Brain Advance Access originally published online on April 22, 2003
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Brain, Vol. 126, No. 7, 1671-1682,
July 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg150
Enhanced activation of axonally transported stress-activated protein kinases in peripheral nerve in diabetic neuropathy is prevented by neurotrophin-3
School of Biological Sciences, University of Manchester, Manchester, UK *Present address: Department of Neurology, Stanford University, Stanford, CA, USA
Correspondence to: Dr Paul Fernyhough, 1.124 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK E-mail: paul.fernyhough{at}man.ac.uk
Received October 17, 2002. Revised January 28, 2003. Accepted February 13, 2003.
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The objective was to determine whether stress-activated protein kinases (SAPKs) mediated the transfer of diabetes-induced stress signals from the periphery to somata of sensory neurons. Thus, we characterized axonal transport of SAPKs in peripheral nerve, studied any alteration in streptozotocin (STZ)-diabetic rats and examined effects of neurotrophin-3 (NT-3) on diabetes-induced events. We demonstrate that c-jun N-terminal kinase (JNK) and p38 are bidirectionally axonally transported at fast rates in sciatic nerve. In STZ-diabetic rats the relative levels of retrograde axonal transport of phosphorylated (activated) JNK and p38 were raised compared with age-matched controls (all data are in arbitrary units and expressed as fold increase over control: JNK 54–56 kDa isoforms, control 1.0 ± 0.19, diabetic 2.5 ± 0.26; p38, control 1.0 ± 0.09, diabetic 2.9 ± 0.52; both P < 0.05). Transport of total enzyme levels of JNK and p38 and phosphorylated extracellular signal-regulated kinase (ERK) was not significantly altered and anterograde axonal transport of phosphorylated JNK and p38 was unaffected by diabetes. The transcription factor ATF-2, which is phosphorylated and activated by JNK and p38, also exhibited elevated retrograde axonal transport in STZ-diabetic animals (control 1.0 ± 0.07, diabetic 3.0 ± 0.41; P < 0.05). Treatment of STZ-diabetic animals with 5 mg/kg human recombinant NT-3 prevented activation of JNK and p38 in sciatic nerve (phosphorylated JNK, control 1.0 ± 0.09, diabetic 1.95 ± 0.35, diabetic + NT-3 1.09 ± 0.12; P < 0.05 diabetic versus others; phosphorylated p38, control 1.0 ± 0.16, diabetic 4.7 ± 0.9, diabetic + NT-3 1.19 ± 0.18; P < 0.05 diabetic versus others). The results show that JNK and p38 are transported axonally and may mediate the transfer of diabetes-related stress signals, possibly triggered by loss of neurotrophic support, from the periphery to the neuronal soma.
Keywords: DRG; neurotrophic factors; NT-3; peripheral neuropathy; SAPKs
Abbreviations: CGRP = calcitonin gene-related peptide; DRG = dorsal root ganglia; ERK = extracellular signal-regulated kinase; JIP-1 = JNK inhibitory protein 1; JNK = c-jun N-terminal kinase; NGF = nerve growth factor; NT-3 = neurotrophin-3; SAPK = stress-activated protein kinase; STZ = streptozotocin
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Introduction |
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Diabetic sensory neuropathy in humans is associated with a spectrum of structural changes in peripheral nerve that includes axonal degeneration, paranodal demyelination and loss of myelinated fibres, the latter probably the result of dying back of distal axons (Thomas and Tomlinson, 1993
; Yagihashi, 1997). In the streptozotocin (STZ)-diabetic rat and Bio-Breeding rat animal models of type I diabetes, similar structural abnormalities in peripheral nerve have been observed (Yagihashi, 1997). The Diabetes Control and Com plications Trial concluded that control of hyperglycaemia is still the ideal means of preventing the appearance of complications in diabetes, such as peripheral neuropathy (Diabetes Control and Complications Trial Research Group, 1993). However, such a goal remains an unrealized ideal and research continues to focus on biochemical transducers downstream from hyperglycaemia that may directly induce neuropathic sensory nerve damage.
