Elisabetta Daré

Methylmercury-induced neurotoxicity and apoptosis

Methylmercury is a widely distributed environmental toxicant with detrimental effects on the developing and adult nervous system. Due to its accumulation in the food chain, chronic exposure to methylmercury via consumption of fish and sea mammals is still a major concern for human health, especially developmental exposure that may lead to neurological alterations, including cognitive and motor dysfunctions. Mercury-induced neurotoxicity and the identification of the underlying mechanisms has been a main focus of research in the neurotoxicology field. Three major mechanisms have been identified as critical in methylmercury-induced cell damage including (i) disruption of calcium homeostasis, (ii) induction of oxidative stress via overproduction of reactive oxygen species or reduction of antioxidative defenses and (iii) interactions with sulfhydryl groups. In vivo and in vitro studies have provided solid evidence for the occurrence of neural cell death, as well as cytoarchitectural alterations in the nervous system after exposure to methylmercury. Signaling cascades leading to cell death induced by methylmercury involve the release of mitochondrial factors, such as cytochrome c and AIF with subsequent caspase-dependent or -independent apoptosis, respectively; induction of calcium-dependent proteases calpains; interaction with lysosomes leading to release of cathepsins. Interestingly, several pathways can be activated in parallel, depending on the cell type. In this paper, we provide an overview of recent findings on methylmercury-induced neurotoxicity and cell death pathways that have been described in neural and endocrine cell systems.

Modulation of glial cell functions by adenosine receptors

Adenosine is an endogenous neuromodulator, acting on four distinctive G-protein-coupled receptors, the A1, A2A, A2B and A3 adenosine receptors. Increased neuronal activity and, hypoxia or ischemia, result in elevated levels of adenosine reflecting changes of the metabolic state. This increases activation of the adenosine receptors. It is well appreciated that adenosine has a neuroprotective role in brain injuries. Although adenosine effects have been explained mainly by actions on nerve cells, modulation of glial functions by adenosine is likely to be important as discussed in this minireview. Thus, in astrocytes adenosine receptors modulate inter alia glycogen metabolism, glutamate transporters, astrogliosis and astrocyte swelling. Microglial cells appear to be important in regulating adenosine formation from ATP and adenosine can affect many microglial signaling pathways. Adenosine receptors on oligodendrocytes regulate white matter development.

Antioxidants J811 and 17?-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death

Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well‐known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 μM to 1.5 μM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high‐molecular‐weight DNA fragments. Nevertheless, caspase‐3‐like activity was not significantly induced, and the broad caspase inhibitor Z‐VAD‐FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg‐induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase‐3 and α‐fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17β‐estradiol (10 μM) and the Δ8,9‐dehydro derivative of 17α‐estradiol J811 (10 μM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17β‐estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication. J. Neurosci. Res. 62:557–565, 2000.

Prenatal exposure to methylmercury changes dopamine-modulated motor activity during early ontogeny: age and gender-dependent effects.

We have shown previously that prenatal exposure of rats to 0.5 mg/kg/day of methylmercury (MeHg) produces gender-dependent changes in motor activity in adulthood. In the present study we have investigated whether changes in motor activity could also be found during early ontogeny of the offspring. Pregnant rats were treated with MeHg from day 7 of pregnancy to day 7 of lactation. The habituation to a novel environment (spontaneous activity) and the response to stimulation of the dopaminergic system were studied on postnatal day 14 and 21. Measures of spontaneous activity showed a slight increase in MeHg-prenatal exposed male and female rats at 14 days, but not at 21 days. Following administration of U91356A, a selective dopamine D(2) receptor agonist, a significantly lower dopamine-mediated locomotor activity was observed in the 21 day old MeHg-treated males, but not in females. These results show that prenatal exposure to MeHg alters postjunctional dopaminergic activity during the period of maturation of the dopamine system in the brain. Moreover, the gender-dependent susceptibility previously found in adulthood is already evident at the prepubertal stage.

Methylmercury and H2O2 provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 microM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H(2)O(2)) exposure (100 microM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H(2)O(2) preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H(2)O(2) stimulated divergent pathways, with caspases being activated only by H(2)O(2). The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H(2)O(2), suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H(2)O(2). The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.

Sex differences in mouse heart rate and body temperature and in their regulation by adenosine A1 receptors

Aim:  To examine cardiac function, body temperature and locomotor behaviour in the awake adenosine A1 receptor knock out mouse of both sexes.

