Chemotherapy-induced peripheral neuropathy (CIPN) develops in about a half of all cancer patients receiving anti-neoplastic drugs, such as the microtubule stabilizer paclitaxel (PTX). Because there is no effective treatment, painful CIPN is one of the most common causes of early termination of chemotherapy. We found, in time-lapse imaging studies of cultured mouse sensory neurons expressing mitochondria-targeting fluorescent protein (mitoDendra), that (i) the reduction of distal neurite mitochondrial density, emerging within 3 hours of PTX exposure, constitutes one of the earliest signs of PTX-CIPN (PCIPN) in vitro and precedes subsequent neurite degeneration; and (ii) the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxyamide ribonucleotide (AICAR), which promotes mitochondrial biogenesis via activation of peroxisome proliferatoractivated receptor-γ coactivator (PGC) 1α, mitigates these phenotypes. In this R21 application, we will take advantage of our in vitro and in vivo PCIPN models to determine, with particular focus on the AMPK-PGC1α axis, the mechanism by which AICAR counteracts this condition. SA-I: To assess the impact of AICAR and PGC1α activation on PCIPN in vitro - AICAR acts on PGC1α via activation of AMPK, which also modulates tau and glycogen synthase kinase 3β involved in the regulation of microtubule stability and thus may affect the PCIPN phenotypes. To gain insights into the disease pathogenesis and the mechanism of action of AICAR, we will assess temporal changes of these proteins in cultured sensory neurons treated with PTX, AICAR, or both. To correlate the molecular data obtained with the PCIPN phenotypes, we will grow mitoDendra sensory neurons in a compartment culture system, which allows us to spatiotemporally evaluate changes of neurites and their mitochondria. To determine the significance of activating PGC1α, the neurons will also be treated with nicotinamide riboside, which activates PGC1α independent of AMPK. SA-II: To evaluate the therapeutic potential of AICAR and PGC1α overexpression for PCIPN in vivo - To compare therapeutic efficacy at different stages of PCIPN, AICAR or vehicle will be given before, at the time of, or after inducing PCIPN in the mice. Outcome measures will include histological and a series of nociception assays. To determine the significance of the PGC1α pathway, PCIPN in mice overexpressing PGC1α in sensory neurons under the inducible promotor will be compared with PCIPN in non-induced littermates treated with or without AICAR. Study Impact: The proposed studies will provide novel insights into the molecular and cellular mechanisms and new therapeutic strategies for PCIPN. The results obtained will be advanced, in our future research, to new preclinical studies in animals, targeting more disease-specific molecules, and further elucidating the disease mechanisms. Upon in vivo validation of its efficacy and safety in our pre-clinical study, AICAR, which is readily-available as a nutritional supplement and has anti-tumor effects, would be translated rapidly to the clinic.