Long-term objective: Identify novel small molecule targets for preventing neuronal death in neurodegenerative disease. Hypothesis: The LAR tyrosine phosphatase regulates neuronal death and differentiation by modulating signal transduction mechanisms relevant to NGF function and apoptosis. Potential signaling components targeted by LAR include the MEK/ERK, PI-3/AKT, TrkA and JNK kinases. Elucidation of cause-and-effect mechanisms by which LAR counter-balances NGF signaling and promotes apoptosis will lead to novel approaches for inhibiting neuronal death and augmenting neurotrophin effects.
Specific aim 1. Establish functional interactions between LAR and TrkA signaling in PC12 cells. Establish the time course for increased NGF-induced TrkA autophosphorylation in LAR-deficient cells. Determine if decreased TrkA dephosphorylation is a candidate mechanism preventing apoptosis in serum-deprived LAR-deficient cells. Determine if co-immunoprecipitation of LAR and TrkA is regulated by NGF or induction of apoptosis. Use confocal microscopy to confirm LAR and TrkA co-localization. Use in vitro enzymatic assays to determine if LAR dephosphorylates TrkA.
Specific aim 2. Determine if LAR modulates activation of NGF signaling intermediates regulating PC12 neurite outgrowth. Determine whether baseline and NGF-induced activation states of key NGF signaling intermediates triggering neurite outgrowth are altered in LAR-deficient cells. Establish time courses for NGF-induced activation and deactivation of MEK and its substrate, ERK.
Specific aim 3. Determine if LAR modulates activation of signaling intermediates regulating PC12 survival. Determine if activation states of key signaling intermediates known to regulate PCl2 cell survival are altered following serum withdrawal. Measure phosphorylation and dephosphorylation of the following signaling intermediates at multiple time points following withdrawal: MEK, ERK, AKT and JNK.
Specific aim 4. Establish cause-and-effect relationships between LAR deficiency, changes in signaling intermediate activation and apoptosis. Measure the effects of the Trk inhibitors K252a and AG879, the MEK inhibitors PD098059 and U0126 and the PI-3/AKT inhibitor LY294002 on serum withdrawal-induced cell death in control and LAR-deficient cells. Further assess LAR-TrkA interactions by determining if LAR deficiency prevents apoptosis in PC12nnr5 cells lacking functional TrkA receptors.
Specific aim 5. Determine whether LAR deficiency inhibits neuronal apoptosis in vivo. Determine if the previously established increase in the number of DRG neurons in adult LAR -/- mice is due to decreased developmental cell death. Measure neuronal precursor proliferation rates and developmental death rates by quantitating BrdU- and TUNEL-positive cells in LAR +/+ and -/- DRG.
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