UC Berkeley

Epilepsy, one of the most common neurological disorders, affects between 0.5 and 2 percent of the population worldwide. Post-traumatic epilepsy is one of the most difficult forms of epilepsy to treat and the mechanism leading to the characteristic hypersynchronous activity has yet to be elucidated. Previous clinical studies have shown that perturbations in the blood-brain barrier seen after brain injury may be associated with epileptic activity in these patients. We previously demonstrated that albumin is critical in the generation of epilepsy following blood-brain barrier compromise and in this thesis TGF-Beta pathway activation is identified as the underlying mechanism.

We demonstrate that direct activation of the TGF-Beta pathway by TGF-Beta1 results in epileptiform activity similar to that following exposure to albumin. Co-immunoprecipitation revealed binding of albumin to TGF-Beta receptor II and Smad2 phosphorylation confirmed downstream activation of this pathway. Transcriptome profiling demonstrated similar expression patterns following BBB breakdown, albumin and TGF-Beta1 exposure, including modulation of genes associated with the TGF-Beta pathway, early astrocytic activation, inflammation, and reduced inhibitory transmission. Importantly, TGF-Beta pathway blockers suppressed most albumin-induced transcriptional changes and prevented the generation of epileptiform activity. Microarray data also revealed changes in many astrocytic genes following BBB disruption and albumin treatment including downregulation of glutamate transporters, glutamine synthetase, the potassium channel Kcnj10, and several connexins. Primary cortical cultures enriched for astrocytes were treated with albumin and confirmed these changes in gene expression, indicating a disruption in astrocytic glutamate and potassium buffering. Finally cell type specific changes in TGF-Beta signaling pathways were evaluated with primary cortical cultures enriched for astrocytes or neurons. In astrocytes, treatment with albumin resulted in preferential activation of the canonical TGF-Beta pathway mediated by the TGF-Beta type I receptor Alk5. Treatment resulted in an increase in Smad2 phosphorylation at 4 hours and an increase in Smad1 phosphorylation as well as Alk5 expression at 24 hours. In neurons, albumin treatment resulted in preferential activation of an alternate TGF-Beta pathway mediated by the TGF-Beta type I receptor Alk1. Treatment resulted in an increase in Smad1 phosphorylation at 4 and 24 hours as well as a small increase in Alk1 expression at 24 hours. In addition, TGF-BetaR2 expression was decreased in both cell types and TGF-Beta pathway blockers prevented astrocytic Smad2 phosphorylation. Our present data identifies the TGF-Beta pathway as a novel putative epileptogenic signaling cascade and therapeutic target for the prevention of injury-induced epilepsy.