Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices

The effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices have been examined. Methylmercury induced a concentration-dependent increase in the spontaneous release of glutamate, aspartate, gamma-aminobutyric acid, and taurine from mouse cerebellar slices. Glycine release was slightly increased, but not in a concentration-dependent manner. The spontaneous release of glutamine from mouse cerebellar slices was not altered by any concentration of methylmercury examined (10, 20, and 50 microM). The tissue content of glutamate, gamma-aminobutyric acid, glutamine, and taurine decreased after exposure to methylmercury. Exposure of cerebellar slices to 20 microM methylmercury resulted in a significant enhancement in glutamate release during stimulation with 35 mM K+. This increase could be accounted for by the methylmercury-induced increase in spontaneous glutamate release. The increase in spontaneous release of glutamate and gamma-aminobutyric acid was independent of the availability of extracellular calcium. These results suggest that methylmercury increases the release of neurotransmitter amino acids, particularly gamma-aminobutyric acid and glutamate, by acting at intracellular sites to increase release from a neurotransmitter pool. The increase in the potassium-stimulated release of glutamate may reflect an increased sensitivity of the cerebellar granule cell to the effects of methylmercury. It is suggested that alterations in amino acid neurotransmitter function in the cerebellum may contribute to some of the neurological symptoms of methylmercury intoxication.     

Effects of methylmercury on the morphogenesis of the rat cerebellum

Developing rat cerebellums were examined following continuous methylmercury exposure via maternal drinking water at 12.5 ppm during gestation and the suckling period. The continuous exposure resulted in reductions of the total cerebellar cell population and higher mercury tissue burdens than previous acute-dose studies. Cell necrosis was not evident, but rather alterations in the pattern of mitotic figures were observed. A decreased number of cells in the methylmercury exposed cerebellums was associated with an increased number of mitotic figures in the early stages of mitosis and a decrease in the number in the middle and late stages. These in vivo exposure observations are consistent with in vitro cell cycle studies in which the cells were found to have accumulated in G2 and early M phases. Impaired cell proliferation is suggested to be a major mechanism of developmental neurotoxicity following continuous low-dose exposure to methylmercury.     

Human-induced pluripotent stems cells as a model to dissect the selective neurotoxicity of methylmercury

Methylmercury (MeHg) is a potent neurotoxicant affecting both the developing and mature central nervous system (CNS) with apparent indiscriminate disruption of multiple homeostatic pathways. However, genetic and environmental modifiers contribute significant variability to neurotoxicity associated with human exposures. MeHg displays developmental stage and neural lineage selective neurotoxicity. To identify mechanistic-based neuroprotective strategies to mitigate human MeHg exposure risk, it will be critical to improve our understanding of the basis of MeHg neurotoxicity and of this selective neurotoxicity. Here, we propose that human-based pluripotent stem cell cellular approaches may enable mechanistic insight into genetic pathways that modify sensitivity of specific neural lineages to MeHg-induced neurotoxicity. Such studies are crucial for the development of novel disease modifying strategies impinging on MeHg exposure vulnerability.     

Gain-of-function screen identifies a role of the Sec61α translocon in Drosophila postmitotic neurotoxicity

To elucidate the intrinsic mechanisms of neurotoxicity induction, including those underlying neural cell death and neurodegeneration, we developed a gain-of-function screen for gene products causing neural cell loss. To identify novel genes with a cell-death-related function in neurons, we screened 4,964 Drosophila GS lines, in which one or two genes from much of the Drosophila genome can be overexpressed. Approximately 0.68% of the GS lines produced phenotypes involving a loss of postmitotic neurons. Of these, we identified and characterized the endd2 gene, which encodes the Drosophila ortholog of Sec61α (DSec61α), an endoplasmic reticulum protein with protein translocation activity. Ectopic expression of DSec61α caused neural cell death accompanied by the accumulation of ubiquitinated proteins, which was mediated by DSec61α's translocon activity. This supported our previous observation that the DSec61α translocon contributes to expanded polyglutamine-mediated neuronal toxicity, which is also associated with ubiquitinated protein accumulation. These data suggest that the translocon may be a novel component of neural cell death and degeneration pathways. Our approach can be used to identify potential neurotoxic factors within the whole genome, which will increase our understanding of the molecular mechanisms of various types of cell death, including those associated with human neurodegenerative diseases.     

