Resveratrol Activates Antioxidant Protective Mechanisms in Cellular Models of Alzheimer's Disease Inflammation.

Resveratrol is a natural phenolic compound with known benefits against neurodegeneration. We analyzed in vitro the protective mechanisms of resveratrol against the proinflammatory monomeric C-reactive protein (mCRP). mCRP increases the risk of AD after stroke and we previously demonstrated that intracerebral mCRP induces AD-like dementia in mice. Here, we used BV2 microglia treated with mCRP for 24 h in the presence or absence of resveratrol. Cells and conditioned media were collected for analysis. Lipopolysaccharide (LPS) has also been implicated in AD progression and so LPS was used as a resveratrol-sensitive reference agent. mCRP at the concentration of 50 µg/mL activated the nitric oxide pathway and the NLRP3 inflammasome pathway. Furthermore, mCRP induced cyclooxygenase-2 and the release of proinflammatory cytokines. Resveratrol effectively inhibited these changes and increased the expression of the antioxidant enzyme genes Cat and Sod2. As central mechanisms of defense, resveratrol activated the hub genes Sirt1 and Nfe2l2 and inhibited the nuclear translocation of the signal transducer NF-ĸB. Proinflammatory changes induced by mCRP in primary mixed glial cultures were also protected by resveratrol. This work provides a mechanistic insight into the protective benefits of resveratrol in preventing the risk of AD induced by proinflammatory agents.

New insights in animal models of neurotoxicity-induced neurodegeneration.

The high prevalence of neurodegenerative diseases is an unintended consequence of the high longevity of the population, together with the lack of effective preventive and therapeutic options. There is great pressure on preclinical research, and both old and new models of neurodegenerative diseases are required to increase the pipeline of new drugs for clinical testing. We review here the main models of neurotoxicity-based animal models leading to central neurodegeneration. Our main focus was on studying how changes in neurotransmission and neuroinflammation, mainly in rodent models, contribute to harmful processes linked to neurodegeneration. The majority of the models currently in use mimic Parkinson's disease (PD) and Alzheimer's disease (AD), which are the most common neurodegenerative conditions in older adults. AD is the most common age-related dementia, whereas PD is the most common movement disorder with also cases of dementia. Several natural toxins and xenobiotic agents induce dopaminergic neurodegeneration and can reproduce neuropathological traits of PD. The literature analysis of MPTP, 6-OH-dopamine, and rotenone models suggested the latter as a useful model when specific doses of rotenone were administrated systemically to C57BL/6 mice. Cholinergic neurodegeneration is mainly modelled with the toxin scopolamine, which is a useful rodent model for the screening of protective drugs against cognitive decline and AD. Several agents have been used to model neuroinflammation-based neurodegeneration and dementia in AD, including lipopolysaccharide (LPS), streptozotocin, and monomeric C-reactive protein. The bacterial agent LPS makes a useful rodent model for testing anti-inflammatory therapies to halt the development and severity of AD. However, neurotoxin models might be more useful than genetic models for drug discovery in PD but that is not the case in AD where they cannot beat the new developments in transgenic mouse models. Overall, we should work using all available models, either in vivo, in vitro, or in silico, considering the seriousness of the moment and urgency of developing effective drugs.

Neuroprotective Epigenetic Changes Induced by Maternal Treatment with an Inhibitor of Soluble Epoxide Hydrolase Prevents Early Alzheimer's Disease Neurodegeneration.

