Abstracts

RATIONALE: Tuberous Sclerosis Complex (TSC) is a multi-system genetic disease characterized by abnormal cellular proliferation and differentiation. Neurologic manifestations are usually quite severe and include epilepsy, developmental delay, mental retardation, and autism. While these features are generally attributed to dysfunction of neurons, we postulated a more central role for astrocytes in the pathophysiology of TSC. Our laboratory recently developed a mouse model of TSC utilizing Cre-LoxP technology to generate an astrocyte-specific conditional knockout of [italic]Tsc1[/italic] ([italic]Tsc1[/italic]^GFAP cKO). These animals have frequent seizures, hippocampal pathology and increased astrocyte proliferation (See accompanying abstract I). Our objective in this study was to explore astrocyte medicated mechanisms of epilepsy in TSC. We hypothesized that the loss of [italic]Tsc1[/italic] in astrocytes triggers epileptogenesis via alterations of synaptic processing in the hippocampus. We further hypothesized that this mechanism involves abnormal astrocytic glutamate uptake.
METHODS: Hippocampal brain slices from 1-3 month old [italic]Tsc1[/italic]^GFAP cKO and control mice were prepared for extracellular recordings using standard methods. Synaptic responses from CA3 and CA4 pyramidal layers were evoked by mossy fiber stimulation. Whole brain and derivative astrocyte cultures were also used to study expression patterns of proteins critical for glutamate transport by Western blot and immunohistochemistry.
RESULTS: Electrical stimulation of dentate gyrus mossy fibers in control hippocampal slices produced paired-pulse facilitation in regions CA3 and CA4 as expected. Paired-pulse facilitation however, was not seen in CA3 and CA4 regions from [italic]Tsc1[/italic]^GFAP cKO mice. The convulsant 4-aminopyridine produced comparable epileptiform activity in [italic]Tsc1[/italic]^GFAP cKO and control slices. To investigate a possible role of altered glutamate transport, we examined expression in derivative cell cultures and intact brain slices. We observed differential down-regulation of glutamate transporter proteins in derivative cultured astrocytes from [italic]Tsc1[/italic]^GFAP cKO mice. Whole brain lysates and intact slices also had down-regulation of glutamate transporters by Western blot and immunohistochemistry.
CONCLUSIONS: [italic]Tsc1[/italic]^GFAP cKO mice develop progressive epilepsy at an early age. Our data demonstrate abnormalities in hippocampal synaptic transmission and down-regulation of astrocyte glutamate transporters. These results support a primary role for astrocytes in TSC epileptogenesis and suggest impaired glutamate homeostasis as one possible mechanism.
[Supported by: Tuberous Sclerosis Alliance (DHG, HO, DJK, LB), NIH NS36996 (DHG), NIH NS31535 & NS24279 (DJK), NIH NRSA (EJU), NIH 5K12NS0169004 (MW)]