Mingchao Liu

Manganese induces dopaminergic neurodegeneration via microglial activation in a rat model of manganism

Manganese is an essential trace element required for normal development and bodily functions. However, exposure of the brain to excessive amounts of manganese results in neurotoxicity. Although previous studies examining manganese neurotoxicity have focused on neuronal injury, especially direct injury to dopaminergic neurons, the effects of manganese-induced neurotoxicity on glial cells have not been reported. The current study was designed to examine the effect of manganese on microglial activation, and the underlying mechanism of manganese-induced dopaminergic neuronal injury in vivo. We established an animal model of manganism by intrastriatal injection of MnCl(2).4H(2)O into male Sprague-Dawley rats. One day after administration of manganese, a few microglial cells in the substantia nigra (SN) were activated, although the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the SN was unaffected. Seven days after administration of manganese, a marked reduction in the number of TH-immunoreactive neurons was observed in the SN, and the majority of microglial cells were activated. We found that manganese upregulated inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-alpha) gene expression, as well as iNOS, TNF-alpha, and interleukin-1beta (IL-1beta) protein levels in the SN. Furthermore, treatment with minocycline, an inhibitor of microglial activation, attenuated microglial activation and mitigated IL-1beta, TNF-alpha, and iNOS production as well as dopaminergic neurotoxicity induced by manganese. These results suggested that dopaminergic neurons could be damaged by manganese neurotoxicity, and that the activated microglial cells and their associated activation products played an important role in this neurodegenerative process.

Involvement of microglia activation in the lead induced long-term potentiation impairment.

Exposure of Lead (Pb), a known neurotoxicant, can impair spatial learning and memory probably via impairing the hippocampal long-term potentiation (LTP) as well as hippocampal neuronal injury. Activation of hippocampal microglia also impairs spatial learning and memory. Thus, we raised the hypothesis that activation of microglia is involved in the Pb exposure induced hippocampal LTP impairment and neuronal injury. To test this hypothesis and clarify its underlying mechanisms, we investigated the Pb-exposure on the microglia activation, cytokine release, hippocampal LTP level as well as neuronal injury in in vivo or in vitro model. The changes of these parameters were also observed after pretreatment with minocycline, a microglia activation inhibitor. Long-term low dose Pb exposure (100 ppm for 8 weeks) caused significant reduction of LTP in acute slice preparations, meanwhile, such treatment also significantly increased hippocampal microglia activation as well as neuronal injury. In vitro Pb-exposure also induced significantly increase of microglia activation, up-regulate the release of cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) in microglia culture alone as well as neuronal injury in the co-culture with hippocampal neurons. Inhibiting the microglia activation with minocycline significantly reversed the above-mentioned Pb-exposure induced changes. Our results showed that Pb can cause microglia activation, which can up-regulate the level of IL-1β, TNF-α and iNOS, these proinflammatory factors may cause hippocampal neuronal injury as well as LTP deficits.

Changes in the synaptic structure of hippocampal neurons and impairment of spatial memory in a rat model caused by chronic arsenite exposure

Many epidemiological studies and in vitro experiments have found that chronic arsenic exposure may influence memory formation. The goal of this study was to create an animal model of memory impairment induced by chronic arsenite exposure and to study the underlying mechanisms. Sixty male Sprague-Dawley (SD) male rats were randomly divided into a control group, a low-dose sodium arsenite exposure group and a high-dose sodium arsenite exposure group. Sodium arsenite was administered by adding it to drinking water for 3 months. Then, the spatial memory of the rats was examined with Morris water maze and Y maze. The concentration of arsenic in the blood and the brain was determined by an atomic fluorescence absorption spectrometer. The ultra-structure of hippocampal neurons was observed by an electron microscope. Timm staining was used for observing mossy fibers. We found that the concentration of arsenic in the blood and the brain increased in a dose-response manner (P<0.05). The performance of rats in the arsenite exposed group (15 mg/kg) was significantly impaired in the Morris water maze and Y maze tasks than those in the control group (P<0.05). Sodium arsenite exposure resulted in abnormal structural changes in the myelin sheaths of nerve fibers and decreases in the terminals of mossy fibers. Together, chronic sodium arsenite exposure through drinking water results in detrimental changes in the neuronal synapses, which may contribute to the arsenite-induced impairment of spatial memory.