Modulation of Alzheimer's disease (AD) risk begins early in life. During embryo development and postnatal maturation, the brain receives maternal physiological influences and establishes epigenetic patterns that build its level of resilience to late-life diseases. The soluble epoxide hydrolase inhibitor N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl] urea (TPPU), reported as ant-inflammatory and neuroprotective against AD pathology in the adult 5XFAD mouse model of AD, was administered to wild-type (WT) female mice mated to heterozygous 5XFAD males during gestation and lactation. Two-month-old 5XFAD male and female offspring of vehicle-treated dams showed memory loss as expected. Remarkably, maternal treatment with TPPU fully prevented memory loss in 5XFAD. TPPU-induced brain epigenetic changes in both WT and 5XFAD mice, modulating global DNA methylation (5-mC) and hydroxymethylation (5-hmC) and reducing the gene expression of some histone deacetylase enzymes (Hdac1 and Hdac2), might be on the basis of the long-term neuroprotection against cognitive impairment and neurodegeneration. In the neuropathological analysis, both WT and 5XFAD offspring of TPPU-treated dams showed lower levels of AD biomarkers of tau hyperphosphorylation and microglia activation (Trem2) than the offspring of vehicle-treated dams. Regarding sex differences, males and females were similarly protected by maternal TPPU, but females showed higher levels of AD risk markers of gliosis and neurodegeneration. Taken together, our results reveal that maternal treatment with TPPU impacts in preventing or delaying memory loss and AD pathology by inducing long-term modifications in the epigenetic machinery and its marks.

Antibody Protection against Long-Term Memory Loss Induced by Monomeric C-Reactive Protein in a Mouse Model of Dementia.

Monomeric C-reactive protein (mCRP), the activated isoform of CRP, induces tissue damage in a range of inflammatory pathologies. Its detection in infarcted human brain tissue and its experimentally proven ability to promote dementia with Alzheimer's disease (AD) traits at 4 weeks after intrahippocampal injection in mice have suggested that it may contribute to the development of AD after cerebrovascular injury. Here, we showed that a single hippocampal administration of mCRP in mice induced memory loss, lasting at least 6 months, along with neurodegenerative changes detected by increased levels of hyperphosphorylated tau protein and a decrease of the neuroplasticity marker Egr1. Furthermore, co-treatment with the monoclonal antibody 8C10 specific for mCRP showed that long-term memory loss and tau pathology were entirely avoided by early blockade of mCRP. Notably, 8C10 mitigated Egr1 decrease in the mouse hippocampus. 8C10 also protected against mCRP-induced inflammatory pathways in a microglial cell line, as shown by the prevention of increased generation of nitric oxide. Additional in vivo and in vitro neuroprotective testing with the anti-inflammatory agent TPPU, an inhibitor of the soluble epoxide hydrolase enzyme, confirmed the predominant involvement of neuroinflammatory processes in the dementia induced by mCRP. Therefore, locally deposited mCRP in the infarcted brain may be a novel biomarker for AD prognosis, and its antibody blockade opens up therapeutic opportunities for reducing post-stroke AD risk.

Revisiting Astrocytic Roles in Methylmercury Intoxication.

Intoxication by heavy metals such as methylmercury (MeHg) is recognized as a global health problem, with strong implications in central nervous system pathologies. Most of these neuropathological conditions involve vascular, neurotransmitter recycling, and oxidative balance disruption leading to accelerated decline in fine balance, and learning, memory, and visual processes as main outcomes. Besides neurons, astrocytes are involved in virtually all the brain processes and perform important roles in neurological response following injuries. Due to astrocytes' strategic functions in brain homeostasis, these cells became the subject of several studies on MeHg intoxication. The most heterogenous glial cells, astrocytes, are composed of plenty of receptors and transporters to dialogue with neurons and other cells and to monitor extracellular environment responding tightly through fluctuation of cytosolic ions. The overall toxicity of MeHg might be determined on the basis of the balance between MeHg-mediated injury to neurons and protective responses from astrocytes. Although the role of neurons in MeHg intoxication is relatively well-established, the role of the astrocytes is only beginning to be understood. In this review, we update the information on astroglial modulation of the MeHg-induced neurotoxicity, providing remarks on their protective and deleterious roles and insights for future studies.

Manganese-induced neurotoxicity in cerebellar granule neurons due to perturbation of cell network pathways with potential implications for neurodegenerative disorders.

