高铁对大鼠大脑皮层二价金属转运蛋白1表达的影响

目的 探讨在脑铁代谢中发挥重要生理作用的二价金属转运蛋白1(DMT1)的表达及其调控机制.方法 大鼠(n=6)侧脑室注射右旋糖酐铁3d和7d后,采用铁组织化学法检测脑内铁含量的变化,免疫组织化学技术检测大脑皮层中DMT1的两种亚型,即DMT1(+IRE)和DMT1(-IRE)蛋白表达的变化.结果 铁组织化学染色结果显示,大鼠侧脑室注射右旋糖酐铁500μg/(只·d)7d后,大脑皮层中二价铁和三价铁均显著增高.同时,免疫组织化学结果表明,与对照组相比,脑内达高铁状态时大脑皮层DMT1(+IRE)蛋白表达显著升高,而DMT1(-IRE)蛋白表达无显著变化.结论 在大鼠大脑皮层中,DMT1(+IRE)蛋白对铁水平的升高更为敏感,其表达与脑铁水平(尤其是二价铁)呈正相关.高铁对脑内不同区域内不同亚型DMT1表达的影响存在特异性.     

Effects of Intracerebroventricular Injection of Iron Dextran on the Iron Concentration and Divalent Metal Transporter 1 Expression in the Caudate Putamen and Substantia Nigra of Rats

The cellular localization of DMT1 and its functional characterization suggest that DMT1 may play an important role in the physiological brain iron transport. But the regulation of DMT1 expression by iron in the brain is still not clearly understood. In this study, both the contents of ferric and ferrous iron as well as DMT1 expression were evaluated in CPu and SN after ICV of 500 μg iron dextran/rat/day for 3 or 7 days. It was found that the iron levels in CPu and SN were not altered obviously until ICV for 7 days. Immunohistochemistry results indicated that the expression of DMT1 (?IRE) in CPu and SN was not altered significantly after 3 days of ICV. Whereas the expression of DMT1 (?IRE) decreased significantly after 7 days of ICV when ferrous iron was increased significantly. Contrary to that of DMT1 (?IRE) in the same regions, there were no significant alterations in DMT1 (+IRE) expression in CPu and SN in spite of the existence of the altered iron levels, compared with that of control groups. The results demonstrate that DMT1 (?IRE) expression was correlated probably with brain iron levels; especially, its regulation was correlated with ferrous iron (not ferric iron) in CPu and SN in adult rats, compared with those of saline‐injected control rats. The effect of ferrous iron on the expression of DMT1 (?IRE) in the brain also suggests that it might play a major physiological role in brain iron uptake and transport, but further studies are needed to clarify these issues. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.     

Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

The H⁺ -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H⁺ electrochemical gradient. At low extracellular pH (pH_o), H⁺ binding preceded binding of Fe²⁺ and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pH_o, and at pH_o 7.4 we observed Fe²⁺ transport that was not associated with H⁺ influx. His²⁷² Ala substitution uncoupled the Fe²⁺ and H⁺ fluxes. At low pH_o, H272A mediated H⁺ uniport that was inhibited by Fe²⁺. Meanwhile H272A-mediated Fe²⁺ transport was independent of pH_o. Our data indicate (i) that H⁺ coupling in DMT1 serves to increase affinity for Fe²⁺ and provide a thermodynamic driving force for Fe²⁺ transport and (ii) that His-272 is critical in transducing the effects of H⁺ coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe²⁺ transport in the absence of a H⁺ gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H⁺ -uncoupled facilitative Fe²⁺ transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.     

铁转运相关蛋白在肌萎缩性侧索硬化症转基因鼠脊髓中的表达变化

目的 探讨肌萎缩性侧索硬化症(ALS)转基因鼠脊髓内铁转运相关蛋白表达变化与铁稳态失衡的关联.方法 选取hSOD1G93A转基因鼠(ALS鼠)和同窝野生型鼠(WT鼠),分别于生后70、95和122 d分离脊髓,每时间点每组各9只实验动物.Western blotting检测脊髓组织内铁转运蛋白二价金属转运蛋白-1(DM...     

