Oxidative Stress Regulated Iron Regulatory Protein IRP2 Through FBXL5-Mediated Ubiquitination-Proteasome Way in SH-SY5Y Cells

Iron regulatory protein 2 (IRP2) plays a key role in the cellular iron homeostasis and could be regulated by a variety of factors, such as oxidative stress, hypoxia and iron, etc. IRP2 depletion results in neurodegenerative movement disorder with the loss of neurons and accumulations of iron. Since oxidative stress extensively exists in several neurodegenerative diseases where iron accumulation also exists, it is important to clarify the mechanisms underlying the effects of oxidative stress on IRP2 expression and its consequence. 200 and 300 μM H2O2 could result in the reduced cell viability in SH-SY5Y cells. The intracellular levels of reactive oxygen species (ROS) were increased by 52.2 and 87.3% with 200 and 300 μM H2O2 treatments, respectively. The decreased levels of mitochondrial transmembrane potential (ΔΨm) were only observed in 300 μM H2O2-treated group. The protein levels of IRP2, but not for its mRNA levels, were observed decreased in both groups, which resulted in the lower TfR1 expression and decreased iron uptake in these cells. Pretreatment with MG132, the decreased IRP2 levels caused by H2O2 treatment could be antagonized. The protein levels of F box and leucine-rich repeat protein 5 (FBXL5), the only E3 ligase of IRP2, were observed decreased accordingly. When knockdown the intracellular FBXL5 levels by si-FBXL5, the protein levels of IRP2 were found increased with H2O2 treatment. Our results suggest that FBXL5 is involved in the degradation of IRP2 under oxidative stress in dopaminergic-like neuroblastoma cells, which implies that its role in the neuronal regulation of IRP2 in neurodegenerative diseases

Glut9-mediated Urate Uptake Is Responsible for Its Protective Effects on Dopaminergic Neurons in Parkinson’s Disease Models

Considerable evidence has shown that elevated plasma or cerebrospinal fluid (CSF) urate levels correlated with a reduced risk of Parkinson’s disease (PD). Based on its anti-oxidative properties, urate might serve as one of promising neuroprotective candidates for PD. However, how urate is transported through cell membranes to exert its effects inside the cells in PD is largely unknown. To elucidate this, we showed that increased intracellular urate exerted its neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity in MES23.5 cells and elevated urate could antagonize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neuronal death in urate oxidase (UOx) knockout (KO) mice. Its transporter, glucose transporter type 9 (Glut9), was observed up-regulated, which was caused by the activation of p53. These protective effects could be abolished by Glut9 blocker and p53 inhibitor. These results suggested that Glut9 was a functional urate transporter, whose up-regulation by activation of p53 resulted in the increased intracellular urate levels in PD models. Our findings suggest that Glut9 could be modified to modulate urate levels in dopaminergic neurons and urate-elevating strategies without increasing systemic levels to avoid side effects might serve as a potential therapeutic target for PD

High Dietary Iron Supplement Induces the Nigrostriatal Dopaminergic Neurons Lesion in Transgenic Mice Expressing Mutant A53T Human Alpha-Synuclein

Both alpha-synuclein aggregation and iron deposits are neuropathological hallmarks of Parkinson’s disease (PD). We are particularly interested in whether iron could synergize with alpha-synuclein pathology in vivo, especially in the nigrostriatal system. In the present study, we reported transgenic mice with overexpressing human A53T alpha-synuclein, as well as WT mice with high dietary iron displayed hyperactive motor coordination and impaired colonic motility, compared with those with basal dietary iron. Only A53T mice, but not WT mice with high dietary iron exhibited nigral dopaminergic neuronal loss, lower levels of tyrosine hydroxylase (TH) in the substantia nigra (SN) and decreased dopamine contents in the striatum. Although there was no obvious elevation of iron contents in the SN in WT mice with high dietary iron, we observed iron contents in the SN were especially higher than the other brain regions in 12-month aged mice with either high or basal dietary iron. These results suggested high dietary iron supplement could induce nigral dopaminergic neurons lesion in A53T mice, which might be due to the vulnerability of SN to accumulate iron

Biometal Dyshomeostasis and Toxic Metal Accumulations in the Development of Alzheimer’s Disease

Biometal dyshomeostasis and toxic metal accumulation are common features in many neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease. The neurotoxic effects of metal imbalance are generally associated with reduced enzymatic activities, elevated protein aggregation and oxidative stress in the central nervous system, in which a cascade of events lead to cell death and neurodegeneration. Although the links between biometal imbalance and neurodegenerative disorders remain elusive, a major class of endogenous proteins involved in metal transport has been receiving increasing attention over recent decades. The abnormal expression of these proteins has been linked to biometal imbalance and to the pathogenesis of AD. Here, we present a brief overview of the physiological roles of biometals including iron, zinc, copper, manganese, magnesium and calcium, and provide a detailed description of their transporters and their synergistic involvement in the development of AD. In addition, we also review the published data relating to neurotoxic metals in AD, including aluminum, lead, cadmium, and mercury

