Abstracts

Rationale: Understanding the molecular basis of epilepsy remains a primary goal in the pursuit of efficacious pharmaceutical treatments. Currently, all drugs target the relief of symptoms, with 30% of patients refractory to any medication. We have shown that signaling via brain-derived neurotrophic factor (BDNF) induction of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway alters GABA signaling in a Temporal Lobe Epilepsy (TLE) animal model. Although many JAK inhibitors exist on the market to treat cancer and autoimmune disease, their use in epilepsy therapy has not been explored. Previous studies show that certain JAK inhibitors can prevent development of spontaneous seizures in TLE animal models. However, a detailed description of how the BDNF and JAK/STAT pathways interact is missing and could be critical for our understanding of epileptogenesis and its future treatment and prevention. Recently, our laboratory discovered what may be a unique intracellular association between JAK2 and the BDNF receptor p75NTR in primary cultured neurons treated with BDNF. Our studies presented here are aimed at identifying the binding domain of this JAK2/p75NTR complex, its cellular localization, and potential role in BDNF-mediated JAK/STAT activation. Canonical JAK/STAT signaling predicts that the interaction between JAK2 and p75NTR should take place at the intracellular domain of membrane bound p75NTR, however, our results do not support this classical model and indicate a novel role of intracellular p75NTR in the sequestration of JAK2 through an alternate interaction surface. Methods: Primary cultured rat cortical neurons were used to generate proteins for co-immunoprecipitation (co-IP) assays to demonstrate endogenous p75NTR binding to JAK2. In order to determine the critical region of p75NTR or JAK2 necessary for binding to its partner, we co-transfected human embryonic kidney cells (HEK-293) with a series of full length and truncated forms of p75NTR and JAK2 and performed Co-IP. Studies are underway to identify key amino acids involved in the formation of the p75NTR/JAK2 protein complex via site directed mutagenesis of full-length transgenes so that we can directly test the function of these protein interactions in epileptogenesis. Results: Endogenous co-IP experiments in neurons in addition to previously presented work with Proximity Ligation Assays (PLA) demonstrate that the full-length p75NTR BDNF receptor associates with JAK2. PLA showed that this binding occurred not at the membrane, as would be expected, but rather, inside the cytoplasm and even in the nucleus. In Canonical JAK/STAT signaling, JAK binds to the C-terminus of a membrane bound receptor. However, we now show that overexpression of this fragment of p75NTR does not associate with JAK2 as seen with the full-length transgene (n=4, p < .0001).  Conclusions: Our results suggest that the BDNF-induced JAK/STAT pathway may display novel binding and cellular localization features in neurons that remain to be discovered and may inform more effective drug design for TLE.  Funding: NIGMS T32GM008541, NINDS R01NS051710, NINDS R21 NS083057, CURE Epilepsy Multidisciplinary Award