Abstracts

Rationale: CNS infection is a common cause of epilepsy that is often refractory to established antiseizure drugs. We have developed a novel mouse model of infection-induced epilepsy that offers a unique opportunity to study the molecular mechanism(s) underlying epileptogenesis and to identify novel therapeutic strategies. Theiler’s murine encephalomyelitis virus (TMEV) infected C57BL/6J mice show acute behavioral seizures between 3 and 7 days post-infection (dpi), exhibit pathological and physiological changes (gliosis, neurodegeneration in CA1, and increased excitation in CA3 pyramidal neurons) in the hippocampus, survive the infection, and develop epilepsy after a latent period. TMEV infection increases mRNA expression of proinflammatory cytokines, especially tumor necrosis factor-alpha (TNF-α), in the brain during the acute seizure period. In several systems, TNF-α has been shown to increase excitatory neurotransmission by upregulating the insertion of AMPAR subunits on the post-synaptic membrane via TNFR1. Therefore, we hypothesized that an increase in TNF-α following TMEV infection would increase the levels of AMPAR subunits on the neuronal membrane and contribute to hyperexcitability in TMEV-infected mice.Methods: Mice were injected with either TMEV or PBS and monitored for acute seizure behavior. In one group of mice, we microdissected hippocampi (n=5-6) at 5 dpi and measured the protein expression of various inflammatory modulators including TNF-α by multiplex electroluminescence immunoassay. In a second group of mice, we measured the membrane expression of GluA1 and GluA2 subunits of AMPAR by cell surface biotinylation assays in acute hippocampal slices (n=6) at 5 dpi followed by SDS-PAGE and immunoblotting.Results: We found >200 fold increase in TNF-α protein levels in the hippocampus obtained from TMEV-infected mice compared to control. The levels of other inflammatory modulators such as IFNγ, IL6, IL1β, and IL10 were also increased significantly in the TMEV-infected group. In addition, biotinylation assays revealed that the ratios of surface/total protein expressions of GluA1 and GluA2 were elevated in the TMEV group, while the total protein levels of both subunits were decreased.Conclusions: These data suggest that increased levels of TNF-α may contribute to synaptic scaling of AMPARs in the hippocampus, which in turn may contribute to increased seizure susceptibility following TMEV infection. Future work will evaluate the role of TNF-α. This work was funded by R01 NS065434 (KSW & HSW), Margolis Foundation (KSW & JAW), Skaggs Scholar Program Grant (KSW & MP), and American Epilepsy Society (DCP).