Morphine Protects against Methylmercury Intoxication: A Role for Opioid Receptors in Oxidative Stress?

Mercury is an extremely dangerous environmental contaminant responsible for episodes of human intoxication throughout the world. Methylmercury, the most toxic compound of this metal, mainly targets the central nervous system, accumulating preferentially in cells of glial origin and causing oxidative stress. Despite studies demonstrating the current exposure of human populations, the consequences of mercury intoxication and concomitant use of drugs targeting the central nervous system (especially drugs used in long-term treatments, such as analgesics) are completely unknown. Morphine is a major option for pain management; its global consumption more than quadrupled in the last decade. Controversially, morphine has been proposed to function in oxidative stress independent of the activation of the opioid receptors. In this work, a therapeutic concentration of morphine partially protected the cellular viability of cells from a C6 glioma cell line exposed to methylmercury. Morphine treatment also reduced lipid peroxidation and totally prevented increases in nitrite levels in those cells. A mechanistic study revealed no alteration in sulfhydryl groups or direct scavenging at this opioid concentration. Interestingly, the opioid antagonist naloxone completely eliminated the protective effect of morphine against methylmercury intoxication, pointing to opioid receptors as the major contributor to this action. Taken together, the experiments in the current study provide the first demonstration that a therapeutic concentration of morphine is able to reduce methylmercury-induced oxidative damage and cell death by activating the opioid receptors. Thus, these receptors may be a promising pharmacological target for modulating the deleterious effects of mercury intoxication. Although additional studies are necessary, our results support the clinical safety of using this opioid in methylmercury-intoxicated patients, suggesting that normal analgesic doses could confer an additional degree of protection against the cytotoxicity of this xenobiotic.     

Ethanol exposure alters neurotrophin receptor expression in the rat central nervous system: Effects of neonatal exposure

The detrimental effects of ethanol exposure during nervous system development have been well established. The cellular mechanisms of ethanol neurotoxicity, however, have not been clearly defined. Recent studies suggest that neurotrophin signaling pathways may be involved in ethanol-mediated neuronal death. The present investigation, therefore, was designed to examine ethanol-induced alterations in neurotrophin receptor protein levels in the developing central nervous system (CNS) following chronic ethanol treatment administered during the early neonatal period. For this study, rats were exposed to ethanol via vapor inhalation from postnatal day 4 (P4) to P10. Brains were then dissected on P10 or P21, and Western blots used to quantify expression of neurotrophin receptors TrkA, TrkB, TrkC, and p75. This early postnatal ethanol treatment produced significant alterations in receptor levels in hippocampus, septum, cerebral cortex, and cerebellum. The alterations seen were variable, with decreases generally found in hippocampus and cerebellum, increases noted in septum, and changes in both directions occurring in cortex. These alterations were generally more prevalent in males than in females. While most of the receptor changes observed were transient, sustained or delayed alterations were occasionally seen in hippocampus, cortex, and cerebellum. These results suggest that developmental ethanol exposure modulates expression of these neurotrophin receptors throughout the CNS, alterations which could have wide-ranging effects on functional CNS development. The possible linkage between such changes and abnormalities encountered in the fetal alcohol syndrome are considered.     

Insufficient Apaf-1 expression in early stages of neural differentiation of human embryonic stem cells might protect them from apoptosis

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.     

