Limin Shi

Peptide hormone ghrelin enhances neuronal excitability by inhibition of Kv7/KCNQ channels

The gut-derived orexigenic peptide hormone ghrelin enhances neuronal firing in the substantia nigra pars compacta, where dopaminergic neurons modulate the function of the nigrostriatal system for motor coordination. Here we describe a novel mechanism by which ghrelin enhances firing of nigral dopaminergic neurons by inhibiting voltage-gated potassium Kv7/KCNQ/M-channels through its receptor GHS-R1a and activation of the PLC-PKC pathway. Brain slice recordings of substantia nigra pars compacta neurons reveal that ghrelin inhibits native Kv7/KCNQ/M-currents. This effect is abolished by selective inhibitors of GHS-R1a, PLC and PKC. Transgenic suppression of native Kv7/KCNQ/M-channels in mice or channel blockade with XE991 abolishes ghrelin-induced hyperexcitability. In vivo, intracerebroventricular ghrelin administration causes increased dopamine release and turnover in the striatum. Microinjection of ghrelin or XE991 into substantia nigra pars compacta results in contralateral dystonic posturing, and attenuation of catalepsy elicited by systemic administration of the D2 receptor antagonist haloperidol. Our findings indicate that the ghrelin/KCNQ signalling is likely a common pathway utilized by the nervous system.

6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes

It is well known that disrupted brain iron homeostasis was involved in Parkinson’s disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10xa0μM 6-OHDA treatment for 24xa0h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12xa0h, however, down-regulation at 24xa0h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1xa0+xa0IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.

Ghrelin and Neurodegenerative Disorders—a Review

Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor 1a (GHS-R1a), is a gut-derived, orexigenic peptide hormone that primarily regulates growth hormone secretion, food intake, and energy homeostasis. With the wide expression of GHS-R1a in extra-hypothalamic regions, the physiological role of ghrelin is more extensive than solely its involvement in metabolic function. Ghrelin has been shown to be involved in numerous higher brain functions, such as memory, reward, mood, and sleep. Some of these functions are disrupted in neurodegenerative disorders, including Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease (HD). This link between ghrelin and these neurodegenerative diseases is supported by numerous studies. This review aims to provide a comprehensive overview of the most recent evidence of the novel neuromodulatory role of ghrelin in PD, AD, and HD. Moreover, the changes in circulating and/or central ghrelin levels that are associated with disease progression are also postulated to be a biomarker for clinical diagnosis and therapy.

Nesfatin-1 Decreases Excitability of Dopaminergic Neurons in the Substantia Nigra

Nesfatin-1, a newly discovered satiety molecule which reduces feeding behavior, has been recognized as a unique regulatory neuropeptide with its multiple roles, both central and peripheral. However, whether it had neuronal modulation effect on dopaminergic neurons is largely unknown. In the present study, using whole-cell patch clamp under current-clamp mode, we investigate the effects of nesfatin-1 on the electrical activity of rat nigral dopaminergic neurons. Nesfatin-1 could produce a resting membrane potential hyperpolarization on the majority of dopaminergic neurons tested. The spike frequency decreased by 23.13u2009±u20095.93 and 43.20u2009±u20095.56xa0% in 5-nM and 10-nM nesfatin-1 groups, respectively. These effects persisted in the presence of ionotropic glutamate and GABA receptor antagonists. Our study suggests that nesfatin-1 postsynaptically inhibits the electrical activity of nigral dopaminergic neurons.

