Mechanisms of Neuronal Death Induced by Environmental Toxicants in Murine Cortical Culture

This study was directed at examining the neurotoxic mechanisms of several classes of environmental toxicants implicated in neurodegenerative disease. Primary cortical cultures were exposed to organophosphorus pesticides, heavy metals and the cyanobacterial toxin, beta-N-methylamino-L-alanine (BMAA). Several components relating to neuronal injury were assessed in each study and novel aspects are described. The main action of organophosphorous insecticides is generally believed to be the inhibition of acetylcholinesterase. However, these compounds are now recognized to inhibit many other enzymes and cause neuronal death through a variety of mechanisms. I found that exposure to chlorpyrifos or diazinon caused concentration-dependent neurotoxicity that could not be attributed to acetylcholinesterase inhibition. Chlorpyrifos exposure increased extracellular glutamate and induced a diffuse nuclear staining characteristic of necrosis; the toxicity was sensitive to ionotropic glutamate receptor antagonists. Diazinon toxicity was blocked by caspase inhibitors. Additionally, diazinon induced punctuate chromatin staining characteristic of apoptosis. These results represent two distinct, novel mechanisms of organophosphorous neurotoxicity. Heavy metals are ubiquitous in the environment and are of significant health concern worldwide. Exposure to lead, iron, mercurials (inorganic mercury, methylmercury, or thimerosal, i.e. ethylmercury) or other heavy metals is implicated as a risk factor for neurodegenerative disease. I found that the toxicity of these metals may be enhanced when interacting with chelators used to treat metal intoxication. As well, my studies describe a new role for mercury-induced oxidative stress as a cytoprotective signal to enhance glutathione levels. My data also suggests an obligate role for MRP1 in the detoxification of methylmercury. Neurodegenerative diseases likely involve complex interactions between genetic predisposition and multiple environmental factors. My final study tested the interaction of the methylmercury and BMAA. Importantly, concentrations of BMAA that caused no toxicity by themselves potentiated methyl mercury toxicity. BMAA plus methylmercury, at concentrations that had no effect by themselves, depleted cellular glutathione. The combined toxicity was attenuated by glutathione monoethyl ester, and the free radical scavenger, trolox, but not by the NMDA receptor antagonist, MK-801. The results indicate a synergistic neurotoxic interaction targeting the cellular redox state. This finding may have implications for neurodegenerative disease caused by environmental toxicant exposure

Glutathione-Mediated Neuroprotection Against Methylmercury Neurotoxicity in Cortical Culture is Dependent on MRP1

Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5 μM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30 min and a late increase at 6 h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3 μM) caused an increase in system xc− mediated 14C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system xc−. Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux

Iron overload accelerates neuronal amyloid-β production and cognitive impairment in transgenic mice model of Alzheimer's disease

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The amyloid-β oligomer Aβ∗56 induces specific alterations in neuronal signaling that lead to tau phosphorylation and aggregation

Oligomeric forms of amyloid-forming proteins are believed to be the principal initiating bioactive species in many neurodegenerative disorders, including Alzheimer's disease (AD). Amyloid-β (Ab) oligomers are implicated in AD-associated phosphorylation and aggregation of the microtubule-associated protein tau. To investigate the specific molecular pathways activated by different assemblies, we isolated various forms of Aβ from Tg2576 mice, which are a model for AD. We found that Aβ∗56, a 56-kDa oligomer that is detected before patients develop overt signs of AD, induced specific changes in neuronal signaling. In primary cortical neurons, Aβ∗56 interacted with N-methyl-D-aspartate receptors (NMDARs), increased NMDAR-dependent Ca influx, and consequently increased intracellular calcium concentrations and the activation of Ca-dependent calmodulin kinase IIa (CaMKIIa). In cultured neurons and in the brains of Tg2576 mice, activated CaMKIIa was associated with increased site-specific phosphorylation and missorting of tau, both of which are associated with AD pathology. In contrast, exposure of cultured primary cortical neurons to other oligomeric Aβ forms (dimers and trimers) did not trigger these effects. Our results indicate that distinct Aβ assemblies activate neuronal signaling pathways in a selective manner and that dissecting the molecular events caused by each oligomer may inform more effective therapeutic strategies. 201

Optimization of <i>Aspergillus versicolor</i> Culture and Aerosolization in a Murine Model of Inhalational Fungal Exposure

Aspergillus versicolor is ubiquitous in the environment and is particularly abundant in damp indoor spaces. Exposure to Aspergillus species, as well as other environmental fungi, has been linked to respiratory health outcomes, including asthma, allergy, and even local or disseminated infection. However, the pulmonary immunological mechanisms associated with repeated exposure to A. versicolor have remained relatively uncharacterized. Here, A. versicolor was cultured and desiccated on rice then placed in an acoustical generator system to achieve aerosolization. Mice were challenged with titrated doses of aerosolized conidia to examine deposition, lymphoproliferative properties, and immunotoxicological response to repeated inhalation exposures. The necessary dose to induce lymphoproliferation was identified, but not infection-like pathology. Further, it was determined that the dose was able to initiate localized immune responses. The data presented in this study demonstrate an optimized and reproducible method for delivering A. versicolor conidia to rodents via nose-only inhalation. Additionally, the feasibility of a long-term repeated exposure study was established. This experimental protocol can be used in future studies to investigate the physiological effects of repeated pulmonary exposure to fungal conidia utilizing a practical and relevant mode of delivery. In total, these data constitute an important foundation for subsequent research in the field