New mechanistic pathways in the development of Schwann cell Injury in diabetes : role of the NADPH oxidases, LXR and mTOR pathways -

Abstract

Background: Diabetic neuropathy (DN) is the most common debilitating complication of diabetes affecting more than 50percent of patients. It is associated with impaired nerve conduction, abnormal thermal perception, axonal atrophy, demyelination, blunted regenerative potential, and loss of nerve fibers. However, the exact mechanisms underlying such complications are still not known. Although reactive oxygen species have been established as the main pathway of cellular injury in diabetic neuropathy, the mechanisms by which they cause their effects need to be more elucidated. Aim: We will investigate the role of ROS generated by the NADPH oxidases family of enzymes in mediating biological responses in Schwann cells including phenotypic changes such as deregulation of myelin gene expression (P0 and PMP22) and apoptosis. We will study the role of ROS production in the alteration of the signaling pathways involving LXR and mTOR and the crosstalk among these pathways in the mediation of cell injury.Methods: Raised beam walking test was used to assess behavioral malfunction in diabetic animals. Dihydroethidium (DHE) and 2’, 7’-dichlorodihydrofluorescin (DCF) diacetate were used for the detection of intracellular ROS in sciatic nerves and Schwann cells (MSC80) respectively, while NADPH oxidase activity assay helped in the specification of the source of ROS. RT-PCR allowed the measurement of mRNA levels of Nox3, Nox4, PMP22, P0, and LXR-β. Western blots were used to assess the protein expression levels of NADPH oxidases and myelin proteins as well as mTOR, P70S6K and LXR- β. DNA fragmentation-ELISA test was used to study apoptosis in Schwann cells. Results: Hyperglycemia causes motor defects at the level of the peripheral nerves and is associated with an alteration in P0 and PMP22 levels. NADPH oxidases levels and activity are increased and result in the production of ROS. High glucose induces the activation of mTOR-P70S6K suggested to play a role in Schwann cell injury. LXR expression is decreased whic