Microglial Hyperreactivity Evolved to Immunosuppression in the Hippocampus of a Mouse Model of Accelerated Aging and Alzheimer's Disease Traits.

Neuroinflammation is a risk factor for Alzheimer's disease (AD). We sought to study the glial derangement in AD using diverse experimental models and human brain tissue. Besides classical pro-inflammatory cytokines, we analyzed chitinase 3 like 1 (CHI3L1 or YKL40) and triggering receptor expressed on myeloid cells 2 (TREM2) that are increasingly being associated with astrogliosis and microgliosis in AD, respectively. The SAMP8 mouse model of accelerated aging and AD traits showed elevated pro-inflammatory cytokines and activated microglia phenotype. Furthermore, 6-month-old SAMP8 showed an exacerbated inflammatory response to peripheral lipopolysaccharide in the hippocampus and null responsiveness at the advanced age (for this strain) of 12 months. Gene expression of TREM2 was increased in the hippocampus of transgenic 5XFAD mice and in the cingulate cortex of autosomal dominant AD patients, and to a lesser extent in aged SAMP8 mice and sporadic early-onset AD patients. However, gene expression of CHI3L1 was increased in mice but not in human AD brain samples. The results support the relevance of microglia activation in the pathways leading to neurodegeneration and suggest diverse neuroinflammatory responses according to the AD process. Therefore, the SAMP8 mouse model with marked alterations in the dynamics of microglia activation and senescence may provide a complementary approach to transgenic mouse models for the study of the neuroinflammatory mechanisms underlying AD risk and progression.

Oxidative Stress Is a Central Target for Physical Exercise Neuroprotection Against Pathological Brain Aging.

Physical exercise is suggested for preventing or delaying senescence and Alzheimer's disease (AD). We have examined its therapeutic value in the advanced stage of AD-like pathology in 3xTg-AD female mice through voluntary wheel running from 12 to 15 months of age. Mice submitted to exercise showed improved body fitness, immunorejuvenation, improvement of behavior and cognition, and reduced amyloid and tau pathology. Brain tissue analysis of aged 3xTg-AD mice showed high levels of oxidative damage. However, this damage was decreased by physical exercise through regulation of redox homeostasis. Network analyses showed that oxidative stress was a central event, which correlated with AD-like pathology and the AD-related behaviors of anxiety, apathy, and cognitive loss. This study corroborates the importance of redox mechanisms in the neuroprotective effect of physical exercise, and supports the theory of the crucial role of oxidative stress in the switch from normal brain aging to pathological aging and AD.

In vitro caloric restriction induces protective genes and functional rejuvenation in senescent SAMP8 astrocytes.

Astrocytes are key cells in brain aging, helping neurons to undertake healthy aging or otherwise letting them enter into a spiral of neurodegeneration. We aimed to characterize astrocytes cultured from senescence-accelerated prone 8 (SAMP8) mice, a mouse model of brain pathological aging, along with the effects of caloric restriction, the most effective rejuvenating treatment known so far. Analysis of the transcriptomic profiles of SAMP8 astrocytes cultured in control conditions and treated with caloric restriction serum was performed using mRNA microarrays. A decrease in mitochondrial and ribosome mRNA, which was restored by caloric restriction, confirmed the age-related profile of SAMP8 astrocytes and the benefits of caloric restriction. An amelioration of antioxidant and neurodegeneration-related pathways confirmed the brain benefits of caloric restriction. Studies of oxidative stress and mitochondrial function demonstrated a reduction of oxidative damage and partial improvement of mitochondria after caloric restriction. In summary, caloric restriction showed a significant tendency to normalize pathologically aged astrocytes through the activation of pathways that are protective against the age-related deterioration of brain physiology.

Lenti-GDNF gene therapy protects against Alzheimer's disease-like neuropathology in 3xTg-AD mice and MC65 cells.