Nicotine and brain development

Preclinical studies, using primarily rodent models, have shown acetylcholine to have a critical role in brain maturation via activation of nicotinic acetylcholine receptors (nAChRs), a structurally diverse family of ligand-gated ion channels. nAChRs are widely expressed in fetal central nervous system, with transient upregulation in numerous brain regions during critical developmental periods. Activation of nAChRs can have varied developmental influences that are dependent on the pharmacologic properties and localization of the receptor. These include regulation of transmitter release, gene expression, neurite outgrowth, cell survival, and synapse formation and maturation. Aberrant exposure of fetal and neonatal brain to nicotine, through maternal smoking or nicotine replacement therapy (NRT), has been shown to have detrimental effects on cholinergic modulation of brain development. These include alterations in sexual differentiation of the brain, and in cell survival and synaptogenesis. Long-term alterations in the functional status and pharmacologic properties of nAChRs may also occur, which result in modifications of specific neural circuitry such as the brainstem cardiorespiratory network and sensory thalamocortical gating. Such alterations in brain structure and function may contribute to clinically characterized deficits that result from maternal smoking, such as sudden infant death syndrome and auditory-cognitive dysfunction. Although not the only constituent of tobacco smoke, there is now abundant evidence that nicotine is a neural teratogen. Thus, alternatives to NRT should be sought as tobacco cessation treatments in pregnant women.     

Molecular and cellular mechanisms of excitotoxic neuronal death

Glutamate receptor-mediated excitatory neurotransmission plays a key role in neural development, differentiation and synaptic plasticity. However, excessive stimulation of glutamate receptors induces neurotoxicity, a process that has been defined as excitotoxicity. Excitotoxicity is considered to be a major mechanism of cell death in a number of central nervous system diseases including stroke, brain trauma, epilepsy and chronic neurodegenerative disorders. Unfortunately clinical trials with glutamate receptor antagonists, that would logically prevent the effects of excessive receptor activation, have been associated with untoward side effects or little clinical benefit. Therefore, uncovering molecular pathways involved in excitotoxic neuronal death is of critical importance to future development of clinical treatment of many neurodegenerative disorders where excitotoxicity has been implicated. This review discusses the current understanding of the molecular and cellular mechanisms of excitotoxicity and their roles in the pathogenesis of diseases of the central nervous system.     

Cell death: insights into the ultrastructure of mitochondria

An essential step in many forms of cell death is the release from mitochondria of “death effectors” which once in the cytoplasm activate signalling pathways leading to cellular demise. In this context mitochondria are known as regulators of cell death functioning as a node where signals are integrated. The discovery that alterations and remodelling of ultrastructural architecture of mitochondria are required to trigger the complete release of cytochrome c in the cytoplasm and the notion that mitochondrial architecture determines/influences the function of this organelle has fostered investigations on mitochondrial dynamics and on the machinery that regulates this process during cell death. In this review I shall summarize the current knowledge of mitochondrial inner membrane remodelling during cell death and discuss the role of mitochondrial proteins in governing structural alterations. I shall then discuss the role of the adaptor protein p66Shc as a regulator of mitochondrial metabolism during apoptosis.     

The effect of hyperthermia on the induction of cell death in brain, testis, and thymus of the adult and developing rat

Stressful stimuli can elicit 2 distinct reactive cellular responses, the heat shock (stress) response and the activation of cell death pathways. Most studies on the effects of hyperthermia on the mammalian nervous system have focused on the heat shock response, characterized by the transient induction of Hsps, which play roles in repair and protective mechanisms. This study examines the effect of hyperthermia on the induction of cell death via apoptosis, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and active caspase 3 cytochemistry, in the adult rat brain, testis, and thymus. Results show that a fever-like increase in temperature triggered apoptosis in dividing cell populations of testis and thymus, but not in mature, postmitotic cells of the adult cerebellum. These differential apoptotic responses did not correlate with whole-tissue levels of Hsp70 induction. We further investigated whether dividing neural cells were more sensitive to heat-induced apoptosis by examining the external granule cell layer of the cerebellum at postnatal day 7 and the neuroepithelial layers of the neocortex and tectum at embryonic day 17. These proliferative neural regions were highly susceptible to hyperthermia-induced apoptosis, suggesting that actively dividing cell populations are more prone to cell death induced by hyperthermia than fully differentiated postmitotic neural cells.     