The stress-activated protein kinase (SAPK) subgroup of mitogen-activated protein kinases are activated by a range of cellular stresses. Among the most commonly studied of these are hyperglycaemia, hypertonicity, treatment with cytokines such as interleukin-1ß (IL-1ß) and tumour necrosis factor 
(TNF-), oxidative stress and ultraviolet irradiation (Whitmarsh and Davis, 1996; Davis, 2000; Weston and Davis, 2002). In PC12 cells, SH-SY5Y cells, embryonic cortical neurons and embryonic peripheral neurons, the members of the SAPK family are activated by glutamate (Ackerley et al., 2000; Brownlees et al., 2000), hyperglycaemia (Cheng and Feldman, 1998), hyperosmolarity (Giasson and Mushynski, 1997) and changes in neurotrophic support (Xia et al., 1995; Deshmukh and Johnson, 1997). The SAPK family includes extracellular signal-regulated kinase 1/2 (ERK 1/2), c-jun N-terminal kinase (JNK; comprising three genes, termed JNK1, 2 and 3) and p38/HOG (Whitmarsh and Davis, 1996; Davis, 2000; Harper and LoGrasso, 2001; Weston and Davis, 2002). Upon activation, this family of kinases bind to the transcription factors c-jun (JNK only), ATF2 and ELK-1 and phosphorylate the activation domains favouring DNA binding (Whitmarsh and Davis, 1996; Davis, 2000; Weston and Davis, 2002). Events downstream of transcription factor activation are not well defined in neurons, but in PC12 cells and sympathetic neurons the activation of JNK and p38, triggered by removal of nerve growth factor (NGF), induces apoptosis (Xia et al., 1995; Deshmukh and Johnson, 1997).
JNK1 and JNK2 combine to regulate apoptosis during development of the mouse brain (Kuan et al., 1999). In the adult CNS there is heterogeneous expression of JNK and activation in response to extracellular stress (Carletti et al., 1995; Xu et al., 1997); for example, kainate-induced excitotoxicity in the hippocampus is mediated via activation of JNK3 (Yang et al., 1997). In the PNS, axotomy of sensory neurons induces rapid and long-term activation of JNK within the lumbar dorsal root ganglia (DRG) (Kenney and Kocsis, 1998). The mechanism of JNK activation is unknown but proinflammatory cytokines, including IL-1ß and TNF-, are expressed in nerve trunks after axotomy and these may activate neuronal SAPKs (Rotshenker et al., 1992; Murphy et al., 1995). Following axotomy, neurons are also exposed to a relative loss of neurotrophic support (Richardson, 1991).
Our previous studies in STZ-diabetic and Bio-Breeding rats show that JNK, p38 and ERK are activated in lumbar DRG and sural nerve and could mediate neurodegeneration (Fernyhough et al., 1999). We have proposed that activation of JNK and ERK may mediate aberrant neurofilament phosphorylation in sensory neurons in diabetes and induce loss of axon calibre and eventual dying back of nerve endings (Fernyhough et al., 1999). Furthermore, activation of p38 and ERK in cultured sensory neurons was shown to be associated with oxidative stress, and protection from cell death could be afforded by inhibition of this activation (Purves et al., 2001).
The transport of signalling molecules downstream from tyrosine kinase receptors has been demonstrated previously in peripheral nerve. For example, ERK and the transcription factor ATF2 are transported bidirectionally in sciatic nerve (Delcroix et al., 1999; Averill et al., 2001; Reynolds et al., 2001). The SAPKs have been localized to the axons of peripheral nerve (Fernyhough et al., 1999) and, therefore, the present study was designed to test whether SAPKs were transported in axons of the sciatic nerve of adult rats. Additionally, the aim was to determine whether the activation state of transported SAPKs was affected by type I diabetes, and if so, to examine effects of treatment with neurotrophin-3 (NT-3), which is known to offer protection against experimental diabetic neuropathy (Mizisin et al., 1999).