Differential effects of overexpression of wild-type and mutant human α-synuclein on MPP-induced neurotoxicity in PC12 cells

The genetic background and the pathogenesis of familial Parkinson's disease (PD) have not been fully elucidated. Two missense mutations in the α-synuclein gene, A30P and A53T, have been linked to familial PD. Increasing evidence suggests the involvement of α-synuclein, the dopamine transporter (DAT), and neurotoxins in the pathogenesis of PD, but their relationships to the death of nigral cells remains poorly understood. In the present study, we used the PC12 cell line, a well recognized model of the nigral cell, to investigate the effects of overexpression of wild-type (WT) and mutant human α-synuclein on MPP⁺-induced neurotoxicity. We found that overexpression of mutant α-synuclein enhanced the toxicity of MPP⁺ to PC12 cells and elevated intracellular reactive oxygen species (ROS) levels, whereas overexpression of WT α-synuclein protected PC12 cells against MPP⁺ toxicity and lowered ROS levels. Furthermore, assays of ¹³¹I-FP-β-CIT binding with DAT membrane fractions showed that WT and mutant α-synuclein had different effects on the expression of DAT on the cell membrane following exposure to MPP⁺. WT α-synuclein reduced the toxic effect of MPP⁺ by facilitating DAT internalization, while both A30P and A53T α-synuclein aggravated the toxic effect of MPP⁺ by reducing DAT internalization. These data indicate that α-synuclein regulates ROS levels and DAT surface expression in dopaminergic neurons, and thus changes the response of these cells to MPP⁺.     

Transduced PEP-1-PON1 proteins regulate microglial activation and dopaminergic neuronal death in a Parkinson's disease model

Parkinson's disease (PD) is an oxidative stress-mediated neurodegenerative disorder caused by selective dopaminergic neuronal death in the midbrain substantia nigra. Paraoxonase 1 (PON1) is a potent inhibitor of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) against oxidation by destroying biologically active phospholipids with potential protective effects against oxidative stress-induced inflammatory disorders. In a previous study, we constructed protein transduction domain (PTD) fusion PEP-1-PON1 protein to transduce PON1 into cells and tissue. In this study, we examined the role of transduced PEP-1-PON1 protein in repressing oxidative stress-mediated inflammatory response in microglial BV2 cells after exposure to lipopolysaccharide (LPS). Moreover, we identified the functions of transduced PEP-1-PON1 proteins which include, mitigating mitochondrial damage, decreasing reactive oxidative species (ROS) production, matrix metalloproteinase-9 (MMP-9) expression and protecting against 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurotoxicity in SH-SY5Y cells. Furthermore, transduced PEP-1-PON1 protein reduced MMP-9 expression and protected against dopaminergic neuronal cell death in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model. Taken together, these results suggest a promising therapeutic application of PEP-1-PON1 proteins against PD and other inflammation and oxidative stress-related neuronal diseases.     

Okadaic acid protects human neuroblastoma SH-SY5Y cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis

1-Methyl-4-phenylpyridinium ion (MPP⁺) has been shown to selectively inhibit mitochondrial function and induce a parkinsonism-like syndrome. MPP⁺ stimulates the production of reactive oxygen species (ROS) and induces cell death in vitro. In this study, we investigated the protective effects of okadaic acid on MPP⁺-induced cell death in SH-SY5Y neuroblastoma cells. We found that MPP⁺-induced apoptosis and -ROS generation were blocked by okadaic acid. MPP⁺-mediated activation of AKT was also inhibited by okadaic acid. Taken together, these results demonstrate that okadaic acid protects against MPP⁺-induced apoptosis by blocking ROS stimulation and ROS-mediated signaling pathways in SH-SY5Y cells. These data indicated that okadaic acid could provide a therapeutic strategy for the treatment of neurodegenerative diseases including Parkinson's disease.     

Protection against neurotoxicity by an autophagic mechanism

The objective of the present study was to investigate the effects of 3-n-butylphthalide (NBP) on a 1-methyl-4-phenylpyridinium (MPP⁺)-induced cellular model of Parkinson''s disease (PD) and to illustrate the potential mechanism of autophagy in this process. For this purpose, rat PC12 pheochromocytoma cells were treated with MPP⁺ (1 mM) for 24 h following pretreatment with NBP (0.1 mM). Cell metabolic viability was determined by the MTT assay and cell ultrastructure was examined by transmission electron microscopy. The intracellular distribution and expression of α-synuclein and microtubule-associated protein light chain 3 (LC3) were detected by immunocytochemistry and Western blotting. Our results demonstrated that: 1) NBP prevented MPP⁺-induced cytotoxicity in PC12 cells by promoting metabolic viability. 2) NBP induced the accumulation of autophagosomes in MPP⁺-treated PC12 cells. 3) Further study of the molecular mechanism demonstrated that NBP enhanced the colocalization of α-synuclein and LC3 and up-regulated the protein level of LC3-II. These results demonstrate that NBP protects PC12 cells against MPP⁺-induced neurotoxicity by activating autophagy-mediated α-synuclein degradation, implying that it may be a potential effective therapeutic agent for the treatment of PD.     

