Diabetic peripheral neuropathy: should a chaperone accompany our therapeutic approach?

Abstract

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes that is associated with axonal atrophy, demyelination, blunted regenerative potential, and loss of peripheral nerve fibers. The development and progression of DPN is due in large part to hyperglycemia but is also affected by insulin deficiency and dyslipidemia. Although numerous biochemical mechanisms contribute to DPN, increased oxidative/nitrosative stress and mitochondrial dysfunction seem intimately associated with nerve dysfunction and diminished regenerative capacity. Despite advances in understanding the etiology of DPN, few approved therapies exist for the pharmacological management of painful or insensate DPN. Therefore, identifying novel therapeutic strategies remains paramount. Because DPN does not develop with either temporal or biochemical uniformity, its therapeutic management may benefit from a multifaceted approach that inhibits pathogenic mechanisms, manages inflammation, and increases cytoprotective responses. Finally, exercise has long been recognized as a part of the therapeutic management of diabetes, and exercise can delay and/or prevent the development of painful DPN. This review presents an overview of existing therapies that target both causal and symptomatic features of DPN and discusses the role of up-regulating cytoprotective pathways via modulating molecular chaperones. Overall, it may be unrealistic to expect that a single pharmacologic entity will suffice to ameliorate the multiple symptoms of human DPN. Thus, combinatorial therapies that target causal mechanisms and enhance endogenous reparative capacity may enhance nerve function and improve regeneration in DPN if they converge to decrease oxidative stress, improve mitochondrial bioenergetics, and increase response to trophic factors.

Fig. 1.

Overview of various pathogenetic components contributing to DPN.

Fig. 2.

Activation of the heat-shock response by KU-32 and exercise. KU-32 is a C-terminal Hsp90 inhibitor that may increase the expression of Hsp70 by promoting the release of HSF1 from Hsp90. After trimerization, phosphorylation and translocation to the nucleus, HSF1 interacts with two heat-shock elements (HSE) on the Hsp70 promoter to increase the expression of Hsp70. Exercise may similarly increase Hsp70 expression but via indirect effects on Hsp90/HSF1. The binding of HSF1 to sequences within a portion of one HSE of the human Hsp70.1 promoter is shown (Calamini et al., 2012). Solid arrow, direct effect; dashed arrow, multiple steps.

Fig. 3.

Induction of Hsp70 but not Hsc70 by KU-32. A, 50B11 cells, an immortalized sensory neuron cell line (Chen et al., 2007) were transfected with a luciferase reporter linked to the human Hsp70.1 promoter, which contains two heat-shock response elements (Calamini et al., 2012). After 24 h, the transfected cells were seeded into a 96-well plate and maintained in culture for an additional 6 h before treatment with vehicle (0.1% dimethyl sulfoxide) or the indicated concentrations of KU-32 for 24 h. The cells were harvested, and luciferase activity was assessed and normalized to total protein per well. Some wells were treated with 250 nM geldanamycin as a positive control. Results are mean ± S.E.M. from 18 replicate wells in two experiments. B, primary neonatal mouse sensory neurons were isolated and grown in culture for 1 week (Urban et al., 2010). The cells were treated with vehicle or 1 μM KU-32 for 24 h in the absence or presence of heat shock (HS; 30 min at 42°C). Cell lysates were prepared, and the induction of Hsp70 was determined by immunoblot analysis. C, primary neonatal mouse sensory neurons were isolated, grown in culture for 1 week, and treated for the indicated time with vehicle or 1 μM KU-32. Cell lysates were prepared, and Hsc70 levels were determined by immunoblot analysis. The levels of β-actin served as a control for protein loading.

Fig. 4.

Hsp70 is required for KU-32 to protect against neuregulin-induced demyelination. Myelinated mouse SC-DRG neuron cocultures were prepared from wild-type (A and B) or Hsp70 knockout (KO; C and D) mice and treated overnight with vehicle or 1 μM KU-32. The cultures were treated with PBS or 200 ng/ml neuregulin-1 for 4 days, and myelin segments were visualized by staining the cultures for myelin basic protein. Total cell number was assessed by staining nuclei with 4,6-diamidino-2-phenylindole. Cell Profiler (http://www.cellprofiler.org) was used to calculate the total myelin segment area from six fields per six coverslips per treatment. The results are expressed as a fold of the untreated control and are an average of three experiments per genotype. *, p < 0.05 versus KU-32, ^∧, p < 0.05 versus NRG. Arrows, examples of myelin internodes. [Modified from Urban MJ, Li C, Yu C, Lu Y, Krise JM, McIntosh MP, Rajewski RA, Blagg BS, and Dobrowsky RT (2010) Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice. ASN Neuro 2:e00040. Open Access from Portland Press Limited and the American Society for Neurochemistry.]

Fig. 5.

Hsp70 is required for the in vivo efficacy of KU-32 in reversing mechanical hypoalgesia. Wild-type (A) and Hsp70 knockout (KO; B) mice were rendered diabetic for 12 weeks and then treated with weekly doses of vehicle or 20 mg/kg KU-32 for 6 weeks. Beginning 2 weeks after the induction of diabetes, mechanical sensitivity was assessed weekly. Twelve weeks of diabetes produced a significant mechanical hypoalgesia, and weekly treatment with KU-32 induced a time-dependent improvement to near control levels in the wild-type (A) but not the Hsp70 KO (B) mice. *, p < 0.01 compared with time-matched untreated controls. ^∧, p < 0.01 compared with time-matched Veh + KU-32. [Modified from Urban MJ, Li C, Yu C, Lu Y, Krise JM, McIntosh MP, Rajewski RA, Blagg BS, and Dobrowsky RT (2010) Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice. ASN Neuro 2:e00040. Open Access from Portland Press Limited and the American Society for Neurochemistry.]

Fig. 6.

Potential mechanisms by which modulating chaperones may increase mitochondrial function in DPN. Diabetes-induced increases in mitochondrial ROS may promote formation of oxidatively modified proteins, which contribute to a decreased respiratory capacity. Increasing transport of proteins to mitochondria may aid in replacing damaged proteins. Hsp70 is involved in protein transport to mitochondria and contributes to the internalization of multispanning inner mitochondrial membrane proteins via TOM70. Although not required, Hsp70 may also facilitate the transfer of preproteins containing a mitochondrial targeting sequence, such as MnSOD, to TOM22. mtHsp70 is a requisite chaperone for internalization of preproteins before final proteolytic processing. Within the organelle, Hsp60 and mtHsp70 may aid the refolding of oxidatively damaged mitochondrial proteins. In the presence of sufficient levels of NADPH, Hsp70 may also increase the GSH/GSSG ratio in the cytoplasm by increasing the activity of glutathione peroxidase and reductase. TOM and TIM, translocases of the outer and inner mitochondrial membrane, respectively; Pi, inorganic phosphate; CoQ, coenzyme Q.