Apoptotic cell death in neural stem cells exposed to toxic stimuli

University dissertation from Stockholm : Karolinska Institutet, Institute of Enviromental Medicine

Abstract: Neural stem cells (NSCs) play an important role in the developing nervous system and in the adult brain, where mitotic regions such as the subventricular zone (SVZ) remain active. In spite of the intense ongoing research on NSCs, we still need to expand the knowledge on biochemical regulation by which NSCs undergo cell death in the course of normal physiology or in response to neurotoxic insults. Also, before the full potential of NSCs can be appreciated, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. We have studied the general apoptotic machinery in NSCs. As experimental models we used primary cultures of adult NSCs (aNSCs) from the SVZ of the adult rat brain, and the neural stem cell line C17.2, initially derived from the developing mouse cerebellum. Our data show that NSCs undergo apoptosis in response to the pan-kinase inhibitor staurosporine, or to agents inducing oxidative stress such as 2,3-dimethoxy-1,4-naphthoquinone. Exposed cells exhibit apoptotic nuclear morphology, phosphatidylserine translocation to the outer leaf of the plasma membrane, cytochrome c release, caspase activation and DNA fragmentation. Additionally our results suggest that extensive oxidative stress causes p53 accumulation and activation of caspase-2, which in turn regulates the mitochondrial apoptotic signaling. Our findings show the importance of the intrinsic mitochondria-mediated pathway in NSC apoptosis induced by toxic stimuli. Both aNSCs and C17.2 cells express the Fas receptor, but exposure to agonistic antibodies fails to induce apoptosis. It is known that Fas not only induces apoptosis, but also can deliver growth stimulatory signals through activation of the extracellular-signal regulated kinase (ERK) pathway. The Fas-induced ERK phosphorylation that we detect in C17.2 cells, suggests that in NSCs Fas may function as a mediator of growth rather than death. There is still little understanding about how neurotoxicants affect the developing nervous system, especially at low-dose exposures. Hence, we have investigated the toxic effects of the environmental neurotoxicants methylmercury (MeHg) and manganese (Mn) in C17.2 cells and primary embryonic cortical NSCs (cNSCs). Our results show that NSCs are more sensitive to both MeHg and Mn than differentiated neuronal or glial cells. Both toxicants induce apoptosis via Bax-activation, cytochrome c release, and activation of downstream caspases. In addition, a parallel calpain-dependent cell death pathway could be detected upon MeHg exposure. Remarkably, exposure to MeHg at concentrations lower than observed in cord blood of Swedish pregnant women inhibits spontaneous neuronal differentiation of NSCs, via activation of the Notch signaling pathway. In conclusion this study shows that NSCs are a highly sensitive model system for in vitro developmental neurotoxicity studies and offer new perspectives for evaluating the biological significance of low level exposures to neurotoxicants.

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