AIMS: Glial cell-derived neurotrophic factor (GDNF) is emerging as a potent neurotrophic factor with therapeutic potential against a range of neurodegenerative conditions including Alzheimer's disease (AD). We assayed the effects of GDNF treatment in AD experimental models through gene-therapy procedures. METHODS: Recombinant lentiviral vectors were used to overexpress GDNF gene in hippocampal astrocytes of 3xTg-AD mice in vivo, and also in the MC65 human neuroblastoma that conditionally overexpresses the 99-residue carboxyl-terminal (C99) fragment of the amyloid precursor protein. RESULTS: After 6 months of overexpressing GDNF, 10-month-old 3xTg-AD mice showed preserved learning and memory, while their counterparts transduced with a green fluorescent protein vector showed cognitive loss. GDNF therapy did not significantly reduce amyloid and tau pathology, but rather, induced a potent upregulation of brain-derived neurotrophic factor that may act in concert with GDNF to protect neurons from atrophy and degeneration. MC65 cells overexpressing GDNF showed an abolishment of oxidative stress and cell death that was at least partially mediated by a reduced presence of intracellular C99 and derived amyloid ß oligomers. CONCLUSIONS: GDNF induced neuroprotection in the AD experimental models used. Lentiviral vectors engineered to overexpress GDNF showed to be safe and effective, both as a potential gene therapy and as a tool to uncover the mechanisms of GDNF neuroprotection, including cross talk between astrocytes and neurons in the injured brain.

Rcor2 underexpression in senescent mice: a target for inflammaging?

BACKGROUND: Aging is characterized by a low-grade systemic inflammation that contributes to the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). However, little knowledge is currently available on the molecular processes leading to chronic neuroinflammation. In this context, recent studies have described the role of chromatin regulators in inflammation and longevity including the REST corepressor (Rcor)-2 factor, which seems to be involved in an inflammatory suppressive program. METHODS: To assess the impact of Rcor2 in age-related inflammation, gene expression levels were quantified in different tissues and ages of the spontaneous senescence-accelerated P8 mouse (P8) using the SAMR1 mouse (R1) as a control. Specific siRNA transfection in P8 and R1 astrocyte cultures was used to determine Rcor2 involvement in the modulation of neuroinflammation. The effect of lipopolysaccharide (LPS) treatment on Rcor2 levels and neuroinflammation was analyzed both in vivo and in vitro. RESULTS: P8 mice presented a dramatic decrease in Rcor2 gene expression compared with R1 controls in splenocytes, an alteration also observed in the brain cortex, hippocampus and primary astrocytes of these mice. Rcor2 reduction in astrocytes was accompanied by an increased basal expression of the interleukin (Il)-6 gene. Strikingly, intraperitoneal LPS injection in R1 mice downregulated Rcor2 in the hippocampus, with a concomitant upregulation of tumor necrosis factor (Tnf-α), Il1-ß and Il6 genes. A negative correlation between Rcor2 and Il6 gene expression was also verified in LPS-treated C6 glioma cells. Knock down of Rcor2 by siRNA transfection (siRcor2) in R1 astrocytes upregulated Il6 gene expression while siRcor2 further increased Il6 expression in P8 astrocytes. Moreover, LPS activation provoked a further downregulation of Rcor2 and an amplified induction of Il6 in siRcor2-tranfected astrocytes. CONCLUSIONS: Data presented here show interplay between Rcor2 downregulation and increased inflammation and suggest that Rcor2 may be a key regulator of inflammaging.

Physical exercise neuroprotects ovariectomized 3xTg-AD mice through BDNF mechanisms.

