Nuclear accumulation of histone deacetylase 4 (HDAC4) by PP1-mediated dephosphorylation exerts neurotoxicity in Pb-exposed neural cells.

Lead (Pb) is an environmental contaminant that primarily affects the central nervous system, particularly the developing brain. Recently, increasing evidence indicates the important roles of histone deacetylases (HDACs) in Pb-induced neurotoxicity. However, the precise molecular mechanisms involving HDAC4 remains unknown. The purpose of this study was to investigate the role of HDAC4 in Pb-induced neurotoxicity both in vivo and in vitro. In vitro study, PC12 cells were exposed to Pb (10 µM) for 24 h, then the mRNA and protein levels of HDAC4 were analyzed. In vivo study, pregnant rats and their female offspring were treated with lead (50 ppm) until postnatal day 30. Then the pups were sacrificed and the mRNA and protein levels of HDAC4 in the hippocampus were analyzed. The results showed that HDAC4 was significantly increased in both PC12 cells and rat hippocampus upon Pb exposure. Blockade of HDAC4 with either LMK-235 (an inhibitor of HDAC4) or shHDAC4 (HDAC4-knocking down plasmid) ameliorated the Pb-induced neurite outgrowth deficits. Interestingly, HDAC4 was aberrantly accumulated in the nucleus upon Pb exposure. By contrast, blocking the HDAC4 shuffling from the cytosol to the nucleus with ΔNLS2-HDAC4 (the cytosol-localized HDAC4 mutant) was able to rescue the neuronal impairment. In addition, Pb increased PP1 (protein phosphatase 1) expression which in turn influenced the subcellular localization of HDAC4 by dephosphorylation of specific serine/threonine residues. What's more, blockade of PP1 with PP1-knocking down construct (shPP1) ameliorated Pb-induced neurite outgrowth deficits. Taken together, nuclear accumulation of HDAC4 by PP1-mediated dephosphorylation involved in Pb-induced neurotoxicity. This study might provide a promising molecular target for medical intervention with environmental cues.

Developmental lead (Pb)-induced deficits in redox and bioenergetic status of cerebellar synapses are ameliorated by ascorbate supplementation.

Neurotoxicity induced by exposure to heavy metal lead (Pb) is a concern of utmost importance particularly for countries with industrial-based economies. The developing brain is especially sensitive to exposure to even minute quantities of Pb which can alter neurodevelopmental trajectory with irreversible effects on motor, emotive-social and cognitive attributes even into later adulthood. Chemical synapses form the major pathway of inter-neuronal communications and are prime candidates for higher order brain (motor, memory and behavior) functions and determine the resistance/susceptibility for neurological disorders, including neuropsychopathologies. The synaptic pathways and mechanisms underlying Pb-mediated alterations in neuronal signaling and plasticity are not completely understood. Employing a biochemically isolated synaptosomal fraction which is enriched in synaptic terminals and synaptic mitochondria, this study aimed to analyze the alterations in bioenergetic and redox/antioxidant status of cerebellar synapses induced by developmental exposure to Pb (0.2 %). Moreover, we test the efficacy of vitamin C (ascorbate; 500 mg/kg body weight), a neuroprotective and neuromodulatory antioxidant, in mitigation of Pb-induced neuronal deficits. Our results implicate redox and bioenergetic disruptions as an underlying feature of the synaptic dysfunction observed in developmental Pb neurotoxicity, potentially contributing to consequent deficits in motor, behavioral and psychological attributes of the organisms. In addition, we establish ascorbate as a key ingredient for therapeutic approach against Pb induced neurotoxicity, particularly for early-life exposures.

The Neuroprotective Role of Coenzyme Q10 Against Lead Acetate-Induced Neurotoxicity Is Mediated by Antioxidant, Anti-Inflammatory and Anti-Apoptotic Activities.