Manganese (Mn) is essential for living organisms, playing an important role in nervous system function. Nevertheless, chronic and/or acute exposure to this metal, especially during early life stages, can lead to neurotoxicity and dementia by unclear mechanisms. Thus, based on previous works of our group with yeast and zebrafish, we hypothesized that the mechanisms mediating manganese-induced neurotoxicity can be associated with the alteration of protein metabolism. These mechanisms may also depend on the chemical speciation of manganese. Therefore, the current study aimed at investigating the mechanisms mediating the toxic effects of manganese in primary cultures of cerebellar granule neurons (CGNs). By exposing cultured CGNs to different chemical species of manganese ([[2-[(dithiocarboxy)amino]ethyl]carbamodithioato]](2-)-kS,kS']manganese, named maneb (MB), and [[1,2-ethanediylbis[carbamodithioato]](2-)]manganese mixture with [[1,2-ethanediylbis[carbamodithioato]](2-)]zinc, named mancozeb (MZ), and manganese chloride (MnCl2)), and using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, we observed that both MB and MZ induced similar cytotoxicity (LC50∼ 7-9 µM), which was higher than that of MnCl2 (LC50∼ 27 µM). Subsequently, we applied systems biology approaches, including metallomics, proteomics, gene expression and bioinformatics, and revealed that independent of chemical speciation, for non-cytotoxic concentrations (0.3-3 µM), Mn-induced neurotoxicity in CGNs is associated with metal dyshomeostasis and impaired protein metabolism. In this way, we verified that MB induced more post-translational alterations than MnCl2, which can be a plausible explanation for cytotoxic differences between both chemical species. The metabolism of proteins is one of the most energy consuming cellular processes and its impairment appears to be a key event of some cellular stress processes reported separately in other studies such as cell cycle arrest, energy impairment, cell signaling, excitotoxicity, immune response, potential protein accumulation and apoptosis. Interestingly, we verified that Mn-induced neurotoxicity shares pathways associated with the development of Alzheimer's disease, Amyotrophic Lateral Sclerosis, Huntington's disease, and Parkinson's disease. This has been observed in baker's yeast and zebrafish suggesting that the mode of action of Mn may be evolutionarily conserved.

Glutathione in Chlorpyrifos-and Chlorpyrifos-Oxon-Induced Toxicity: a Comparative Study Focused on Non-cholinergic Toxicity in HT22 Cells.

Chlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide widely used for agricultural purposes. CPF-mediated neurotoxicity is mainly associated with its anticholinesterase activity, which may lead to a cholinergic syndrome. CPF metabolism generates chlorpyrifos-oxon (CPF-O), which possesses higher anticholinesterase activity and, consequently, plays a major role in the cholinergic syndrome observed after CPF poisoning. Recent lines of evidence have also reported non-cholinergic endpoints of CPF- and CPF-O-induced neurotoxicities, but comparisons on the non-cholinergic toxic properties of CPF and CPF-O are lacking. In this study, we compared the non-cholinergic toxicities displayed by CPF and CPF-O in cultured neuronal cells, with a particular emphasis on their pro-oxidant properties. Using immortalized cells derived from mouse hippocampus (HT22 line, which does present detectable acetylcholinesterase activity), we observed that CPF-O was 5-fold more potent in decreasing cell viability compared with CPF. Atropine, a muscarinic acetylcholine receptor antagonist, protected against acetylcholine (ACh)-induced toxicity but failed to prevent the CPF- and CPF-O-induced cytotoxicities in HT22 cells. CPF or CPF-O exposures significantly decreased the levels of the antioxidant glutathione (GSH); this event preceded the significant decrease in cell viability. Pretreatment with N-acetylcysteine (NAC, a GSH precursor) protected against the cytotoxicity induced by both CPF and CPF-O. The present study indicates that GSH depletion is a non-cholinergic event involved in CPF and CPF-O toxicities. The study also shows that in addition of being a more potent AChE inhibitor, CPF-O is also a more potent pro-oxidant molecule when compared with CPF, highlighting the role of CPF metabolism (bioactivation to CPF-O) in the ensuing non-cholinergic toxicity.