Effect of Microglia Activation on Dopaminergic Neuronal Injury Induced by Manganese, and Its Possible Mechanism

Manganese (Mn) is an essential trace element. It is known to have various functions, such as participating in enzymatic synthesis, and promoting hematopoiesis. On the other hand, it can cause toxic injury upon excess intake. However, toxic effects and its mechanism on glial cells are unclear. In the present study, we demonstrated that MnCl2 can activate microglia, and that this can cause dopaminergic neuronal injury. Investigation of the underlying mechanisms showed that inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) was induced and highly expressed following Mn treatment. Moreover, pretreatment with S-methylisothiourea (SMT. iNOS inhibitor), Mn-induced iNOS expression and dopaminergic neuronal injury were partly reverse. Pretreatment with minocycline (microglia activation inhibitor), Mn-induced activation of microglia and dopaminergic neuronal injury was partly reverse. Taken together, our results showed that Mn can cause microglia activation, which can up-regulate the level of IL-1β, TNF-α and iNOS, and these inflammatory factors can cause dopaminergic neuronal injury. SMT and minocycline prevent Mn-induced dopaminergic neuronal injury.

Anti-cancer effects of JKA97 are associated with its induction of cell apoptosis via a Bax-dependent and p53-independent pathway.

p53, one of the most commonly mutated genes in human cancers, is thought to be associated with cancer development. Hence, screening and identifying natural or synthetic compounds with anti-cancer activity via p53-independent pathway is one of the most challenging tasks for scientists in this field. Compound JKA97 (methoxy-1-styryl-9H-pyrid-[3,4-b]-indole) is a small molecule synthetic anti-cancer agent, with unknown mechanism(s). In this study we have demonstrated that the anti-cancer activity of JKA97 is associated with apoptotic induction via p53-independent mechanisms. We found that co-incubation of human colon cancer HCT116 cells with JKA97 inhibited HCT116 cell anchorage-independent growth in vitro and tumorigenicity in nude mice and also induced a cell apoptotic response, both in the cell culture model and in a tumorigenesis nude mouse model. Further studies showed that JKA97-induced apoptosis was dramatically impaired in Bax knock-out (Bax-/-) HCT116 cells, whereas the knock-out of p53 or PUMA did not show any inhibitory effects. The p53-independent apoptotic induction by JKA97 was confirmed in other colon cancer and hepatocarcinoma cell lines. In addition, our results showed an induction of Bax translocation and cytochrome c release from the mitochondria to the cytosol in HCT116 cells, demonstrating that the compound induces apoptosis through a Bax-initiated mitochondria-dependent pathway. These studies provide a molecular basis for the therapeutic application of JKA97 against human cancers with p53 mutations.

The changes of miRNA expression in rat hippocampus following chronic lead exposure.

miRNAs have been found to contribute to normal brain functions, nervous system diseases, as well as neurotoxicities induced by external agents. However, whether they are involved in lead-induced neurotoxicities is still not clear. To identify that, a lead-induced chronic neurotoxicity model of rats was built. Both miRNA microarray analysis and qRT-PCR were performed to determine the change of miRNA expression in hippocampus. Then 3 bioinformatics databases were used to analyze the relative target genes of these miRNA, which were further confirmed by qRT-PCR and Western blot. In the present study, lead exposure resulted in the changed expression of 7 miRNAs: miR-204, miR-211, miR-448, miR-449a, miR-34b, and miR-34c were greatly up-regulated while miR-494 was greatly down-regulated. Bioinformatics analysis results showed that the target genes of 6 up-regulated miRNAs were related to neural injury and neurodegeration, axon and synapse function, neural development and regeneration. Correspondingly, the expression levels of mature mRNAs and proteins of three target genes (Bcl-2, Itpr1, and Map2k1) were greatly repressed, verifying the results of bioinformatics analysis. Taken together, our results showed that the expression of several miRNAs reported to be associated with neurophysiological pathways and neurodegenerative diseases changed in rat hippocampus following chronic lead exposure. These miRNAs may play important roles in lead-induced neurotoxicity.

Effects of acetazolamide on cognitive performance during high-altitude exposure

High-altitude hypoxia impedes cognitive performance. It is not well known whether the prophylactic use of acetazolamide for altitude sickness can influence cognitive performance at high altitude. When ascending to high altitude locations, one may face medical risks, including cognitive impairment, which may significantly hinder climbing abilities or exploratory behavior. Effective prophylactic drugs have rarely been reported. Because acetazolamide is commonly used to treat acute mountain sickness (AMS), we assessed the potential effects of acetazolamide on cognitive performance during high-altitude exposure. Twenty-one volunteers aged 22-26 years were randomized to receive a 4-day treatment of acetazolamide (125 mg Bid, n=11) or placebo (n=10) before and after air travel from Xianyang (402 m) to Lhasa (3561 m). Neuropsychological performance was assessed using the digit symbol substitution test (DSST), paced auditory serial addition test (PASAT), operation span task, and free recall test at 6, 30, and 54 h after arrival at Lhasa. The Lake Louise Score (LLS) was used to diagnose AMS. At high altitude, acetazolamide impaired rather than improved neuropsychological measures of concentration, cognitive processing speed, reaction time, short-term memory, and working memory, which were assessed by DSST, PASAT, and operation span task at 6 and 30 h after arrival (p<0.05). However, the prophylactic use of acetazolamide was found to reduce the incidence of AMS compared to the placebo (p<0.05). In conclusion, acetazolamide impairs neuropsychological function, at least in part, shortly after the ascent to high altitude.