Heavy metal exposure, in lead (Pb) particularly, is associated with severe neuronal impairment though oxidative stress mediated by reactive oxygen species, and antioxidants may be used to abolish these adverse effects. This study investigated the potential neuroprotective role of coenzyme Q10 (CoQ10) against lead acetate (PbAc)-induced neurotoxicity. Twenty-eight male Wistar albino rats were divided into four equal groups (n = 7) and treated as follows: the control group was injected with physiological saline (0.9% NaCl); the CoQ10 group was injected with CoQ10 (10 mg/kg); PbAc group was injected with PbAc (20 mg/kg); PbAc + CoQ10 group was injected first with PbAc, and after 1 h with CoQ10. All groups were injected intraperitoneally for seven days. PbAc significantly increased cortical lipid peroxidation, nitrate/nitrite levels, and inducible nitric oxide synthase expression, and decreased glutathione content, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase activity and mRNA expression, as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and homoxygenase-1 (HO-1) expression. PbAc also promoted the secretion of interleukin-1ß and tumor necrosis factor-α, inhibited interleukin-10 production, triggered the activation of pro-apoptotic proteins, and suppressed anti-apoptotic proteins. Additionally, PbAc increased the cortical levels of serotonin, dopamine, norepinephrine, GABA, and glutamate, and decreased the level of ATP. However, treatment with CoQ10 rescued cortical neurons from PbAc-induced neurotoxicity by restoring the balance between oxidants and antioxidants, activating the Nrf2/HO-1 pathway, suppressing inflammation, inhibiting the apoptotic cascade, and modulating cortical neurotransmission and energy metabolism. Altogether, our findings indicate that CoQ10 has beneficial effects against PbAc-induced neuronal damage through its antioxidant, anti-inflammatory, anti-apoptotic, and neuromodulatory activities.

Pb exposure reduces the expression of SNX6 and Homer1 in offspring rats and PC12 cells.

Lead (Pb) is a widespread environmental heavy metal toxicant and chronic Pb exposure can have irreversible effects on memory and cognitive function, which is closely related to dendritic spines. Studies have shown that SNX6 and Homer1 can regulate the growth of dendritic spines. We aimed to investigate the effect of Pb exposure on the dendritic spines in hippocampus, the expression of SNX6 and Homer1 in rats and PC12 cells. The animals were randomly divided to three groups: control group, low lead group and high lead group. PC12 cells were divided into 3 groups: 0 µM, 1 µM and 100 µM Pb acetate. The results showed that the Pb levels in blood and hippocampus of all exposure groups were significantly higher than that of the control group. The morphology of dendritic spines in hippocampus after Pb treatment was changed and the density of dendritic spines was reduced. The expression of SNX6 and Homer1 was decreased in Pb exposed groups compared with the control group. Furthermore, up-regulation of SNX6 expression could reverse the down-regulation of Pb exposure on Homer1. These results indicate that Pb exposure can reduce the expression of SNX6 and lead to a decrease in Homer1 expression, which affects the changes in dendritic spines causing learning and memory impairment.

Regulatory Roles of Histone Deacetylases 1 and 2 in Pb-induced Neurotoxicity.

Lead (Pb) prevails among the environmental hazards against human health. Although increasing evidence highlights the epigenetic roles underlying the Pb-induced neurotoxicity, the exact mechanisms concerning histone acetylation and its causative agents are still at its infancy. In the present study, the roles of histone deacetylases 1 and 2 (HDAC1/2), as well as acetylation of Lys9 on histone H3 (Ac-H3K9), in Pb-induced neurotoxicity were investigated. Pb was administered to PC12 cells at 10 µM for 24 h. And Sprague Dawley rats were chronically exposed to Pb through drinking water containing 250 ppm Pb for 2 months. Owing to Pb exposure, it indicated that HDAC2 was up-regulated accompanied by Ac-H3K9 down-regulation. Meanwhile, chromatin immunoprecipitation assay revealed that the changes in HDAC2 were attributed to histone H3 Lys27 trimethylation occupancy on its promoter. Blockade of HDAC2 with either Trichostatin A or HDAC2-knocking down construct (shHDAC2) resulted in amelioration of neurite outgrowth deficits via increasing Ac-H3K9 levels. It implied that HDAC2 plays essential regulatory roles in Pb-induced neurotoxicity. And, coimmunoprecipitation trials revealed that HDAC2 colocalized with HDAC1, forming a so-called HDAC1/2 complex. Subsequently, it was shown that HDAC1/2 repression could markedly prevent neurite outgrowth impairment and rescue the spatial memory deficits caused by Pb exposure, unequivocally implicating this complex in the studied toxicological process. Furthermore, Notch2 maybe the functional target of the HDAC1/2 and Ac-H3K9 alterations. Our study provided insight into the precise roles of HDAC1/2 in Pb-induced neurotoxicity, and thereby provided a promising molecular target for medical intervention of neurological disorders with environmental etiology.