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Induction of diabetes
Male Wistar rats were made diabetic for 8 weeks (or for 14 weeks in the NT-3 study; see below) by a single intraperitoneal injection of STZ (55 mg/kg; Sigma, St Louis, MO, USA). The level of glucose in tail vein blood was measured using a reflectance meter operated by a glucose oxidase strip (Reflolux II BCL; Boehringer Mannheim), and it was >27 mmol/l for all STZ-injected rats. At death, plasma glucose levels for all groups of diabetic rats were >20 mmol/l (plasma glucose levels for control rats ranged from 10 to 11 mmol/l). In the axonal transport study, the body weights for age-matched control rats ranged between 450 and 500 g, while all groups of diabetic rats weighed 300–350 g at the end of the study. All animal procedures followed the strict guidelines of the UK Home Office Regulations and were in accordance with the UK Animals (Scientific Procedures) Act 1986.
NT-3 treatment study
Two groups of Wistar rats were made diabetic for 14 weeks. One group of diabetic rats received subcutaneous injections of 5 mg/kg human recombinant NT-3 (a gift from Regeneron Pharmaceuticals, Tarrytown, NY, USA) three times weekly for the final 10 weeks of the 14-week diabetes study. Body weights for age-matched control rats ranged from 530 to 710 g, while all groups of diabetic rats weighed 290–445 g at study end. For all diabetic animals, the motor nerve conductance velocity and sensory nerve conductance velocity (as measured by the H-reflex) were significantly reduced. The efficacy of NT-3 treatment was confirmed by reversal of the sensory nerve conductance velocity deficit, as described previously (Tomlinson et al., 1996; Mizisin et al., 1998, 1999) (data not shown). Animals were killed by a blow to the head and exsanguination and 1-cm segments of sciatic nerve were isolated and frozen immediately on dry ice in preparation for quantitative western blotting.
Quantification of axonal transport using western blotting
Animals were anaesthetized using halothane, the sciatic nerve was exposed unilaterally and double ligatures 1 cm apart were placed at mid-thigh. This double ligature procedure for the measurement of axonal transport has been described by Raivich et al. (1991). Briefly, the double ligatures were applied for 6 or 12 h, the rats were killed, and 0.5 cm of sciatic nerve proximal (for anterograde accumulation), distal (for retrograde accumulation) and intermediate to each ligature was extracted and frozen in liquid nitrogen. For calculation of retrograde axonal transport, the western blot signal for the intermediate nerve segment was subtracted from the signal for the distal or retrograde accumulation. The intermediate value was derived mainly from local synthesis between the ligatures (and induced by the ligature), whereas the distal (retrograde) accumulation included local synthesis and the accumulated material. These samples and the sciatic nerve segments from the NT-3 study were homogenized using a Polytron (Kinematica, Lucerne, Switzerland) in 0.1 mM PIPES (pH 6.9), 5.0 mM MgCl2, 5.0 mM EGTA, 0.5% Triton X-100, 20% glycerol, 1.0 mM phenylmethylsulphonyl fluoride and a mixture of protease inhibitors (1.0 µg/ml pepstatin A, 1.0 µg/ml leupeptin, 10 µg/ml benzoyl-L-arginine methyl ester, 10 µg/ml p-tosyl-L-arginine methyl ester, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml L-1-tosylamide-2 phenylethylchloromethyl ketone and 7 µg/ml aprotinin). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (10% acrylamide) was performed on 10 µg of protein, and the separated proteins were transferred to nitrocellulose [Amersham ECL (enhanced chemiluminescence) membrane; Amersham Biosciences, Little Chalfont, UK] using a graphite blotter. Primary antibodies used were a polyclonal to total JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA, code FL; diluted 1 : 1000) and a monoclonal antibody to phosphorylated JNK (Santa Cruz, code G-7; 1 : 200; phosphorylated on Thr-183 and Tyr-185); antibodies to total p38 (New England Biolabs, Beverley, MA, USA, code 9212; 1 : 1000) and phosphorylated p38 (Biosource International, Camarillo, CA, USA, code 44–684; 1 : 2000; phosphorylated on Thr-180 and Tyr-182); polyclonal antibodies to total ATF2 (New England Biolabs, code 9222; 1 : 1000) and phosphorylated ATF2 (New England Biolabs, code 9225; 1 : 1000; phosphorylated on Thr-69/71); and polyclonal antibody to phosphorylated ERK (New England Biolabs, code 9101; 1 : 4000; phosphorylated on Thr-202 and Tyr-204). Detection was achieved using the New England Biolabs phototope-HRP system. The relative levels of protein were determined using an image analyser (AI, Cambridge, UK). Following ECL detection, all blots were stained with Indian ink to confirm even loading of protein.