α7 Nicotinic receptor activation reduces β-amyloid-induced apoptosis by inhibiting caspase-independent death through phosphatidylinositol 3-kinase signaling

The neurotoxicity of amyloid-β (Aβ) involves caspase-dependent and -independent programmed cell death. The latter is mediated by the nuclear translocation of the mitochondrial flavoprotein apoptosis inducing factor (AIF). Nicotine has been shown to decrease Aβ neurotoxicity via inhibition of caspase-dependent apoptosis, but it is unknown if its neuroprotection is mediated through caspase-independent pathways. In the present study, pre-treatment with nicotine in rat cortical neuronal culture markedly reduced Aβ(1-42) induced neuronal death. This effect was accompanied by a significant reduction of mitochondrial AIF release and its subsequent nuclear translocation as well as significant inhibition of cytochrome c release and caspase 3 activation. Pre-treatment with selective α7nicotinic acetylcholine receptor(nAChR) antagonist (methyllycaconitine), but not the α4 nAChR antagonist (dihydro-β-erythroidine), could prevent the neuroprotective effect of nicotine on AIF release/translocation, suggesting that nicotine inhibits the caspase-independent death pathway in a α7 nAChR-dependent fashion. Furthermore, the neuroprotective action of nicotine on AIF release/translocation was suppressed by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Pre-treatment with nicotine significantly restored Akt phosphorylation, an effector of PI3K, in Aβ(1-42) -treated neurons. These findings indicate that the α7 nAChR activation and PI3K/Akt transduction signaling contribute to the neuroprotective effects of nicotine against Aβ-induced cell death by modulating caspase-independent death pathways.     

Utilization of Neural Stem Cell-Derived Models to Study Anesthesia-Related Toxicity and Preventative Approaches

Early-life stress has been shown in both preclinical and clinical studies to cause neuroanatomical and biological alterations and disruptions in homeostasis. These alterations can lead to dysfunction in critical regulatory systems and concomitant increases in risk for the development of pathology. The existing data from research using in vivo animal models have implicated some general anesthetics as being toxic to the developing brain and causing cognitive deficits later in life. Because of obvious limitations, it is not possible to thoroughly explore the effects of early-life stress—e.g., prolonged exposure to anesthetic agents—on neurons in vivo in human infants or children. However, the availability of stem cell-derived models, especially human embryonic neural stem cells, along with their capacity for proliferation and ability to differentiate, has provided a potentially invaluable tool for examining the developmental effects of anesthetic agents in vitro. This review focuses on how embryonic neural stem cells, when combined with biochemical, pathological, and pharmacokinetic assessments, might serve as a bridging platform to provide the most expeditious approaches toward decreasing the uncertainty in extrapolating preclinical data to the human condition. This review presents key concepts in stem cell biology with respect to the nervous system, presents an overview of neural development, and summarizes the involvement of neural cell types in developmental neurotoxicity associated with anesthetic exposure.     

Roles of Wnt proteins in neural development and maintenance

Many constituents of Wnt signaling pathways are expressed in the developing and mature nervous systems. Recent work has shown that Wnt signaling controls initial formation of the neural plate and many subsequent patterning decisions in the embryonic nervous system, including formation of the neural crest. Wnt signaling continues to be important at later stages of development. Wnts have been shown to regulate the anatomy of the neuronal cytoskeleton and the differentiation of synapses in the cerebellum. Wnt signaling has been demonstrated to regulate apoptosis and may participate in degenerative processes leading to cell death in the aging brain.     