Mitochondria dysfunction was involved in copper-induced toxicity in MES23.5 cells

To investigate the toxicity of copper on MES23.5 dopaminergic cells and the probable mechanisms involved in this process. Methods MES23.5 dopaminergic cells were selected as our experimental model. [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide] (MTT) assay was used to detect the influence of copper on the cell viability. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting and the high performance liquid chromatography-electrochemical detection (HPLC-ECD) have been used to detect the tyrosine hydroxlase (TH) mRNA and protein expression and the dopamine content in MES23.5 cells. The flow cytometry have been used to detect the changes of mitochondrial transmembrane potential. 100 and 200 μmol/L copper had no effect on the MES23.5 cell viability, whereas 400 and 800 μmol/L of copper could decrease the cell viability (P < 0.01). Treating cells with 200 μmol/L copper for 24 h decreased the TH mRNA expression, the TH expression and the dopamine content compared with the control (P < 0.01, P < 0.01, P < 0.05, respectively). Besides, the mitochondrial transmembrane potential also decreased with the treatment of 200 μmol/L copper for 24 h (P < 0.01). Copper could exert the toxic effects on MES23.5 dopaminergic cells and decrease the cell function. The dysfunction of mitochondria may be the mechanism of this toxicity effect. 探讨铜离子对MES23.5多巴胺能细胞的毒性作用及可能机制. 用MTT法检测铜离子对细胞存活率的影响; 用半定量逆转录聚合酶链反应 (RT-PCR), Western blotting以及高效液相色谱电化学检测法 (HPLC-ECD)检测细胞内酪氨酸羟化酶 (tyrosine hydroxlase, TH) mRNA、 蛋白的表达以及多巴胺含量的改变; 用流式细胞仪检测线粒体跨膜电位的改变. 100 μmol/L和200 μmol/L铜离子对细胞存活率没有影响, 400 μmol/L和800 μmol/L铜离子可以造成细胞存活率降低 (P < 0.01). 200 μmol/L铜离子孵育细胞24 h, TH mRNA、蛋白表达量以及多巴胺含量较正常对照组降低(P < 0.01, P < 0.01, P < 0.05). 200 μmol/L铜离子孵育细胞24 h, 线粒体跨膜电位较正常对照组明显降低 (P < 0.01). 铜离子对MES23.5 多巴胺能细胞具有毒性作用, 降低细胞功能的表达, 其机制可能与线粒体功能障碍有关.ObjectiveTo investigate the toxicity of copper on MES23.5 dopaminergic cells and the probable mechanisms involved in this process. Methods MES23.5 dopaminergic cells were selected as our experimental model. [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide] (MTT) assay was used to detect the influence of copper on the cell viability. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting and the high performance liquid chromatography-electrochemical detection (HPLC-ECD) have been used to detect the tyrosine hydroxlase (TH) mRNA and protein expression and the dopamine content in MES23.5 cells. The flow cytometry have been used to detect the changes of mitochondrial transmembrane potential.Results100 and 200 μmol/L copper had no effect on the MES23.5 cell viability, whereas 400 and 800 μmol/L of copper could decrease the cell viability (P < 0.01). Treating cells with 200 μmol/L copper for 24 h decreased the TH mRNA expression, the TH expression and the dopamine content compared with the control (P < 0.01, P < 0.01, P < 0.05, respectively). Besides, the mitochondrial transmembrane potential also decreased with the treatment of 200 μmol/L copper for 24 h (P < 0.01).ConclusionCopper could exert the toxic effects on MES23.5 dopaminergic cells and decrease the cell function. The dysfunction of mitochondria may be the mechanism of this toxicity effect.摘要目的探讨铜离子对MES23.5多巴胺能细胞的毒性作用及可能机制.方法用MTT法检测铜离子对细胞存活率的影响; 用半定量逆转录聚合酶链反应 (RT-PCR), Western blotting以及高效液相色谱电化学检测法 (HPLC-ECD)检测细胞内酪氨酸羟化酶 (tyrosine hydroxlase, TH) mRNA、 蛋白的表达以及多巴胺含量的改变; 用流式细胞仪检测线粒体跨膜电位的改变.结果100 μmol/L和200 μmol/L铜离子对细胞存活率没有影响, 400 μmol/L和800 μmol/L铜离子可以造成细胞存活率降低 (P < 0.01). 200 μmol/L铜离子孵育细胞24 h, TH mRNA、蛋白表达量以及多巴胺含量较正常对照组降低(P < 0.01, P < 0.01, P < 0.05). 200 μmol/L铜离子孵育细胞24 h, 线粒体跨膜电位较正常对照组明显降低 (P < 0.01).结论铜离子对MES23.5 多巴胺能细胞具有毒性作用, 降低细胞功能的表达, 其机制可能与线粒体功能障碍有关.