Neuroprotective effect of morin on lead acetate- induced apoptosis by preventing cytochrome c translocation via regulation of Bax/Bcl-2 ratio.

Lead (Pb) intoxication is a prevalent type of environmental toxicity as well as minimal amount of lead exposure is liable for neurobehavioral or perhaps intelligence defects. The present study was undertaken to investigate the beneficial effects of morin in protecting the lead acetate (PbAc)-induced oxidative stress in rat brain. PbAc intoxication resulted in motor deficit, memory impairment and oxidative stress Further, PbAc administration alters Bax/Bcl-2 expression thereby increases cytochrome c release from the mitochondria. Treatment with morin at a dose of 40 mg/kg b.wt. significantly restored back the abnormal changes that were noticed in PbAc intoxicated rats. Histopathological sections of cortex, cerebellum and hippocampus showed the extent of neuronal loss in PbAc induced rats and its restoration upon administration of morin. These outcomes imply that morin might be employed therapeutically to chelate toxic metals like Pb, thus possibly lowering PbAc-induced neurotoxicity and tissue damage.

Effects of curcumin and tannic acid on the aluminum- and lead-induced oxidative neurotoxicity and alterations in NMDA receptors.

Exposure to aluminum (Al) and lead (Pb) can cause brain damage. Also, Pb and Al exposure alters N-methyl-d-aspartate receptor (NMDAR) subunit expression. Polyphenols such as tannic acid and curcumin are very efficient chelator for metals. The effects of curcumin and tannic acid (polyphenols) on Al(3+)- and Pb(2+)-induced oxidative stress were examined by investigating lipid peroxidation (LPO) levels, antioxidant enzyme activities, acetyl cholinesterase (AChE) activity and also NMDA receptor subunits 2A and 2B concentrations in the brain tissue of rats sub-chronically. Rats were divided into seven groups as control, Al, Pb, aluminum-tannic acid treatment (AlT), aluminum-curcumin treatment (AlC), lead-tannic acid treatment (PbT) and lead-curcumin treatment (PbC). After 16 weeks of treatment, LPO levels in the brain and hippocampus were higher in Al(3+)-exposed rats than that of Pb(2+)-exposed group. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in brain tissue of Al- and Pb-exposed rats increased significantly compared with control, while catalase (CAT) and AChE activities decreased. It was observed that metal exposure affected NR2A concentrations more than NR2B concentrations and also that polyphenol treatments increased these receptor protein concentrations.

Enhanced taupathy and AD-like pathology in aged primate brains decades after infantile exposure to lead (Pb).

Late Onset Alzheimer Disease (LOAD) constitutes the majority of AD cases (∼90%). Amyloidosis and tau pathology, which are present in AD brains, appear to be sporadic in nature. We have previously shown that infantile lead (Pb) exposure is associated with a change in the expression and regulation of the amyloid precursor protein (APP) and its beta amyloid (Aß) products in old age. Here we report that infantile Pb exposure elevated the mRNA and protein levels of tau as well as its transcriptional regulators namely specificity protein 1 and 3 (Sp1 and Sp3) in aged primates. These changes were also accompanied by an enhancement in site-specific tau phosphorylation as well as an increase in the mRNA and protein levels of cyclin dependent kinase 5 (cdk5). There was also a change in the protein ratio of p35/p25 with more Serine/Threonine phosphatase activity present in aged primates exposed to Pb as infants. These molecular alterations favored abundant tau phosphorylation and immunoreactivity in the frontal cortex of aged primates with prior Pb exposure. These findings provide more evidence that neurodegenerative diseases may be products of environmental influences that occur during the development.

Effect of lead sulfide nanoparticles exposure on calcium homeostasis in rat hippocampus neurons.