Immunohistochemistry for JNK and p38 in sciatic nerve
Three normal rats and three rats that had undergone unilateral sciatic nerve ligature for 12 h (as described above) were used for immunohistochemical analysis of axonal transport and SAPK localization to axons using standard techniques (Priestley, 1997). The animals were anaesthetized with sodium pentobarbital (60 mg/kg) and perfused through the ascending aorta with saline followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The mid-sciatic nerve (covering ligatured area) and intact nerve from unoperated animals were removed, postfixed for 2 h in the same fixative and cryoprotected in 15% sucrose overnight. Tissues were frozen and sectioned at 8 µm either longitudinally (for SAPK accumulation at crush site) or transversely (for localization of SAPKs to axon). Sections were stained using indirect immunofluorescence histochemistry with polyclonal antibodies directed against total JNK (Santa Cruz Biotechnology, code FL; diluted 1 : 100); phosphorylated JNK (Promega, Madison, WI, USA, code v1211; 1 : 200; phosphorylated on Thr-183 and Tyr-185); antibodies to total p38 (Santa Cruz Biotechnology, code FL; diluted 1 : 100) and phosphorylated p38 (Biosource International, Camarillo, CA, USA, code 44–684; 1 : 250; phosphorylated on Thr-180 and Tyr-182). Calcitonin gene-related peptide (CGRP) was detected using a sheep polyclonal antibody at 1 : 2000 (Michael et al., 1997) and mouse anti-S100ß was obtained from Sigma. For detection of primary polyclonal antibody, a donkey anti-rabbit FITC (fluorescein isothiocyanate)-conjugated secondary antiserum (Jackson Immunoresearch, West Grove, PA, USA; 1 : 200) was used; for detection of monoclonal antibody a sheep anti-mouse TRITC (tetramethyl rhodamine isothiocyanate) conjugate was used (Jackson Immunoresearch). Double labelling of phosphorylated JNK or total p38 together with CGRP was achieved using standard indirect immunofluorescence. After final washes in phosphate-buffered saline, sections were coverslipped in a phosphate-buffered saline/glycerol solution (1 : 3) containing 2.5% 1,4-diazobicyclo(2,2,2)octane (antifading agent; Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was visualized on a Leica epifluorescence microscope using the Y3 and L4 filter block.
Data analysis
The axonal transport data derived from western blots are presented normalized to control as mean ± SEM (n = 3). Data from western blots were derived by subtracting the intermediate nerve segment value (relates possible local changes in synthesis of proteins induced by the ligature) from either the proximal or retrograde accumulation value. Values were also adjusted for background levels of signal derived from intact nerve (Delcroix et al., 1998, 1999). Single comparisons between groups were made using Student’s t-test. The NT-3 study data, where appropriate and allowing for homogeneity of variances according to the Levene test, were subjected to one-way ANOVA (analysis of variance) using the Statistical Package for the Social Sciences (SPSS/PC+; SPSS, Chicago, IL, USA). Where the F ratio gave P < 0.05, comparisons between individual group means were made using Tukey’s HSD (highly significant difference) multiple range test at significance levels of P = 0.05.
