Category A. Neuroscience and Neurodegenerative Diseases
A1. KU596 Improves Mitochondrial Bioenergetics and Decreases Oxidative Stress in Diabetic Sensory Neurons via Hsp70
Zhenyuan You1, Brian Blagg2, Rick T. Dobrowsky1
1Department of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, USA; 2Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA
Neuronal mitochondrial dysfunction is a key pathophysiologic mechanism of diabetic peripheral neuropathy (DPN). In vivo treatment with the heat shock protein (Hsp) 90 modulator KU596 can reverse symptoms of DPN and this correlates with improving mitochondrial bioenergetics (mtBE) in diabetic sensory neurons in an Hsp70 dependent manner. However, we have not determined the mechanism by which KU596 improves mtBE. The goal of the current work is to determine if KU596 improves mtBE by decreasing glucose-induced oxidative stress. Primary adult sensory neurons from non-diabetic or 14 week diabetic wild type (WT) and Hsp70 knock out mice were isolated and treated +/- 1μM KU596 overnight in medium containing 6.1 mM glucose. Hyperglycemia was then induced in some cultures for 24 hrs by raising the total glucose concentration to 26.1 mM. We then studied the drug effect on cellular respiration and oxidative stress. In WT neurons, KU596 significantly improved mtBE in non-diabetic and diabetic sensory neurons under both normal and hyperglycemic conditions. Surprisingly, although KU596 increased mtBE in Hsp70 KO neurons isolated from diabetic mice and maintained under normal glucose conditions, the drug was not able to improve mtBE when these neurons where placed in high glucose medium. These results suggest that KU596 does not require Hsp70 to improve mtBE in cells incubated in low glucose but that induction of hyperglycemia requires Hsp70 for the drug to improve mtBE. We next determined if this effect was related to an inability of KU596 to decrease oxidative stress under hyperglycemic conditions. Sensory neurons from the diabetic WT and Hsp70 KO mice were incubated with KU596 under low and high glucose conditions and the levels of mitochondrial superoxide assessed by fluorescent microscopy. In WT and Hsp70 KO neurons, superoxide levels were significantly increased in cell incubated with 26.1 mM glucose. Although KU596 decreased the ability of hyperglycemia to increase superoxide levels in WT neurons, this effect was lost in the hyperglycemically stressed Hsp70 KO neurons. Collectively, these data suggest that the ability of KU596 to increase mtBE in diabetically stressed neurons is linked to an Hsp70-dependent inhibition of glucose-induced superoxide production.
A2. Modulating Molecular Chaperones with KU-596 Suppresses c-jun Expression and Improves Motor Function in MPZ-Raf Mice
Xinyue Zhang1, Chengyuan Li1, Brain S. Blagg2, and Rick T. Dobrowsky1
1Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS, USA; 2Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA
Demyelinating neuropathies can result from Schwann cell (SC) dedifferentiation upon loss of axonal contact or injury. Recent evidence suggests that c-jun is at the center of promoting Schwann cell dedifferentiation. Elevated c-jun levels have been detected in a variety of human neuropathies suggesting that it may be a potential target for preventing or slowing the demyelination process. We previously demonstrated that modulation of heat shock protein 90 (Hsp90) with the small molecule Hsp90 modulator KU-32 could decrease c-jun expression and demyelination in SC-neuron co-cultures in an Hsp70-dependent manner. In the current study, we utilized a transgenic mouse model (MPZ-Raf) in which injection of tamoxifen (TMX) leads to activation of the Raf kinase pathway in SCs, elevated c-jun expression, demyelination and subsequent motor dysfunction. With this model, we sought to determine whether modulating heat shock proteins with KU596, a second generation Hsp90 modulator was sufficient to improve the motor neuropathy that develops in these mice. Treating MPZ-Raf mice with KU-596 decreased the induction of c-jun, improved motor performance on the rotorod, decreased the onset of rear-limb paralysis and improved the extent of peripheral nerve demyelination. Collectively, our data indicate that modulating heat shock proteins could be beneficial in attenuating the onset and severity of c-jun-induced demyelinating neuropathies.