Mol #71308 Mol #71308 2 Running Title: Abl Kinases Regulate Nicotinic Signaling in Autonomic Neurons Mol #71308 3

1 Title: Abelson family tyrosine kinases regulate the function of nicotinic acetylcholine receptors and nicotinic synapses on autonomic neurons. Abbreviations: Abelson family tyrosine kinase (AFK), Abelson family tyrosine kinase 1 (Abl1, c-Abl), Abelson family tyrosine kinase 2 (Abl2, Arg), non receptor tyrosine kinase (NRTK), Src family tyrosine kinase (SFK), postsynaptic density (PSD), nicotinic acetylcholine receptor (nAChR), Imatinib Mesylate (STI571), ciliary ganglion (CG), platelet derived growth factor receptor (PDGFR), stem cell factor receptor (c-Kit), Jasplakinolide, Jaspis johnstoni; C 36 H 45 BrN 4 O 6 (Jasplakinolide). Abstract Abelson family kinases (AFKs; Abl1, Abl2) are non-receptor tyrosine kinases (NRTKs) implicated in cancer, but they also have important physiological roles that include regulating synaptic structure and function. Recent studies using Abl-deficient mice and the anti-leukaemia drug, STI571 (Imatinib Mesylate, Gleevec; Novartis) which potently and selectively blocks Abl kinase activity, implicate AFKs in regulating presynaptic neurotransmitter release in hippocampus and postsynaptic clustering of nicotinic acetylcholine receptors (nAChRs) in muscle. Here, we tested whether AFKs are relevant for regulating nAChRs and nAChR-mediated synapses on autonomic neurons. AFK immunoreactivity was detected in ciliary ganglion (CG) lysates and neurons, and STI571 application blocked endogenous Abl tyrosine kinase activity. With similar potency, STI571 specifically reduced whole-cell current responses generated by both nicotinic receptor subtypes present on CG neurons (α3*-and α7-nAChRs) and lowered the frequency and amplitude of α3*-nAChR mediated excitatory postsynaptic currents. Quantal analysis indicated the synaptic perturbations were postsynaptic in origin, and confocal imaging experiments revealed they were unaccompanied by changes in nAChR clustering or alignment with presynaptic terminals. The results indicate that in autonomic neurons Abl kinase activity normally supports postsynaptic nAChR function to sustain nAChR-mediated neurotransmission. Such consequences contrast with the influence of Abl kinase activity on presynaptic function and synaptic structure in hippocampus and muscle, respectively, demonstrating a cell-specific mechanism of action. Finally, since STI571 potently inhibits Abl kinase activity, the autonomic dysfunction side effects associated with its use as a chemotherapeutic agent may result from perturbed α3*-and/or α7-nAChR function.