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Dysregulated IGFBP5 expression causes axon degeneration and motoneuron loss in diabetic neuropathy.

Simon CM, Rauskolb S, Gunnersen JM, Holtmann B, Drepper C, Dombert B, Braga M, Wiese S, Jablonka S, Pühringer D, Zielasek J, Hoeflich A, Silani V, Wolf E, Kneitz S, Sommer C, Toyka KV, Sendtner M - Acta Neuropathol. (2015)

Bottom Line: IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP.These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration.Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University of Würzburg, Versbacher-Str. 5, 97078, Würzburg, Germany.

ABSTRACT
Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes. The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor (IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.

No MeSH data available.


Related in: MedlinePlus

Igfbp5 tg+ motoneurons show decreased survival when cultured with IGF1. a qRT-PCR revealed a 1.8-fold increase of the Igfbp5 mRNA level in the spinal cord of 6-month-old Igfbp5 tg+ mice (Bp5 tg+). Scored values were obtained by normalizing to β-actin mRNA levels. A.U. arbitrary units. b Igfbp5 protein elevation was detected in sciatic nerve, but not in spinal cord and brain of 6-month-old Igfbp5 tg+ mice compared to controls. Actin was used as loading control. c Quantification of Igfbp5 protein levels. QL quantitative labeling. d Motoneurons stained for Tau (red) and Igfbp5 (green) 7 days after plating. Igfbp5 was increased in the soma and neurites of Igfbp5 tg+ motoneurons. Igfbp5 appeared associated with the cell membrane. Scale bar 5 μm. e The positive effect of IGF1 on motoneuron survival is reduced by 37 % in Igfbp5 tg+ motoneurons. Motoneuron survival was unchanged in the presence of BDNF
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Fig3: Igfbp5 tg+ motoneurons show decreased survival when cultured with IGF1. a qRT-PCR revealed a 1.8-fold increase of the Igfbp5 mRNA level in the spinal cord of 6-month-old Igfbp5 tg+ mice (Bp5 tg+). Scored values were obtained by normalizing to β-actin mRNA levels. A.U. arbitrary units. b Igfbp5 protein elevation was detected in sciatic nerve, but not in spinal cord and brain of 6-month-old Igfbp5 tg+ mice compared to controls. Actin was used as loading control. c Quantification of Igfbp5 protein levels. QL quantitative labeling. d Motoneurons stained for Tau (red) and Igfbp5 (green) 7 days after plating. Igfbp5 was increased in the soma and neurites of Igfbp5 tg+ motoneurons. Igfbp5 appeared associated with the cell membrane. Scale bar 5 μm. e The positive effect of IGF1 on motoneuron survival is reduced by 37 % in Igfbp5 tg+ motoneurons. Motoneuron survival was unchanged in the presence of BDNF