Postmenopausal women may be more vulnerable to cognitive loss and Alzheimer's disease (AD) than premenopausal women because of their deficiency in estrogens, in addition to their usually older age. Aerobic physical exercise has been proposed as a therapeutic approach for maintaining health and well-being in postmenopausal women, and for improving brain health and plasticity in populations at high risk for AD. To study the neuroprotective mechanisms of physical exercise in a postmenopausal animal model, we submitted previously ovariectomized, six-month old non-transgenic and 3xTg-AD mice to three months of voluntary exercise in a running wheel. At nine months of age, we observed lower grip strength and some exacerbation of the behavioral and psychological symptoms of dementia (BPSD)-like involving active exploratory activities. A similar major cognitive impairment was observed of ovariectomized 3xTg-AD mice in comparison with sham-operated 3xTg-AD mice. A reduction of bodily fitness and lack of retention of memory were observed in the ovariectomized non-transgenic mice. Physical exercise protected against all deleterious behaviors and normalized learning and memory. It also protected against body frailty, as expected. Analyses of hippocampal key markers of antioxidant and neuroplasticity signaling pathways, showed that ovariectomy impairs the activation of CREB through physical exercise. Furthermore, molecular and behavioral correlates suggested a central role of BDNF in the neuroprotection mediated by physical exercise therapy against apathy and memory loss induced by ovariectomy and the AD-genotype.

Physical exercise improves synaptic dysfunction and recovers the loss of survival factors in 3xTg-AD mouse brain.

Physical exercise has become a potentially beneficial therapy for reducing neurodegeneration symptoms in Alzheimer's disease. Previous studies have shown that cognitive deterioration, anxiety and the startle response observed in 7-month-old 3xTg-AD mice were ameliorated after 6 months of free access to a running wheel. Also, alterations in synaptic response to paired-pulse stimulation were improved. The present study further investigated some molecular mechanisms underlying the beneficial effects of 6 months of voluntary exercise on synaptic plasticity in 7-month-old 3xTg-AD mice. Changes in binding parameters of [(3)H]-flunitrazepam to GABAA receptor and of [(3)H]-MK-801 to NMDA receptor in cerebral cortex of 3xTgAD mice were restored by voluntary exercise. In addition, reduced expression levels of NMDA receptor NR2B subunit were reestablished. The synaptic proteins synaptophysin and PSD-95 and the neuroprotective proteins GDNF and SIRT1 were downregulated in 3xTgAD mice and were recovered by exercise treatment. Overall, in this paper we highlight the fact that different interrelated mechanisms are involved in the beneficial effects of exercise on synaptic plasticity alterations in the 3xTg-AD mouse model.

Melatonin plus physical exercise are highly neuroprotective in the 3xTg-AD mouse.

Alzheimer's disease (AD) is a devastating age-related neurodegenerative disease with no specific treatment at present. Several healthy lifestyle options and over-the-counter drugs that it has been suggested delay the onset of the disease are in an experimental phase, but it is unclear whether they will have any therapeutic value against AD. We assayed physical exercise and melatonin in 3xTg-AD male mice aged from 6 to 12 months, therefore from moderate to advanced phases of AD pathology. Analysis of behavior and brain tissue at termination showed differential patterns of neuroprotection for the 2 treatments. Both treatments decreased soluble amyloid ß oligomers, whereas only melatonin decreased hyperphosphorylated tau. Melatonin was effective against the immunosenescence that 3xTg-AD mice present. Voluntary physical exercise protected against behavioral and psychological symptoms of dementia such as anxiety, a lack of exploration, and emotionality. Both treatments protected against cognitive impairment, brain oxidative stress, and a decrease in mitochondrial DNA (mtDNA). Interestingly, only the combined treatment of physical exercise plus melatonin was effective against the decrease of mitochondrial complexes. Therefore, melatonin plus physical exercise may exert complementary, additive, or even synergistic effects against a range of disturbances present in AD.

Neurons from senescence-accelerated SAMP8 mice are protected against frailty by the sirtuin 1 promoting agents melatonin and resveratrol.