Involvement of DMT1 +IRE in the transport of lead in an in vitro BBB model

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB). The BBB is particularly vulnerable to lead (Pb) insults. This study was designed to test the hypothesis that divalent metal transporter 1 (DMT1), which is a divalent cation membrane transporter, was involved in transcellular transport across the BBB. An in vitro BBB model, which was a co-culture system of human umbilical vascular endothelial cells (ECV304) and rat glioma cells (C6), was established. Transendothelial electrical resistance (TEER) and fluoresceinisothiocyanate (FITC)-dextran permeability results showed that Pb exposure at the tested concentrations had no significant effects on intercellular tightness. Pb transport displayed properties that were associated with iron response element (IRE) positive isoform of DMT1. Accordingly, Pb transport was significantly blocked by iron (Fe). Moreover, ECV304 cells that were depleted of Fe with the chelator deferoxamine (DFO) demonstrated increased Pb transport. By transfecting ECV-304 cells with a DMT1 expression vector, overexpression of DMT1 promoted an increase in Pb transport. Treatment of ECV304 cells with DMT1 antisense oligonucleotides (ASONs) MA1 significantly inhibited the transport of Pb. Our results suggest that Pb is transported in the in vitro BBB model by a transporter with biochemical properties similar to those of the DMT1 IRE-positive isoform.

Acute cold exposure and rewarming enhanced spatial memory and activated the MAPK cascades in the rat brain

Cold is a common stressor that is likely to occur in everyday occupational or leisure time activities. Although there is substantial literature on the effects of stress on memory from behavioral and pharmacologic perspectives, the effects of cold stress on learning and memory were little addressed. The aims of the present work were to investigate the effects of acute cold exposure on Y-maze learning and the activation of cerebral MAPK cascades of rats. We found that the 2-hour cold exposure (-15 degrees C) and a subsequent 30-min rewarming significantly increased the performance of the rats in the Y-maze test. Serum corticosterone (CORT) level was increased after the cold exposure. After a transient reduction following the cold exposure, the P-ERK levels in the hippocampus and PFC drastically increased 30 min later. The levels of P-JNK increased gradually after the cold exposure in all the three brain regions we investigated, but the level of P-p38 only increased in the PFC. The levels of GABAA receptor alpha1 subunit remained unchanged after the cold exposure. Furthermore, the performance of rats treated with cold plus muscimol or bicuculline in the Y-maze test was similar to that of the rats treated with those GABAergic agents alone. These results demonstrated that acute cold exposure and the subsequent rewarming could result in enhanced performance of spatial learning and memory, and the activation of MAPKs in the brain. However, GABAA receptor may not be involved in the acute cold exposure-induced enhancement of memory.

Microglia activation regulates GluR1 phosphorylation in chronic unpredictable stress-induced cognitive dysfunction

Abstract Chronic stress is considered to be a major risk factor in the development of psychopathological syndromes in humans. Cognitive impairments and long-term potentiation (LTP) impairments are increasingly recognized as major components of depression, anxiety disorders and other stress-related chronic psychological illnesses. It seems timely to systematically study the potentially underlying neurobiological mechanisms of altered cognitive and synaptic plasticity in the course of chronic stress. In the present study, a rat model of chronic unpredictable stress (CUS) induced a cognitive impairment in spatial memory in the Morris water maze (MWM) test and a hippocampal LTP impairment. CUS also induced hippocampal microglial activation and attenuated phosphorylation of glutamate receptor 1 (GluR1 or GluA1). Moreover, chronic treatment with the selective microglial activation blocker, minocycline (120 mg/kg per day), beginning 3 d before CUS treatment and continuing through the behavioral testing period, prevented the CUS-induced impairments of spatial memory and LTP induction. Additional studies showed that minocycline-induced inhibition of microglia activation was associated with increased phosphorylation of GluR1. These results suggest that hippocampal microglial activation modulates the level of GluR1 phosphorylation and might play a causal role in CUS-induced cognitive and LTP disturbances.