PbS nanoparticles (NPs) is an important nanomaterial for biomedical imaging in living tissues. However, concerning the high toxicity, especially neurotoxicity, of Pb element, it is crucial that the toxicity assessment of "naked" PbS NPs should be adequately studied. In the current study, we systematically explored the neurotoxicity of PbS NPs in rats by measuring the body weight and brain coefficient changes, testing memory behaviors in Y-electric maze, and studying the neuronal ultrastructure and pathology in hippocampus. Furthermore, in order to study the toxic mechanism, we performed Pb and Ca content measurements in various organs, and investigated Ca(2+)-ATPase activity and L-type calcium channel subunit expression. Our results confirmed that PbS NPs showed high neurotoxicity, while a possible mechanism was suggested to be due to the PbS NPs-induced calcium homeostasis disorder which was caused by the abnormal calcium transportation.

The protective effect of green tea extract on lead induced oxidative and DNA damage on rat brain.

The role of green tea in protection against neurotoxicity induced by lead acetate was investigated in rats. Five equal groups, each of ten rats were used. The first group was served as control, the second, third, and fourth groups were given lead acetate, lead acetate and green tea, and green tea only, respectively, for one month, the fifth group was administered lead acetate for one month followed by green tea for 15 days. Lead acetate was given orally at a dose of 100 mg/kg b. wt, while green tea was given in drinking water at a concentration of 5 g/L. Lead acetate administration induced loss of body weight and decreased concentration of reduced glutathione and SOD activity in brain tissues as well as significantly high DNA fragmentation and pathological changes. Co-administration of green tea with lead acetate significantly alleviated these adverse effects.

Effects of flaxseed oil on lead acetate-induced neurotoxicity in rats.

It is well known that chronic exposure to lead (Pb(+2)) alters a variety of behavioral tasks in rats and mice. Here, we investigated the effect of flaxseed oil (1,000 mg/kg) on lead acetate (20 mg/kg)-induced brain oxidative stress and neurotoxicity in rats. The levels of Pb(+2), lipid peroxidation, nitric oxide (NO), and reduced glutathione (GSH) and the activity of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GPx) were determined in adult male albino rats. The level of Pb(+2) was markedly elevated in brain and blood of rats. This leads to enhancement of lipid peroxidation and NO production in brain with concomitant reduction in GSH, CAT, SOD, GR, GST, and GPx activities. These findings were associated with DNA fragmentation. In addition, lead acetate induced brain injury as indicated by histopathological changes of the brain. Treatment of rats with flaxseed oil resulted in marked improvement in most of the studied parameters as well as histopathological features. These findings suggest to the conclusion that flaxseed oil significantly decreased the adverse harmful effects of lead acetate exposure on the brain as well as Pb(+2)-induced oxidative stress.

The role of metabotropic glutamate receptor 5 in developmental lead neurotoxicity.

A complete explanation of the mechanisms of lead-induced developmental neurotoxicity remains unknown. The glutamate receptor is one of the most important targets of lead. More recently, metabotropic glutamate receptor 5 (mGluR5) has been shown to have a functional relationship with learning and memory. We investigated the impact of developmental lead exposure on hippocampal mGluR5 expression and its potential role in lead neurotoxicity. Both in vitro model of lead exposure with Pb(2+) concentrations of 0, 10 nM, 1 microM, and 100 microM in cultured rat embryonic hippocampal neurons, and the in vivo model of rat maternal lead exposure involving both gestational and lactational exposure with 0, 0.05%, 0.2%, and 0.5% lead acetate were utilized. Immunoperoxidase and immunofluorescent analyses, quantitative PCR and western blotting were used. In vitro studies revealed that expression of mGluR5 mRNA and protein was decreased dose-dependently after lead exposure, which was further confirmed by the results of in vivo studies. These data suggest that mGluR5 might be involved in lead-induced neurotoxicity by disturbing mGluR5-induced long-term depression and decreasing N-methyl-D-aspartic acid receptor (NMDAR)-dependent or protein synthesis-dependent long-term potentiation. These results might improve the understanding of the mechanism and potential treatments for moderate to severe lead poisoning in children.

Effects of lead on cholinergic SN56 neuroblastoma cells.

