Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis

Abstract

Death of neuronal cells during development and following deprivation of trophic factors is known to occur via an active mechanism requiring RNA and protein synthesis, known as apoptosis. Apoptosis is a form of cell "suicide" whereby the cell decides its own fate by activating a genetic programme of cell death. In contrast, necrosis is a passive uncontrolled form of cell death often observed in response to a toxic insult. Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying neurotoxin-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which 6-hydroxydopamine, a specific neurotoxin for catecholaminergic cells, induces neuronal cell death in vitro. We report that 6-hydroxydopamine induces cell death in the neuronal PC12 cell line via a mechanism which has the characteristic morphological and biochemical hallmarks of apoptosis. PC12 cells induced to die by 6-hydroxydopamine treatment exhibited cell shrinkage, classical chromatin condensation and membrane blebbing. Analysis of DNA integrity from 6-hydroxydopamine-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. 6-Hydroxydopamine-induced apoptosis of PC12 cells was suppressed by desipramine, a monoamine uptake inhibitor, suggesting that 6-hydroxydopamine is initiating apoptosis via a specific intracellular mechanism. Aurintricarboxylic acid, a general inhibitor of nucleases, also suppressed 6-hydroxydopamine-induced apoptosis, suggesting the involvement of an endonuclease in the death pathway. The aetiology of idiopathic Parkinson's disease remains uncertain, although evidence suggests that endogenous and/or exogenous toxins may initiate neuronal cell death in this disease. The dopaminergic neurotoxin 6-hydroxydopamine is used to generate animal models of Parkinson's disease in vivo. We have demonstrated that this neurotoxin kills neuronal cells in vitro by an active process of apoptosis. Thus, the possibility exists that cell death in neurodegenerative diseases such as Parkinsonism also occurs in an active manner initiated by as yet unidentified environmental or metabolic toxins. Cell death that involves activation of an apoptotic programme can be modulated by addition of extracellular trophic factors, and is also controlled by the levels of intracellular factors. If neurotoxin-induced apoptosis plays a role in Parkinson's disease the implication is that the neuronal degeneration may be prevented by pharmacological manipulations.