NEW evidences for fractalkine/CX3CL1 involved in substantia nigral microglial activation and behavioral changes in a rat model of Parkinson's disease

Activated microglia are instrumental to neurodegeneration in Parkinson's disease (PD). Fractalkine, as an exclusive ligand for CX3CR1 expressed on microglia, has recently been reported to be released out by neurons, and induce microglial activation as a neuron-to-glia signal in the spinal cord. However, the role of fractalkine-induced microglial activation in PD remains unknown. In our study, we injected 1-methyl-4-phenylpyridinium (MPP⁺) into unilateral substantia nigra (SN) which induced ipsilateral endogenous fractalkine expression on neuron and observe the increase of CX3CR1 expression in response to MPP⁺ by Western blotting analysis. Moreover, pre-administration of anti-CX3CR1 neutralizing antibody which potentially blocked microglial activation can promote rotation behaviors. To further investigate the role of fractalkine in PD, we injected exogenous fractalkine in unilateral SN, and observed microglial activation, dopaminergic cell depletion, and motor dysfunction. All these effects can be totally abolished by cerebroventricular administration of anti-CX3CR1. Intracerebroventricular administration of minocycline, a selective microglia inhibitor, can prevent fractalkine-induced rotation behaviors, and inhibit dopaminergic neurons from degeneration in the way of dose-dependent. Our studies demonstrate that fractalkine-induced microglial activation plays an important role in the development of PD, and provide an evidence of fractalkine and CX3CR1 as new therapeutic targets for PD treatment.     

Tetrahydroxystilbene glucoside attenuates MPP-induced apoptosis in PC12 cells by inhibiting ROS generation and modulating JNK activation

It is known that oxidative stress plays a major role in the progression of Parkinson's disease (PD). Previous studies have suggested that 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), an active component extracted from a traditional Chinese herb Polygonum multiflorum Thunb., has significant antioxidant and free radical-scavenging activities. This is the first study that investigated the protective effects of TSG against MPP⁺-induced apoptosis in PC12 cells and determined the underlying mechanism. The results showed that incubation of PC12 cells with TSG before exposing them to MPP⁺ could significantly decrease cell viability loss and reverse cell apoptosis in a dose-dependent manner. The anti-apoptotic effects of TSG were probably mediated via the inhibition of ROS generation and modulation of JNK activation because TSG blocked ROS increase and JNK phosphorylation induced by MPP⁺. Taken together, these results indicated that TSG may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases such as PD.     

Acute toxicity of MPTP and MPP+ in the brain of embryo and newborn mice

1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) causes damage to dopaminergic neurons in the nigrostriatal system, similar to that seen in Parkinson disease (PD). Recently, a few reports have confirmed neuroblastic apoptosis in the subventricular zone (SVZ) of adult C57BL/6J mice by i.p. injection of MPTP, and concluded that MPTP is also toxic to neuroblasts in the SVZ. While there have been many researches on the neurotoxicity of MPTP in adult mice, there have been only a few in fetal mice. In the present study, we assessed the toxicity of MPTP to embryonic and newborn mice after a single injection into pregnant or newborn mice. MPTP and 1-methyl-4-phenylpyridinium (MPP⁺), a metabolite of MPTP, caused loss of tyrosine hydroxylase (TH)-positive cells or fibers and increased apoptotic cells in embryonic and newborn mice. In addition, MPTP and MPP⁺ induced a marked increase of apoptotic cells in the SVZ compared to the nigrostriatal system. The present results may indicate that MPTP and MPP⁺ pass through the placenta and blood–brain barrier (BBB) and that a different mechanism may be involved in MPTP- or MPP⁺-induced toxicity in the SVZ and in the nigrostriatal system of embryonic and newborn mice.     