DIFFERENTIALLY EXPRESSED GENES AND THEIR SIGNIFICANCE THE SUBSTANTIA NIGRA OF MICE WITH 1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE-INDUCED PARKINSON’S DISEASE

Objective To investigate the differentially expressed genes (DEGs) in the substantia nigra of mice with Parkinson’s disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) based on bioinformatics, as well as their role in the pathological and progression of PD. Methods GEO database was used to obtain the gene expression microarray data in the substantia nigra of mice in the normal group and the PD model group, and the bioinformatics method was used to screen for DEGs in the substantia nigra between the normal group and the PD model. Metascape tool was used to perform GO functional enrichment analysis and KEGG pathway enrichment analysis. Quantitative real-time PCR was used to measure the mRNA expression levels of key functional genes regulating neuronal apoptosis and prion transmission. Results There were 174 upregulated DEGs and 173 downregulated DEGs in the substantia nigra of mice with MPTP-induced PD (P<0.05, fold change >0.26). The upregulated genes were significantly enriched in the pathways regulating prion transmission, neuronal development, iron homeostasis, and export across the plasma membrane, while the downregulated genes were significantly enriched in the pathways regulating synaptic vesicle release, transsynaptic complex transmission, neuronal apoptosis, and actin in cytoskeleton. Compared with the normal group, the PD model group had significant reductions in the expressions of Bdnf and Fbxw7 in the substantia nigra of mice (t=2.25,2.39,P<0.05), while there was no significant difference in the expression of sod1 between the two groups (P>0.05). Conclusion There are significant changes in the key functional genes Bdnf and Fbxw7 that regulate neuronal apoptosis in PD model, which lays a foundation for further clarifying their role in the pathological progression of PD

Investigation of Behavioral Dysfunctions Induced by Monoamine Depletions in a Mouse Model of Parkinson's Disease

Parkinson's disease (PD) is characterized not only by typical motor symptoms, but also by nonmotor symptoms in the early stages. In addition to the loss of dopaminergic (DAergic) neurons, progressive degenerations of noradrenergic (NA) and serotonergic (5-HT) neurons were also observed. However, the respective effects and interactions of these monoamine depletions on certain nonmotor symptoms are still largely unknown. In the present study, we performed selective depletions of NA, 5-HT and DA in mice by intraperitioneal injection of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), 4-chloro-L-phenylalanine (pCPA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively. DSP-4 led to a 34% decrease in the number of NAergic neurons in the locus coeruleus, and MPTP led to a 30% decrease in the number of DAergic neurons in the substantia nigra. Although there was no obvious change in the number of 5-HTergic neurons in the dorsal raphe nucleus after pCPA treatment, the levels of 5-HT and its metabolite in the frontal cortex and hippocampus were reduced, respectively. Locomotor activity deficit was induced by DA depletion and a decrease in traveled distance was potentiated by additional NA depletion. Despair-associated depressive-like behavior could be observed in every group. Anxiety states emerged only from the combined depletion of two or three monoamines. However, combined depletion of the three monoamines dramatically induced anhedonia, and it could also aggravate the depressive-like and anxiety behavior. Furthermore, NA depletion significantly reduced spatial learning and memory ability, which was not enhanced by additional 5-HT or DA depletion. Our data highlighted the interactive role of NA, 5-HT and DA in the motor, emotional and cognitive deficits, providing new insight into the complex orchestration of impaired monoaminergic systems that related to the pathology of PD

Deubiquitylase OTUD3 Mediates Endoplasmic Reticulum Stress through Regulating Fortilin Stability to Restrain Dopaminergic Neurons Apoptosis

OTU domain-containing protein 3 (OTUD3) knockout mice exhibited loss of nigral dopaminergic neurons and Parkinsonian symptoms. However, the underlying mechanisms are largely unknown. In this study, we observed that the inositol-requiring enzyme 1α (IRE1α)-induced endoplasmic reticulum (ER) stress was involved in this process. We found that the ER thickness and the expression of protein disulphide isomerase (PDI) were increased, and the apoptosis level was elevated in the dopaminergic neurons of OTUD3 knockout mice. These phenomena were ameliorated by ER stress inhibitor tauroursodeoxycholic acid (TUDCA) treatment. The ratio of p-IRE1α/IRE1α, and the expression of X-box binding protein 1-spliced (XBP1s) were remarkably increased after OTUD3 knockdown, which was inhibited by IRE1α inhibitor STF-083010 treatment. Moreover, OTUD3 regulated the ubiquitination level of Fortilin through binding with the OTU domain. OTUD3 knockdown resulted in a decrease in the interaction ability of IRE1α with Fortilin and finally enhanced the activity of IRE1α. Taken together, we revealed that OTUD3 knockout-induced injury of dopaminergic neurons might be caused by activating IRE1α signaling in ER stress. These findings demonstrated that OTUD3 played a critical role in dopaminergic neuron neurodegeneration, which provided new evidence for the multiple and tissue-dependent functions of OTUD3