Grantee Research Project Results
2000 Progress Report: Mechanism of Manganese-Induced Neurotoxicity
EPA Grant Number: R826248Title: Mechanism of Manganese-Induced Neurotoxicity
Investigators:
Institution: The State University of New York at Buffalo
EPA Project Officer: Chung, Serena
Project Period: October 1, 1997 through September 30, 2000
Project Period Covered by this Report: October 1, 1999 through September 30, 2000
Project Amount: $355,000
RFA: Ambient Air Quality (1997) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Air
Objective:
Atmospheric levels of manganese (Mn) have significantly increased in urban cities since its introduction as a fuel additive. This is particularly relevant based on recent studies indicating that people with compromised liver function may be at considerably greater risk than the normal population to the toxic actions of Mn. Mn is a potent neurotoxin, which is capable of producing a variety of neurological symptoms characterized by severe extrapyramidal dysfunction resembling the dystonic movements associated with Parkinson's disease. With the realization of increased environmental exposure to Mn, it becomes necessary to delineate the fundamental biochemical and molecular mechanisms responsible for its selective neurotoxic actions in order to prevent and identify individuals with Mn toxicity.Progress Summary:
Our laboratory has chosen to employ an in vitro cell culture system as a paradigm to study the selective biological processes affected by exposure to Mn. For these studies, we initially selected the rat pheochromocytoma cells (PC12) as our model system since it possesses much of the biochemical machinery associated with dopaminergic neuron. Rat PC12 cells have served as a model for studying the molecular mechanisms promoting neuronal differentiation induced by nerve growth factor (NGF) and other growth promoting agents, and these cells have recently been proposed to serve as an effective in vitro model for studying the mechanisms of apoptosis and neurotoxicity. We have observed that Mn can similarly induce neuronal differentiation in PC12 cells similar to that produced by NGF. However, in contrast to NGF treatment, exposure of these cells to Mn invariably leads to cell death in a time and concentration dependent manner. The mechanism by which Mn induces neurite outgrowth appears to be similar for that which promotes NGF-induced neuronal differentiation of PC12 cells (i.e., induction of the phosphorylation of the MAP kinases, ERK1 and 2 [p42 and p44]). Activation of both ERK1 and 2 is dependent on the interaction of the cell surface integrin receptors with that of the basement membrane proteins, fibronectin and vitronectin. Function-blocking antisera specific for ?1 integrins block the neurite-promoting activity of Mn by 90-95 percent. Bioassays and biochemical studies with antisera specific for the av, a5, or a8 integrin subunit suggest that the av ?1 heterodimer is one of the principal ?1 integrins mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express av at levels detectable by immunoprecipitation or immunocytochemistry. SDS PAGE analysis of biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells indicate that Mn increases the surface expression of av integrins. These data indicate that Mn induces neurite outgrowth in PC12 cells by upregulating av integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.
PC12 cell death induced by Mn is both time and concentration dependent with an IC50 value of approximately 0.6 mM after 24 hours. Oxidative stress does not appear to contribute to Mn-induced cytotoxicity since Mn did not increase lipid peroxidation. However, increases in the stress activated protein kinase, p38, and capspase-3 activity were observed in cell treated with Mn suggesting that apoptosis may play a role in its cytotoxic actions. Inhibitors of p38, SKF86002 and SB203580, prevent Mn-induced PC12 cell death, but only at 24 hours of exposure and not at longer exposure times. These results suggest that both a p38-dependent and independent mechanism contribute to PC12 cell death. Selective and non-selective inhibitors of caspase-3 activity, DEVD-CHO and Z-VAD-FMK, respectively, failed to prevent Mn-induced cell death demonstrating that activation of the caspases is not involved in PC12 cell death. Mn has previously been shown to effect mitochondrial function and in agreement with this, we have demonstrated that production of ATP is inhibited in PC12 cells treated with Mn. In summary, these results demonstrate that the cytotoxic effects of Mn in PC12 cells are not brought about by oxidative stress but, most likely, generated by disruption of mitochondrial oxidative phosphorylation. We hypothesize that both apoptosis as well as necrosis contribute to Mn-induced PC12 cell death although the necrotic events prevail even when the apoptotic signaling is inhibited.
