Abstracts

Rationale: The mammalian Target of Rapamycin (mTOR) signaling pathway integrates growth factor and nutritional signals to regulate protein sythesis. Upregulation of the mTOR pathway in the brain occurs in tuberous sclerosis complex (TSC) due to mutations in either Tsc1 or Tsc2 genes, in which brain involvement causes early-onset epilepsy, mental retardation and autism. However, the mTOR pathway also plays an important role in neuronal plasticity in normal brain. mTOR activation increases surface expression of AMPA receptors, while inhibition of mTOR with rapamycin blocks late LTP. BDNF-dependent activation of mTOR increases translation of GluR1, NR1 and NR3A. The immature brain has increased susceptibility to seizures and later epilepsy, and demonstrates higher activity of the mTOR pathway. Here we aimed to determine whether seizure-induced upregulation of the mTOR signaling pathway might play a role in epileptogenesis in normal non-TSC immature brain using an established rodent model of neonatal hypoxic seizures. Methods: Long Evans rat pups were exposed to global hypoxia (graded decline from 7-4% over 15 min) at postnatal day (P)10 generating tonic clonic seizures. Pups were euthanized at 1h, 3h, 12h, 24h, and 48h after seizures. Expression of mTOR signaling cascade proteins in cortex and hippocampus was evaluated using Western blot techniques and immunocytochemistry. Results: Our Western blot data showed that cortical BDNF was increased 1h after seizure, compared to littermate controls (n=8, p<0.01). Phospho-Erk (Thr202/Tyr204) and phospho-Akt (Thr308), which activate the mTOR pathway, were upregulated at 3h after seizure in both cortex and hippocampus (n=4 in cortex, n=8, 6 in hippocampus, respectively, p<0.05). Rheb and phospho-mTOR (Ser2448) (p-mTOR), which regulate activation of protein translation, were also increased 3h after seizure in cortex (n=5, 9, respectively, p<0.05). Both p-mTOR targets, phospho-4E binding protein 1 (Thr37/46), the inactivated form of the translational repressor, and phospho-p70 S6 kinase (Thr389) (p-p70S6K), were increased at 12h in both cortex and hippocampus (n=11, 8, respectively, p<0.05). The ribosomal protein phospho-S6 (Ser235/236), which is the critical marker indicating mTOR pathway activation and is activated by p-p70S6K, was increased 24h post-seizure in both cortex and hippocampus (n=9, 12, respectively, p<0.01). Immunocytochemical double labeling with pS6 and neuronal marker NeuN on coronal brain sections indicated increases in pS6 reactivity and distribution in CA1 and CA3 regions of hippocampus, motor and sensory cortex, especially in pyramidal cell layer V and the subplate 24h after seizure (n=5).