Correlation between lead-induced changes in cerebral ornithine decarboxylase and protein kinase C activities during development and in cultured PC 12 CELLS

Exposure to lead (Pb) interferes with neurodevelopment and disturbs ornithine decarboxylase (ODC) activity. ODC the key regulatory enzyme of the polyamine pathway, is a potential substrate for protein kinase C (PKC). Therefore, we examined developmental changes in PKC activity and its relationship to ODC activity. Male rats were lactationally exposed to 0.2% Pb-acetate from birth to weaning. PKC and ODC activity were measured on postnatal days (PND) 3, 5, 10, 20 and 30. We found that the basal patterns of ODC and PKC activities resembled each other in both the neocortex and cerebellum and Pb-exposure attenuated both enzymes in a similar manner. To determine whether any link existed between these enzymes, ODC and PKC activities were induced to increase using nerve growth factor (NGF) in the presence and/or absence of ODC (difluoromethylornithine, DFMO) and PKC (staurosporine) inhibitors, in control and Pb-exposed Pheochromocytoma (PC-12) cells. Staurosporine decreased both ODC activity and PKC activity, while DFMO had no effect on PKC activity. These data suggest that ODC may be regulated by PKC and that Pb-induced developmental alterations in ODC activity may be secondary to changes in the integrity of PKC.     

PKC isoforms were reduced by lead in the developing rat brain

A plethora of protein kinase C (PKC) isoforms play important roles in regulating synaptic plasticity and neurotransmitter release. Even though, most PKC isoforms are involved in Pb-induced neuronal toxicity, its mechanism is still unclear. The current study addresses the effect of Pb on PKC isoforms in different regions of the developing rat brain. Sprague-Dawley (SD) pregnant rats were exposed to 0.1% Pb as lead acetate dissolved in distilled deionized water (DDW) from gestation day 6 through 21 postnatal day (PND). Control rats were allowed to drink DDW. Pups were sacrificed on PND 1, 5, 10 and 45. Rat brain was immediately excised and separated into the brain stem (BS), the cerebellum (CB), the hippocampus (HC) and the frontal cortex (FC). The Pb level in different regions of the brain was determined using an analytical graphite tube atomizer (Varian). Typical PKC (alpha, beta, gamma), novel PKC (epsilon) and atypical PKC (mu) in the above brain regions were enriched by immunoprecipitation and later were assayed by Western blotting. The total, calcium-dependent and -independent PKC activities were determined by the radioactivity of total gamma-32P transferred to histone. The results indicated that on PND 1, Pb reduced the PKC-gamma protein in HC and FC, whereas on PND 5 the proteins of PKC isoforms (alpha, beta, gamma, epsilon, mu) in HC and FC were significantly reduced. These reductions in PKC proteins were higher in membrane fractions than in cytosolic fractions. On PND 10, Pb reduced all PKC isoforms. However, on PND 45, Pb had no significant effect on all PKC isoforms except epsilon. Pb inhibited the total PKC activity by 70% on PND 1 and 5, the bulk of these PKC activities were calcium-dependent. The results suggest that during early stages of the rat brain development, Pb exposure decreased PKC activities and also reduced PKC isoforms including PKC-gamma and epsilon which are reported to have roles in the memory formation and long-term potentiation (LTP).     

Developmental stage-dependent protective effect of NGF against lead cholinotoxicity in the rat septum

The ability of nerve growth factor (NGF) to ameliorate developmental cholinotoxicity of inorganic lead (Pb) for the septal neurons was investigated by making intracerebroventricular injections of single doses of 30 microg 2.5S NGF into maternally lead-exposed suckling rats on postnatal days P2, P4, P11, or P18. Administration of NGF on P4 or later induced septal choline acetyltransferase (ChAT) activity to the same relative extent in both Pb-exposed as in control rats but failed to reverse the net reductions of ChAT activity induced by Pb. In contrast, injection of NGF at P2 completely restored ChAT activity in Pb-exposed pups to control levels by preventing the loss of ChAT-immunoreactive cells in the septum. It is concluded that although NGF retains the capacity to upregulate ChAT throughout the period of Pb exposure, it protects against the Pb-induced loss of septal cholinergic neurons only when applied within the critical period of Pb-vulnerability between postnatal days 2 and 4.     

