Abstracts

Rationale: Human hypothalamic hamartoma (HH) is a developmental malformation often characterized by gelastic seizures that are refractory to antiepileptic drugs. Our recent study using surgically-resected HH tissues has revealed that a proportion of HH neurons are non-GABAergic neurons with an immature phenotype. This suggests that these HH neurons may have relatively low expression of the outwardly-directed K+-Cl- co-transporter (KCC2) and a relatively high intracellular Cl- concentration. As a result, the inhibitory neurotransmitter GABA rather plays an excitatory role in these immature HH neurons, potentially leading to gelastic seizures. Thus, to elucidate the molecular mechanisms underlying GABA-induced neuronal excitation and epileptogenesis in HH will aid the selection of superior strategies for drug development to better manage this disease. Methods: Frozen human hypothalamic tissues (controls) were acquired through Harvard Brain Tissue Resource Center. HH tissues were obtained from patients who were surgically treated through the Barrow HH Program in our hospital. Total proteins were extracted from these tissues, quantitated, and resolved by SDS-PAGE. Immunoblotting (Western blot) was performed with commercially available antibodies using standard methods. Immunoblotting signals were detected with chemiluminescent substrates, and images were captured using a Kodak Molecular Imager. Total RNAs were extracted from tissues and used for cDNA synthesis. Quantitative PCR was performed using Platinum SYBR Green qPCR Supermix UDG Kit (Invitrogen) on an iQ5 Real-Time PCR Detection System (Bio-Rad). Beta-actin was used as an internal control. Results: Proteins including KCC2, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) (p44 and p42 mitogen-activated protein kinases (MAPKs)) were expressed in both human hypothalamic and HH tissues, which were readily detected by immunoblotting with anti-KCC2 and anti-ERK antibodies, respectively. When assessing the phosphorylation signals by immunoblotting with anti-phosphotyrosine (pTyr) and anti-phospho-ERK (pERK) antibodies, elevated overall tyrosine phosphorylation and activation of ERK1/2 were found in HH tissues compared to normal hypothalamic control tissues. Furthermore, both real-time RT-PCR and immunoblotting results showed that KCC2 expression (mRNA and protein) levels were significantly lower in HH than those in control tissues (P<0.05). Conclusions: Our findings suggest that reduced KCC2 expression in HH could result in an elevated intracellular Cl- concentration and consequent GABA-mediated neuronal excitation in HH. Activation of the Ras/Raf/MEK/ERK pathway and other phosphorylation signaling cascades could be one of the mechanisms underlying KCC2 down-regulation in HH, which is currently under investigation in our laboratories.