differentiation direction of NSCs after 7 days from DPYSL2 transfection, as well as those from DPYSL2-knockout rats. RESULTS: Seven differentially expressed protein spots were detected by PD Quest, and DPYSL2 was found as one of the key factors of NSC differentiation in a PNS-treated condition. The results of immunostaining further showed that mainly Tuj1 and GFAP-positive cells increased in the DPYSL2-overexpressed group, while both were depressed in the hippocampal NSCs in the DPYSL2-knockout rat. CONCLUSIONS: The present study revealed that the differentiation direction of NSCs could be enhanced through PNS administration, and the DPYSL2 is a key regulator in promoting NSC differentiation. These results not only emphasized the effect of PNS but also indicated DPYSL2 could be a novel target to enhance the NSC differentiation in future clinical trials.

MicroRNA339 Targeting PDXK Improves Motor Dysfunction and Promotes Neurite Growth in the Remote Cortex Subjected to Spinal Cord Transection.

Spinal cord injury (SCI) is a fatal disease that can cause severe disability. Cortical reorganization subserved the recovery of spontaneous function after SCI, although the potential molecular mechanism in this remote control is largely unknown. Therefore, using proteomics analysis, RNA interference/overexpression, and CRISPR/Cas9 in vivo and in vitro, we analyzed how the molecular network functions in neurological improvement, especially in the recovery of motor function after spinal cord transection (SCT) via the remote regulation of cerebral cortex. We discovered that the overexpression of pyridoxal kinase (PDXK) in the motor cortex enhanced neuronal growth and survival and improved locomotor function in the hindlimb. In addition, PDXK was confirmed as a target of miR-339 but not miR-124. MiR-339 knockout (KO) significantly increased the neurite outgrowth and decreased cell apoptosis in cortical neurons. Moreover, miR-339 KO rats exhibited functional recovery indicated by improved Basso, Beattie, and Bresnehan (BBB) score. Furthermore, bioinformatics prediction showed that PDXK was associated with GAP43, a crucial molecule related to neurite growth and functional improvement. The current research therefore confirmed that miR-339 targeting PDXK facilitated neurological recovery in the motor cortex of SCT rats, and the underlying mechanism was associated with regulating GAP43 in the remote cortex of rats subjected to SCT. These findings may uncover a new understanding of remoting cortex control following SCI and provide a new therapeutic strategy for the recovery of SCI in future clinical trials.

Iron-induced oxidative stress contributes to α-synuclein phosphorylation and up-regulation via polo-like kinase 2 and casein kinase 2.

α-Synuclein plays a central role in synucleinopathies pathogenesis such as Parkinson's disease (PD). Phosphorylation is the most common and important protein modification linked to α-synuclein pathologies. There is mounting evidence suggested iron and α-synuclein are closely related in PD. We previously reported iron up-regulated α-synuclein mRNA levels and induced α-synuclein aggregation. In the present study, we aimed to investigate whether and how phosphorylation was involved in iron-induced α-synuclein regulations. The results showed that iron could induce pS129 α-synuclein (phosphorylation at Ser129) and α-synuclein upregulation in the substantia nigra of iron-overloaded rats and iron-treated SH-SY5Y cells, accompanied by the elevated levels of polo-like kinase 2 (PLK2) and casein kinase 2 (CK2). Over-expression of CK2 or PLK2 induced pS129 α-synuclein up-regulation and inhibitors of CK2 or PLK2 could suppress iron-induced α-synuclein phosphorylation. Antioxidant NAC could fully block iron-induced upregulation of CK2, PLK2 and pS129 α-synuclein levels, indicating oxidative stress plays a critical role in iron-induced α-synuclein phosphorylation. However, iron-induced α-synuclein up-regulation could only be partially blocked by CK2/PLK2 inhibitor or NAC. These findings demonstrate that iron-induced oxidative stress is largely responsible for α-synuclein phosphorylation and upregulation via CK2 and PLK2, and α-synuclein upregulation is not fully phosphorylation-dependent.