Sindbis Virus-Induced Neuronal Death Is both Necrotic and Apoptotic and Is Ameliorated by N-Methyl-d-Aspartate Receptor Antagonists

Neuronal death is a tightly regulated process that is necessary for proper development of the nervous system. However, neuronal death that is inappropriate, either in timing or extent, is also involved in production of disease associated with neurodegeneration, stroke, and trauma (50). Similar to cell death in other tissues, neuronal death can be characterized as either apoptotic or necrotic. Apoptotic cell death, a caspase-dependent programmed cell death, is important for the elimination of unnecessary or potentially harmful cells and involves nuclear and cytoplasmic condensation, intranucleosomal DNA cleavage, and blebbing of the cell into membrane-bound apoptotic bodies. Necrotic, or lytic, cell death occurs following intense cellular injury and is associated with swelling of the cell body, increases in cellular volume, changes in plasma membrane permeability, and release of cellular contents into the extracellular space. The types of morphological changes that occur during neuronal death depend on the developmental state of the neuron and on the cell death stimulus (34, 50). Sindbis virus (SV) is an enveloped, single-stranded, positive-sense RNA alphavirus related to eastern, western, and Venezuelan equine encephalitis viruses, important causes of acute mosquito-borne encephalitis in the Americas (55). SV causes fever, rash, and arthritis in humans but causes an age-dependent encephalitis in mice and serves as a model for studying viral encephalitis and neuronal damage caused by the encephalitic alphaviruses (18). SV induces apoptotic cell death in vitro and in vivo (35–37, 45, 60), but characteristic apoptotic changes are not always evident in neurons induced to die by alphavirus infection (17, 19, 24, 51). As determined by caspase-3 activation, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling positivity, and morphological changes, apoptotic neurons are present in the hippocampi of infected animals (24; T. Kimura and D. E. Griffin, submitted for publication). However, swollen neurons without condensed apoptotic nuclei can also be detected in this region, and SV-induced motor neuron death does not appear to be apoptotic (24). Therefore, the mechanism of SV-induced death appears to differ according to the type and maturity of the infected neuron (24, 36). Neuronal excitotoxicity is mediated by excessive or prolonged activation of excitatory amino acid receptors and is involved in the pathogeneses of ischemic brain injury, epilepsy, and neurodegenerative diseases. Glutamate is an excitatory amino acid neurotransmitter that triggers neuronal death when it is present in excess quantities (11, 49). Excess glutamate overstimulates α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-, kainate-, and N-methyl-d-aspartate (NMDA)-type glutamate receptors, resulting in an influx of Ca2+, Na+, and Zn2+ ions through channels gated by these receptors. The resulting elevation in intracellular Ca2+ activates phospholipases, oxidases, nitric oxide synthase, proteases, and phosphatases and leads to lethal metabolic derangements (references 34 and 47 and references therein). In many types of mature neurons, glutamate-induced Ca2+ influx is mediated predominantly by NMDA receptors, and the treatment of primary neuronal cultures with NMDA receptor antagonists protects the cells from glutamate-induced death (10). Furthermore, the ischemic release of glutamate can cause lethal excitation of surrounding neurons. Frequently, a mix of both apoptotic and necrotic morphological changes follow ischemic injury (34). Excitotoxicity has been implicated in the pathogeneses of some virus-induced diseases of the central nervous system. Lentivirus- and measles virus-induced central nervous system damage may result, at least in part, from excitotoxic neuronal death (4, 15, 16, 20, 39–41, 54, 57, 58). Interestingly, virus antigen-negative apoptotic neurons can be detected following alphavirus infection, suggesting that uninfected cells also die during the infection process (2; Kimura and Griffin, submitted). Using SV infection of primary cortical neurons as an in vitro system of SV-induced neuronal death, the morphological changes that occur in infected and uninfected neurons in the same culture were examined. Treatment with NMDA receptor antagonists revealed that the excitatory amino acid neurotransmitter glutamate contributes to SV-induced neuronal death.