Neuropathy is the most frequent symptomatic complication of diabetes, affecting 50-60% of the greater than 100 million patients worldwide. A large and still confusing body of work suggests that hyperglycemia leading to increased polyol flux and oxidant stress and formation of advanced glycation products as well as depletion of neurotrophic factors in the absence of insulin signaling may initiate diabetic neuropathy. The objective of this proposal is to test the role of the different families of mitogen-activated protein kinases (MAPK's) as mediators of diabetic neuropathy. Members of the superfamily of MAPK's, especially the cJun N-terminal kinases (JNK's) are potently activated by diverse cell stresses and published studies provide compelling evidence for a pro-apoptotic role of the JNK's in neurons. We hypothesize that MAPK's, especially the JNK's, are activated in the setting of diabetes and signal neuronal dysfunction and apoptosis which is manifested as neuropathy. To test this hypothesis, we will employ readily-transfected PC-12 cells as well as primary sympathetic and sensory neurons from mice in which specific JNK and MEKK genes have been disrupted to: 1) characterize the ability of trophic factor withdrawal, hyperglycemia, oxidant stress and different combinations of these stresses to induce dysfunction or apoptosis in neuronal PC-12 cells, cultured dorsal root ganglion (DRG) and superior cervical ganglion (SCG) neurons as well as their ability to regulate different MAPK pathways including the extracellular signal-regulated kinases (ERK's), JNK's, p38 MAPK's and ERK5; 2) define the role of the diabetic stress-regulated MAPK's in neuronal apoptosis and dysfunction using cultured DRG and SCG neurons isolated from JNK and MEKK-deficient mice as well as recombinant viral expression vectors encoding inhibitory forms of MAPK pathway components; 3) employ JNK/MEKK-knockout mice to test the in vivo role of the JNK pathway in diabetes-associated neuropathy. Physiologic endpoints including the hyperalgesia response as well as in vivo measurements of neuronal morphology and apoptosis will be assessed. Completion of this project will define MAPK pathways that are regulated in the setting of diabetes and potentially serve as mediators of the multiple cell stresses that putatively initiate diabetic neuropathy. As a result, the studies will highlight specific MAPK pathways as novel targets for therapeutic intervention in this highly debilitating complication of diabetes.
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