Role of Resveratrol and Selenium on Oxidative Stress and Expression of Antioxidant and Anti-Aging Genes in Immortalized Lymphocytes from Alzheimer's Disease Patients.

Oxidative damage is involved in the pathophysiology of age-related ailments, including Alzheimer's disease (AD). Studies have shown that the brain tissue and also lymphocytes from AD patients present increased oxidative stress compared to healthy controls (HCs). Here, we use lymphoblastoid cell lines (LCLs) from AD patients and HCs to investigate the role of resveratrol (RV) and selenium (Se) in the reduction of reactive oxygen species (ROS) generated after an oxidative injury. We also studied whether these compounds elicited expression changes in genes involved in the antioxidant cell response and other aging-related mechanisms. AD LCLs showed higher ROS levels than those from HCs in response to H2O2 and FeSO4 oxidative insults. RV triggered a protective response against ROS under control and oxidizing conditions, whereas Se exerted antioxidant effects only in AD LCLs under oxidizing conditions. RV increased the expression of genes encoding known antioxidants (catalase, copper chaperone for superoxide dismutase 1, glutathione S-transferase zeta 1) and anti-aging factors (sirtuin 1 and sirtuin 3) in both AD and HC LCLs. Our findings support RV as a candidate for inducing resilience and protection against AD, and reinforce the value of LCLs as a feasible peripheral cell model for understanding the protective mechanisms of nutraceuticals against oxidative stress in aging and AD.

Reduced cortical serotonin 5-HT2A receptor binding and glutamate activity in high compulsive drinker rats.

Serotonin2A receptors and glutamate signaling have been implicated in the pathophysiology and treatment of compulsive spectrum disorders. Schedule-Induced Polydipsia (SIP), characterized by excessive drinking under intermittent food reinforcement schedules, is a valid model for studying the compulsive phenotype in rats. We explored the expression, function, and neurochemistry of 5-HT2A receptors in the frontal cortex (FC) of rats with individual differences to compulsivity. Rats were selected for high (HD) versus low (LD) drinking on SIP. First, we measured 5-HT2A, 5-HT1A, and mGlu2/3 receptors and serotonin transporter binding in different brain regions. Second, we assessed the effect of microinfusion into the medial prefrontal cortex (mPFC) of the 5-HT2A/C receptor agonist DOI, the mGlu2/3 agonist LY379268, and the combination of DOI with the 5-HT2A receptor antagonist M100907 and the 5-HT2C receptor antagonist SB242084. Finally, we measured the serotonin and glutamate efflux in mPFC in basal condition and after DOI local application. The compulsive HD rats showed a specific reduction of 5-HT2A receptor binding in FC compared to LD rats. The highest dose of DOI reduced compulsive drinking in HD rats on SIP, whereas LY379268 did not induce any significant effect. The 5-HT2A receptor antagonist M100907 reversed the DOI induced reduction on compulsive drinking in HD rats while blocking the 5-HT2C receptor did not affect SIP. Compulsive HD rats showed increased serotonin and decreased glutamate efflux in basal conditions that were modified by the DOI application. These findings indicate that reduced 5-HT2A receptor binding and glutamate neurochemical mechanisms may underlie compulsive behavior vulnerability.

Neurotransmitter amines and antioxidant agents in neuronal protection against methylmercury-induced cytotoxicity in primary cultures of mice cortical neurons.

