PFOS induces behavioral alterations, including spontaneous hyperactivity that is corrected by dexamfetamine in zebrafish larvae

Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae

NRXN1 deletion and exposure to methylmercury increase astrocyte differentiation by different notch-dependent transcriptional mechanisms

Controversial evidence points to a possible involvement of methylmercury (MeHg) in the etiopathogenesis of autism spectrum disorders (ASD). In the present study, we used human neuroepithelial stem cells from healthy donors and from an autistic patient bearing a bi-allelic deletion in the gene encoding for NRXN1 to evaluate whether MeHg would induce cellular changes comparable to those seen in cells derived from the ASD patient. In healthy cells, a subcytotoxic concentration of MeHg enhanced astroglial differentiation similarly to what observed in the diseased cells (N1), as shown by the number of GFAP positive cells and immunofluorescence signal intensity. In both healthy MeHg-treated and N1 untreated cells, aberrations in Notch pathway activity seemed to play a critical role in promoting the differentiation toward glia. Accordingly, treatment with the established Notch inhibitor DAPT reversed the altered differentiation. Although our data are not conclusive since only one of the genes involved in ASD is considered, the results provide novel evidence suggesting that developmental exposure to MeHg, even at subcytotoxic concentrations, induces alterations in astroglial differentiation similar to those observed in ASD

Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy

Background: Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. Methods: To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. Results: The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1alpha (HIF-1alpha) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. Conclusion: In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways

Blocking interleukin-1 signalling in the brain-structural and functional outcomes.

The aims of this thesis were to evaluate the consequences of blocking interleukin (IL)-1 signalling in the brain. The studies used a transgenic mouse model with brain-directed overexpression of the human soluble isoform of IL-1 receptor antagonist with the GFAP promoter (Tg hsIL-1ra). In the first study we investigated the impact of blocking IL-1 signalling in the brain, and we found no compensatory increase in expression of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha). Moreover, ageing had a much stronger effect on cytokine expression than blocking IL-1 signalling per se.Similarly, we found that APP and PS1 levels varied in opposite directions with ageing in both transgenic and WT animals. Direct volumetric measurements showed that Tg hsIL-1ra mice have smaller brains than age-matched wild-type (WT) animals. In the second study we analysed the role of IL-1 signalling in regulating neurogenesis and gliosis. The experimental setting consisted of either acute (seizures) or chronic (ageing) neuroinflammation. We found that overexpressing hsIL-ra in the brain impairs the generation of new neurones in the subgranular zone of the dentate gyrus, and that the step most tightly controlled by IL-1 is the terminal neuronal differentiation. Similarly, blocking IL-1 signalling resulted in much attenuated variations in the levels of expression of glial activation markers in both acute and chronic neuroinflammation. In addition, the Tg hsIL-1ra mice displayed higher levels of microglial activation markers under basal conditions, but no variations either following seizures or in ageing as compared to WT mice. For the third study we used both littermate and non-littermate mice in order to control for the effect of prenatal exposure to hsIL-1ra, and analysed the behavioural profile at the ages of 6 and 12 months, as well as the morphology of the brain at 12 months of age. Multivariate data analysis was used for detecting significant separation according to the genotype (WT vs. Tg hsIL-1ra) and parenting (littermates vs. non-littermates), as well as for studying the correlation between brain morphology and behavioural pattern. We found that continuous overexpression of hsIL-1ra in the brain results in an altered behavioural profile characterised by lower anxiety and higher exploratory activity. The prenatal exposure to hsIL-1ra induced alterations in the behavioural pattern detectable at the age of 6 months, but not at 12 months. The differences in brain morphology explained most of the altered behavioural profile, and the brain-restricted overexpression of hsIL-1ra and the prenatal exposure to the transgene product had cumulative effects. In the fourth study we investigated the mechanisms by which interfering with IL-1 signalling results in learning deficits. We found that the consolidation of long-term memory is impaired both in the dentate gyrus and in the retrosplenial cortex. The underlying mechanisms are related to synaptic consolidation, including BDNF expression and synaptic plasticity. In conclusion, we have shown that IL-1 signalling is essential for the appropriate development of the brain, but its blocking does not result in compensatory increase in the expression of proinflammatory cytokines in the adult brain. IL-1 is also playing an important role in synaptic plasticity, and the deficit induced by prenatal exposure to IL-1ra results in an altered behavioural profile that can be recovered from, provided the IL-1 signalling is intact

