Modulating Molecular Chaperones Improves Mitochondrial Bioenergetics and Decreases the Inflammatory Transcriptome in Diabetic Sensory Neurons

Abstract

We have previously demonstrated that modulating molecular chaperones with KU-32, a novobiocin derivative, ameliorates physiologic and bioenergetic deficits of diabetic peripheral neuropathy (DPN). Replacing the coumarin core of KU-32 with a meta-fluorinated biphenyl ring system created KU-596, a novobiocin analogue (novologue) that showed neuroprotective activity in a cell-based assay. The current study sought to determine whether KU-596 offers similar therapeutic potential for treating DPN. Administration of 2–20 mg/kg of KU-596 improved diabetes induced hypoalgesia and sensory neuron bioenergetic deficits in a dose-dependent manner. However, the drug could not improve these neuropathic deficits in diabetic heat shock protein 70 knockout (Hsp70 KO) mice. To gain further insight into the mechanisms by which KU-596 improved DPN, we performed transcriptomic analysis of sensory neuron RNA obtained from diabetic wild-type and Hsp70 KO mice using RNA sequencing. Bioinformatic analysis of the differentially expressed genes indicated that diabetes strongly increased inflammatory pathways and that KU-596 therapy effectively reversed these increases independent of Hsp70. In contrast, the effects of KU-596 on decreasing the expression of genes regulating the production of reactive oxygen species were more Hsp70-dependent. These data indicate that modulation of molecular chaperones by novologue therapy offers an effective approach toward correcting nerve dysfunction in DPN but that normalization of inflammatory pathways alone by novologue therapy seems to be insufficient to reverse sensory deficits associated with insensate DPN.

Schwann cell interactions with axons and microvessels in diabetic neuropathy

“…Macrophage infiltration and proliferation have been observed in nerves from mouse models of diabetes 97,98 , and upregulation of proinflammatory genes during activation of inflammatory pathways in the PNS [99][100][101] further implicates involvement of RAGE-mediated Schwann cell involvement in the inflammatory response. One up regulated gene encodes the S100 calcium binding protein A8/A9 (REF.…”

Promoting Neuronal Tolerance of Diabetic Stress

“…Recent work from our lab has shown that Hsp70 may play a role in affecting the expression of TXNIP. RNA-seq analysis of mRNA from DRG of Type 1 diabetic mice showed a 2-fold elevated expression of TXNIP expression (Ma et al, 2015) and treating diabetic mice with the chaperone modulator, KU-596 (see section 5.0), suppressed TXNIP expression to control levels (Fig. 1A).…”

“…Importantly, the efficacy of modulating chaperones is not limited to neurodegenerative diseases driven by the formation of protein aggregates. Hsp70 has also been shown to downregulate inflammatory signaling, decrease oxidative stress and improve mitochondrial function in DPN and other conditions whose etiology is not linked to the formation of protein aggregates (Ianaro et al, 2003; Jones et al, 2011; Li et al, 2012; Ma et al, 2015; Madden et al, 2008; Saibil, 2013; Zhang et al, 2012). However, an important aspect of Hsp70 biology that must be considered in moving forward in therapy development is how modulating the intra- versus extracellular pools of Hsp70 may affect disease progression.…”

“…For example, the superoxide generating NADPH oxidases (NOX) are negatively regulated by iHsp70. In diabetic sensory neurons, NOX2 mRNA overexpression was reversed in an Hsp70-dependent manner by treatment with the C-terminal Hsp90 inhibitor, KU-596 and this correlated with an improvement in DPN (see Section 5.0) (Ma et al, 2015). Further, in vascular smooth muscle cells, co-immunoprecipitation, and Hsp70 knockdown studies demonstrate iHsp70 trafficking of NOX4 regulates degradation of the protein (Bocanegra et al, 2010; Gil Lorenzo et al, 2014).…”

“…Interestingly, RNA-Seq analysis indicated that the mitochondrial transcriptome was unaltered by pharmacological modulation of Hsp70 in diabetic mice, despite an improvement in mitochondrial bioenergetics. Therefore iHsp70’s protection of mitochondrial bioenergetics may not be significantly mediated through gene transcription (Ma et al, 2015). iHsp70-dependent influx of mitochondrial proteins may offer an explanation for at least part of this mitochondrial bioenergetics protection (Afolayan et al, 2014).…”