Age-dependent accumulation of lead in brain has been implicated in the pathomechanisms of Alzheimer's disease. The aim of this work was to investigate whether cholinotoxic effects of lead may result from alterations in acetyl-CoA metabolism. One day exposure of differentiated SN56 cholinergic neuroblastoma cells to 0.5 micromol/L lead or 0.01 mmol/L amyloid-beta1-42, increased fraction of nonviable cells to about 2%. Suppression of choline acetyltransferase activity occurred only in the presence of fresh amyloid-beta1-42, whereas lead was ineffective. All agents in combination caused suppression of acetyl-CoA in cytoplasm and mitochondria down to 19% and 34% of controls, respectively. Inverse correlation was observed between whole cell acetyl-CoA level and fraction of nonviable cells at different combinations of lead and other neurotoxic compounds. It indicates that lead had no primary suppressive effect on cholinergic phenotype but, at least in part, exerted cytotoxic influence on cholinergic neurons through the decrease of their acetyl-CoA.

Long-term consequences of prenatal exposure to lead on brain development in rats.

Administration of singe doses of lead citrate (200 mg/kg) to pregnant rats (on day 18 of pregnancy) was followed by the appearance of destructive changes in brains at age 40 days, with cysts, foci of gliocyte proliferation, pyknotic neurons, and decreases in NADH and NADPH diaphorase activities in neocortical and hippocampal neurons. Decreases in the density of neurons in the cortex and decreases in cortical thickness were also observed. The intensity of free-radical oxidation in the cortex increased three-fold, along with a 3.9-fold increase in the concentration of lipid peroxides, providing evidence of oxidative stress. The possible mechanisms by which these alterations develop are analyzed.

Postnatal lead poisoning impairs behavioral recovery following brain damage.

Lead is a potent environmental toxicant with well-known effects on intelligence, school achievement and behavior. Lead exposure is also associated with an increased risk of a variety of health problems including cancer, hypertension, cardiovascular disease, and renal disease. Considering the risk of hypertension, cardiovascular problems, and stroke following lead exposure, the current research assessed the extent to which postnatal exposure to environmentally relevant levels of lead could impair the recovery from a later occurring brain injury. Using a photochemical thrombotic stroke model we found that postnatal lead exposure significantly impaired post-stroke recovery of beam walking ability and proprioceptive limb placing. Considering the increased risk for hypertension and cardiovascular disease in lead-exposed humans, diminished capacity for repair or adaptation following lead exposure needs to now be examined in greater detail.

Inflammation-like glial response in lead-exposed immature rat brain.

Numerous studies on lead (Pb) neurotoxicity have indicated this metal to be a dangerous toxin, particularly during developmental stages of higher organisms. Astrocytes are responsible for sequestration of this metal in brain tissue. Activation of astroglia may often lead to loss of the buffering function and contribute to pathological processes. This phenomenon is accompanied by death of neuronal cells and may be connected with inflammatory events arising from the production of a wide range of cytokines and chemokines. The effects of prolonged exposure to Pb upon glial activation are examined in immature rats to investigate this potential proinflammatory effect. When analyzed at the protein level, glial activation is observed after Pb exposure, as reflected by the increased level of glial fibrillary acidic protein and S-100beta proteins in all parts of the brain examined. These changes are associated with elevation of proinflammatory cytokines. Production of interleukin (IL)-1beta and tumor necrosis factor-alpha is observed in hippocampus, and production of IL-6 is seen in forebrain. The expression of fractalkine is observed in both hippocampus and forebrain but inconsiderably in the cerebellum. In parallel with cytokine expression, signs of synaptic damage in hippocampus are seen after Pb exposure, as indicated by decreased levels of the axonal markers synapsin I and synaptophysin. Obtained results indicate chronic glial activation with coexisting inflammatory and neurodegenerative features as a new mechanism of Pb neurotoxicity in immature rat brain.

Investigation of acute lead poisoning on apoptosis in rat hippocampus in vivo.

