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

Saleh A, Chowdhury SK, Smith DR, Balakrishnan S, Tessler L, Schartner E, Bilodeau A, Van Der Ploeg R, Fernyhough P - Mol Brain (2013)

Bottom Line: Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1β 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.Endogenous synthesis of IL-1β 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Neurodegenerative Disorders, St, Boniface Hospital Research Centre, R4048 - 351 Tache Ave, Winnipeg, MB R2H 2A6, Canada. asaleh@sbrc.ca.

ABSTRACT

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-1β to be significantly depressed. We, therefore, tested the hypothesis that impaired IL-1α and IL-1β 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-1β 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-1β 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-1β 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-1β expression completely blocked neurite outgrowth. Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1β 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-1β 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.

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DRG sensory neurons express IL-1β and its blockade inhibits neurite outgrowth. In (a) DRG sensory neurons from normal rats were cultured for 1 day in the presence of low dose of neurotrophic growth factors and were fixed and immunostained for IL-1β. Top panels are untreated cells and bottom panels are treated with IL-1β (10 ng/ml) for 24 h. The right panels are co-stained for β-tubulin III. In (b) DRG cultures were infected with GFP-expressing lentivirus containing scrambled (scr) or shRNA to IL-1β. After 48 h of culture images of GFP fluorescence were acquired and neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs shRNA-IL-1β (Student’s t-Test). In addition, in (c) infected cultures were examined by Western blot for IL-1β expression and P-STAT3-Tyr and revealed significantly reduced expression in presence of shRNA to IL-1β. In (d) DRG neurons over-expressed shRNA to IL-1β for 48 h and then were treated ± IL-1β. Neurons were stimulated with/without IL-1β for 24 h and then neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p < 0.05 vs shRNA for IL-1β + IL-1β (Student’s t-Test). In (e) IL-1β treatment for 24 h up-regulates IL-1β luciferase promoter activity in cultured DRG sensory neurons. Co-transfection with plasmid expressing GFP and carrying shRNA to IL-1β blocked the effect of exogenous IL-1β. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs all groups (oneway ANOVA with Dunnett’s test).
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Figure 5: DRG sensory neurons express IL-1β and its blockade inhibits neurite outgrowth. In (a) DRG sensory neurons from normal rats were cultured for 1 day in the presence of low dose of neurotrophic growth factors and were fixed and immunostained for IL-1β. Top panels are untreated cells and bottom panels are treated with IL-1β (10 ng/ml) for 24 h. The right panels are co-stained for β-tubulin III. In (b) DRG cultures were infected with GFP-expressing lentivirus containing scrambled (scr) or shRNA to IL-1β. After 48 h of culture images of GFP fluorescence were acquired and neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs shRNA-IL-1β (Student’s t-Test). In addition, in (c) infected cultures were examined by Western blot for IL-1β expression and P-STAT3-Tyr and revealed significantly reduced expression in presence of shRNA to IL-1β. In (d) DRG neurons over-expressed shRNA to IL-1β for 48 h and then were treated ± IL-1β. Neurons were stimulated with/without IL-1β for 24 h and then neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p < 0.05 vs shRNA for IL-1β + IL-1β (Student’s t-Test). In (e) IL-1β treatment for 24 h up-regulates IL-1β luciferase promoter activity in cultured DRG sensory neurons. Co-transfection with plasmid expressing GFP and carrying shRNA to IL-1β blocked the effect of exogenous IL-1β. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs all groups (oneway ANOVA with Dunnett’s test).

Mentions: We cultured adult rat DRG sensory neurons for 24 h with low dose neurotrophic factors in the presence or absence of IL-1β at 10 ng/ml. Cultures were fixed and stained using anti-IL-1β antibody and antibody to neuron-specific β-tubulin III (Figure 5a). Immunocytochemical staining for IL-1β revealed that the majority of DRG neurons expressed IL-1β in the control rat cultures. To inhibit endogenous IL-1β activity, cultured DRG neurons were transduced with lentivirus carrying GFP and either scrambled or shRNA for IL-1β (Figure 5b). GFP fluorescence signal was used to quantify axonal outgrowth, demonstrating that neurite outgrowth in neurons over-expressing shRNA to IL-1β was significantly decreased (Figure 5b). We also examined the knockdown effect of shRNA to IL-1β on IL-1β and P-STAT3-Tyr705 expression in DRG cultures by Western blot (Figure 5c). The delivery of shRNA to IL-1β was very effective in lowering endogenous IL-1β and P-STAT3-Tyr705 expression in these cultures. In DRG neurons over-expressing shRNA to IL-1β for 48 h and stimulated with IL-1β for the final 12 h there was a significant effect of exogenous IL-1β on neurite outgrowth (Figure 5d). Another approach was to use promoter reporter technology to study IL-1β transcriptional activity. IL-1β promoter reporter construct (IL-1β-Luc-pr) and/or shRNA to IL-1β were simultaneously transfected into adult sensory neuron cultures and luciferase activity assessed after 48 h. A sub-group of cultures were treated with 10 ng/ml of IL-1β for the final 12 h. Figure 5e shows that IL-1β raised IL-1β promoter activity by approximately 2-fold and shRNA to IL-1β significantly decreased this activity suggesting auto-regulation of IL-1β promoter.


