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Abstract. Mercury is one of the most toxic substances known to humans. It has been introduced into the human environment and has also been widely used in medicine. Since circumstantial evidence exists that the pathology of Alzheimer's disease (AD) might be in part caused or exacerbated by inorganic mercury, we conducted a systematic review using a comprehensive search strategy. Studies were screened according to a pre-defined protocol. Two reviewers extracted relevant data independent of each other. One thousand and forty one references were scrutinized, and 106 studies fulfilled the inclusion criteria. Most studies were case control or comparative cohort studies. Thirty-two studies, out of 40 testing memory in individuals exposed to inorganic mercury, found significant memory deficits. Some autopsy studies found increased mercury levels in brain tissues of AD patients. Measurements of mercury levels in blood, urine, hair, nails, and cerebrospinal fluid were inconsistent. In vitro models showed that inorganic mercury reproduces all pathological changes seen in AD, and in animal models inorganic mercury produced changes that are similar to those seen in AD. Its high affinity for selenium and selenoproteins suggests that inorganic mercury may promote neurodegenerative disorders via disruption of redox regulation. Inorganic mercury may play a role as a co-factor in the development of AD. It may also increase the pathological influence of other metals. Our mechanistic model describes potential causal pathways. As the single most effective public health primary preventive measure, industrial, and medical usage of mercury should be eliminated as soon as possible.

Many studies indicate a crucial role for the vitamin B12 and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B12 status in the brain across the lifespan has not been previously investigated. Vitamin B12 (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B12 status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.

Canonically, opioids influence cells by binding to a G proteincoupled opioid receptor, initiating intracellular signaling cascades, such as protein kinase, phosphatidylinositol 3-kinase, and extracellular receptor kinase pathways. This results in several downstream effects, including decreased levels of the reduced form of glutathione (GSH) and elevated oxidative stress, as well as epigenetic changes, especially in retrotransposons and heterochromatin, although the mechanism and consequences of these actions are unclear. We characterized the acute and long-term influence of morphine on redox and methylation status (including DNA methylation levels) in cultured neuronal SH-SY5Y cells. Acting via m-opioid receptors, morphine inhibits excitatory amino acid transporter type 3-mediated cysteine uptake via multiple signaling pathways, involving different G proteins and protein kinases in a temporal manner. Decreased cysteine uptake was associated with decreases in both the redox and methylation status of neuronal cells, as defined by the ratios of GSH to oxidized forms of glutathione and S-adenosylmethionine to S-adenosylhomocysteine levels, respectively. Further, morphine induced global DNA methylation changes, including CpG sites in long interspersed nuclear elements (LINE-1) retrotransposons, resulting in increased LINE-1 mRNA. Together, these findings illuminate the mechanism by which morphine, and potentially other opioids, can influence neuronal-cell redox and methylation status including DNA methylation. Since epigenetic changes are implicated in drug addiction and tolerance phenomenon, this study could potentially extrapolate to elucidate a novel mechanism of action for other drugs of abuse.

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Methylation events play a critical role in the ability of growth factors to promote normal development. Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt growth factor signaling, raising the possibility that they might exert adverse effects on methylation. We found that insulin-like growth factor-1 (IGF-1)-and dopamine-stimulated methionine synthase (MS) activity and folate-dependent methylation of phospholipids in SH-SY5Y human neuroblastoma cells, via a PI3-kinase-and MAP-kinase-dependent mechanism. The stimulation of this pathway increased DNA methylation, while its inhibition increased methylationsensitive gene expression. Ethanol potently interfered with IGF-1 activation of MS and blocked its effect on DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potently affected IGF-1 and dopamine-stimulated MS activity, as well as folate-dependent phospholipid methylation: Cu 2 þ promoted enzyme activity and methylation, while Cu þ , Pb 2 þ , Hg 2 þ and Al 3 þ were inhibitory. The ethylmercury-containing preservative thimerosal inhibited both IGF-1-and dopamine-stimulated methylation with an IC 50 of 1 nM and eliminated MS activity. Our findings outline a novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation. The potent inhibition of this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an important target of neurodevelopmental toxins.

