Opposing actions of hippocampus TNFα receptors on limbic seizure susceptibility

Abstract

Resected epileptic tissues exhibit elements of chronic neuroinflammation that include elevated TNFα and increased TNFα receptor activation, but the seizure related consequences of chronic TNFα expression remain unknown. Twenty four hours after acute limbic seizures the rat hippocampus exhibited a rapid upregulation of TNFR1, but a simultaneous downregulation of TNFR2. These limbic seizures also evoked significant increases in measures of neuroinflammation and caused significant neuronal cell death in both the hilus and CA3 of the hippocampus. In order to mimic a state of chronic TNFα exposure, adeno-associated viral vectors were packaged with a TNF receptor 1 (TNFR1) specific agonist, human TNFα, or a TNF receptor 1/2 agonist, rat TNFα. Subsequently, chronic hippocampal overexpression of either TNFR ligand caused microglial activation and blood-brain barrier compromise, a pattern similar to limbic seizure-induced neuroinflammation. However, no evidence was found for neuronal cell death or spontaneous seizure activity. Thus, chronic, in vivo TNFα expression and the subsequent neuroinflammation alone did not cause cell death or elicit seizure activity. In contrast, chronic hippocampal activation of TNFR1 alone significantly increased limbic seizure sensitivity in both amygdala kainic acid and electrical amygdala kindling models, while chronic activation of both TNFR1 and TNFR2 significantly attenuated the amygdala kindling rate. With regard to endogenous TNFα, chronic hippocampal expression of a TNFα decoy receptor significantly reduced seizure-induced cell death in the hippocampus, but did not alter seizure susceptibility. These findings suggest that blockade of endogenous TNFα could attenuate seizure related neuropathology, while selective activation of TNFR2 could exert beneficial therapeutic effects on in vivo seizure sensitivity.

Keywords: AAV; Adeno-associated virus; Epilepsy; Neuroimmune; Neuropathology; Seizures; TNF receptors; TNF-alpha; TNF-α; TNFR; adeno-associated virus; tumor necrosis factor receptor; tumor necrosis factor-alpha.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1. Seizures alter TNFR1 and TNFR2 protein expression and immune activation in the hippocampus

Panels A and B show immunostaining of TNFR1 and TNFR2, respectively, in the dorsal hippocampus in control and kainic-acid treated rats 24 h post-treatment. Panel C shows western blots probing TNFR1 and TNFR2 expression in whole hippocampus homogenates in rats at 0, 3, 8, or 24 h post-kainic acid. β-actin expression was used to determine protein loading. Panel D shows average relative TNF-α receptor expression, based on densitometric analysis. The inset indicates a strong inverse correlation of TNFR1 protein expression to TNFR2 expression across all animals. Panel E shows western blots probing MHC-II and CD11-b expression in whole hippocampus homogenates in rats at 0, 3, 8, or 24 h post-kainic acid. Panels F and G show average relative MHC-II and CD11-b expression, respectively, based on densitometric analysis. ***, *, Significantly different from untreated control group (P

Figure 2. Rat TNFR2 is targeted by rat, but not human TNFα

Panel A, top: diagram of the chimeric rat TNFR2 extracellular domain linked to an IgG Fc domain. A linker peptide allows for flexibility of the receptor extracellular domain. Panel A, bottom left: Rat TNFR2:Fc binds to rat TNFα (rTNFα), preventing luciferase expression in κB-luciferase transfected HeLa cells. Panel A, bottom right: Rat TNFR2:Fc does not bind to human TNFα (hTNFα), which activates luciferase expression in κB-luciferase transfected HeLa cells. Panel B shows relative light units read in HeLa cell lysates. GFP/GFP refers to the condition in which conditioned media from GFP transfected cells (control for rTNFα or hTNFα) was incubated with conditioned media from GFP transfected cells (control for TNFR2:Fc). ***, Significantly different from GFP/GFP group (P

Figure 3. Effect of chronic TNFα expression on hippocampal inflammation, IgG extravasation and neuronal viability

Representative images show the dorsal hippocampi from AAV5-GFP, rTNFα, or hTNFα-treated rats immunostained for MHC-II, CD11-b, IgG, or NeuN. There was very high MHC-II expression in rTNFα-treated rats, throughout, but limited to, the hippocampus. There was much less relative MHC-II expression in the hippocampus of hTNFα-treated rats, but MHC-II-positive cells still were present throughout the AAV-transduced area. No MHC-II-positive cells were found in GFP-treated rats. CD11b showed a similar pattern as MHC-II, but immunoreactivity was less robust. IgG extravasation was markedly elevated in both TNFα-treated groups as compared to the GFP-treated control. An arrow indicates the needle tract seen in the GFP-treated animal which illustrates the relative lack of any injection effect alone on IgG extravasation. However, there was no evidence of cell death (NeuN). Panel B shows that IgG was significantly elevated in both the rTNF-α and hTNF-α overexpressing groups versus the GFP controls. ***, *, Significantly different from untreated control group (P

Figure 4. Effects of rat or human TNF-α expression on hippocampal TNFR1 and TNFR2

Panel A shows western blots probing TNFR1 and TNFR2 expression in whole hippocampus homogenates in rats 1 week post-AAV delivery. The AAV-based transgenes are listed as column heads. Panel B shows the densitometric analysis of western blot results, which clearly show a relative decrease of TNFR1 protein after chronic rTNFα or hTNFα expression, but no change in TNFR2 expression. ***, *, Significantly different (P

Figure 5. Effect of chronic TNFα expression on limbic seizures

Panel A shows that after amygdala delivery of kainic acid to AAV5-GFP, rTNF-α, or hTNF-α treated rats, hTNF-α treated rats exhibited significantly more class IV seizures than either GFP or rTNF-α treated rats. Panel B shows that compared to GFP treated rats a greater number of stimulations was necessary to elicit three class IV seizures in rTNF-α treated rats, whereas fewer stimulations were necessary to elicit three class IV seizures in hTNFα-treated rats. Panel C shows that the average duration of class IV seizures in kindled rats was longer in hTNFα-treated rats. ***, *, Significantly different from GFP group. Error bars represent the standard error of the mean.

Figure 6. In vitro validation and in vivo effect of soluble decoy TNFR1:Fc expression on seizure-induced neuronal cell death and limbic seizures

Panel A shows that in vitro, TNFR1:Fc blocks the function of rat and human TNFα, preventing apoptosis in WEHI 164 clone 13 cells. ***, Significantly different from GFP/GFP group (P < 0.01). Panel B shows that kainic acid-induced cell death is reduced in the hilus and CA3 in rats previously administered AAV5-TNFR1:Fc in the dorsal hippocampus as compared to AAV5-GFP- treated rats. Panel C shows that kainic acid-induced seizures, amygdala kindling, and average kindled seizure duration were similar in AAV5-TNFR1:Fc-treated and –GFP-treated rats. ***, *, Significantly different from GFP control (P < 0.01, < 0.05, respectively). Error bars represent the standard error of the mean.