C-Terminal Heat Shock Protein 90 Inhibitor Decreases Hyperglycemia-induced Oxidative Stress and Improves Mitochondrial Bioenergetics in Sensory Neurons

Diabetic peripheral neuropathy (DPN) is a common complication
of
diabetes in which hyperglycemia-induced mitochondrial dysfunction
and enhanced oxidative stress contribute to sensory neuron pathology.
KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular
chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple
sensory deficits associated with the progression of DPN and protects
unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically,
KU-32 increased the expression of Hsp70, and this protein was critical
for drug efficacy in reversing DPN. However, it remained unclear if
KU-32 had a broader effect on chaperone induction and if its efficacy
was linked to improving mitochondrial dysfunction. Using cultures
of hyperglycemically stressed primary sensory neurons, the present
study investigated whether KU-32 had an effect on the translational
induction of other chaperones and improved mitochondrial oxidative
stress and bioenergetics. A variation of stable isotope labeling with
amino acids in cell culture called pulse SILAC (pSILAC) was used to
unbiasedly assess changes in protein translation. Hyperglycemia decreased
the translation of numerous mitochondrial proteins that affect superoxide
levels and respiratory activity. Importantly, this correlated with
a decrease in mitochondrial oxygen consumption and an increase in
superoxide levels. KU-32 increased the translation of Mn superoxide
dismutase and several cytosolic and mitochondrial chaperones. Consistent
with these changes, KU-32 decreased mitochondrial superoxide levels
and significantly enhanced respiratory activity. These data indicate
that efficacy of modulating molecular chaperones in DPN may be due
in part to improved neuronal mitochondrial bioenergetics and decreased
oxidative stress.

糖尿病周围神经病变（Diabetic peripheral neuropathy, DPN）是糖尿病的常见并发症，高血糖诱导的线粒体功能障碍与氧化应激增强共同介导了感觉神经元的病理损伤。KU-32是一种基于新生霉素（novobiocin）的分子伴侣热休克蛋白90（heat shock protein 90, Hsp90）C端抑制剂。KU-32可改善糖尿病周围神经病变进展过程中出现的多种感觉功能缺陷，并保护无髓鞘感觉神经元免受葡萄糖诱导的毒性损伤。机制研究显示，KU-32可上调热休克蛋白70（Hsp70）的表达，该蛋白对于KU-32逆转糖尿病周围神经病变的药效发挥至关重要。然而，目前仍不清楚KU-32是否对伴侣蛋白的诱导表达具有更广泛的调控作用，也不确定其药效是否与改善线粒体功能障碍相关。本研究采用高糖应激原代感觉神经元培养模型，探究了KU-32对其他伴侣蛋白翻译诱导的调控效应，以及其对线粒体氧化应激与生物能学的改善作用。本研究使用了一种改良的细胞培养稳定同位素氨基酸标记技术——脉冲稳定同位素标记细胞培养（pulse SILAC, pSILAC），以无偏倚的方式评估蛋白质翻译水平的变化。实验结果表明，高血糖可降低多种影响超氧化物水平与呼吸活性的线粒体蛋白的翻译效率。值得注意的是，该现象与线粒体耗氧量降低及超氧化物水平升高呈显著相关性。KU-32可上调锰超氧化物歧化酶以及多种胞质与线粒体伴侣蛋白的翻译水平。与上述变化一致，KU-32可降低线粒体超氧化物水平，并显著增强呼吸活性。上述数据表明，在糖尿病周围神经病变中调控分子伴侣的药效，可能部分源于神经元线粒体生物能学的改善与氧化应激的减轻。