High glucose concentration suppresses a SIRT2 regulated pathway that enhances neurite outgrowth in cultured adult sensory neurons.

In peripheral nerve under hyperglycemic conditions high flux of d-glucose through the polyol pathway drives an aberrant redox state contributing to neurodegeneration in diabetic sensorimotor polyneuropathy (DSPN). Sirtuins, including SIRT2, detect the redox state via the NAD+/NADH ratio to regulate mitochondrial function via, in part, AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). In adult dorsal root ganglia (DRG) sensory neurons mitochondrial dysfunction has been proposed as an etiological factor in dying-back neuropathy in diabetes. We tested the hypothesis that a high concentration of d-glucose depleted SIRT2 expression via enhancement of polyol pathway activity. We posited that this would lead to impaired mitochondrial function and suppression of neurite outgrowth in cultured sensory neurons. The use of dominant negative mutants or neurons from SIRT2 knockout (KO) mice to block SIRT2 signaling revealed that neurons derived from control or type 1 diabetic rodents required SIRT2 for optimal neurite outgrowth. Over-expression of WT-SIRT2 elevated neurite outgrowth in normal and diabetic cultures. SIRT2 protein isoforms 2.1 and 2.2 were reduced by 20-30% in DRG of type 1 diabetic mice (p < .05). After 72 h exposure to high d-glucose (25 mM vs 5 mM) cultured sensory neurons showed a significant 2-fold (p < .05) decrease in SIRT2 expression, P-AMPK, levels of respiratory Complexes II/III and respiratory capacity. DRG neurons expressed aldose reductase and the aforementioned deficits were prevented by treatment with aldose reductase inhibitors (lidorestat or sorbinil) or sorbitol dehydrogenase inhibitor (SDI-158). In cultures derived from type 1 diabetic rats treatment with SDI-158 elevated expression of SIRT2, P-AMPK/PGC-1α and neurite outgrowth (p < .05). SIRT2 KO neurons exhibited deficits in the LKB-1/AMPK/PGC-1α pathway and mitochondrial function. In cultured neurons the SIRT2 pathway enhances axonal outgrowth and this signaling axis encompassing activation of AMPK/PGC-1α is impaired in DSPN, in part, due to enhanced polyol pathway activity caused by hyperglycemia.

Diabetes impairs an interleukin-1ß-dependent pathway that enhances neurite outgrowth through JAK/STAT3 modulation of mitochondrial bioenergetics in adult sensory neurons.

BACKGROUND: A luminex-based screen of cytokine expression in dorsal root ganglia (DRG) and nerve of type 1 diabetic rodents revealed interleukin-1 (IL-1α) and IL-1ß to be significantly depressed. We, therefore, tested the hypothesis that impaired IL-1α and IL-1ß expression in DRG may contribute to aberrant axon regeneration and plasticity seen in diabetic sensory neuropathy. In addition, we determined if these cytokines could optimize mitochondrial bioenergetics since mitochondrial dysfunction is a key etiological factor in diabetic neuropathy. RESULTS: Cytokines IL-1α and IL-1ß were reduced 2-fold (p<0.05) in DRG and/or nerve of 2 and 5 month streptozotocin (STZ)-diabetic rats. IL-2 and IL-10 were unchanged. IL-1α and IL-1ß induced similar 2 to 3-fold increases in neurite outgrowth in cultures derived from control or diabetic rats (p<0.05). STAT3 phosphorylation on Tyr705 or Ser727 was depressed in DRG from STZ-diabetic mice and treatment of cultures derived from STZ-diabetic rats with IL-1ß for 30 min raised phosphorylation of STAT3 on Tyr705 and Ser727 by 1.5 to 2-fold (p<0.05). shRNA-based or AG490 inhibition of STAT3 activity or shRNA blockade of endogenous IL-1ß expression completely blocked neurite outgrowth. Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1ß and maximal oxygen consumption rate and spare respiratory capacity, both key measures of bioenergetic fidelity that were depressed in diabetic compared with control neurons, were enhanced 2-fold. This effect was blocked by AG490. CONCLUSIONS: Endogenous synthesis of IL-1ß is diminished in nerve tissue in type 1 diabetes and we propose this defect triggers reduced STAT3 signaling and mitochondrial function leading to sup-optimal axonal regeneration and plasticity.