Apoptotic morphology does not always require caspase activity in rat cerebellar granule neurons

The death of a cell via apoptosis is characterized by morphological changes including cell shrinkage and nuclear condensation. Intracellularly, proteases, including caspases, are activated. In the present article we have compared the ability of three different neurotoxic agents to induce caspase activity in cerebellar granule cells (CGC). These compounds are the micro-tubule-disrupting agent colchicine and the oxidative stress-inducing agents hydrogen peroxide and meth-ylmercury (MeHg). We have previously shown that each of these agents causes nuclear changes that are consistent with apoptosis, i.e., induction of chromatin condensation and DNA cleavage into fragments of regular size (700, 300 and 50 kbp). However, only colchicine causes a large increase in caspase activity, as monitored by the ability of whole cell extracts to cleave the synthetic caspase substrate DEVD-MCA. In contrast, MeHg and hydrogen peroxide do not induce any significant increase of DEVDase activity as compared to control cells. Immunocytochemistry confirms that active caspase-3 is abundant only in colchicine-exposed cells. In agreement with these findings, the pan-caspase inhibitor, z-VAD-fmk, is efficient in protecting CGC against colchicine, but not against hydrogen peroxide or MeHg. These data suggest that in CGC the activation of caspases is not always required to induce morphological changes and pattern of DNA fragmentation consistent with apoptosis.

Methylmercury induces neurite degeneration in primary culture of mouse dopaminergic mesencephalic cells

Summary. Methylmercury cation (MeHg) is an hazardous environmental pollutant with neurotoxic action. Little is known about the effects of MeHg on catecholaminergic neurons. In the present study we have used epifluorescence microscopy and confocal microscopy to investigate the alterations induced by MeHg in primary DA (dopaminergic) cells isolated from the ventral mesencephalon of CD-1 embryonic mice and cultured for six days in vitro. DA cells were identified in the multi-culture by immunocytochemistry using a tyrosine-hydroxylase antibody. The morphometric analysis of DA neurons exposed to 1 μM MeHg demonstrated a striking decrease in the number of neurites, indicative of cytoskeletal alteration. In addition, DA neurons displayed cell shrinkage and a significant increase of nuclei with chromatin condensation. Based on these results it is concluded that MeHg is highly toxic to primary DA neurons.

Decreased behavioral activation following caffeine, amphetamine and darkness in A3 adenosine receptor knock-out mice

We have examined behavioral consequences of genetic deletion of the adenosine A3 receptors in mice. The open field behavior of A3 adenosine receptor knock-out (A3R KO) mice was investigated both under basal conditions and after stimulation with psychostimulants. Adolescent (21 day-old) and adult A3R KO males showed an increase in overall motor activity compared to wild type (WT) males, but the type of activity differed. The motor activity, especially rearing, was also higher in A3R KO compared to WT adult females. A3 receptors have a low affinity for caffeine and it was therefore surprising to find a decreased response to stimulation with either caffeine or amphetamine in A3R KO as compared to WT mice in males as well as females. Telemetry recordings also showed a significantly smaller increase in activity upon darkness in A3R KO. There were no compensatory changes in the mRNA expression of any other adenosine receptor subtypes (A1, A2A and A2B) or any changes in dopamine D1 and D2 receptor binding in A3R KO brains. Challenge with the developmental toxicant methylmercury (1 microM in drinking water) during pregnancy and lactation did not cause any behavioral alterations in adolescent and adult WT female offspring. In contrast, the A3R KO female offspring displayed changes in locomotion indicating an interaction between perinatal methylmercury and adenosine A3 receptors. In conclusion, despite low expression of A3 receptors in wild type mouse brain we observed several behavioral consequences of genetic elimination of the adenosine A3 receptors. The possibility that this is due to a role of A3 receptors in development is discussed.

Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro.

The neurotoxic effects of carbon monoxide (CO) are well known. Brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct effect of CO on intracellular targets remains poorly understood. In the present study, we have investigated the pathways leading to neural cell death induced by in vitro exposure to CO using a gas exposure chamber that we have developed. Mouse hippocampal neurons (HT22) and human glial cells (D384) were exposed to concentrations of CO ranging from 300 to 1000 ppm in the presence of 20% oxygen. Cytotoxicity was observed after 48 h exposure to 1000 ppm, corresponding to approximately 1 microM CO in the cultured medium, as measured by gas chromatography. CO induced cell death with characteristic features of apoptosis. Exposed cells exhibited loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, nuclei with chromatin condensation, and exposure of phosphatidyl serine on the external leaflet of the plasma membrane. CO also triggered activation of caspase and calpain proteases. Pre-incubation with either the pancaspase inhibitor Z-VAD-fmk (20 microM) or the calpain inhibitor E64d (25 microM) reduced by 50% the occurrence of apoptosis. When pre-incubating the cells with the two inhibitors together there was an additional reduction in the number of cells with apoptotic nuclei. These data suggest that CO causes apoptosis via activation of parallel proteolytic pathways involving both caspases and calpains. Furthermore, pre-treatment with the antioxidant MnTBAP (100 microM) significantly reduced the number of apoptotic nuclei, pointing to a critical role of oxidative stress in CO toxicity.