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Immunohistochemical demonstration of axonal transport of JNK and p38 in sciatic nerve
The first aim was to use immunohistochemistry to characterize axonal transport of SAPKs in the sciatic nerve of normal rats. Adult rats underwent unilateral sciatic nerve ligature at mid-thigh for 12 h. Figures 1 and 2 show immunohistochemistry for total and phosphorylated JNK and p38 in ligated and intact sciatic nerve. The detection of phosphorylated JNK and p38 represented the populations of JNK and p38 enzyme that had undergone activation (this also applies for the antibody detection of phosphorylated ERK and ATF2 presented later). Nerve ligation induced bidirectional accumulation of phosphorylated JNK (Fig. 1A and B), total JNK (Fig. 1C and D), phosphorylated p38 (Fig. 2A and B) and total p38 (Fig. 2C and D). Double labelling of the proximal segment (anterograde transport) of nerve showed colocalization of phosphorylated JNK with the neuronal marker CGRP (Fig. 1A and F). The same was true for phosphorylated p38 (Fig. 2A and F). Phosphorylated JNK and p38 were localized to axons but could also be visualized in a limited number of non-neurons, probably Schwann cells (arrowheads in Figs 1A and 2A). Staining for total JNK and p38 was more widespread, with localization to axons but also extensive labelling of non-neuronal elements (Figs 1C, D and 2C, D). Intact sciatic nerve was also stained for phosphorylated JNK (Fig. 1E) and phosphorylated p38 (Fig. 2E), confirming that the staining observed in ligated nerve was not simply an artefact generated by the operation. Ligated nerve tissues from STZ-diabetic rats were also analysed using immunohistochemistry and the pattern of staining for JNK and p38 was comparable to that seen in control tissues.
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JNK and p38 accumulate at a nerve crush with kinetics indicative of fast axonal transport
The identity and quantity of JNK and p38 (total and phosphorylated enzyme) immunoreactivity was examined by western blotting. At the ligature sites, Fig. 3 shows the westerns blots for the SAPK proteins. The antibodies to total and phosphorylated JNK detected protein species of 56, 54 and 46 kDa, and this most likely reflects the expression of all three of the JNK genes (Gupta et al., 1996
). The anti-p38 antibodies detected a single species at 
38–40 kDa. Western blots were quantified and anterograde and retrograde accumulation was calculated by subtracting the intermediate nerve segment values from the values in segments proximal or distal to the ligatures. The rate of retrograde accumulation of phosphorylated JNK and p38 was 1.0 µm/s and was linear for up to 12 h, which is consistent with accumulation by fast axonal transport (Table 1).
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Effect of STZ diabetes on levels of axonal transport of JNK and p38 in sciatic nerve
Age-matched control rats and 8-week STZ-diabetic rats underwent unilateral double crushes of the sciatic nerve of 6 h duration in order to quantify the fast axonal transport levels of the SAPKs [and the downstream target ATF2 (transcription factor); see later]. Nerve segments were homogenized and subjected to western blotting; the levels of accumulation of the SAPKs were determined and the amounts of fast axonal transport of these proteins calculated using the double-ligature paradigm described previously. Figures 3 and 4 show that STZ diabetes induced a significant 2.5- to 3-fold increase in the retrograde axonal transport of the phosphorylated forms of JNK (46 and 54–56 kDa isoforms) and p38 compared with age-matched control rats (all data are in arbitrary units and expressed as fold increase over control: JNK 54–56 kDa isoforms, control 1.0 ± 0.19 versus diabetic 2.5 ± 0.26; p38, control 1.0 ± 0.09 versus diabetic 2.9 ± 0.52; both P < 0.05). Retrograde axonal transport of phosphorylated ERK was not similarly affected (see western blot in Fig. 3). The levels of total JNK undergoing retrograde axonal transport were also raised
1.5-fold compared with controls. For all the SAPKs the levels of anterograde axonal transport were not affected by STZ diabetes (Table 2).
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Diabetes induces elevated axonal transport of a SAPK-targeted transcription factor
The effect of diabetes on the axonal transport of the transcription factor ATF2 was also analysed. Both JNK and p38, when activated, induce the phosphorylation of ATF2 and enhance transcriptional activity (Whitmarsh and Davis, 1996
; Davis, 2000; Harper and LoGrasso, 2001; Weston and Davis, 2002). Figures 3 and 4 show that diabetes induced a significant 3-fold elevation in the retrograde axonal transport of phosphorylated ATF2 (control 1.0 ± 0.07 versus diabetic 3.0 ± 0.41; P < 0.05). Levels of retrograde transport of total ATF2 were unaffected, as was anterograde transport of phosphorylated ATF2 (data not shown).