Receptor for advanced glycation end-products (RAGE) activates divergent signaling pathways to augment neurite outgrowth of adult sensory neurons.

BACKGROUND: The receptor for advanced glycation end-products (RAGE) is implicated in neuronal differentiation during embryogenesis and in regulation of peripheral nerve regeneration. However, the role of RAGE ligands and the signaling pathways utilized by activated RAGE in mediating axon regeneration in adult neurons remain unknown. We tested the hypothesis that RAGE signaling modulated neurotrophin-induced neurite outgrowth in cultured adult sensory neurons. RESULTS: Dorsal root ganglia (DRG) neurons from adult rats in vitro were exposed to specific RAGE ligands, signal transduction inhibitors and function blocking anti-RAGE IgG to assess their impact on neurite outgrowth. RAGE ligands including human glycated albumin (HGA), S100 calcium binding protein (S100B) and high mobility group 1 protein (HMGB1; alternatively termed amphoterin) in the presence of neurotrophins elevated neurite outgrowth 2-fold (p<0.05). shRNA to RAGE or anti-RAGE IgG blockade of RAGE inhibited neurite outgrowth by 40-90% (p<0.05). Western blotting and gene reporter analysis showed RAGE ligands activated NF-κB, JAK-STAT and ERK pathways. RAGE ligand induction of neurite outgrowth was blocked by inhibition of NF-κB, JAK-STAT or ERK pathways revealing the necessity for combined activation for optimal growth. RAGE ligands rapidly elevated NF-κB p65 expression in the cytoplasm while triggering translocation of NF-κB p50 to the nucleus. shRNA blockade of p50 demonstrated that translocation of p50 to the nucleus was implicated in driving axonal outgrowth. CONCLUSIONS: RAGE signaling is a complex mediator of neurotrophin-dependent neurite outgrowth, operating through divergent but partly inter-dependent pathways.

Ciliary neurotrophic factor activates NF-κB to enhance mitochondrial bioenergetics and prevent neuropathy in sensory neurons of streptozotocin-induced diabetic rodents.

Diabetes causes mitochondrial dysfunction in sensory neurons that may contribute to peripheral neuropathy. Ciliary neurotrophic factor (CNTF) promotes sensory neuron survival and axon regeneration and prevents axonal dwindling, nerve conduction deficits and thermal hypoalgesia in diabetic rats. In this study, we tested the hypothesis that CNTF protects sensory neuron function during diabetes through normalization of impaired mitochondrial bioenergetics. In addition, we investigated whether the NF-κB signal transduction pathway was mobilized by CNTF. Neurite outgrowth of sensory neurons derived from streptozotocin (STZ)-induced diabetic rats was reduced compared to neurons from control rats and exposure to CNTF for 24 h enhanced neurite outgrowth. CNTF also activated NF-κB, as assessed by Western blotting for the NF-κB p50 subunit and reporter assays for NF-κB promoter activity. Conversely, blockade of NF-κB signaling using SN50 peptide inhibited CNTF-mediated neurite outgrowth. Studies in mice with STZ-induced diabetes demonstrated that systemic therapy with CNTF prevented functional indices of peripheral neuropathy along with deficiencies in dorsal root ganglion (DRG) NF-κB p50 expression and DNA binding activity. DRG neurons derived from STZ-diabetic mice also exhibited deficiencies in maximal oxygen consumption rate and associated spare respiratory capacity that were corrected by exposure to CNTF for 24 h in an NF-κB-dependent manner. We propose that the ability of CNTF to enhance axon regeneration and protect peripheral nerve from structural and functional indices of diabetic peripheral neuropathy is associated with targeting of mitochondrial function, in part via NF-κB activation, and improvement of cellular bioenergetics.