Mn is primarily taken up into cells via the same transport protein responsible for iron uptake (i.e., the divalent metal transporter, DMT1). This membrane carrier has very broad substrate specificity and is responsible for the cellular transport of other divalent cations as well, including Cd+2, Zn+2, Co+2, Ni+2, Cu+2 and Pb+2. Two forms of DMT1 are present in mammalian cells, which result from post-transcriptional modification of a single gene product. The two forms differ primarily in their carboxy terminal end such that only one of the two forms contains an iron response element (IRE) motif on the mRNA. The presence of the IRE within the message promotes the binding and subsequent iron-dependent-activation of the iron response protein (IRP) causing stabilization of the message and increased expression of the protein. Accordingly, the form of DMT1 containing the +IRE is negatively regulated by iron status such that if iron levels are low, DMT1 expression is elevated resulting in the increased transport of heavy metals. Thus, changes in iron levels have the potential to influence heavy metal cytotoxicity.
Recent studies have revealed that the subcellular distribution of the two
isoforms of DMT1 is distinct and the ?IRE species accumulates in the nucleus of
neuronal or neuronal-like cells. RT-PCR and Western blot analysis of PC12 cells
reveal that these cells express both forms of DMT1. Immunofluorescence and
immunoblotting studies, using immunospecific antibodies to the ?IRE forms of
DMT1, demonstrate that this form of the transporter, in PC12 cells, is
predominantly localized in the nucleus, cell membrane and neurites with only
weak staining of the cell body. Studies, using antibodies to the +IRE form,
indicate this species of DMT1 is distributed within vesicles in the cell body
and neurite projections, with minimal nuclear staining. Similar staining
patterns are observed for the two forms of DMT1 in cultures of sympathetic
ganglion neurons isolated from perinatal rat pups. To determine whether nuclear
localization of the ?IRE form of DMT1 is constrained to neuronal or
neuronal-like cells, immunocytochemical studies were performed with human
HEK293T, HEP2G hepatoma and medulloblastoma and rat Schwann cells. The ?IRE
specific antibodies stained nuclei from medulloblastoma whereas little nuclear
staining was observed with HEK293T, hepatoma or Schwann cells. The unexpected
finding that the ?IRE species of DMT1 selectively accumulates in the nucleus of
neuronal and neuronal-like cells leads us to postulate that the two proteins may
have different functions in vivo.
Future Activities:
Recent studies have focused on the role of DMT1 in controlling Mn-induced PC12 cell death. It is known that iron can influence Mn levels in vivo and it is possible that DMT1 may play a major role in regulating the neurotoxic actions of this metal.Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 16 publications | 10 publications in selected types | All 10 journal articles |
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Larsen KE, Pacheco M, Roth J, Aletta JM. Increased MAP1B expression without increased phosphorylation in manganese-treated PC12Mn cells. Experimental Cell Research 1998;245(1):105-115. |
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Lein P, Gallagher PJ, Amodeo J, Howie H, Roth JA. Manganese induces neurite outgrowth in PC12 cells via upregulation of αv integrins. Brain Research 2000;885(2):220-230. |
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Roth JA, Feng L, Walowitz J, Browne RW. Manganese-induced rat pheochromocytoma (PC12) cell death is independent of caspase activation. Journal of Neuroscience Research 2000;61(2):162-171. |
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Roth JA, Horbinski C, Feng L, Dolan KG, Higgins D, Garrick MD. Differential localization of divalent metal transporter 1 with and without iron response element in rat PC12 and sympathetic neuronal cells. Journal of Neuroscience 2000;20(20):7595-7601. |
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Walowitz JL, Roth JA. Activation of ERK1 and ERK2 is required for manganese-induced neurite outgrowth in rat pheochromocytoma (PC12) cells. Journal of Neuroscience Research 1999;57(6):847-854. |
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Supplemental Keywords:
biology, heavy metals, manganese, urban exposure, human health, metabolism, neurotoxicology, apoptosis., Health, Scientific Discipline, PHYSICAL ASPECTS, Air, air toxics, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Disease & Cumulative Effects, Biochemistry, Physical Processes, Molecular Biology/Genetics, Biology, acute effects, ambient air quality, health effects, urban air toxics, exposure and effects, air pollutants, cellular metabolism, effects assessment, hazardous air pollutants, airway disease, ambient air, exposure, urban air pollutants, enzyme systems, neurotoxicity, human exposure, chronic effects, manganese, toxicity, inner city toxicants, public health, acute toxicity, hazardous air pollutants (HAPs), environmental toxicant, harmful environmental agents, toxicodynamics, human health, fuel additives, Parkinson's Disease, acute exposure, atmospheric chemistry, chronic toxicity, disease, heavy metals, exposure assessmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.