Mentions: To study this effect on motoneurons and sensory neurons in vivo, we generated mice with neuron-specific overexpression of IGFBP5. An 8-kb fragment including the human NF-L promoter, mouse Igfbp5 cDNA, the polyA signal from pMC-Cre and exons 2–4 of the NF-L gene was injected into fertilized mouse eggs (for details see electronic supplementary Fig. A1, supplementary methods section). Real-time RT-PCR analysis revealed that the levels of Igfbp5 mRNA in the spinal cord of 6 months old Igfbp5 transgenic mice (Bp5 tg+) were up-regulated 1.8-fold compared with controls (P < 0.001, two-tailed Student’s t test) (Fig. 3a). Western blot analysis for IGFBP5 in the spinal cord, sciatic nerve and brain of 6-month-old Igfbp5 transgenic mice exhibited a fivefold IGFBP5 protein up-regulation in the sciatic nerve (P < 0.001, two-tailed Student’s t test) (Fig. 3b, c), indicating that the IGFBP5 protein is anterogradely transported in axons. Isolated motoneurons from Igfbp5 transgenic embryos (E13.5) also showed increased IGFBP5 immunoreactivity in cell bodies and axons after 7 days in vitro (Fig. 3d). IGFBP5 was predominantly localized on the surface of these neurons. This correlates with previous data showing that IGFBP5 binds to the cell surface and the surrounding extracellular matrix [2]. Igfbp5 transgenic motoneurons showed a different survival response to IGF1 than wild-type motoneurons. While survival of Igfbp5-overexpressing motoneurons was unchanged in the presence of BDNF (P > 0.05, one-way ANOVA), their survival was significantly reduced by 37 % in the presence of IGF1 compared to wild-type motoneurons (P < 0.001, one-way ANOVA) (Fig. 3e). When we compared the dose response for IGF1 on motoneuron survival in Igfbp5 transgenic and wild-type motoneurons, a shift was observed (0.2 ng/ml IGF1; P < 0.05; 1 ng/ml IGF1; P < 0.001; 5 ng/ml IGF1; P < 0.001; one-way ANOVA) (Fig. 4a), indicating that motoneurons overexpressing IGFBP5 need more IGF1 for their survival than non-transgenic motoneurons. This result provides further evidence that IGFBP5 inhibits IGF1 actions on motoneurons. We then compared the phosphorylation levels of IGF1R and downstream AKT in Igfbp5 transgenic motoneurons relative to wild-type controls. A strong reduction in IGF1R and AKT phosphorylation levels was observed in Igfbp5 transgenic motoneurons compared to controls (pIGF1R: P < 0.001; pAKT: P < 0.01, two-tailed Student’s t test) grown for 5 days with 5 ng/ml BDNF, then starved overnight and pulsed for 20 min with 20 ng/ml IGF1 (Fig. 4b–d). Motoneurons overexpressing IGFBP5 also exhibited reduced axon growth (Fig. 4e, f), when cultured with IGF1 at a concentration of 5 ng/ml (P < 0.001, one-way ANOVA). Axon growth was normal when these motoneurons were cultured with 5 ng/ml BDNF (P > 0.05, one-way ANOVA), indicating that IGFBP5 not only reduces survival but also specifically inhibits the effects of IGF1 on axon growth.Fig. 3


Dysregulated IGFBP5 expression causes axon degeneration and motoneuron loss in diabetic neuropathy.

Simon CM, Rauskolb S, Gunnersen JM, Holtmann B, Drepper C, Dombert B, Braga M, Wiese S, Jablonka S, Pühringer D, Zielasek J, Hoeflich A, Silani V, Wolf E, Kneitz S, Sommer C, Toyka KV, Sendtner M - Acta Neuropathol. (2015)

