Diabetic neuropathy is a major complication of diabetes mellitus and the leading cause of nontraumatic limb amputations. Many different biochemical perturbations have been implicated in the pathogenesis of diabetic neuropathy, e.g., oxidative stress, hypoxia and ischemia, activation of the polyol pathway, increase advanced glycation end products, activation of protein kinase C and NFkB and mitogen-activated kinases, and growth factor deficiency. We recently found that diabetes is associated with the appearance of proinsulin-positive cells in the bone marrow (Kojima et al., 2004). These proinsulin-positive bone marrow-derived cells (PI-BMDCs) also coexpress tumor necrosis factor (TNF)-a. In diabetes, the hyperglycemia-induced PI-BMDCs circulate to the liver where they form fusion cells with hepatocytes (Fujimiya et al., 2007). In mice with streptozotocin- induced diabetes, the PI-BMDCs also migrate to and fuse with neurons in the sciatic nerve and dorsal root ganglion (DRG), leading to abnormal nerve function, premature apoptosis of neurons and diabetic neuropathy (Terashima et al., 2005). The goal of this proposal is to investigate the role of the PI-BMDCs using 4 Specific Aims: Aim 1. To examine the effect of the inhibition/inactivation of the pro-oxidative stress enzyme, poly(ADP- ribose) polymerase (PARP), by [a] an inhibitor, and [b] bone marrow transfer (BMT) of PARP-1-/- BM cells on diabetic neuropathy. The hypothesis to be tested is that the protective effect of PARP inhibition is mediated partly via bone marrow-derived cells and partly via a direct action on neurons. Aim 2: To develop a strategy that allows us to selectively ablate PI-BMDCs in streptozotocin-induced diabetic mice to examine its effect on the development of diabetic neuropathy. The hypothesis to be tested is that depletion of PI-BMDCs in diabetic mice partially protects against diabetic neuropathy. Aim 3: To examine the role of PI-BMDC-derived TNF-a in diabetic neuropathy by performing BMT from TNF-a-/- donor mice. The hypothesis to be tested is that TNF-a expressed by PI-BMDCs in diabetes contributes to neuronal fusion, dysfunction and premature apoptosis, events that are important to diabetic neuropathy. Aim 4: To develop an in vitro system that recapitulates the effect of high glucose on PI-BMDC formation and PI-BMDC-neuron cell fusion in vivo. The availability of a robust assay system will enable us to study PI-BMDC-neuron fusion under both in vivo and controlled in vitro conditions and to dissect the underlying mechanisms involved. The proposed research on the role and mechanism of high glucose-induced PI-BMDCs and PI-BMDC-neuron cell fusion may lead to a fundamental change in our understanding of the pathogenesis of diabetic neuropathy. PUBLIC HEALTH RELEVANCE: Multiple biochemical perturbations have been proposed to underlie diabetic neuropathy. We hypothesize that abnormal cell differentiation in the form of high glucose-induced proinsulin-producing bone marrow cells and oxidative stress processes and proinflammatory cytokines produced by these abnormal cells play important roles. We will study the molecular and cellular mechanisms involved in these processes and events.