The neurological effects of ghrelin in brain diseases: Beyond metabolic functions

HighlightsGhrelin is attracted more attention for its neuroprotective and neurogenesis roles.Ghrelin modulates numerous neuronal behaviors in pathological conditions.Ghrelin promotes neurogenesis through stimulating neural stem/progenitor cells proliferation, differentiation and migration.Ghrelin regulates synaptic plasticity and electrophysiological activity which may influence advanced brain functions. &NA; Ghrelin, a peptide released by the stomach that plays a major role in regulating energy metabolism, has recently been shown to have effects on neurobiological behaviors. Ghrelin enhances neuronal survival by reducing apoptosis, alleviating inflammation and oxidative stress, and accordingly improving mitochondrial function. Ghrelin also stimulates the proliferation, differentiation and migration of neural stem/progenitor cells (NS/PCs). Additionally, the ghrelin is benefit for the recovery of memory, mood and cognitive dysfunction after stroke or traumatic brain injury. Because of its neuroprotective and neurogenic roles, ghrelin may be used as a therapeutic agent in the brain to combat neurodegenerative disease. In this review, we highlight the pre‐clinical evidence and the proposed mechanisms underlying the role of ghrelin in physiological and pathological brain function.

Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro

Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson’s disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe2+-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment.

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.

Potassium channels are involved in zinc-induced apoptosis in MES23.5 cells

There is evidence that zinc may be involved in the pathogenesis of Parkinsons disease by an apoptotic pathway. However, the mechanisms underlying zinc‐induced apoptosis are unknown. Previous studies showed that 6‐hydroxydopamine (6‐OHDA)‐enhanced potassium channels are involved in apoptosis of dopaminergic neurons. Our study was designed to test whether zinc‐induced apoptosis was mediated by potassium channels. First we demonstrated cell apoptosis with zinc treatment by Hoechst staining assay. The results showed that 13.38% ± 0.6% of MES23.5 cells were apoptotic after 24 hr of incubation with 60 μM zinc sulfate. Then we observed that the tyrosine hydroxylase (TH) protein expression and the dopamine content decreased, as detected by Western blots and high‐performance liquid chromatography‐electrochemical detection (HPLC‐ECD). We further elucidated the mechanism of cell apoptosis by using whole‐cell patch clamp recording. The data demonstrated that MES23.5 cells exhibited a tetraethylammonium (TEA)‐sensitive outward K+ current with delayed rectifier characteristics. Increases of K+ current density were recorded following the treatment with 60 μM zinc for 4–8 hr. After incubation with 20 mM TEA, the zinc‐induced enhancement of K+ currents was fully blocked. Furthermore, incubation with TEA blocked zinc‐mediated caspase‐3 activation and cell apoptosis. These data suggest that zinc‐induced apoptosis of MES23.5 dopaminergic cells may due to the enhancement of TEA‐sensitive K+ channel activity.

Potassium Channels: A Potential Therapeutic Target for Parkinson’s Disease

The pathogenesis of the second major neurodegenerative disorder, Parkinson’s disease (PD), is closely associated with the dysfunction of potassium (K+) channels. Therefore, PD is also considered to be an ion channel disease or neuronal channelopathy. Mounting evidence has shown that K+ channels play crucial roles in the regulations of neurotransmitter release, neuronal excitability, and cell volume. Inhibition of K+ channels enhances the spontaneous firing frequency of nigral dopamine (DA) neurons, induces a transition from tonic firing to burst discharge, and promotes the release of DA in the striatum. Recently, three K+ channels have been identified to protect DA neurons and to improve the motor and non-motor symptoms in PD animal models: small conductance (SK) channels, A-type K+ channels, and KV7/KCNQ channels. In this review, we summarize the physiological and pharmacological effects of the three K+ channels. We also describe in detail the laboratory investigations regarding K+ channels as a potential therapeutic target for PD.