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Comparative Study
. 2004 Aug 25;24(34):7566-75.
doi: 10.1523/JNEUROSCI.2002-04.2004.

Viral-induced spinal motor neuron death is non-cell-autonomous and involves glutamate excitotoxicity

Jessica Darman  1 , Stephanie Backovic, Sonny Dike, Nicholas J Maragakis, Chitra Krishnan, Jeffrey D Rothstein, David N Irani, Douglas A Kerr
Affiliations
Comparative Study

Viral-induced spinal motor neuron death is non-cell-autonomous and involves glutamate excitotoxicity

Jessica Darman et al. J Neurosci. .

Abstract

Neuroadapted Sindbis virus (NSV) is a neurotropic virus capable of inducing the death of spinal motor neurons in mice and rats. In this study we investigated the mechanisms that underlie NSV-induced motor neuron death. We found that many degenerating spinal motor neurons were not infected directly with NSV, suggesting that bystander cell death occurs. An excitotoxic mechanism was confirmed when blockade of calcium-permeable AMPA receptors attenuated motor neuron death both in vitro and in vivo. Blockade of astroglial glutamate reuptake potentiated NSV-induced motor neuron loss in vivo, suggesting that astrocyte-mediated removal of perisynaptic glutamate is important in limiting NSV-induced excitotoxic injury. Astroglial glutamate transport was reduced markedly in the spinal cord during NSV infection, in advance of motor neuron injury in susceptible mice. In contrast, we found 5.6-fold elevated glutamate uptake in the spinal cords of mice resistant to NSV-induced paralysis. Likewise, minocycline markedly increased spinal cord glutamate transport and protected mice from NSV-induced motor neuron death. These studies suggest that NSV infection triggers a cascade of events in the spinal cord resulting in impaired astrocytic glutamate transport and excitotoxic injury of motor neurons mediated via calcium-permeable AMPA receptors. Similar changes may occur in other motor neuron disorders such as amyotrophic lateral sclerosis or West Nile Virus-induced poliomyelitis, suggesting a common tissue injury pathway.