Trasferrin receptor 2 gene regulation by microRNA 221 in SH-SY5Y cells treated with MPP as Parkinson's disease cellular model

Parkinson's disease (PD) is one of the most frequent human neurodegenerations. The neurodegeneration in PD is related to cellular iron increase but the mechanisms involved in iron accumulation remain unclear. Transferrin receptor type 2 (TFR2) is a protein expressed on cell membrane and involved in the cellular iron uptake. We hypothesized that microRNA 221 could regulate the expression of TfR2 in an in vitro model of Parkinson's disease, SH-SY5Y cells treated with MPP⁺.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuroblastic apoptosis in the subventricular zone is caused by 1-methy-4-phenylpiridinium (MPP+) converted from MPTP through MAO-B

Intraperitoneal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration induces apoptosis of subventricular zone (SVZ) doublecortin (Dcx)-positive neural progenitor cells (migrating neuroblasts, A cells). Actually, a metabolite of MPTP, 1-methy-4-phenylpiridinium (MPP⁺), is responsible for neural progenitor cell toxicity. In the present study, to examine whether the MPTP-induced SVZ cell apoptosis is caused directly by MPP⁺ metabolized through monoamine oxidase B (MAO-B), MPTP or MPP⁺ was intracerebroventricularly (icv) injected into C57BL/6 mice. At Day 1 postinjection, many terminal deoxynucleotidyl transferase-mediated dUTP endlabeling (TUNEL)-positive cells were observed in the SVZ of both low (36 μg) and high (162 μg) dose MPTP- and MPP⁺-injected mice. The number of Dcx-positive A cells showed a significant decrease following high dose of MPTP- or MPP⁺-injection on Days 1 and 3, respectively, whereas that of EGFR-positive C cells showed no change in mice with any treatment. In addition, prior icv injection of a MAO-B inhibitor, R(?)-deprenyl (deprenyl), inhibited MPTP-induced apoptosis, but not MPP⁺-induced apoptosis. MAO-B- and GFAP-double positive cells were detected in the ependyma and SVZ in all mice. It is revealed from these results that icv injection of MPTP induces apoptosis of neural progenitor cells (A cells) in the SVZ via MPP⁺ toxicity. In addition, it is suggested that the conversion from MPTP to MPP⁺ is caused mainly by MAO-B located in ependymal cells and GFAP-positive cells in the SVZ.     

DMT1 polymorphism and risk of Parkinson's disease

Growing evidence suggests that iron accumulation in the substantia nigra (SN) is involved in the pathology of Parkinson’s diseases (PD). Divalent metal transporter 1 (DMT1) is an endogenous transporter for ferrous iron, the levels of which are significantly increased in the SN in postmortem PD brains. To study the possible association of DMT1 gene with PD occurrence, one mutation (1303C/A) and two single nucleotide polymorphisms (SNPs) (1254T/C and IVS4 + 44C/A) in DMT1 gene were investigated in 192 PD patients in a Han Chinese population and 193 healthy controls by method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Direct sequencing was performed in 10% of the samples to validate the genotyping results. Our results failed to find any significant association between the tested genotypes, alleles or mutation and PD, however, a haplotype (C alleles of 1254T and IVS4 + 44C/A polymorphisms) occurred at greater frequencies in PD subjects compared with that of control (18.2% versus 11.4%, OR = 1.72, 95% CI = 1.15-2.59, P = 0.01). These results suggest that CC haplotype in DMT1 gene is a possible risk factor for PD in this Han Chinese population.     

Functional applications of novel Semliki Forest virus vectors are limited by vector toxicity in cultures of primary neurons in vitro and in the substantia nigra in vivo

The Semliki Forest virus (SFV) system has been shown to be highly efficient in transduction of cell lines and primary cells. We employed a novel noncytotoxic SFV(PD) vector for transduction of primary ventral midbrain floor cultures in vitro and rat substantia nigra in vivo. Rapid protein expression was noted with preferential transduction of neuronal cells including the dopaminergic subpopulation. To examine the suitability of the SFV vector system for functional gene expression, SFV(PD) vectors encoding for antiapoptotic proteins Bcl-X_L and XIAP were designed. Despite effective transgene expression, SFV(PD) vectors were unable to rescue dopaminergic neurons from MPP⁺-induced apoptosis. In vivo, virus injection into substantia nigra resulted in fast onset of transgene expression, but elicited an activation of microglia and an inflammation response. We conclude that the use of novel SFV(PD) vectors is currently limited by persistent neurotoxicity of the vector system. Although SFV(PD) vectors may be useful for protein localization studies in dopaminergic neurons, functional applications will require the development of even less cytopathic vector systems.Funded by Deutsche Forschungsgemeinschaft through the DFG Research Center for Molecular Physiology of the Brain     

miR-29c-3p regulates TET2 expression and inhibits autophagy process in Parkinson's disease models

