We have two goals in our neurobiological studies. Our first goal has been to identify, in neurons, depolarization-induced immediate-early genes (IEGs) that play a role in synaptic plasticity and to characterize the biochemical mechanisms by which these IEGs mediate synaptic function. Our second goal has been to identify, in neuronal precursors, IEGs induced preferentially by Nerve Growth Factor (NGF) versus other stimulating ligands and to characterize the biochemical mechanisms by which these NGF-induced IEGs mediate NGF-driven neuronal differentiation. We identified synaptotagmin IV (Syt IV) as an IEG induced by depolarization in the hippocampus, prepared anti-Syt IV antibodies and showed that Syt IV is a labile protein incorporated into synaptic vesicles after induced synthesis, where it can form oligomers with Syt I. We created a Syt IV knock-out mouse, and demonstrated that Syt IV (-/-) mice are deficient in acquiring two hippocampal-dependent learning tasks, but not in acquiring two amygdala-associated learning tasks. We subsequently identified four protein kinases (KID-l, PIM-1, SIK, MAPKAP-2) and two transcription factors (nurr1, rTLE3) induced by depolarization. These genes are additional candidate IEGs whose products may mediate synaptic plasticity. We also identified six messages preferentially induced by NGF (versus Epidermal Growth Factor) in the PC12 cell model system of neuronal differentiation. We demonstrated that one of these IEGs, the urokinase plasminogen activator receptor (UPAR), is required for NGF-driven PC12 cell morphological differentiation and secondary response gene expression, using UPAR antisense oligonucleotides and anti-UPAR antibody. We will determine the biochemical basis for Syt IV modulation of synaptic function and collaborate on studies of electrophysiological correlates of the Syt IV (-/-) behavioral deficits. We will determine if Syt IV and our depolarization-induced IEGs are induced via the CREB pathway, and whether they modulate depolarization-induced, calcium-dependent exocytosis. For those IEGs that appear to be good candidates for modulators of synaptic function, we will prepare knock-out mice and analyze their behavioral characteristics. To determine the role of UPAR in NGF-driven neuronal maturation, we will examine development of sympathetic and sensory ganglia in UPAR null mice. We will also use cultured peripheral ganglion cells from BAX/NGF null mice to characterize the role of UPAR in NGF-driven differentiation. Finally, we will determine mechanisms by which (i) NGF induces UPAR and (ii) UPAR modulates NGF-driven neuron differentiation.
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