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Targeting apoptosis signalling kinase-1 (ASK-1) does not prevent the development of neuropathy in streptozotocin-induced diabetic mice.

Newton VL, Ali S, Duddy G, Whitmarsh AJ, Gardiner NJ - PLoS ONE (2014)

Bottom Line: Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38.As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy.Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n) mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy.

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Phospho-p38 increases in the sciatic nerve from 4-week diabetic C57 mice.Representative images of levels of phospho (pp38), total p38 (tp38;) and total ERK (tERK) are displayed in i) from (A) sciatic nerve (B) DRG and (C) spinal cord protein samples from control and diabetic C57 mice (4-week). (A–C) Densitometric analysis of bands are presented as mean pp38 intensity relative to ERK1 (ii) and tp38 band intensity relative to ERK1 (iii). There was increased pp38 in sciatic nerve from 4-week diabetic mice compared with controls (**p<0.01 in a Student’s t-test, n = 5–6) and no difference in total p38. However, there was no significant difference in pp38 in DRG or spinal cord between control and diabetic mice at 4 weeks (p>0.05; Student’s unpaired t-tests. n = 5–6). There was no difference in pp38 or tp38 in sciatic nerve, DRG or spinal cord from 12-week diabetic compared with control C57 mice (data, not shown for 12 weeks). Western blots probed for pp38, tp38 and ERK (i) of sciatic nerve (D) and spinal cord (E) from 12-week C57 and ASK1n diabetic and control mice, with densitometric analysis of pp38 (ii) and tp38 (iii) relative to ERK1, confirmed that p38 activation is unchanged in nerve tissue from 12-week diabetic mice, and that overall, p38 activation is significantly reduced in ASK1n mice compared with C57 mice (p<0.05 in sciatic nerve; p<0.01 in spinal cord overall in a 2-way ANOVA; *p<0.05 in Bonferroni post hoc tests, n = 5–6). Data represents mean intensities + SD.
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pone-0107437-g005: Phospho-p38 increases in the sciatic nerve from 4-week diabetic C57 mice.Representative images of levels of phospho (pp38), total p38 (tp38;) and total ERK (tERK) are displayed in i) from (A) sciatic nerve (B) DRG and (C) spinal cord protein samples from control and diabetic C57 mice (4-week). (A–C) Densitometric analysis of bands are presented as mean pp38 intensity relative to ERK1 (ii) and tp38 band intensity relative to ERK1 (iii). There was increased pp38 in sciatic nerve from 4-week diabetic mice compared with controls (**p<0.01 in a Student’s t-test, n = 5–6) and no difference in total p38. However, there was no significant difference in pp38 in DRG or spinal cord between control and diabetic mice at 4 weeks (p>0.05; Student’s unpaired t-tests. n = 5–6). There was no difference in pp38 or tp38 in sciatic nerve, DRG or spinal cord from 12-week diabetic compared with control C57 mice (data, not shown for 12 weeks). Western blots probed for pp38, tp38 and ERK (i) of sciatic nerve (D) and spinal cord (E) from 12-week C57 and ASK1n diabetic and control mice, with densitometric analysis of pp38 (ii) and tp38 (iii) relative to ERK1, confirmed that p38 activation is unchanged in nerve tissue from 12-week diabetic mice, and that overall, p38 activation is significantly reduced in ASK1n mice compared with C57 mice (p<0.05 in sciatic nerve; p<0.01 in spinal cord overall in a 2-way ANOVA; *p<0.05 in Bonferroni post hoc tests, n = 5–6). Data represents mean intensities + SD.

Mentions: At 4 weeks of diabetes there was an increase in phosphorylated, but not total p38 levels in sciatic nerve from C57 diabetic mice compared with control mice (Fig. 5A). At 4 weeks there were no significant changes in p38 levels in the DRG (Fig. 5B) or spinal cord (Fig. 5C). Whilst, there was no significant difference in the levels of phosphorylated p38 at 12 weeks between control and diabetic mice of the same genotype, overall p38 activation levels were lower in sciatic nerve and spinal cord of ASK1n mice compared to C57 mice (Fig. 5D,E). Therefore ASK1n mice show reduced downstream p38 activation.


