PROJECT SUMMARY/ABSTRACT The rationale for this proposal is that there is currently no specific medication that prevents or reverses diabetic neuropathy in humans and this is a major gap in scientific knowledge. Oxidative stress and mitochondrial (Mt) dysfunction are recognized as important causative factors in neurodegenerative disease and in diabetic neuropathy. Our central hypothesis is that nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are precursors that in the presence of nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) increase tissue NAD+ levels in the peripheral nervous system. This in turn activates SIRT1, and in turn downstream transcription factors, which differentially regulate specific Mt complexes to optimize Mt respiration and prevent Mt degeneration. Our objectives are to determine if NR or NMN can be used as a therapy for experimental diabetic neuropathy, determine if the SIRT1- PGC-1α signaling pathway provides molecular targets for treatment of neuropathy, identify potential Mt respiratory chain targets that may respond to treatment, determine if the axonal enzyme that converts NMN to NAD, Nmnat2, is present in regenerating axons in skin biopsies from diabetic animals and human subjects and if measurement of Nmnat2 may be useful as a marker for response to NR. In the Methods, we will use a variety of molecular, electrophysiology, and pathology tools to achieve the aims of the study. Animal models of type 1 and 2 diabetes will be used to determine the effect of NAD+ and SIRT1 overexpression on neuropathy. In isolated Mt or whole DRG neurons we will manipulate NAD+ and SIRT1 to assess the effect on overall Mt function and specific Mt respiratory complexes. Nmnat2 levels will be determined in control and age and gender matched skin biopsies from subjects with different severity of diabetes and diabetic neuropathy. The preliminary findings support the overall objectives of the proposal and provide promising evidence that NR and NMN would provide a potential therapy for diabetic neuropathy and that NAD+ activation of the SIRT1- PGC-1α signaling pathway is important in regulating Mt respiration. The status of the project based on our recent manuscripts shows that PGC-1α has a key role in regulating Mt function in diabetic neuropathy and that knockdown of PGC-1α intensifies the neuropathy. This novel research will examine the upstream activator of PGC-1α, namely SIRT1 in diabetic neuropathy and help further explore a new potential therapy (NR) that can be taken into clinical studies in a timely manner.