The senescence-accelerated prone 8 (SAMP8) mouse strain shows early cognitive loss that mimics the deterioration of learning and memory in the elderly and is widely used as an animal model of aging. SAMP8 mouse brain suffers oxidative stress, as well as tau- and amyloid-related pathology. Mitochondrial dysfunction and the subsequent increase in cellular oxidative stress are central to the aging processes of the organism. Here, we examined the mitochondrial status of neocortical neurons cultured from SAMP8 and senescence-accelerated-resistant (SAMR1) mice. SAMP8 mouse mitochondria showed a reduced membrane potential and higher vulnerability to inhibitors and uncouplers than SAMR1 mitochondria. DL-buthionine-[S,R]-sulfoximine (BSO) caused greater oxidative damage in neurons from SAMP8 mice than in those from SAMR1 mice. This increased vulnerability, indicative of frailty-associated senescence, was protected by the anti-aging agents melatonin and resveratrol. The sirtuin 1 inhibitor, sirtinol, demonstrated that the neuroprotection against BSO was partially mediated by increased sirtuin 1 expression. Melatonin, like resveratrol, enhanced sirtuin 1 expression in neuron cultures of SAMR1 and SAMP8 mice. Therefore, a deficiency in the neuroprotection and longevity of the sirtuin 1 pathway in SAMP8 neurons may contribute to the early age-related brain damage in these mice. This supports the therapeutic use of sirtuin 1-enhancing agents against age-related nerve cell dysfunction and brain frailty.

Physical exercise protects against Alzheimer's disease in 3xTg-AD mice.

Physical exercise is considered to exert a positive neurophysiological effect that helps to maintain normal brain activity in the elderly. Expectations that it could help to fight Alzheimer's disease (AD) were recently raised. This study analyzed the effects of different patterns of physical exercise on the 3xTg-AD mouse. Male and female 3xTg-AD mice at an early pathological stage (4-month-old) have had free access to a running wheel for 1 month, whereas mice at a moderate pathological stage(7-month-old) have had access either during 1 or 6 months. The non-transgenic mouse strain was used as a control. Parallel animal groups were housed in conventional conditions. Cognitive loss and behavioral and psychological symptoms of dementia (BPSD)-like behaviors were present in the 3xTg-AD mice along with alteration in synaptic function and ong-term potentiation impairment in vivo. Brain tissue showed AD-pathology and oxidative-related changes. Disturbances were more severe at the older age tested. Oxidative stress was higher in males but other changes were similar or higher in females. Exercise treatment ameliorated cognitive deterioration and BPSD-like behaviors such as anxiety and the startle response. Synaptic changes were partially protected by exercise. Oxidative stress was reduced. The best neuroprotection was generally obtained after 6 months of exercise in 7-month-old 3xTg-AD mice. Improved sensorimotor function and brain tissue antioxidant defence were induced in both 3xTg-AD and NonTg mice. Therefore, the benefits of aerobic physical exercise on synapse, redox homeostasis, and general brain function demonstrated in the 3xTg-AD mouse further support the value of this healthy life-style against neurodegeneration.

Gender-Specific Neuroimmunoendocrine Response to Treadmill Exercise in 3xTg-AD Mice.

The 3xTg-AD mouse develops a progressive Alzheimer's disease- (AD-) like brain pathology that causes cognitive- and neuropsychiatric-like symptoms of dementia. Since its neuroimmunoendocrine axis is likewise impaired, this mouse is also useful for modelling complex age-related neurodegeneration. This study analyzed behavioral, physiological, neurochemical, pathological and immunoendocrine alterations in male and female 3xTg-AD mice and assayed the effects of a short therapy of forced physical exercise at the moderate pathology stage of 6 months of age. Gender effects were observed in most AD-related pathology and dysfunctions. Five weeks of treadmill training produced beneficial effects, such as the reduction of brain oxidative stress and GABA-A receptor dysfunction in males and improvement of sensorimotor function in females. In both sexes, exercise decreased the brain amyloid ß 42/40 ratio levels. The results highlight the importance of analyzing experimental therapies in both mouse model genders in order to improve our understanding of the disease and develop more appropriate therapies.

In vitro and in vivo activation of astrocytes by amyloid-beta is potentiated by pro-oxidant agents.