Localization of SAPKs to axon and/or Schwann cell
We next confirmed the localization of activated JNK and p38 to the axon in normal and diabetic nerve. Figure 5 shows immunohistochemical staining for activated JNK and p38 in transverse sections of sciatic nerve from normal (Fig. 5A and C) and STZ-diabetic (Fig. 5B and D) animals. The staining for activated JNK was highly enriched in the axonal area, with little staining of Schwann cells in normal and diabetic animals (Fig. 5A and B). The staining for activated p38 was more widespread, with significant staining of Schwann cells and weak axonal staining in normal animals (S100 staining in Fig. 5C and F). In STZ-diabetic animals the p38 staining showed a stronger axonal signal (Fig. 5D). The level of staining for both activated JNK and p38 was clearly higher in the sciatic nerve of STZ-diabetic animals.
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When analysing whole-nerve expression of SAPKs, we believe it is correct to assume that JNK expression has a mainly axonal origin, whereas the p38 signal has contributions from axon and Schwann cell. This fact should be understood when interpreting the studies presented below on NT-3 effects on SAPK activation in diabetes.
NT-3 prevents the diabetes-induced elevation of JNK and p38 activation in sciatic nerve
In order to determine if the activation of SAPKs in diabetes was related to loss of neurotrophic support, we treated 14-week STZ-diabetic animals with 5 mg/kg human recombinant NT-3 for the final 10 weeks of the study. Sciatic nerve homogenates were subjected to quantitative western blotting and probed for the SAPKs (Fig. 6). Phosphorylation of JNK and p38 was significantly elevated 1.95- and 4.7-fold, respectively, in sciatic nerve of STZ-diabetic animals. The 56/54 and 46 kDa isoforms of JNK were elevated, the 56/54 kDa isoforms being most clearly raised (Fig. 6A). Treatment with NT-3 significantly prevented this diabetes-induced activation of JNK and p38 (Fig. 6) (phosphorylated JNK 46 kDa isoform, control 1.0 ± 0.09, diabetic 1.95 ± 0.35, diabetic + NT-3 1.09 ± 0.12; P < 0.05 diabetic versus others; phosphorylated p38, control 1.0 ± 0.16, diabetic 4.7 ± 0.9, diabetic + NT-3 1.19 ± 0.18; P < 0.05 diabetic versus others). STZ diabetes also increased the levels of total JNK (54–56 kDa isoform; Fig. 6B), although this was not statistically significant (control 1.0 ± 0.21, diabetic 1.69 ± 0.26, diabetic + NT-3 1.41 ± 0.34). Total levels of JNK (46 kDa isoform) and p38 were not affected by STZ diabetes (Fig. 6B and D) (data not shown).
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The results demonstrate for the first time that SAPKs undergo fast axonal transport in peripheral nerve and that the activation status of these enzymes can be elevated in an animal model of sensory neuropathy, the STZ-diabetic rat. A critical observation is that the diabetes-induced enhancement of JNK and p38 activation is restricted to the retrograde component of axonal transport; this gives directionality to the stress signal. Furthermore, activation of JNK and p38 was specific, with no effect on ERK. These results are novel because they highlight one mechanism whereby stress signals that are generated peripherally can be translocated to the sensory neuron soma. Interestingly, as part of the stress response we demonstrate that a target transcription factor of JNK and p38, namely ATF2, is activated peripherally and transported axonally to the neuronal cell body. Treatment of STZ-diabetic rats with NT-3 prevented activation of JNK and p38 in sciatic nerve, implying a role for neurotrophin withdrawal in the mechanism of activation of the SAPKs.