Methylmercury (MeHg) is an environmental toxicant with detrimental effects on the developing brain and adult nervous system. The main mechanisms identified include oxidative stress, changes in intracellular calcium, mitochondrial changes, inhibition of glutamate uptake, of protein synthesis and disruption of microtubules. However, little is known about mechanisms of protection against MeHg neurotoxicity. We found that resveratrol (10 µM) and ascorbic acid (200 µM) protected MeHg-induced cell death in primary cultures of cortical neurons. In this work, we aimed at finding additional targets that may be related to MeHg mode of action in cell toxicity with special emphasis in cell protection. We wonder whether neurotransmitters may affect the MeHg effects on neuronal death. Our findings show that neurons exposed to low MeHg concentrations exhibit less mortality if co-exposed to 10 µM dopamine (DA). However, DA metabolites, HVA (homovanillic acid) and DOPAC (3,4-dihydroxyphenylacetic acid) are not responsible for such protection. Furthermore, both DA D1 and D2 receptors agonists showed a protective effect against MeHg toxicity. It is striking though that DA receptor antagonists SKF83566 (10 µM) and haloperidol (10 µM) did not inhibit DA protection against MeHg. In addition, the protective effect of 10 µM DA against MeHg-induced toxicity was not affected by additional organochlorine pollutants exposure. Our results also demonstrate that cells exposed to MeHg in presence of 100 µM acetylcholine (ACh), show an increase in cell mortality at the "threshold value" of 100 nM MeHg. Finally, norepinephrine (10 µM) and serotonin (20 µM) also had an effect on cell protection. Altogether, we propose to further investigate the additional mechanisms that may be playing an important role in MeHg-induced cytotoxicity.

Biosafety assessment of conducting nanostructured materials by using co-cultures of neurons and astrocytes.

Neural electrode implants are made mostly of noble materials. We have synthesized a nanostructured material combining the good electrochemical properties of iridium oxide (IrOx) and carbon-nanotubes (CNT) and the properties of poly(3,4-ethylenedioxythiophene) (PEDOT). IrOx-CNT-PEDOT charge storage capacity was lower than that of IrOx and IrOx-CNT, but higher than that of other PEDOT-containing hybrids and Pt. Cyclic voltammetry, SEM, XPS and micro-Raman spectroscopy suggest that PEDOT encapsulates IrOx and CNT. In our search for a cell culture platform that could optimize modelling the in vivo environment, we determined cell viability, neuron and astrocyte functionality and the response of astrocytes to an inflammatory insult by using primary cultures of neurons, of astrocytes and co-cultures of both. The materials tested (based on IrOx, CNT and PEDOT, as well as Pt as a reference) allowed adhesion and proliferation of astrocytes and full compatibility for neurons grown in co-cultures. Functionality assays show that uptake of glutamate in neuron-astrocyte co-culture was significantly higher than the sum of the uptake in astrocytes and neurons. In co-cultures on IrOx, IrOx-CNT and IrOx-CNT-PEDOT, glutamate was released by a depolarizing stimulus and induced a significant increase in intracellular calcium, supporting the expression of functional NMDA/glutamate receptors. LPS-induced inflammatory response in astrocytes showed a decreased response in NOS2 and COX2 mRNA expression for IrOx-CNT-PEDOT. Results indicate that neuron-astrocyte co-cultures are a reliable model for assessing the biocompatibility and safety of nanostructured materials, evidencing also that hybrid IrOx-CNT-PEDOT nanocomposite materials may offer larger resistance to inflammatory insults.

Clarified Açaí (Euterpe oleracea) Juice as an Anticonvulsant Agent: In Vitro Mechanistic Study of GABAergic Targets.

Seizures affect about 50 million people around the world. Approximately 30% of seizures are refractory to the current pharmacological arsenal, so, the pursuit of new therapeutic alternatives is essential. Clarified Euterpe oleracea (EO) juice showed anticonvulsant properties similar to diazepam in an in vivo model with pentylenetetrazol, a GABAA receptor blocker. This study investigated the effects of EO on the main GABAergic targets for anticonvulsant drugs, analyzing the effect on the GABA receptor's benzodiazepine and picrotoxinin binding sites and the GABA uptake. Primary cultures of cortical neurons and astrocytes were treated with EO (0-25%) for up to 90 min. [3H]Flunitrazepam and [3H]TBOB binding, [3H]GABA uptake, cell viability, and morphology were assayed. Nonlethal concentrations of EO increased agonist binding and decreased antagonist binding in cortical neurons. Low concentrations significantly inhibited GABA uptake, especially in astrocytes, suggesting an accumulation of endogenous GABA in the synaptic cleft. The results demonstrate, for the first time, that EO can improve GABAergic neurotransmission via interactions with GABAA receptor and modulation of GABA uptake. Understanding these molecular mechanisms will help in the treatment of seizures and epilepsy, especially in developing countries where geographic isolation and low purchasing power are the main barriers to access to adequate treatment.