Alzheimer's disease (AD) is a devastating age-related neurodegenerative disease. Age is the main risk factor for sporadic AD, which is the most prevalent type. Amyloid-beta peptide (Abeta) neurotoxicity is the proposed first step in a cascade of deleterious events leading to AD pathology and dementia. Glial cells play an important role in these changes. Astrocytes provide vital support to neurons and modulate functional synapses. Therefore, the toxic effects of Abeta on astrocytes might promote neurodegenerative changes that lead to AD. Aging reduces astrocyte antioxidant defenses and induces oxidative stress. We studied the effects of Abeta(42) on cultures of human astrocytes in the presence or absence of the following pro-oxidant agents: buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, and FeSO(4), which liberates redox active iron. Pro-oxidant conditions potentiated Abeta toxicity, as shown by the generation of free radicals, inflammatory changes, and apoptosis. Similar treatments were assessed in rats in vivo. A combination of Abeta(40) and Abeta(42) or Abeta(42) alone was infused intracerebroventricularly for 4 weeks. Other animal groups were also infused with BSO and FeSO(4). A long-term analysis that ended 4 months later showed greater cognitive impairment in the Morris water maze task, which was induced by Abeta plus pro-oxidant agent treatments. Pro-oxidant agents also potentiated brain tissue pathology. This was demonstrated in histological studies that showed highly increased astrocyte reactivity in AD-vulnerable areas, Abeta deposits, and oxidative damage of AD-sensitive hippocampal neurons. To increase our understanding of AD, experimental models should be used that mimic age-related brain changes, in which age-related oxidative stress potentiates the effects of Abeta.

Self-assembled multifunctional Fe/MgO nanospheres for magnetic resonance imaging and hyperthermia.

A one-step process for the production of nanoparticles presenting advanced magnetic properties can be achieved using vapor condensation. In this article, we report on the fabrication of Fe particles covered by a uniform MgO epitaxial shell. MgO has a lower surface energy than Fe, which results in a core-shell crystal formation. The particles satisfy a few of technical requirements for the practical use in real clinics, such as a high biocompatibility in living cells in-vitro, an injection through blood vessels without any clothing problems in murine model, a high absorption rate for magnetic hyperthermia at small particle concentration, and the potential to be used as contrast agent in the field of diagnostic magnetic imaging. They are also able to be used in drug delivery and magnetic-activated cell sorting. FROM THE CLINICAL EDITOR: In this paper, the authors report on the synthesis of Fe particles covered by a uniform MgO epitaxial shell resulting in a core-shell crystal formation. The particles are proven to be useful as contrast agents for magnetic resonance imaging and have the potential to be useful as heating mediators for cancer therapy through hyperthermia. They also might be used in drug delivery and magnetic-activated cell sorting.

Proteomic study of neuron and astrocyte cultures from senescence-accelerated mouse SAMP8 reveals degenerative changes.

Senescence-accelerated prone (SAMP) strain 8 mice suffer an earlier development of cognitive age-related pathologies and a shorter life span than conventional mice. Protein alterations in astrocytes, in addition to those in neurons, may contribute to neurodegenerative damage. We applied proteomics techniques to study cell-specific early markers of brain aging-related degeneration in SAMP8. The two-dimensional protein expression patterns of the SAMP8 neuron and astrocyte cultures were compared with those obtained from senescence-accelerated resistant mouse strain 1 cultures. Differentially expressed spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Proteins belonged to cell pathways of energy metabolism, biosynthesis, cell transduction and signaling, stress response, and the maintenance of cytoskeletal functions. Most of the changes were cell type specific. However, there was a general increase in cell transduction, signaling, and stress-related proteins and a decrease in cytoskeletal proteins. In addition, neurons showed an increased expression of proteins involved in biosynthetic pathways. A number of the protein alterations have been previously reported in the brain tissue proteome of SAMP8, aged brain or Alzheimer's disease brain. Alterations in neuron and astrocyte proteoma indicated that both cell types are involved in the brain degenerative changes of SAMP8 mice. However, network analysis suggests that neuronal changes are more complex and have a greater influence.