Long-term consequences of prenatal stress and neurotoxicants exposure on neurodevelopment

There is a large consensus that the prenatal environment determines the susceptibility to pathological conditions later in life. The hypothesis most widely accepted is that exposure to insults inducing adverse conditions in-utero may have negative effects on the development of target organs, disrupting homeostasis and increasing the risk of diseases at adulthood. Several models have been proposed to investigate the fetal origins of adult diseases, but although these approaches hold true for almost all diseases, particular attention has been focused on disorders related to the central nervous system, since the brain is particularly sensitive to alterations of the microenvironment during early development. Neurobiological disorders can be broadly divided into developmental, neurodegenerative and neuropsychiatric disorders. Even though most of these diseases share genetic risk factors, the onset of the disorders cannot be explained solely by inheritance. Therefore, current understanding presumes that the interactions of environmental input, may lead to different disorders. Among the insults that can play a direct or indirect role in the development of neurobiological disorders are stress, infections, drug abuse, and environmental contaminants. Our laboratories have been involved in the study of the neurobiological impact of gestational stress on the offspring (Dr. Antonelli´s lab) and on the effect of gestational exposure to toxicants, mainly methyl mercury (MeHg) and perfluorinated compounds (PFCs) (Dr. Ceccatelli´s lab). In this focused review, we will review the specialized literature but we will concentrate mostly on our own work on the long term neurodevelopmental consequences of gestational exposure to stress and neurotoxicants.Fil: Antonelli, Marta Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Pallares, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Ceccatelli, Sandra. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Spulber, Stefan. Karolinska Huddinge Hospital. Karolinska Institutet; Sueci

Molecular Hydrogen Reduces LPS-Induced Neuroinflammation and Promotes Recovery from Sickness Behaviour in Mice

<div><p>Molecular hydrogen has been shown to have neuroprotective effects in mouse models of acute neurodegeneration. The effect was suggested to be mediated by its free-radical scavenger properties. However, it has been shown recently that molecular hydrogen alters gene expression and protein phosphorylation. The aim of this study was to test whether chronic <em>ad libitum</em> consumption of molecular hydrogen-enriched electrochemically reduced water (H-ERW) improves the outcome of lipopolysaccharide (LPS)-induced neuroinflammation. Seven days after the initiation of H-ERW treatment, C57Bl/6 mice received a single injection of LPS (0.33 mg/kg i.p.) or an equivalent volume of vehicle. The LPS-induced sickness behaviour was assessed 2 h after the injection, and recovery was assessed by monitoring the spontaneous locomotor activity in the homecage for 72 h after the administration of LPS. The mice were killed in the acute or recovery phase, and the expression of pro- and antiinflammatory cytokines in the hippocampus was assessed by real-time PCR. We found that molecular hydrogen reduces the LPS-induced sickness behaviour and promotes recovery. These effects are associated with a shift towards anti-inflammatory gene expression profile at baseline (downregulation of TNF- α and upregulation of IL-10). In addition, molecular hydrogen increases the amplitude, but shortens the duration and promotes the extinction of neuroinflammation. Consistently, molecular hydrogen modulates the activation and gene expression in a similar fashion in immortalized murine microglia (BV-2 cell line), suggesting that the effects observed <em>in vivo</em> may involve the modulation of microglial activation. Taken together, our data point to the regulation of cytokine expression being an additional critical mechanism underlying the beneficial effects of molecular hydrogen.</p> </div

Analysis of activation and gene expression profile in BV-2 cells after 2 h of exposure to LPS.

<p> (A) Quantification of activation. The BV-2 cells cultured in H-ERW-DMEM display a smaller proportion of activated cells and a larger proportion of resting cells after exposure to LPS for 2 h. (B) Gene expression regulation after 2 h exposure to LPS. In BV-2 cells grown in H-ERW-DMEM, the upregulation of TNF-α is higher, while HO-1 is not downregulated by LPS (* - p<0.05 two-tailed t-test).</p