SAPKs and neurodegeneration
Axotomy of adult sensory neurons results in a perikaryal response highlighted by the phosphorylation of JNK and related elevated activation and expression of c-jun; however, a significant level of cell death does not occur (Jenkins et al., 1993; Gold et al., 1994; Kenney and Kocsis, 1998). The loss of neurotrophic support following axotomy may be instrumental in this JNK activation. In rat models of sensory neuropathy there is activation of JNK, p38 and ERK in DRG and sural nerve (Fernyhough et al., 1999). This could be derived from diminished neurotrophic support (Fernyhough et al., 1995, 1998). In cultured adult sensory neurons, high concentrations (>25 mM) of glucose triggered activation of JNK. This suggests that, in STZ-diabetic or Bio-Breeding diabetic rats, a critical factor controlling the neuronal stress response is also hyperglycaemia (Tomlinson, 1999; Purves et al., 2001).
Hyperglycaemia, neurotrophic support and activation of SAPKs
In kidney, in response to high concentrations of glucose the GTPase Cdc42 is activated via an adapter protein, Gene 33, and mediates sustained activation of JNK (Makkinje et al., 2000). The mobilization of GTP-Cdc42 by high glucose is JNK-dependent, i.e. JNK triggers its own long-term activation. In neurons there is evidence that high glucose can induce oxidative stress and may be a central mediator of SAPK activation (Tomlinson, 1999; Purves et al., 2001). High glucose concentrations result in elevated oxidative phosphorylation and the subsequent production of reactive oxygen species may induce diabetic complications (Nishikawa et al., 2000). The reactive oxygen species-dependent activation of SAPKs may be one critical pathway leading to neurodegeneration (Tomlinson, 1999; Purves et al., 2001). Furthermore, in neurons the loss of trophic support may result in lack of ligand binding to trk receptors and p75NTR. This can induce activation of the death domain of p75NTR and activation of JNK (Kaplan and Miller, 2000; Harrington et al., 2002). Additionally, neurotrophins activate protein kinase B/Akt, which is a negative modulator of the scaffold protein JNK inhibitory protein 1 (JIP-1), and lack of neurotrophin action will therefore lead to downregulation of Akt and consequently the enhancement of JNK activation (Kim et al., 2002).
We have described a retrograde stress signal that reflects a neurodegenerative process that is operating along the length of the axon and/or is related to a nerve ending/synapse interaction with target. Hyperglycaemia will occur along the whole length of the nerve and therefore activation of SAPKs in the axon will occur at any point. The activated SAPKs are loaded specifically onto the retrograde transport machinery (to be discussed later). Impairment in trophic support could also occur along the whole length of the axon. However, it is most likely that a target tissue-related loss of neurotrophic support is involved (Fernyhough and Tomlinson, 1999). NGF and NT-3 expression is downregulated in muscle and skin in STZ-diabetic rats and, therefore, reduced occupancy of trk receptors and p75NTR at nerve endings may contribute to a distally generated stress signal.
NT-3-dependent normalization of SAPK activation
The results show that NT-3 prevented the diabetes-induced elevation in JNK and p38 activation in intact nerve. The complete reversal of activation of JNK and p38 in nerve by NT-3 was surprising. In cultured embryonic cortical neurons, brain-derived neurotrophic factor and NT-3 treatment were shown to elevate neurofilament H phosphorylation, possibly via increased activation of JNK and ERK (Tokuoka et al., 2000). Furthermore, only 20% of sensory neurons in adult DRG express trkC receptors. The nociceptive/mechanoreceptive population, which constitutes 80% of the neuronal DRG cells, do not express trkC receptors (Michael et al., 1999). However, at 5.0 mg/kg, NT-3 may be affecting SAPK activation in a wide range of sensory neuron phenotypes via binding to trkC, trkA and/or p75NTR (Belliveau et al., 1997; Dechant et al., 1997). An alternatively spliced form of trkA has high affinity for NT-3; however, its expression in sensory neurons is unknown (Clary and Reichardt, 1994). The ability of NT-3 to interact with trkA is also partly dependent on p75NTR. Extracellular truncation or loss of expression of p75NTR permits NT-3 binding to trkA (Mischel et al., 2001), and this will be encouraged in sensory neurons of diabetic animals, in which p75NTR levels of expression are reduced (Delcroix et al., 1998).