Dysfunction of astrocytes in senescence-accelerated mice SAMP8 reduces their neuroprotective capacity.

Early onset increases in oxidative stress and tau pathology are present in the brain of senescence-accelerated mice prone (SAMP8). Astrocytes play an essential role, both in determining the brain's susceptibility to oxidative damage and in protecting neurons. In this study, we examine changes in tau phosphorylation, oxidative stress and glutamate uptake in primary cultures of cortical astrocytes from neonatal SAMP8 mice and senescence-accelerated-resistant mice (SAMR1). We demonstrated an enhancement of abnormally phosphorylated tau in Ser(199) and Ser(396) in SAMP8 astrocytes compared with that of SAMR1 control mice. Gsk3beta and Cdk5 kinase activity, which regulate tau phosphorylation, was also increased in SAMP8 astrocytes. Inhibition of Gsk3beta by lithium or Cdk5 by roscovitine reduced tau phosphorylation at Ser(396). Moreover, we detected an increase in radical superoxide generation, which may be responsible for the corresponding increase in lipoperoxidation and protein oxidation. We also observed a reduced mitochondrial membrane potential in SAMP8 mouse astrocytes. Glutamate uptake in astrocytes is a critical neuroprotective mechanism. SAMP8 astrocytes showed a decreased glutamate uptake compared with those of SAMR1 controls. Interestingly, survival of SAMP8 or SAMR1 neurons cocultured with SAMP8 astrocytes was significantly reduced. Our results indicate that alterations in astrocyte cultures from SAMP8 mice are similar to those detected in whole brains of SAMP8 mice at 1-5 months. Moreover, our findings suggest that this in vitro preparation is suitable for studying the molecular and cellular processes underlying early aging in this murine model. In addition, our study supports the contention that astrocytes play a key role in neurodegeneration during the aging process.

Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels.

Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl(-) channels.

Down's syndrome astrocytes have greater antioxidant capacity than euploid astrocytes.

Down's syndrome (trisomy 21) brain tissue is considered to be susceptible to oxidative injury, mainly because its increased Cu/Zn-superoxide dismutase (SOD1) activity is not followed by an adaptive rise in hydrogen peroxide metabolizing enzymes. In vitro, trisomic neurons suffer oxidative stress and degenerate. We studied the response of trisomy 21 neuron and astrocyte cultures to hydrogen peroxide injury and found that they were, respectively, more and less vulnerable than their euploid counterparts. Differences were detected 24 h after exposures in the region of 50 microm and 500 microm hydrogen peroxide for neuron and astrocyte cultures, respectively. Cytotoxicity results were paralleled by a decrease in cellular glutathione. In addition, trisomic astrocytes showed a lower basal content of superoxide ion and a higher clearance of hydrogen peroxide from the culture medium. In the presence of hydrogen peroxide, trisomic astrocytes maintained their concentration of intracellular superoxide and hydroperoxides at a lower level than euploid astrocytes. Consistent with these results, trisomic astrocytes in neuron coculture were more neuroprotective than euploid astrocytes against hydrogen peroxide injury. We suggest that SOD1 overexpression has beneficial effects on astrocytes, as it does in other systems with similarly high disposal of hydroperoxides. In addition to a higher enzymatic activity of SOD1, cultures of trisomic astrocytes showed slightly higher glutathione reductase activity than euploid cultures. Thus, trisomy 21 astrocytes showed a greater antioxidant capacity against hydrogen peroxide than euploid astrocytes, and they partially counteracted the oxidative vulnerability of trisomic neurons in culture.

First direct demonstration of extensive GABA synthesis in mouse cerebellar neuronal cultures.