Igfbp5 tg+ motoneurons show decreased survival when cultured with IGF1. a qRT-PCR revealed a 1.8-fold increase of the Igfbp5 mRNA level in the spinal cord of 6-month-old Igfbp5 tg+ mice (Bp5 tg+). Scored values were obtained by normalizing to β-actin mRNA levels. A.U. arbitrary units. b Igfbp5 protein elevation was detected in sciatic nerve, but not in spinal cord and brain of 6-month-old Igfbp5 tg+ mice compared to controls. Actin was used as loading control. c Quantification of Igfbp5 protein levels. QL quantitative labeling. d Motoneurons stained for Tau (red) and Igfbp5 (green) 7 days after plating. Igfbp5 was increased in the soma and neurites of Igfbp5 tg+ motoneurons. Igfbp5 appeared associated with the cell membrane. Scale bar 5 μm. e The positive effect of IGF1 on motoneuron survival is reduced by 37 % in Igfbp5 tg+ motoneurons. Motoneuron survival was unchanged in the presence of BDNF
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Fig3: Igfbp5 tg+ motoneurons show decreased survival when cultured with IGF1. a qRT-PCR revealed a 1.8-fold increase of the Igfbp5 mRNA level in the spinal cord of 6-month-old Igfbp5 tg+ mice (Bp5 tg+). Scored values were obtained by normalizing to β-actin mRNA levels. A.U. arbitrary units. b Igfbp5 protein elevation was detected in sciatic nerve, but not in spinal cord and brain of 6-month-old Igfbp5 tg+ mice compared to controls. Actin was used as loading control. c Quantification of Igfbp5 protein levels. QL quantitative labeling. d Motoneurons stained for Tau (red) and Igfbp5 (green) 7 days after plating. Igfbp5 was increased in the soma and neurites of Igfbp5 tg+ motoneurons. Igfbp5 appeared associated with the cell membrane. Scale bar 5 μm. e The positive effect of IGF1 on motoneuron survival is reduced by 37 % in Igfbp5 tg+ motoneurons. Motoneuron survival was unchanged in the presence of BDNF
Mentions: To study this effect on motoneurons and sensory neurons in vivo, we generated mice with neuron-specific overexpression of IGFBP5. An 8-kb fragment including the human NF-L promoter, mouse Igfbp5 cDNA, the polyA signal from pMC-Cre and exons 2–4 of the NF-L gene was injected into fertilized mouse eggs (for details see electronic supplementary Fig. A1, supplementary methods section). Real-time RT-PCR analysis revealed that the levels of Igfbp5 mRNA in the spinal cord of 6 months old Igfbp5 transgenic mice (Bp5 tg+) were up-regulated 1.8-fold compared with controls (P < 0.001, two-tailed Student’s t test) (Fig. 3a). Western blot analysis for IGFBP5 in the spinal cord, sciatic nerve and brain of 6-month-old Igfbp5 transgenic mice exhibited a fivefold IGFBP5 protein up-regulation in the sciatic nerve (P < 0.001, two-tailed Student’s t test) (Fig. 3b, c), indicating that the IGFBP5 protein is anterogradely transported in axons. Isolated motoneurons from Igfbp5 transgenic embryos (E13.5) also showed increased IGFBP5 immunoreactivity in cell bodies and axons after 7 days in vitro (Fig. 3d). IGFBP5 was predominantly localized on the surface of these neurons. This correlates with previous data showing that IGFBP5 binds to the cell surface and the surrounding extracellular matrix [2]. Igfbp5 transgenic motoneurons showed a different survival response to IGF1 than wild-type motoneurons. While survival of Igfbp5-overexpressing motoneurons was unchanged in the presence of BDNF (P > 0.05, one-way ANOVA), their survival was significantly reduced by 37 % in the presence of IGF1 compared to wild-type motoneurons (P < 0.001, one-way ANOVA) (Fig. 3e). When we compared the dose response for IGF1 on motoneuron survival in Igfbp5 transgenic and wild-type motoneurons, a shift was observed (0.2 ng/ml IGF1; P < 0.05; 1 ng/ml IGF1; P < 0.001; 5 ng/ml IGF1; P < 0.001; one-way ANOVA) (Fig. 4a), indicating that motoneurons overexpressing IGFBP5 need more IGF1 for their survival than non-transgenic motoneurons. This result provides further evidence that IGFBP5 inhibits IGF1 actions on motoneurons. We then compared the phosphorylation levels of IGF1R and downstream AKT in Igfbp5 transgenic motoneurons relative to wild-type controls. A strong reduction in IGF1R and AKT phosphorylation levels was observed in Igfbp5 transgenic motoneurons compared to controls (pIGF1R: P < 0.001; pAKT: P < 0.01, two-tailed Student’s t test) grown for 5 days with 5 ng/ml BDNF, then starved overnight and pulsed for 20 min with 20 ng/ml IGF1 (Fig. 4b–d). Motoneurons overexpressing IGFBP5 also exhibited reduced axon growth (Fig. 4e, f), when cultured with IGF1 at a concentration of 5 ng/ml (P < 0.001, one-way ANOVA). Axon growth was normal when these motoneurons were cultured with 5 ng/ml BDNF (P > 0.05, one-way ANOVA), indicating that IGFBP5 not only reduces survival but also specifically inhibits the effects of IGF1 on axon growth.Fig. 3

Bottom Line: IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP.These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration.Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University of Würzburg, Versbacher-Str. 5, 97078, Würzburg, Germany.

ABSTRACT
Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes. The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor (IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.

No MeSH data available.


Related in: MedlinePlus