In vivo manganese exposure modulates Erk, Akt and Darpp-32 in the striatum of developing rats, and impairs their motor function

Manganese (Mn) is an essential metal for development and metabolism. However, exposures to high Mn levels may be toxic, especially to the central nervous system (CNS). Neurotoxicity is commonly due to occupational or environmental exposures leading to Mn accumulation in the basal ganglia and a Parkinsonian-like disorder. Younger individuals are more susceptible to Mn toxicity. Moreover, early exposure may represent a risk factor for the development of neurodegenerative diseases later in life. The present study was undertaken to investigate the developmental neurotoxicity in an in vivo model of immature rats exposed to Mn (5, 10 and 20 mg/kg; i.p.) from postnatal day 8 (PN8) to PN12. Neurochemical analysis was carried out on PN14. We focused on striatal alterations in intracellular signaling pathways, oxidative stress and cell death. Moreover, motor alterations as a result of early Mn exposure (PN8-12) were evaluated later in life at 3-, 4- and 5-weeks-of-age. Mn altered in a dose-dependent manner the activity of key cell signaling elements. Specifically, Mn increased the phosphorylation of DARPP-32-Thr-34, ERK1/2 and AKT. Additionally, Mn increased reactive oxygen species (ROS) production and caspase activity, and altered mitochondrial respiratory chain complexes I and II activities. Mn (10 and 20 mg/kg) also impaired motor coordination in the 3^rd, 4^th and 5^th week of life. Trolox™, an antioxidant, reversed several of the Mn altered parameters, including the increased ROS production and ERK1/2 phosphorylation. However, Trolox™ failed to reverse the Mn (20 mg/kg)-induced increase in AKT phosphorylation and motor deficits. Additionally, Mn (20 mg/kg) decreased the distance, speed and grooming frequency in an open field test; Trolox™ blocked only the decrease of grooming frequency. Taken together, these results establish that short-term exposure to Mn during a specific developmental window (PN8-12) induces metabolic and neurochemical alterations in the striatum that may modulate later-life behavioral changes. Furthermore, some of the molecular and behavioral events, which are perturbed by early Mn exposure are not directly related to the production of oxidative stress.     

Neurobehavioral Dysfunction as a Possible Sentinel of Methylmercury Exposure

The main concern regarding methylmercury neurotoxicity relates to adverse effects on the brain during development. Many environmental chemicals may act as developmental neurotoxicants, but solid documentation from epidemiological studies exists only on methylmercury, lead, and polychlorinated biphenyls (PCBs). Neurobehavioral tests may reveal subtle dysfunctions, but the tests chosen must be valid and appropriate for the setting. In a prospective study in the Faroe Islands, the main neuropsychological functions affected by prenatal methylmercury exposure were attention, language and memory. Deficits in visuospatial function were mainly related to postnatal exposures. These associations were stable after adjustment for confounders and exclusion of the children with the highest exposures to methylmercury and PCBs. Tests with good psychometric properties were more likely to show an association with mercury exposure. Greater sensitivity was also seen with tests administered by specialized academic staff rather than a trained technician. Despite highly significant effects on nervous system function, the deficits were subtle, and mercury exposure explained only a small part of the variation. Available evidence suggests that neurotoxicity may have severe implications on public health, but current methods are not amenable to application as sentinels of adverse health effects in environmental health surveillance.     

Manganese activates the mitochondrial apoptotic pathway in rat astrocytes by modulating the expression of proteins of the Bcl-2 family

Manganese induces the central nervous system injury leading to manganism, by mechanisms not completely understood. Chronic exposure to manganese generates oxidative stress and induces the mitochondrial permeability transition. In the present study, we characterized apoptotic cell death mechanisms associated with manganese toxicity in rat cortical astrocytes and demonstrated that (i) Mn treatment targets the mitochondria and induces mitochondrial membrane depolarization followed by cytochrome c release to the cytoplasm, (ii) Mn induces both effector caspases 3/7 and 6 as well as PARP-1 cleavage and (iii) Mn shifts the balance of cell death/survival of Bcl-2 family proteins to favor the apoptotic demise of astrocytes. Our model system using cortical rat astrocytes treated with Mn would emerge as a good tool for investigations aimed to elucidate the role of apoptosis in manganism.