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.

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.

Lead induces COX-2 expression in glial cells in a NFAT-dependent, AP-1/NFκB-independent manner.

Epidemiologic studies have provided solid evidence for the neurotoxic effect of lead for decades of years. In view of the fact that children are more vulnerable to the neurotoxicity of lead, lead exposure has been an urgent public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis.

Over activation of hippocampal serine/threonine protein phosphatases PP1 and PP2A is involved in lead-induced deficits in learning and memory in young rats.

Serine/threonine protein phosphatases regulate several key cellular events in the brain, including learning and memory. These enzymes, when over-activated, are known to function as a constraint on learning and memory. We investigated whether these phosphatases are implicated in lead (Pb)-induced deficits in learning and memory. Wistar rat pups were exposed to 0.2% Pb-acetate via their dams' drinking water from postnatal day (PND) 1-21 and directly in drinking water until PND 30. Pb levels in blood, brain and hippocampus were measured and expression of PP1, PP2A, PP2B and PP5 in hippocampus was analyzed. Total phosphatase activity, and PP1 and PP2A activities were determined. Tau phosphorylation at various epitopes was determined by Western blot. Spatial learning and memory was determined by Morris water maze test. Pb exposure significantly increased levels of Pb in blood, brain and hippocampus, reduced the number of synapses in hippocampus and impaired learning and long-term memory (LTM). Short-term memory (STM) was only affected in rats at PND21. Pb exposure increased the expression and activity of PP1 and decreased phosphorylation of tau at threonine-231 in hippocampus at both PND21 and PND30. Pb-induced phosphorylation of tau at serine-199/202 (AT8) paralleled with PP2A activity; at PND21 PP2A activity increased and AT8 phosphorylation decreased; at PND30 PP2A activity decreased and AT8 phosphorylation increased. Increased PP1 activity in hippocampus by Pb is associated with learning and LTM impairment, whereas, increased PP2A activity is associated with STM impairment. These findings suggest the overactivation of PP1 and PP2A, together with changes in tau phosphorylation, as a potential mechanism of lead-induced deficits in learning and memory.

Lead inhibits in vitro creatine kinase and pyruvate kinase activity in brain cortex of rats.

Lead intoxication is a serious occupational disease that constitutes a major public health problem. Lead, a heavy metal, has been used by humans for many technological purposes, which is the main reason for its widespread distribution. The toxic mechanisms of lead on the molecular machinery of living organisms include metal transport, energy metabolism, diverse enzymatic processes, genetic regulation, and membrane ionic channels and signaling molecules. Since lead is able to cross the blood-brain barrier it may cause neurotoxicity. Creatine kinase and pyruvate kinase are two thiol-containing enzymes that exert a key role for cellular energy homeostasis in brain. Our main objective was to investigate the in vitro effect of lead on pyruvate kinase and creatine kinase activities of extracts and subcellular fractions from the brain cortex of rats in the presence or not of thiol-protecting substances such as glutathione and cysteamine. The results showed that lead inhibited the two enzyme activities and the thiol-protecting substances prevented their inhibition. These results suggest that lead inhibits creatine kinase and pyruvate kinase activity by interaction with their thiol groups. Therefore, lead may disrupt energy homeostasis and this effect may contribute to the neurological dysfunction found in lead exposed individuals.

Monosialoanglioside (GM1) prevents lead-induced neurotoxicity on long-term potentiation, SOD activity, MDA levels, and intracellular calcium levels of hippocampus in rats.