Ghrelin Causes a Decline in GABA Release by Reducing Fatty Acid Oxidation in Cortex.

Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system.

Methylmercury-induced developmental toxicity is associated with oxidative stress and cofilin phosphorylation. Cellular and human studies.

Environmental exposure to methylmercury (MeHg) during development is of concern because it is easily incorporated in children's body both pre- and post-natal, it acts at several levels of neural pathways (mitochondria, cytoskeleton, neurotransmission) and it causes behavioral impairment in child. We evaluated the effects of prolonged exposure to 10-600nM MeHg on primary cultures of mouse cortical (CCN) and of cerebellar granule cells (CGC) during their differentiation period. In addition, it was studied if prenatal MeHg exposure correlated with altered antioxidant defenses and cofilin phosphorylation in human placentas (n=12) from the INMA cohort (Spain). Exposure to MeHg for 9days in vitro (DIV) resulted in protein carbonylation and in cell death at concentrations ≥200nM and ≥300nM, respectively. Exposure of CCN and CGC to non-cytotoxic MeHg concentrations for 5 DIV induced an early concentration-dependent decrease in cofilin phosphorylation. Furthermore, in both cell types actin was translocated from the cytosol to the mitochondria whereas cofilin translocation was found only in CGC. Translocation of cofilin and actin to mitochondria in CGC occurred from 30nM MeHg onwards. We also found an increased expression of cortactin and LIMK1 mRNA in CGC but not in CCN. All these effects were prevented by the antioxidant probucol. Cofilin phosphorylation was significantly decreased and a trend for decreased activity of glutathione reductase and glutathione peroxidase was found in the fetal side of human placental samples from the highest (20-40µg/L) MeHg-exposed group when compared with the low (<7µg/L) MeHg-exposed group. In summary, cofilin dephosphorylation and oxidative stress are hallmarks of MeHg exposure in both experimental and human systems.

Long-term and low-dose malathion exposure causes cognitive impairment in adult mice: evidence of hippocampal mitochondrial dysfunction, astrogliosis and apoptotic events.

The organophosphorus (OP) pesticide malathion is a neurotoxic compound whose acute toxicity is primarily caused by the inhibition of acetylcholinesterase (AChE), leading to cholinergic syndrome-related symptoms. Some lines of evidence indicate that long-term exposure to low levels of OP may produce neuropsychiatric and/or neurobehavioral signs that do not necessarily involve the AChE inhibition. This study evaluated the effects of a repeated (15-day period) and low-dose malathion exposure on spatial memory and discrimination (object location task), as well as on biochemical parameters in the hippocampus of mice [AChE and mitochondrial chain complexes activities; levels of proapoptotic proteins (Bax and Bak) and cholinergic neuronal and astroglial markers (ChAT and GFAP, respectively)]. Malathion treatments (30 and 100 mg/kg, s.c.) did not affect the body weight of animals and caused no evident signs of cholinergic toxicity throughout the treatment, although the highest dose (100 mg/kg) was associated with inhibition of AChE activity. Malathion-exposed animals showed a significant impairment on spatial memory and discrimination, which was correlated with a decrease in the mitochondrial complex I activity in the hippocampus. Moreover, malathion increased the levels of proapoptotic proteins and induced astroglial activation. The results show that long-term malathion exposure, at a dose that does not affect hippocampal AChE activity (30 mg/kg), caused impaired spatial memory and discrimination in mice that was related to hippocampal mitochondrial dysfunctional, astrogliosis and apoptosis. When extrapolated to humans, such results shed light on noncholinergic mechanisms likely related to the neurobehavioral and cognitive deficits observed in individuals chronically exposed to this pesticide.

Attentional performance, impulsivity, and related neurotransmitter systems in apoE2, apoE3, and apoE4 female transgenic mice.