The influence of developmental period of lead exposure on long-term potentiation in the adult rat dentate gyrus in vivo

Previous work has demonstrated an increase in the threshold for induction of long-term potentiation (LTP) in the dentate gyrus of animals chronically exposed to lead (Pb) from birth (Gilbert et al., 1996). The present study sought to extend these findings by evaluating the developmental periods critical for Pb-induced impairment of LTP. Rats were exposed to Pb through maternal milk and/or the drinking water over different developmental intervals: 1) beginning just prior to birth and continuing throughout life (PL); 2) beginning just prior to birth and terminating at weaning (PW); or 3) continuously from the early post-weaning period throughout life (WL). Pregnant dams received 0.2% Pb-acetate in the drinking water on gestational day (GD)16, with male offspring switched to the same solution (PL group) or tap water (PW group) at weaning on postnatal day (PND)21. Postweaning exposure began on PND30 and continued throughout life. As adults (PND130-210), field potentials evoked by perforant path stimulation were recorded in the dentate gyrus under urethane anesthesia, and an ascending series of stimulus trains was administered to induce LTP and to determine its threshold. The magnitude of population spike (PS) LTP was reduced relative to controls in animals exposed throughout life (PL) and in animals exposed after weaning (WL). No impairment in PS LTP was evident in animals removed from Pb at weaning and tested as adults (PW). Similarly, thresholds for induction of PS LTP were elevated relative to controls in the PL and WL groups, but were not affected by Pb exposure limited to the lactational period (PW). Reductions in the magnitude of LTP of the EPSP slope were evident in posttrain I/O functions in all Pb-exposed groups, including the PW group. An elevated LTP threshold was evident in the EPSP slope measure in the continuously exposed group (PL) only. Thus Pb exposure restricted to the lactational period appeared less disruptive to adult LTP in the dentate gyrus than continuous exposure beginning around birth or weaning. However, EPSP slope LTP was impaired in animals exposed to Pb for as little as 30 days in the early postnatal period. An attenuated ability to support neuroplastic change in synaptic function may contribute to cognitive deficits associated with Pb-induced toxicity.     

Brain metabolic effects of Neotrofin in patients with Alzheimer's disease

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.     

Alterations in brain protein kinase C isoforms following developmental exposure to a polychlorinated biphenyl mixture

PCBs have been shown to alter several neurochemical end-points and are implicated in the etiology of some neurological diseases. Recent in vivo studies from our laboratory indicated that developmental exposure to a commercial PCB mixture, Aroclor 1254, caused perturbations in calcium homeostasis and changes in protein kinase C (PKC) activities in rat brain. However, it is not known which molecular substances are targets for PCB-induced developmental neurotoxicity. Since the PKC signaling pathway has been implicated in the modulation of motor behavior as well as learning and memory, and the roles of PKC are subspecies specific, the present study attempted to analyze the effects on selected PKC isozymes in the cerebellum and the hippocampus following developmental exposure (gestational day 6 through postnatal day 21) to a PCB mixture, Aroclor 1254. The results indicated that the developmental exposure to PCBs caused significant hypothyroxinemia and age-dependent alterations in the translocation of PKC isozymes; the effects were greatly significant at postnatal day (PND) 14. Immunoblot analysis of PKC-alpha (alpha) from both cerebellum and hippocampus revealed that developmental exposure to Aroclor 1254 caused a significant decrease in cytosolic fraction and an increase in particulate fraction. There was no significant difference between these two brain regions on the level of fractional changes. However, the ratio between the fractions (particulate/cytosol) from cerebellum only was increased in a dose-dependent manner. Analysis of PKC-gamma (gamma) in cerebellum on PND14 showed a decrease in cytosolic fraction in both dose groups and an increase in particulate fraction at high dose (6 mg/kg) only. The ratio between the two fractions was increased in a dose-dependent manner. In the hippocampus, there was a significant decrease in PKC-gamma in cytosolic fraction of the high-dose group and a significant increase in particulate fraction of the low-dose group. But, the ratio between the fractions showed a significant increase (2.6-fold increase in high dose on PND14). Analysis of PKC-epsilon (epsilon) in cerebellum showed a significant decrease in cytosolic fraction at PND14, while particulate PKand an increase in ratio between fractions at 6 mg/kg on PND14. The results from this study indicate that the patterns of subcellular distributions of PKC isoforms following a developmental PCB exposure were PKC isozyme- and developmental stage-specific. Considering the significant role of PKC signaling in motor behavior, learning and memory, it is suggested that altered subcellular distribution of PKC isoforms at critical periods of brain development may be a possible mechanism of PCB-induced neurotoxic effects and that PKC-alpha, gamma, and epsilon may be among the target molecules implicated with PCB-induced neurological impairments during developmental exposure. It is believed that this is the first report successfully identifying PKC isoforms responding to PCBs during developmental exposure.     