Lead (Pb(2+)) is one of the most common neurotoxic metals present in our environment. Chronic or acute exposure to Pb(2+) causes impairment to the central nervous system (CNS). As one potent useful tool in the attempt to protect against impairment and promote functional recovery of the CNS, gangliosides are hopeful for recovering Pb(2+) neurotoxicity. The aim of this study is to investigate the effects of monosialoganglioside (GM1) on the Pb(2+)-induced impairments of synaptic plasticity, antioxidant system function, and intracellular calcium levels in the hippocampus of acute Pb(2+)-exposed rats. Our study showed that: (1) Acute Pb(2+) exposure impaired synaptic transmission and plasticity in the hippocampus and GM1 preconditioning rescued to some extent this impairment in urethane-anesthetized rats. (2) Superoxide dismutase activities and malondialdehyde levels were significantly increased in the acute Pb(2+)-exposed hippocampus which could be reduced by GM1 preconditioning. (3) Further, acute Pb(2+) exposure caused the internal free Ca(2+) fluctuation in the cultured hippocampal neurons and GM1 preconditioning could abate this fluctuation. Taken together, our results illustrated the possible mechanisms underlying the protective effects of GM1 against Pb(2+) neurotoxicity and might shed light on protection against Pb(2+) toxicity and its treatment.

In vitro vs in vivo Pb effects on brain protein kinase C activity.

Alteration of normal protein kinase C (PKC) function by environmental Pb exposure during neurodevelopment is hypothesized to be an important mechanism of toxicity underlying neurologic impairment. Previous studies have reported widely varying effects of Pb on PKC, possibly in part because of differences in in vitro and in vivo models used in those studies. Therefore, we tested the hypothesis that, with comparable tissue Pb levels, the effects of in vitro Pb exposure on brain PKC are the same as the effects caused by in vivo Pb exposure of intact animals. For chronic in vivo Pb exposure, female Long-Evans rats were exposed to Pb or vehicle from postnatal days 1 to 34-36 (n=10/treatment). For in vitro Pb exposure, homogenate of the frontal cortex region was exposed directly to Pb in an amount comparable to that accumulated in brain during chronic in vivo Pb exposure. Brain Pb levels were measured using ultraclean techniques and inductively coupled plasma mass spectrometry. PKC activity was subsequently determined in cytosolic and membrane subcellular fractions in the frontal cortex, hippocampus, and remaining brain regions. Results indicate that brain Pb levels following in vivo Pb exposure were increased approximately 20-fold above those of nonexposed animals (vehicle group [Pb] approximately 130ng Pb/g dry wt.). However, in vivo Pb exposure did not measurably alter brain PKC activity in the regions tested. In contrast, in vitro Pb exposure significantly increased PKC activity by approximately 20% in the frontal cortex homogenate membrane subcellular fraction. These results indicate that Pb added in vitro caused more dramatic effects than those produced by a comparable amount of Pb in the tissue from in vivo exposure. While the mechanisms underlying these outcomes are not clear, they suggest that in vitro models might not accurately reflect effects of chronic low-level in vivo Pb exposure.

Erythrocyte aminolevulinate dehydratase activity as a lead marker in patients with chronic renal failure.

BACKGROUND: Overexposure to lead may result in an increased risk for developing chronic renal failure (CRF) and hypertension. Subclinical lead poisoning is difficult to identify. Because the heme biosynthetic pathway is highly sensitive to lead, we considered the study of enzymes involved in this pathway as a method to detect an excessive body lead burden. METHODS: Main concerns in assessing the heme pathway in patients with CRF were related to aminolevulinate dehydratase (ALAD) activity. We first selected a number of patients with CRF at a predialysis stage, subsequently dividing them into two groups after the EDTA mobilization test had determined whether lead pools were expanded. The study included 24 healthy controls, 12 patients with clinical plumbism and biochemical demonstration of lead poisoning (Pb-CONT), 18 patients with CRF with no evidence of high lead storage (CRF/-), and 8 patients with CRF with high urinary excretion of lead in contrast to normal blood lead levels (CRF/+). RESULTS: As expected, symptoms of plumbism (Pb-CONT) were accompanied by an increased erythrocyte zinc-protoporphyrin-free protoporphyrin ratio and high urine coproporphyrin excretion, whereas both these values were within the normal range in all patients with CRF. CRF/- patients showed minor abnormalities of erythrocyte heme metabolism, such as low ALAD activity, both baseline and in vitro restored. The ALAD-restored ALAD ratio correlated closely with urine lead excretion; it was normal in healthy controls and CRF/- patients and significantly reduced in Pb-CONT and CRF/+ patients. CONCLUSION: The erythrocyte ALAD-restored ALAD ratio may be a useful tool to show otherwise subclinical lead poisoning in patients with CRF.