Autophagy in dopamine (DA) neurons is concerned to be associated with Parkinson's disease (PD), but the detailed mechanism remains unknown. Herein, we aimed to investigate the function of microRNA (miR)-29c-3p in autophagy in PD models. Intraperitoneal injection of MPTP (20 mg/kg) was given to C57BL/6 mice to establish PD mouse model. SH-SY5Y cells were treated with MPP⁺ (1 mmol/L) to establish in vitro PD model. The results indicated that in the substantia nigra pars compacta (SNpc) DA neurons of PD mice, autophagy was activated accompanied by down-regulated miR-29c-3p and up-regulated ten-eleven translocation 2 (TET2) expression. Up-regulation of miR-29c-3p inhibited TET2 expression and SNpc (including DA neurons) autophagy in PD mice. In vitro PD model confirmed that MPP⁺ treatment markedly down-regulated miR-29c-3p expression and up-regulated TET2 expression in SH-SY5Y cells in a dose/time-dependent manner. Moreover, miR-29c-3p up-regulation also inhibited autophagy and TET2 expression in vitro. Additionally, TET2 was proved to be targeted and down-regulated by miR-29c-3p. TET2 knockdown inhibited MPP⁺-induced autophagy, whereas TET2 over-expression reversed the effects of miR-29c-3p over-expression on SH-SY5Y cell autophagy. Overall, miR-29c-3p over-expression inhibits autophagy in PD models, which may be mediated by TET2. Our finding may provide new insights for regulating autophagy to improve PD progression.     

Oral administration of coenzyme Q10 prevents cytochrome c release from mitochondria induced by 1-methyl-4-phenylpyridinium ion in mouse brain synaptosomes

Coenzyme Q₁₀ (CoQ₁₀) exerts neuroprotective effects in several in vivo and in vitro models of neurodegenerative disorders. However, the mechanisms of action are not fully understood. The aim in this study was to investigate whether oral administration of CoQ₁₀ could inhibit cytochrome c (cyt c) release from mitochondria induced by 1-methyl-4-phenylpyridinium ion (MPP⁺), which causes dopaminergic cell death by selective inhibition of complex I of the electron transport chain, in mouse brain synaptosomes. An increase of cyt c was detected in the cytosolic fraction from mouse brain synaptosomes treated with MPP⁺. Oral administration of CoQ₁₀ prevented the mitochondrial cyt c release in the MPP⁺-treated synaptosomes. In addition, CoQ₁₀ did not affect the MPP⁺-induced decrease in mitochondrial oxidation–reduction activity and membrane potential in brain synaptosomes. Our findings demonstrate that MPP⁺-induced mitochondrial cyt c release in brain synaptosomes is prevented by oral administration of CoQ₁₀ independently of mitochondrial dysfunction prior to the cyt c release.     

dl-3-n-Butylphthalide prevents oxidative damage and reduces mitochondrial dysfunction in an MPP-induced cellular model of Parkinson's disease

The aim of the present study was to explore the neuroprotective effects and mechanisms of action of dl-3-n-butylphthalide (NBP) in a 1-methyl-4-phenylpyridiniumion (MPP⁺)-induced cellular model of Parkinson's disease (PD). NBP was extracted from seeds of Apium graveolens Linn. (Chinese celery). MPP⁺ treatment of PC12 cells caused reduced viability, formation of reactive oxygen, and disruption of mitochondrial membrane potential. Our results indicated that NBP reduced the cytotoxicity of MPP⁺ by suppressing the mitochondrial permeability transition, reducing oxidative stress, and increasing the cellular GSH content. NBP also reduced the accumulation of α-synuclein, the main component of Lewy bodies. Given that NBP is safe and currently used in clinical trials for stroke patients, NBP will likely be a promising chemical for the treatment of PD.