Despite decades of study, the exact mechanism of action of lead, a potent neurotoxic agent, have not been fully elucidated. One of the suggested mechanism of lead neurotoxicity is apoptotic cell death. The present study sought to examine the effect of acute lead poisoning on apoptosis in rat hippocampus. Two to four and 12-14 week old rats were treated for 7 days with 15 mg/kg daily dose of lead acetate intraperitoneally. Control animals received distilled water. In treated groups, the blood lead levels was increased by about 17-19-folds. Histological study of hippocampus revealed apoptotic cells, using light and electron microscopy. In Western blot analysis, the ratio of Bax/Bcl-2 protein expression in hippocampus was significantly increased compared to controls. In conclusion, the lead induced cell death in hippocampus in vivo may partly be due to apoptosis.

Protein kinase C activation is required for the lead-induced inhibition of proliferation and differentiation of cultured oligodendroglial progenitor cells.

Lead (Pb) is a common neurotoxicant of major public health concern. Previous studies revealed that cultured oligodendrocyte progenitor cells (OPCs) are highly vulnerable to Pb toxicity. The present study examines the effect of Pb on the survival, proliferation and differentiation of OPCs in vitro. Dose-response studies showed that> or = l5-10 microM Pb is cytotoxic to OPCs within 24 h. However, 1 microM of Pb was found to inhibit the proliferation and differentiation of OPCs without affecting cell viability. Pb markedly decreased the proliferative capability of OPCs and inhibited cell-intrinsic lineage progression of OPCs at a late progenitor stage. The Pb-induced decrease of proliferation and differentiation was abolished by inhibition of protein kinase C (PKC) with bisindolylmaleimide I, while the effect of the PKC-activating agent phorbol-12,13-didecanoate was potentiated by Pb. Furthermore, Pb exposure of OPCs caused the translocation of PKC from the cytoplasm to membrane without an increase in total cellular PKC enzymic activity. These results indicate that Pb inhibits the proliferation and differentiation of oligodendrocyte lineage cells in vitro through a mechanism requiring PKC activation.

Lead content of brain tissue in diffuse neurofibrillary tangles with calcification (DNTC): the possibility of lead neurotoxicity.

Diffuse neurofibrillary tangles with calcification (DNTC) is a form of presenile dementia, characterized pathologically by fronto-temporal atrophy with neurofibrillary tangles (NFTs), neuropil threads and Fahr-type calcification, in which no senile plaques are observed. As already noted, chronic exposure to lead (Pb) might be one of the etiological factors of Fahr-type calcification. Until now, there have been no reports in which Pb concentration has been quantified in DNTC brains. We examined the concentration of Pb in fresh-frozen brain tissue and in 10% formalin-fixed brain tissue from six cases of DNTC, four cases of Alzheimer's disease, and in nine non-demented elderly controls by flameless atomic absorption spectrometry, and demonstrated a high concentration of Pb in DNTC brains. Although it remains unclear how these findings are related to the formation of NFTs, they suggest that Pb neurotoxicity may be involved in the pathogenesis of DNTC.

Astroglial reaction during the early phase of acute lead toxicity in the adult rat brain.

The developing nervous system is susceptible to lead (Pb) exposure but less is known about the effect of this toxic agent in adult rat brain. Since astrocytes serve as a cellular Pb deposition site, it is of importance to investigate the response of astroglial cells in the adult rat brain in a model of acute lead exposure (25 mg/kg b.w. of lead acetate, i.p. for 3 days). An increased immunoreactivity of glial fibrillary acidic protein (GFAP) on Western blots was noticeable in fractions of astroglial origin-glial plasmalemmal vesicles (GPV) and in homogenates from the hippocampus and cerebral cortex but not in the cerebellum. The features of enhanced astrocytic reactivity (i.e. large accumulation of mitochondria, activated Golgi apparatus and increment of gliofilaments) were observed in electron microscopy studies in the same tissues. Total glutathione levels increased both in GPV fractions and in brain homogenates-in the cerebellum (120% above control) and in hippocampus (30% above control). The results of current studies indicate that acute lead exposure is accompanied by astrocyte activation connected with the presence of the enhanced expression of GFAP. It may indicate lead-induced neuronal injury. At the same time, a regional enhancement of detoxicative mechanisms (GSH) was noticed, suggesting activation of astrocyte-mediated neuroprotection against toxic Pb action.