Targeting apoptosis signalling kinase-1 (ASK-1) does not prevent the development of neuropathy in streptozotocin-induced diabetic mice.

Newton VL, Ali S, Duddy G, Whitmarsh AJ, Gardiner NJ - PLoS ONE (2014)

Phospho-p38 increases in the sciatic nerve from 4-week diabetic C57 mice.Representative images of levels of phospho (pp38), total p38 (tp38;) and total ERK (tERK) are displayed in i) from (A) sciatic nerve (B) DRG and (C) spinal cord protein samples from control and diabetic C57 mice (4-week). (A–C) Densitometric analysis of bands are presented as mean pp38 intensity relative to ERK1 (ii) and tp38 band intensity relative to ERK1 (iii). There was increased pp38 in sciatic nerve from 4-week diabetic mice compared with controls (**p<0.01 in a Student’s t-test, n = 5–6) and no difference in total p38. However, there was no significant difference in pp38 in DRG or spinal cord between control and diabetic mice at 4 weeks (p>0.05; Student’s unpaired t-tests. n = 5–6). There was no difference in pp38 or tp38 in sciatic nerve, DRG or spinal cord from 12-week diabetic compared with control C57 mice (data, not shown for 12 weeks). Western blots probed for pp38, tp38 and ERK (i) of sciatic nerve (D) and spinal cord (E) from 12-week C57 and ASK1n diabetic and control mice, with densitometric analysis of pp38 (ii) and tp38 (iii) relative to ERK1, confirmed that p38 activation is unchanged in nerve tissue from 12-week diabetic mice, and that overall, p38 activation is significantly reduced in ASK1n mice compared with C57 mice (p<0.05 in sciatic nerve; p<0.01 in spinal cord overall in a 2-way ANOVA; *p<0.05 in Bonferroni post hoc tests, n = 5–6). Data represents mean intensities + SD.
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pone-0107437-g005: Phospho-p38 increases in the sciatic nerve from 4-week diabetic C57 mice.Representative images of levels of phospho (pp38), total p38 (tp38;) and total ERK (tERK) are displayed in i) from (A) sciatic nerve (B) DRG and (C) spinal cord protein samples from control and diabetic C57 mice (4-week). (A–C) Densitometric analysis of bands are presented as mean pp38 intensity relative to ERK1 (ii) and tp38 band intensity relative to ERK1 (iii). There was increased pp38 in sciatic nerve from 4-week diabetic mice compared with controls (**p<0.01 in a Student’s t-test, n = 5–6) and no difference in total p38. However, there was no significant difference in pp38 in DRG or spinal cord between control and diabetic mice at 4 weeks (p>0.05; Student’s unpaired t-tests. n = 5–6). There was no difference in pp38 or tp38 in sciatic nerve, DRG or spinal cord from 12-week diabetic compared with control C57 mice (data, not shown for 12 weeks). Western blots probed for pp38, tp38 and ERK (i) of sciatic nerve (D) and spinal cord (E) from 12-week C57 and ASK1n diabetic and control mice, with densitometric analysis of pp38 (ii) and tp38 (iii) relative to ERK1, confirmed that p38 activation is unchanged in nerve tissue from 12-week diabetic mice, and that overall, p38 activation is significantly reduced in ASK1n mice compared with C57 mice (p<0.05 in sciatic nerve; p<0.01 in spinal cord overall in a 2-way ANOVA; *p<0.05 in Bonferroni post hoc tests, n = 5–6). Data represents mean intensities + SD.
Mentions: At 4 weeks of diabetes there was an increase in phosphorylated, but not total p38 levels in sciatic nerve from C57 diabetic mice compared with control mice (Fig. 5A). At 4 weeks there were no significant changes in p38 levels in the DRG (Fig. 5B) or spinal cord (Fig. 5C). Whilst, there was no significant difference in the levels of phosphorylated p38 at 12 weeks between control and diabetic mice of the same genotype, overall p38 activation levels were lower in sciatic nerve and spinal cord of ASK1n mice compared to C57 mice (Fig. 5D,E). Therefore ASK1n mice show reduced downstream p38 activation.

Bottom Line: Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38.As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy.Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n) mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy.

Show MeSH
Related in: MedlinePlus