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Figures

Figure 3.
Figure 3.
In vivo blockade of glutamate transporters and glutamate receptors alters NSV pathogenesis in rats. Sprague Dawley rats with cannulas surgically implanted into their spinal subarachnoid spaces were given NASPM, THA, or PBS at a rate of 0.5 μl/hr for 7 d by an Alzet mini-osmotic pump. A, Hindlimb grip strength of cannulated rats given THA, a specific inhibitor of the astrocyte glutamate transporter GLT-1, is not altered by the drug alone but contributes to a more drastic reduction of grip strength after NSV challenge. B, THA infusion also resulted in significantly increased motor neuron loss in NSV-infected rats when compared with saline-infused controls. C, Hindlimb grip strength of NSV-infected rats administered NASPM, a synthetic analog of JST (inhibitor of GluR2, excluding AMPA receptors); when compared with saline-infused controls, the NSV-infected rats did not develop any hindlimb weakness in response to NSV infection. D, NASPM infusion also resulted in significant preservation of motor neurons after NSV infection.
Figure 4.
Figure 4.
Expression of the astrocyte glutamate transporter GLT-1 in the spinal cords of NSV-infected mice. A, C57BL/6 mice develop paralysis in response to NSV. The decline in hindlimb grip strength correlates with the classic paralysis observed in the NSV model of rodent infection after NSV infection. B, Immunohistochemical detection of astrocyte glutamate transporter GLT-1 (brown staining, left panels) as well as the astrocyte marker GFAP (brown staining, right panels) in the lumbar enlargement of NSV-infected mice. The transient decrease in GLT-1 staining at day 6 after infection (DPI) is not correlated with a loss of GFAP immunoreactivity, implying a selective downregulation of GLT-1 expression. C, GLT-1 expression in lysates from lumbar and cervical spinal cords of NSV-infected mice by Western blot analysis. The intensity of the GLT-1 band decreases over the time course in the lumbar and, to a lesser degree, in cervical spinal cordlysates. GFAP and neurofilament (NF) protein immunoreactivity (SMI-31) was relatively unchanged over this time course while Ponceau S staining of membranes confirmed equivalent loading of lanes (LC). D, Quantitation of GLT-1 immunoreactivity by Western blots in spinal cordlysates of NSV-infected mice. As early as 3 d after infection, the GLT-1 expression was reduced significantly compared with either GFAP or SMI-31 expression. Asterisks indicate that intensity of GLT-2 is significantly different from GFAP at indicated time points (p < 0.05). E, Functional loss of GLT-1-mediated glutamate uptake involved in NSV infection. Spinal cord homogenates were generated at various times after infection from the lumbar region. Total and DHK-sensitive (GLT-1-mediated) glutamate transport was assessed with data normalized to uptake by uninfected, control samples. DHK-sensitive transport was decreased at days 2-6 after infection (*p < 0.05) relative to day 0.
Figure 5.
Figure 5.
GLT-1 expression in a strain of mouse (BALB/cBy) resistant to NSV-induced paralysis. A, Hindlimb grip strength measurements in BALB/cBy mice infected with NSV show that these animals do not develop hindlimb weakness over the time course of NSV infection. B, Motor neuron counts from the lumbar spinal cords of NSV-infected BALB/cBy mice at baseline and day 6 after infection. C, Immunoblot analysis of GLT-1 expression and Ponceau S staining in the BALB/c mouse. D, Quantitative analysis of triplicate immunoblots revealed preservation of GLT-1 expression after NSV infection. E, Baseline levels of functional glutamate uptake in the BALB/cBy mouse are significantly higher than in the C57BL/6 mouse. F, Functional glutamate uptake declines by 6 d after infection in the BALB/cBy mice, although levels remain consistently above those observed in the C57BL/6 mouse. Error bars: A, B, D, F, not significant (p > 0.05); E, p < 0.05.
Figure 1.
Figure 1.
Evidence for bystander destruction of noninfected motor neuron cells during neuroadapted Sindbis virus infection. C57BL/6 mice were infected with an NSV construct expressing GFP. A, B, Lumbar enlargement of an NSV-infected C57BL/6 mouse at 2 d after infection. Infected cells are visible by the expression of GFP (green), while neurons are visualized with the Nissl counterstain (red). C, D, Lumbar enlargement of an NSV-infected C57BL/6 mouse at 4 d after infection showing many abnormally appearing neurons with swollen nuclei and intracellular vacuoles. None of these motor neurons exhibited immunoreactivity to GFP or to NSV capsid proteins (data not shown), suggesting that these neurons have not been infected directly. E, Electron micrograph of an infected lumbar enlargement at 4 d after infection. Long arrow denotes a synapse onto the cell body, confirming its neuronal identity. Swollen cytoplasm and nucleoplasm are observed, along with dilated, disrupted mitochondria (short arrows). F, ChAT staining (red) in an NSV-infected (green) lumbar enlargement at 4 d after infection. G, Quantitation of the percentage of motor neurons infected by GFP-expressing NSV (left axis) compared with the total number of motor neurons per section (right axis in green). Infection status was determined both by GFP expression and immunoreactivity to NSV glycoproteins. Error bars, p < 0.05.
Figure 2.
Figure 2.
Bystander destruction of cultured spinal neurons by neuroadapted Sindbis virus. A, Infection of mixed spinal neurons cultured on a monolayer of astrocytes with NSV-GFP (green) at 24 hr after infection, counter stained with GFAP (red; 20×). Images were deconvolved by Simple PCI software. B, At 24 hr after treatment with NSV-GFP, the cultures were treated with propidium iodide (PI; red). Several cells are PI-positive, yet only one is infected with NSV (green). C, Nitrotyrosine reactivity (red) indicates the presence of reactive oxygen species. At 24 hr after NSV-GFP infection, the cultures were fixed with 4% paraformaldehyde and stained with a nitrotyrosine-specific antibody. D, Mixed cultures were treated with NSV-GFP in the presence of AMPA-type glutamate receptor blocking agents. At 48 hr after infection, those cultures treated with NSV-GFP alone revealed widespread death of spinal neurons, whereas parallel cultures treated with CNQX or JST showed preservation of spinal neurons. E, Quantitation of spinal motor neuron protection mediated by blockage of AMPA-type glutamate receptors. Survival of motor neurons treated with NSV and any of the three AMPA blockers is greater than with NSV alone (*p < 0.01 at 48 and 72 hr after infection).
Figure 6.
Figure 6.
GLT-1 expression in minocycline-treated, NSV-infected C65BL/6 mice. A, Hindlimb grip strength in NSV-infected mice is preserved with minocycline treatment. B, Motor neurons are protected in the lumbar spinal cord in NSV-infected minocycline-treated, NSV-infected mice. C, Immunoblot analysis of GLT-1 expression in minocycline-treated, NSV-infected mice reveals sustained GLT-1 expression with Ponceau S staining as a loading control (LC). D, Quantitative analysis of triplicate immunoblots shows a preservation of GLT-1 expression after NSV infection in minocycline-treated mice. Asterisks indicate that the intensity of GLT-1 from minocycline-treated, NSV-infected mice is significantly different from GLT-1 in untreated, NSV-infected mice at indicated time points (p < 0.05). E, Functional GLT-1-mediated glutamate uptake is increased in the lumbar spinal cord of C57BL/6 mice treated with minocycline (*p < 0.05; DHK day 6 relative to day 0). F, In NSV-infected mice treated with minocycline, functional glutamate uptake is unchanged over time, suggesting that minocycline and NSV have opposite effects that cancel each other.
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