Perfluorooctane sulfonate induces neuronal and oligodendrocytic differentiation in neural stem cells and alters the expression of PPARγ in vitro and in vivo

Perfluorinated compounds are ubiquitous chemicals of major concern for their potential adverse effects on the human population. We have used primary rat embryonic neural stem cells (NSCs) to study the effects of perfluorooctane sulfonate (PFOS) on the process of NSC spontaneous differentiation. Upon removal of basic fibroblast growth factor, NSCs were exposed to nanomolar concentrations of PFOS for 48 h, and then allowed to differentiate for additional 5 days. Exposure to 25 or 50 nM concentration resulted in a lower number of proliferating cells and a higher number of neurite-bearing TuJ1-positive cells, indicating an increase in neuronal differentiation. Exposure to 50 nM also significantly increased the number of CNPase-positive cells, pointing to facilitation of oligodendrocytic differentiation. PPAR genes have been shown to be involved in PFOS toxicity. By q-PCR we detected an upregulation of PPARγ with no changes in PPARα or PPARδ genes. One of the downstream targets of PPARs, the mitochondrial uncoupling protein 2 (UCP2) was also upregulated. The number of TuJ1- and CNPase-positive cells increased after exposure to PPARγ agonist rosiglitazone (RGZ, 3 μM) and decreased after pre-incubation with the PPARγ antagonist GW9662 (5 μM). RGZ also upregulated the expression of PPARγ and UCP2 genes. Meanwhile GW9662 abolished the UCP2 upregulation and decreased Ca2 + activity induced by PFOS. Interestingly, a significantly higher expression of PPARγ and UCP3 genes was also detected in mouse neonatal brain after prenatal exposure to PFOS. These data suggest that PPARγ plays a role in the alteration of spontaneous differentiation of NSCs induced by nanomolar concentrations of PFOS

In vitro models to study mechanisms of neural cell death induced by toxic agents

Neurotoxicity arises when exposure to toxic agents, either naturally occurring or manmade substances, alters the normal activity and/or structure of the nervous system. This can lead to disruption of vital metabolic processes and eventually to cell death. Certain unique features of the nervous system make it particularly vunerable to toxicants, e.g. its high demand of energy, high content of polyunsaturated fatty acids and low levels of antioxidant enzymes. In this thesis, we have investigated the mechanisms leading to neurotoxic cell death and the role of ion channels in neural apoptosis by using different in vitro experimental models. Such models offer unique advantages in elucidating mechanisms of toxicity, and hence are used to understand the consequences of exposure to toxicants. Occupational exposure to styrene in industrial workers has been associated with neurobehavioral deficits. Alterations of neurotransmitters and loss of neurons have also been observed in in vivo models. The main metabolite of styrene, styrene 7,8-oxide (SO), is believed to account for most of styrene s toxicity. Carbon monoxide (CO), an endogenous gas, plays important physiological roles, but CO poisoning due to accidental or intentional exposure, occurs frequently. CO has higher affinity for hemoglobin than oxygen, and brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity. However, the direct effect of CO on intracellular targets is still not well understood. We have characterized the cellular damage induced by SO and CO in different in vitro models. Our data show that SO causes apoptosis via activation of caspases and that its neurotoxic effects are related to mitochondrial damage and oxidative stress. CO induces hypoxia-independent apoptotic cell death via parallel activation of both caspases and calpains. Cell shrinkage is an early morphological feature occurring during apoptosis that is associated with an increased efflux of K+ and Cl- ions. We investigated the role of ion channels in differentiated and non-differentiated neural cells undergoing apoptosis. Our results point to a novel function of the voltage-dependent anion channel in the plasma membrane (pl-VDAC) playing a role in the early phase of neuronal apoptosis. In contrast, pl-VDAC is scarcely seen in apoptotic cortical neural stem cells and instead, an amiloride-sensitive Na+-channel is activated. Thus, it appears that neurons and neural stem cells utilize different apoptotic strategies. With appropriate in vitro models we have been able to characterize the intracellular pathways affected by SO and CO, and to demonstrate activation of different ion channels during apoptosis in undifferentiated and differentiated neural cells. There is an increasing consensus on in vitro models being useful tools to test neurotoxic agents and dissect their mechanisms of action. In fact, by using multiple cell models it is possible to recognize specific patterns of toxicity of different neurotoxicants and use this information for risk assessement

Hippocampal Neurons Exposed to the Environmental Contaminants Methylmercury and Polychlorinated Biphenyls Undergo Cell Death via Parallel Activation of Calpains and Lysosomal Proteases

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants commonly found as contaminants in the same food sources. Even though their neurotoxic effects are established, the mechanisms of action are not fully understood. In the present study, we have used the mouse hippocampal neuronal cell line HT22 to investigate the mechanisms of neuronal death induced by MeHg, PCB 153, and PCB 126, alone or in combination. All chemicals induced cell death with morphological changes compatible with either apoptosis or necrosis. Mitochondrial functions were impaired as shown by the significant decrease in mitochondrial Ca²+ uptake capacity and ATP levels. MeHg, but not the PCBs, induced loss of mitochondrial membrane potential and release of cytochrome c into the cytosol. Also, pre-treatment with the antioxidant MnTBAP was protective only against cell death induced by MeHg. While caspase activation was absent, the Ca²+-dependent proteases calpains were activated after exposure to MeHg or the selected PCBs. Furthermore, lysosomal disruption was observed in the exposed cells. Accordingly, pre-treatment with the calpain specific inhibitor PD150606 and/or the cathepsin D inhibitor Pepstatin protected against the cytotoxicity of MeHg and PCBs, and the protection was significantly enhanced when the two inhibitors were combined. Simultaneous exposures to lower doses of MeHg and PCBs suggested mostly antagonistic interactions. Taken together, these data indicate that MeHg and PCBs induce caspase-independent cell death via parallel activation of calpains and lysosomal proteases, and that in this model oxidative stress does not play a major role in PCB toxicity

Dexamethasone enhances oxidative stress-induced cell death in murine neural stem cells

Contains fulltext : 110873.pdf (publisher's version ) (Closed access)Glucocorticoids (GCs) are essential for normal brain development; however, there is consistent evidence that prenatal exposure of the fetal brain to excess GCs permanently modifies the phenotype of neuronal cells. In this paper, the murine-derived multipotent stem cell line C17.2 was used, as an in vitro model, to investigate the impact of GCs on neural stem cell survival. Our results indicate that dexamethasone (Dex) increases the sensitivity of murine neural stem cells (NSCs) to 2,3-methoxy-1,4-naphthoquinone-induced apoptosis, and this effect could be blocked by the glucocorticoid-receptor (GR) antagonist mifepristone, strongly suggesting the involvement of the GR. Furthermore, our results show that Dex decreases cell number and induces a G1-arrest. We hypothesized that the mitochondria are the main target of Dex. Interestingly, after treatment with Dex, 72% of the investigated genes involved in the mitochondrial respiratory chain are down-regulated, as well as 29% of the genes encoding for antioxidant enzymes. In conclusion, using the C17.2 cell line as a model to study developmental neurotoxicity in vitro, we have shown that GCs can increase cellular sensitivity to oxidative stress and alter the phenotype of NCSs