Methylmercury inhibits TrkA signaling through the ERK1/2 cascade after NGF stimulation of PC12 cells

Using PC12 cells as a model of neuronal differentiation, we have shown that acute exposure to methylmercury (CH3Hg) inhibits nerve growth factor (NGF)-induced activation of TrkA. In the present study, we examined the effects of CH3Hg on pathways activated by NGF. NGF-induced phosphorylation of ERK1/2 in PC12 cells was time-dependent. Concurrent exposure to CH3Hg and NGF for 2.5 min resulted in a concentration-dependent inhibition of ERK1/2 phosphorylation (EC50 = 0.018 microM). However, NGF-stimulated ERK1/2 phosphorylation was not altered after 5 min of exposure to CH3Hg. In vitro studies revealed that CH3Hg did not directly inhibit the ERK kinase MEK. As reported in other neuronal tissue, CH3Hg can inhibit PKC activity in vitro. Incubation of PC12 cell lysates with CH3Hg produced a concentration-dependent inhibition of PKC activity that was significant at 0.3-10 microM. Further studies using recombinant enzymes examined the effect of CH3Hg on PKC isoforms expressed in PC12 cells. CH3Hg inhibited PKCdelta, and zeta activity in a concentration-dependent manner at higher concentrations (3-10 microM), while a significant increase in PKCalpha activity was observed at lower concentrations (0.1 microM). However, CH3Hg had no affect on NGF-induced PKC activity in intact cells. These results show that CH3Hg inhibition of NGF-stimulated TrkA activation in PC12 cells decreases downstream signaling through the Raf/MEK/ERK cascade. In intact cells PKC does not appear to be a primary target for CH3Hg.     

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-induced accumulation of β-amyloid in the choroid plexus: Role of low density lipoprotein receptor protein-1 and protein kinase C

The choroid plexus (CP), constituting the blood–cerebrospinal fluid barrier, has the capacity to remove beta-amyloid (Aβ) from the cerebrospinal fluid. Our previous work indicates that exposure to lead (Pb) results in Aβ accumulation in the CP by decreasing the expression of low density lipoprotein receptor protein-1 (LRP1), a protein involved in the transport and clearance of Aβ. The current study was designed to explore the relationship between Aβ accumulation, protein kinase C (PKC) activity, and LRP1 status in the CP following Pb exposure. Confocal microscopy revealed that LRP1 was primarily localized in the cytosol of the CP in control rats and migrated distinctly towards the apical surface and the microvilli following acute Pb exposure (27 mg Pb/kg, i.p., 24 h). Co-immunostaining revealed a co-localization of both PKC-δ and LRP1 in the cytosol of control rats, with a distinct relocalization of both towards the apical membrane following Pb exposure. Preincubation of the tissues with PKC-δ inhibitor rottlerin (2 μM) prior to Pb exposure in vitro, resulted in abolishing the Pb-induced relocalization of LRP1 to the apical surface. Importantly, a significant elevation in intracellular Aβ levels (p < 0.01) was observed in the cytosol of the CP following Pb exposure, which was abolished following preincubation with rottlerin. In addition, rottlerin caused a relocalization of Aβ from the cytosol to the nucleus in both Pb-treated and control CP tissues. Finally, co-immunoprecipitation studies revealed a strong protein-protein interaction between LRP1 and PKC-δ in the CP. These studies suggest that Pb exposure disrupts Aβ homeostasis at the CP, owing partly to a Pb-induced relocalization of LRP1 via PKC-δ.     

Developmental lead exposure impairs contextual fear conditioning and reduces adult hippocampal neurogenesis in the rat brain

The effects of developmental lead exposure on the emotional reactivity, contextual fear conditioning and neurogenesis in the dentate gyrus of 60-80 days-old rats were studied. Wistar rat pups were exposed to 0.2% lead acetate via their dams' drinking water from postnatal day (PND) 1 to PND 21 and directly via drinking water from weaning until PND 30. At PND 60 and 80 the level of anxiety and contextual fear conditioning were studied, respectively. At PND 80 all animals received injections of BrdU to determine the effects of Pb on the generation of new cells in the dentate gyrus of hippocampus and on their survival and differentiation patterns. The results of the present study demonstrate that developmental lead exposure induces persistent increase in the level of anxiety and inhibition of contextual fear conditioning. Developmental lead exposure reduced generation of new cells in the dentate gyrus and altered the pattern of differentiation of BrdU-positive cells into mature neurons. A lower proportion of BrdU-positive cells co-expressed with the marker for mature neurons, calbindin. In contrast, the proportions of young not fully differentiated neurons and proportions of astroglial cells, generated from newly born cells, were increased in lead-exposed animals. Our results demonstrate that developmental lead exposure induces persistent inhibition of neurogenesis and alters the pattern of differentiation of newly born cells in the dentate gyrus of rat hippocampus, which could, at least partly, contribute to behavioral and cognitive impairments observed in adulthood.     

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.