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

Saleh A, Chowdhury SK, Smith DR, Balakrishnan S, Tessler L, Schartner E, Bilodeau A, Van Der Ploeg R, Fernyhough P - Mol Brain (2013)

DRG sensory neurons express IL-1β and its blockade inhibits neurite outgrowth. In (a) DRG sensory neurons from normal rats were cultured for 1 day in the presence of low dose of neurotrophic growth factors and were fixed and immunostained for IL-1β. Top panels are untreated cells and bottom panels are treated with IL-1β (10 ng/ml) for 24 h. The right panels are co-stained for β-tubulin III. In (b) DRG cultures were infected with GFP-expressing lentivirus containing scrambled (scr) or shRNA to IL-1β. After 48 h of culture images of GFP fluorescence were acquired and neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs shRNA-IL-1β (Student’s t-Test). In addition, in (c) infected cultures were examined by Western blot for IL-1β expression and P-STAT3-Tyr and revealed significantly reduced expression in presence of shRNA to IL-1β. In (d) DRG neurons over-expressed shRNA to IL-1β for 48 h and then were treated ± IL-1β. Neurons were stimulated with/without IL-1β for 24 h and then neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p < 0.05 vs shRNA for IL-1β + IL-1β (Student’s t-Test). In (e) IL-1β treatment for 24 h up-regulates IL-1β luciferase promoter activity in cultured DRG sensory neurons. Co-transfection with plasmid expressing GFP and carrying shRNA to IL-1β blocked the effect of exogenous IL-1β. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs all groups (oneway ANOVA with Dunnett’s test).
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Figure 5: DRG sensory neurons express IL-1β and its blockade inhibits neurite outgrowth. In (a) DRG sensory neurons from normal rats were cultured for 1 day in the presence of low dose of neurotrophic growth factors and were fixed and immunostained for IL-1β. Top panels are untreated cells and bottom panels are treated with IL-1β (10 ng/ml) for 24 h. The right panels are co-stained for β-tubulin III. In (b) DRG cultures were infected with GFP-expressing lentivirus containing scrambled (scr) or shRNA to IL-1β. After 48 h of culture images of GFP fluorescence were acquired and neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs shRNA-IL-1β (Student’s t-Test). In addition, in (c) infected cultures were examined by Western blot for IL-1β expression and P-STAT3-Tyr and revealed significantly reduced expression in presence of shRNA to IL-1β. In (d) DRG neurons over-expressed shRNA to IL-1β for 48 h and then were treated ± IL-1β. Neurons were stimulated with/without IL-1β for 24 h and then neurite outgrowth quantified. Data are mean ± SEM (n = 3 replicates); *p < 0.05 vs shRNA for IL-1β + IL-1β (Student’s t-Test). In (e) IL-1β treatment for 24 h up-regulates IL-1β luciferase promoter activity in cultured DRG sensory neurons. Co-transfection with plasmid expressing GFP and carrying shRNA to IL-1β blocked the effect of exogenous IL-1β. Data are mean ± SEM (n = 3 replicates); *p<0.05 vs all groups (oneway ANOVA with Dunnett’s test).
Mentions: We cultured adult rat DRG sensory neurons for 24 h with low dose neurotrophic factors in the presence or absence of IL-1β at 10 ng/ml. Cultures were fixed and stained using anti-IL-1β antibody and antibody to neuron-specific β-tubulin III (Figure 5a). Immunocytochemical staining for IL-1β revealed that the majority of DRG neurons expressed IL-1β in the control rat cultures. To inhibit endogenous IL-1β activity, cultured DRG neurons were transduced with lentivirus carrying GFP and either scrambled or shRNA for IL-1β (Figure 5b). GFP fluorescence signal was used to quantify axonal outgrowth, demonstrating that neurite outgrowth in neurons over-expressing shRNA to IL-1β was significantly decreased (Figure 5b). We also examined the knockdown effect of shRNA to IL-1β on IL-1β and P-STAT3-Tyr705 expression in DRG cultures by Western blot (Figure 5c). The delivery of shRNA to IL-1β was very effective in lowering endogenous IL-1β and P-STAT3-Tyr705 expression in these cultures. In DRG neurons over-expressing shRNA to IL-1β for 48 h and stimulated with IL-1β for the final 12 h there was a significant effect of exogenous IL-1β on neurite outgrowth (Figure 5d). Another approach was to use promoter reporter technology to study IL-1β transcriptional activity. IL-1β promoter reporter construct (IL-1β-Luc-pr) and/or shRNA to IL-1β were simultaneously transfected into adult sensory neuron cultures and luciferase activity assessed after 48 h. A sub-group of cultures were treated with 10 ng/ml of IL-1β for the final 12 h. Figure 5e shows that IL-1β raised IL-1β promoter activity by approximately 2-fold and shRNA to IL-1β significantly decreased this activity suggesting auto-regulation of IL-1β promoter.

Bottom Line: Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1β 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.Endogenous synthesis of IL-1β 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Neurodegenerative Disorders, St, Boniface Hospital Research Centre, R4048 - 351 Tache Ave, Winnipeg, MB R2H 2A6, Canada. asaleh@sbrc.ca.

ABSTRACT

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-1β to be significantly depressed. We, therefore, tested the hypothesis that impaired IL-1α and IL-1β 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-1β 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-1β 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-1β 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-1β expression completely blocked neurite outgrowth. Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1β 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-1β 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.

Show MeSH
Related in: MedlinePlus