RATIONALE: The apolipoprotein E (apoE) genotype influences cognitive performance in humans depending on age and sex. While the detrimental role of the apoE4 isoform on spatial learning and memory has been well-established in humans and rodents, less is known on its impact on the executive functions. OBJECTIVES: We aimed to evaluate the effect of apoE isoforms (apoE2, apoE3, apoE4) on visuospatial attention and inhibitory control performance in female transgenic mice, and to determine the neurochemical and neuropharmacological basis of this potential relationship. METHODS: Female mice carrying apoE2, apoE3, and apoE4 were trained in the five-choice serial reaction time task (5-CSRTT). Upon a stable performance, we manipulated the inter-trial interval and the stimulus duration to elicit impulsive responding and engage attention respectively. We further performed a pharmacological challenge by administering cholinergic and GABAergic agents. Finally, we analyzed the levels of brain amino acids and monoamines by using reversed phase high-performance liquid chromatography (HPLC). RESULTS: ApoE4 mice showed a deficient inhibitory control as revealed by increased perseveration and premature responding. When attention was challenged, apoE4 mice also showed a higher drop in accuracy. The adverse effect of scopolamine on the task was attenuated in apoE4 mice compared to apoE2 and apoE3. Furthermore, apoE4 mice showed less dopamine in the frontal cortex than apoE2 mice. CONCLUSIONS: We confirmed that the apoE genotype influences attention and inhibitory control in female transgenic mice. The influence of apoE isoforms in the brain neuromodulatory system may explain the cognitive and behavioral differences attributable to the genotype.

Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons.

Fungicides are crucial for food protection as well as for the production of crops of suitable quality and quantity to provide a viable economic return. Like other pesticides, fungicides are widely sprayed on agricultural land, especially in wine-growing areas, from where they can move-off after application. Furthermore, residues of these agrochemicals can remain on crops after harvest and even after some food processing operations, being a major exposure pathway. Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce. In the present study, nine fungicides recently approved for agricultural uses in the EU - ametoctradin, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and pyraclostrobin - have been evaluated for their toxicity in primary cultured mouse cortical neurons. Exposure to 0.1-100µM for 7 days in vitro resulted in a dose-dependent toxicity in the MTT cell viability assay. Strobilurin fungicides kresoxim-methyl (KR) and pyraclostrobin (PY) were the most neurotoxic compounds (lethal concentration 50 were in the low micromolar and nanomolar levels, respectively) causing a rapid raise in intracellular calcium [Ca(2+)]i and strong depolarization of mitochondrial membrane potential. KR- and PY-induced cell death was reversed by the calcium channels blockers MK-801 and verapamil, suggesting that calcium entry through NMDA receptors and voltage-operated calcium channels are involved in KR- and PY-induced neurotoxicity. These results highlight the need for further evaluation of their neurotoxic effects in vivo.

Putative adverse outcome pathways relevant to neurotoxicity.

The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

IrOx-carbon nanotube hybrids: a nanostructured material for electrodes with increased charge capacity in neural systems.

Nanostructured iridium oxide-carbon nanotube hybrids (IrOx-CNT) deposited as thin films by dynamic electrochemical methods are suggested as novel materials for neural electrodes. Single-walled carbon nanotubes (SWCNT) serve as scaffolds for growing the oxide, yielding a tridimensional structure with improved physical, chemical and electrical properties, in addition to high biocompatibility. In biological environments, SWCNT encapsulation by IrOx makes more resistant electrodes and prevents the nanotube release to the media, preventing cellular toxicity. Chemical, electrochemical, structural and surface characterization of the hybrids has been accomplished. The high performance of the material in electrochemical measurements and the significant increase in cathodal charge storage capacity obtained for the hybrid in comparison with bare IrOx represent a significant advance in electric field application in biosystems, while its cyclability is also an order of magnitude greater than pure IrOx. Moreover, experiments using in vitro neuronal cultures suggest high biocompatibility for IrOx-CNT coatings and full functionality of neurons, validating this material for use in neural electrodes.

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.