Summary. The effects of hormonal agonists (norepinephrine, angiotensin, and histamine) on 45Ca efflux from the rabbit aorta were studied using a Ca-EGTA buffered efflux medium. Each caused a transient stimulation of efflux rate which probably reflected the release of an intracellular 45Ca store. The size of the stimulation of efflux correlated with the size of the initial rapid phase of contraction. The norepinephrine-sensitive intracellular Ca fraction was estimated to be greater than 21 gmoles/Kg wet tissue weight. This fraction is separate from intracellular Ca which is accumulated during relaxation. Evidence is presented for the lack of cyclic nucleotide involvement in the release of Ca a+, and possible alternative modes of coupling are discussed.The calcium movements which are responsible for norepinephrine (NE) induced contraction of vascular smooth muscle remain uncertain. Earlier studies by Bohr (1963) described the different sensitivity of the early phasic and latter tonic portions of the contractile response to changes in extracellular Ca. The initial rapid increase in tension was augmented in Ca-free solutions, and reduced in higher than normal Ca (10 raM), while the latter slow increase in tension was reduced in Ca-free solutions and increased in the presence of higher Ca. Hinke, Wilson and Burnham (1964), and later Hudgins (1969) andvan Breemen (1969), noted that the NE response was more resistant to Ca removal than was high K induced contraction, and that NE responsiveness returned at lower Ca levels than high K contractions. Since Briggs (1962) had reported that both NE and high K increased Ca influx, Hinke was led to conclude that NE initially utilizes a more tightly bound Ca fraction, probably located on the outside of the plasma membrane. A number of recent studies have concluded that NE activation involves a combination of cellular and extracellular Ca

Based on the presence of multiple proline-rich motifs in the huntingtin sequence, we tested its possible association with epidermal growth factor (EGF) receptor signaling complexes through SH3 domain-containing modules. We found that huntingtin is associated with Grb2, RasGAP, and tyrosine-phosphorylated EGF receptor. These associations are regulated by activation of the EGF receptor, suggesting that they may be part of EGF receptor-mediated cellular signaling cascade. In vitro binding studies indicate that SH3 domains of Grb2 or RasGAP are required for their binding to huntingtin. Our results suggest that huntingtin may be a unique adapter protein for EGF receptor-mediated signaling and may be involved in the regulation of Ras-dependent signaling pathways.Huntington's disease (HD) 1 is an autosomal dominant inherited neurodegenerative disorder characterized by massive loss of striatal neurons in caudate and putamen (1, 2). The defect in the HD gene locus involves a moderate expansion of the polymorphic CAG trinucleotide repeat near the N terminus of huntingtin (3). The age of onset of HD is roughly correlated to the number of the CAG repeats (4, 5). Although two proteins have been recently identified to interact with huntingtin in vitro (6, 7), the normal function of huntingtin remains a mystery.Identification of associated proteins is an initial step to characterize the biological function of any novel protein. Huntingtin does not contain any kinase, SH2, SH3, PH, or PTB domains, but does possess multiple proline-rich motifs in its sequence which resemble SH3 domain binding motifs (8,9). Most SH3 domain-containing proteins are adapter proteins for tyrosine kinase receptor-mediated signaling (8). We, therefore, examined the possible association of huntingtin with several SH3 domain-containing signaling molecules in EGF receptor signaling complexes. We found that huntingtin is directly associated with both Grb2 and RasGAP and indirectly associated with Shc and the EGF receptor. Huntingtin is not associated with Nck and does not co-exist with Sos in the same signaling complex. Our results suggest that huntingtin may be a unique signaling intermediate for EGF receptors and may be involved in the regulation of Ras-dependent signaling pathways. EXPERIMENTAL PROCEDURESImmunoblotting and Co-immunoprecipitation-Human epithelial carcinoma A431 or embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. These cells, starved in serum-free medium for 24 -48 h, were stimulated with 100 ng/ml EGF for 5 min at 37°C. Cells were then washed once with ice-cold phosphate-buffered saline (PBS) and lysed with Nonidet P-40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1% Nonidet P-40, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 10% glycerol, 100 M phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, 10 g/ml pepstatin A). The antihuntingtin monoclonal antibody 4C8 has been characterized previously (10). The anti-huntingtin pol...