Effects of postnatally administered inorganic lead on the tyrosine hydroxylase immunoreactive norepinephrinergic neurons of the locus ceruleus of the rat.

The neurotoxic effects of inorganic lead are known to include peripheral neuropathy in adults and encephalopathy in children. The purpose of this study was to determine the effect of inorganic lead (PbCl2) administration on norepinephrinergic neurons of the locus ceruleus in neonatal rats by immunocytochemical and electron microscopic analyses. Lead chloride solutions, 0.05%, 0.1% and 0.2% in concentrations, were prepared in distilled water and administered orally via drinking water. After 4, 8, or 12 weeks of continuous administration, the rats were sacrificed and brains were immunostained with the tyrosine hydroxylase antibody. The number of immunoreactive cell bodies in the locus ceruleus was estimated. Densitometric analysis of immunoreactive profiles visualized by electron microscopy was performed using an image analyzer. The numbers of immunoreactive neurons in the locus ceruleus were increased statistically by lead administration. The intensity of the immunoreaction, both under the light and electron microscopes was also increased. Degenerative changes, including intra-axonal vacuole formation and widening of the extracellular spaces, were found by electron microscopy in and around the tyrosine hydroxylase immunoreactive axons. Increased tyrosine hydroxylase immunoreactivity may correlate with the hyper-reactivity of lead intoxicated children. Degenerative changes may account for the reported deficits in intellectual attainment and achievement in lead intoxicated children.

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 exposure affects levels of galactolipid metabolic enzymes in the developing rat brain.

Lead poisoning is known to cause myelin defects. Galactolipids are the major lipid components of myelin and myelin-competent oligodendrocytes. The present study examines the cellular activity of enzymes involved in the galactolipid pathway, tissue concentrations of galactolipids, and the cellular activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in rat pups exposed to lead in utero and subsequently through maternal milk from exposed mothers and in drinking water following weaning. Pups from control and lead-treated groups (500 or 2000 ppm lead in the drinking water) were euthanized by decapitation on postnatal day 7, 14, 21, 35, or 56. Lead decreased levels of galactolipids and the oligodendrocyte marker CNPase in the brain to a similar degree. The ratios of galactocerebrosides/sulfatides and nonhydroxy fatty acid/hydroxy fatty acid forms of the galactolipids were not altered by lead treatment. In contrast, the activities of the galactolipid metabolic enzymes were reduced to a degree significantly greater than that of CNPase or galactolipids. These results are consistent with previously obtained data indicating that in vitro cultured oligodendroglial progenitor cells are a target for Pb toxicity. Chronic Pb exposure may impact on brain development by impairing timely myelin production due to perturbation of the early developmental commitment of oligodendroglial progenitors. It is further suggested that perturbation of the galactolipid pathway during the developmental maturation of oligodendrocytes may represent a contributing mechanism for Pb-induced neurotoxicity.

Alterations in beta-adrenergic receptor density and adenylate cyclase activity in the rat brain treated chronically with lead.

Behavioral and memory impairment resulting from lead exposure is well known but the mechanism is not. We utilized the brain of lead exposed rats to investigate this problem. In an effort to elucidate the mechanism responsible for this alteration we examined blood and brain lead levels, brain beta-adrenoceptor density and cyclic AMP production in lead exposed rats. Wistar rats used in these trials were divided into six groups of ten animals each. Five groups were given drinking water containing 0.05, 0.1, 0.5, 1 and 2% lead acetate for a period of 60 days. One group (control group, 0% lead acetate) was given pure water. Application of a trend test indicated that both blood and brain lead levels increased significantly from group 0% to group 2% (group 0%