Culturing mouse cerebellar neurones (predominantly glutamatergic) in the presence of [1-(13)C]glucose for 7 days resulted in a surprisingly extensive labelling of the inhibitory neurotransmitter GABA, the average content and labelling of which were 20 +/- 4 nmol/mg protein and 20 +/- 4%, respectively. Cultures of neocortical neurones (predominantly GABAergic) had under similar conditions a GABA content and labelling of 32 +/- 2 nmol/mg protein and 21 +/- 2%. The cerebellar cultures contained only 6% glutamate decarboxylase (GAD)-positive neurones when immunolabelled using a GAD67 antibody, while a dense network of neurones in the neocortical cultures stained positively for GAD67. Exposure of the cerebellar cultures to 50 microm kainic acid (KA) which is known to eliminate vesicular release of GABA, only marginally affected GABA labelling and cellular content. Likewise this treatment had no effect on the number of GAD67-positive neurones but a massive punctate immunostaining observed in control cultures was essentially eliminated. Increasing the KA concentration to 0.5 mm in the culture medium for 7 days led to a reduction of GABA labelling and content compared to cerebellar cultures not exposed to KA. Although it is likely that this large capacity for GABA synthesis resides in the relatively few GAD-positive neurones, it seems unlikely that they could account for the large average GABA content in the cultures. Therefore it must be concluded that the newly synthesized GABA is redistributed among the majority of the cells in these cultures, i.e. the glutamatergic neurones.

Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.

Trimethyltin (TMT) and triethyltin (TET) caused cell death in cultures of primary human neurons and astrocytes, rat neurons and human neuroblastoma cell lines. Human neurons and astrocytes showed a delayed response to TMT cytotoxicity. After 24h of TMT exposure, LC50 values were 148.1, 335.5 and 609.7 microM for SK-N-MC neuroblastoma cell line, neurons and astrocytes, respectively. Over 5 days of exposure, the cytotoxic potency of TMT increased about 70-fold in human cortical neurons. Rat hippocampal neurons were the most vulnerable cells to TMT cytotoxicity, exhibiting an LC50 value 30-fold lower (1.4 microM) than that of rat cerebellar granule cells (44.28 microM). With the exception of rat hippocampal neurons, TET was more potent than TMT in inducing cell death (LC50 values of 3.5-16.9 microM). Moreover, TET was more effective than TMT in increasing intracellular free Ca2+ concentration in human and rat neurons. This work shows that human fetal neuron and astrocyte cultures are a useful model for studying the neurotoxic effects of these environmental contaminants and, thus, predicting their impact on human health.

Neurotoxicity of organomercurial compounds.

Mercury is a ubiquitous contaminant, and a range of chemical species is generated by human activity and natural environmental change. Elemental mercury and its inorganic and organic compounds have different toxic properties, but all them are considered hazardous in human exposure. In an equimolecular exposure basis, organomercurials with a short aliphatic chain are the most harmful compounds and they may cause irreversible damage to the nervous system. Methylmercury (CH(3)Hg(+)) is the most studied following the neurotoxic outbreaks identified as Minamata disease and the Iraq poisoning. The first description of the CNS pathology dates from 1954. Since then, the clinical neurology, the neuropathology and the mechanisms of neurotoxicity of organomercurials have been widely studied. The high thiol reactivity of CH(3)Hg(+), as well as all mercury compounds, has been suggested to be the basis of their harmful biological effects. However, there is clear selectivity of CH(3)Hg(+) for specific cell types and brain structures, which is not yet fully understood. The main mechanisms involved are inhibition of protein synthesis, microtubule disruption, increase of intracellular Ca(2+) with disturbance of neurotransmitter function, oxidative stress and triggering of excitotoxicity mechanisms. The effects are more damaging during CNS development, leading to alterations of the structure and functionality of the nervous system. The major source of CH(3)Hg(+) exposure is the consumption of fish and, therefore, its intake is practically unavoidable. The present concern is on the study of the effects of low level exposure to CH(3)Hg(+) on human neurodevelopment, with a view to establishing a safe daily intake. Recommendations are 0.4 micro g/kg body weight/day by the WHO and US FDA and, recently, 0.1 micro g/kg body weight/day by the US EPA. Unfortunately, these levels are easily attained with few meals of fish per week, depending on the source of the fish and its position in the food chain.