Activation of Divergent Neuronal Cell Death Pathways in Different Target Cell Populations during Neuroadapted Sindbis Virus Infection of Mice

Abstract

Infection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apoptotic, as judged by morphologic and biochemical criteria. This may be explained in part by the lack of detectable caspase-3 expression in these cells.Sindbis virus (SV) is an alphavirus, a member of the togavirus family. It is transmitted naturally to a variety of hosts by insect vectors and was originally isolated in 1952 from mosquitoes in Egypt (21). In the laboratory, SV causes an acute encephalomyelitis when inoculated into mice, leading to prominent neuronal infection. A strain of SV more neurovirulent for mice was developed by serial passage of the original isolate in mouse brain (6). Following intracerebral inoculation, this neuroadapted strain (NSV) causes a severe, often fatal, encephalomyelitis accompanied by prominent hindlimb paralysis in adult mice (7,8). Paralysis develops as a result of infection that spreads to motor neurons of the lumbosacral spinal cord which innervate the hindlimb musculature (7). The pathogenesis of this hindlimb paralysis is not completely understood; while some motor neuron degeneration has been observed in NSV-infected mice (8), other animals have been reported to recover neurologic function following infection (4, 7, 13). Our present study was carried out to determine whether paralysis results from the loss of function or the actual degeneration of infected lumbar motor neurons.We find that NSV-induced paralysis typically begins in C57BL/6 mice inoculated with 10 3 PFU of virus after 4 days of infection, and all mice show some degree of paralysis by day 5. Paralysis is initially mild with decreased hindlimb movement, but as the disease worsens, all animals become severely paralyzed and unable to move their hindlimbs in response to a pain stimulus. After 10 days, more than 90% of infected mice had died.To determine how lumbar motor neurons in NSV-infected mice were affected in comparison to the kinetics with which hindlimb paralysis developed, we examined 2-m-thick plasticembedded sections of spinal cord by light microscopy. The cell bodies of motor neurons in uninfected mice exhibited a characteristic morphology, with large nuclei, dispersed chromatin, and prominent nucleoli and Nissl substance (Fig. 1A). At day 3 of infection, most motor neurons had an overall morphology that was similar to that of uninfected cells. However, subtle pathologic changes, including mild swelling with an increase in cytoplasmic vacuolation, were noted in some of these cells (Fig. 1B). These changes progressed to severe abnormalities over several days and correlated with the onset and increasing severi...

The opioid receptor antagonist, naloxone, protects spinal motor neurons in a murine model of alphavirus encephalomyelitis

“…All lumbar spinal cord sections were taken at the same level to ensure the crosssectional area being compared between animals was similar. To assess the fate of lumbar MNs, the entire lumbar spinal column (SC plus adjacent nerve roots) was decalcified (Immunocal, Decal Corporation, Tallman, NY) and embedded in paraffin, after which sections were stained using our modified Bielschowsky silver staining method to label neurofilament proteins present in each nerve axon (Havert et al, 2000;Kerr et al, 2002;Nargi-Aizenman et al, 2004). MN axons were identified and counted in each lumbar ventral nerve root, a site where only MN axons travel after leaving the SC.…”

“…A neuroadapted strain of SV (NSV) produces a lethal disease in susceptible hosts that evolves over a period of 7-10 days (Jackson et al, 1988). Following direct intracerebral inoculation, NSV spreads rapidly from the brain to the spinal cord (SC), where it causes a progressive lower motor neuron (MN) paralysis similar to WNV (Havert et al, 2000;Jackson et al, 1987;Kelley et al, 2003). Like other alphaviruses and flaviviruses, NSV selectively targets neurons with minimal infection of glial cells (Johnson et al, 1972).…”

“…Neuronal fate determines outcome, with the neurovirulence of each SV strain directly related to the extent of neuronal destruction it produces (Lewis et al, 1996). As an added layer of complexity, different neuronal populations undergo different types of cell death following NSV infection (Havert et al, 2000;Lewis et al, 1996), and many non-infected neurons are also damaged via bystander mechanisms, believed to be glutamatemediated excitotoxicity (Nargi-Aizenman and Griffin, 2001). This bystander injury has led to the suggestion that host immune responses somehow contribute to NSV pathogenesis (Liang et al, 1999;Kimura and Griffin, 2000;Kimura and Griffin, 2003), although the degree to which neuronal damage occurs via host-derived rather than direct viral-induced mechanisms remains poorly understood.…”

Alphavirus Encephalomyelitis: Mechanisms and Approaches to Prevention of Neuronal Damage

“…Populations of mature neurons that are particularly susceptible to infection are in the hippocampus and anterior horn of the spinal cord. NSV-infected motor neurons die by a nonapoptotic process [10], and mice develop weakness that progresses to paralysis and death within 7-10 days [11,12].…”

Alternate functions of viral regulators of cell death

“…Sindbis virus infection of cortical neurons exhibits signs of apoptosis, whereas infected spinal cord motor neurons appear necrotic or autophagic and lack markers of apoptosis. [7][8][9][10] Elimination of virus-infected cells by natural killer cells and cytotoxic T-lymphocytes also involves programmed suicide mechanisms of the infected cell. 11 In some virus infections, such as HIV and neuroadapted Sindbis virus, even uninfected cells are prone to cell death.…”