Abstracts

Rationale: Epilepsy is a common, progressive neurological disorder affecting 1-2% of the population. There is no known cure or biomarker of its progression. Although various therapies are available, 30-40% of patients remain medically intractable. In many cases, these patients have an underlying pathology known as cortical dysplasia (CD), a malformation of cortical development. We have recently shown that growth associated protein 43 (GAP-43), a marker of axonal growth and synaptic plasticity, is highly and differentially expressed in tissue resected from patients with CD associated with epileptogenesis. Here, we aimed to better understand the importance of this protein throughout epileptogenesis in a two-hit model of epilepsy. Methods: Pregnant rats were irradiated at embryonic day 17 to produce pups with multifocal CD as a first hit. At postnatal day 55, CD and non-CD rats underwent surgical implantation of EEG electrodes. Half of the rats were also injected with a lentiviral vector containing either shRNA against GAP-43, or a scrambled non-target control. After recovery, all rats received either saline, or 40 mg/kg, i.p. of pentylenetetrazole (PTZ) to trigger an acute seizure as a secondary insult. Animals were chronically monitored with video EEG and sacrificed at time points of 2 days, 15 days, 30 days, and 120 days. Results: The expression of GAP-43 protein was significantly higher in CD brains as compared to controls with saline injections (p=0.000). After PTZ-induced seizures, GAP-43 protein was elevated in both CD and non-CD brains, but it was reduced by day 15 in non-CD brains, and continuously increased in CD brains (p=0.04). The expression of GAP-43 protein within brain samples correlated with interictal spike frequency over the duration of epileptogenesis (R2 = 0.8649). GAP-43 serum levels were heightened in response to spontaneous seizures, which were present in 60% of CD animals by day 120 (p=0.028). None of the non-CD controls exhibited spontaneous seizures. Rats with GAP-43 knockdown had less severe acute seizures, and exhibited a decreased frequency of interictal spikes by day 15 (p=0.000). None of the knockdown animals had spontaneous seizures. GAP-43 proteins primarily co-localized with markers of glutamatergic neurotransmission as compared to GABAergic markers. Knockdown of GAP-43 also showed a decrease in PSD-95 protein, which may explain why acute seizures were inhibited in these animals. Together, these results provide new insight into a potential candidate biomarker for CD and epileptogenesis; as well as a target for the treatment and prevention of progressive epilepsy in CD. Conclusions: This study provides data supporting the role of a novel protein in epileptogenesis. GAP-43 expression is upregulated following an acute seizure in CD rat brains, and continuously increases over the progression of epilepsy. The differential expression of GAP-43 is correlated with interictal spike frequency, providing physiological evidence of its involvement in epileptogenesis. Furthermore, shRNA against GAP-43 leads to a decrease in seizure duration, severity, and interictal spikes over time. GAP-43 positive axons in epileptic dysplastic brains are more likely to create excitatory synapses than inhibitory, which may render the brain prone to seizures. Serum GAP-43 levels were also elevated in CD animals exhibiting spontaneous seizures. Our results advocate GAP-43 as a biomarker for localizing seizure foci in epileptic CD tissue and monitoring disease progression longitudinally. These data also provide support for targeting GAP-43 protein overexpression as a novel therapy and prevention of epilepsy and its progression. Funding: N/A