Mechanism of axonal transport of SAPKs
In fibroblasts, JNK is localized to microtubules and in close proximity to kinesin (Nagata et al., 1998). The scaffold protein JIP-1, an important regulator of JNK function in neurons and non-neurons, is present in axons and growth cones of neurons and undergoes axonal transport in non-neurons (Verhey et al., 2001; Whitmarsh et al., 2001). JIP-1, -2 and -3 bind to the COOH-terminus of the kinesin light chain and regulate JNK function via binding. Therefore, kinesin-dependent transport of a JIP/JNK complex is feasible (Verhey et al., 2001). This would account for the anterograde component; however, no work has been done linking dynein with axonal transport of JIP. Howe and colleagues have recently shown that NGF signalling via endosomes containing aggregates of signalling complexes of the Ras-mitogen-activated protein kinase pathway (Howe et al., 2001). There is no direct evidence that JNK or p38 is retrogradely transported in this manner; however, trkA can bind to cytoplasmic dynein (Yano et al., 2001) and one study demonstrates that JIP-2 can bind to a transmembrane receptor (the reelin receptor ApoER2) (Stockinger et al., 2000). Furthermore, dynein light chain can bind to the neuronal scaffold protein guanylate kinase-associated protein (GKAP) (Fan et al., 2001), which, in turn, can associate with membrane-associated guanylate kinases (MAGUK), which are proteins that have an Src homology 3 domain and may act as a proximal focus of signalling pathways downstream from trkA and upstream from JNK activation (Shin et al., 2000). It can be proposed that retrograde transport of SAPKs is mediated via trk receptor association. Therefore, the diabetes-induced modulation of activation of SAPKs is due to modification of trophic factor activation of trk receptors and alteration of the activation state of the associated signalling complexes.
Downstream consequences of SAPK activation in diabetic neuropathy
Activation of SAPKs, especially JNK and p38, is associated with neurodegeneration in PC12 cells and sympathetic neurons (Xia et al., 1995; Deshmukh and Johnson, 1997). Axotomy of sensory neurons activates JNK, but cell death is not the result; in fact, neurons survive and undergo axonal regeneration (Kenney and Kocsis, 1998). Exposure of cultured adult sensory neurons to high glucose concentrations results in JNK activation but cell death does not occur (Purves et al., 2001). In non-neurons the inhibitor of apoptosis family of proteins protect cells from TNF--induced cell death via activation of JNK1 (Sanna et al., 2002). Therefore, activation of JNK may be a protective pathway against cell death during specific stages of development. The situation with activation of p38 is more straightforward. Exposure of cultured sensory neurons to oxidative stress results in cell death and activation of p38; inhibition of the latter affords protection (Purves et al., 2001). Interestingly, STZ-diabetic rats exhibit allodynia and hyperalgesia, and Ji et al. (2002) have shown that p38 is activated in small-fibre sensory neurons in response to inflammation (Calcutt, 2002; Ji et al., 2002). During inflammatory pain or heat hypersensitivity, the activation of p38 involves enhanced retrograde transport of NGF; therefore, activation of p38 in STZ diabetes is via an alternative mechanism since NGF-dependent trophic support is downregulated in diabetes. Therefore, inhibition of p38 protects not only against oxidative stress-induced neurodegeneration but also against hyperalgesia, and so blockage of p38 activation should be an important target for drug therapy in diabetic neuropathy.
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In summary, we demonstrate for the first time that JNK and p38 are axonally transported in neurons. This transport process is modulated by stressful conditions, such as those found in type I diabetes, and may mediate the transfer of stress signals from periphery to the sensory neuron cell body. The diabetes-induced stress signal may involve a loss of trophic support. Finally, diabetes induces coordinate induction of JNK and p38 in axons, and the level of neurodegeneration may depend on the relative activities of these two signalling pathways.
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Acknowledgement |
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This work was funded by the Juvenile Diabetes Research Foundation (P.F.).
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References |
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Ackerley S, Grierson AJ, Brownlees J, Thornhill P, Anderton BH, Leigh PN, et al. Glutamate slows axonal transport of neurofilaments in transfected neurons. J Cell Biol 2000; 150: 165–75.
Averill S, Delcroix J-D, Michael GJ, Tomlinson DR, Fernyhough P, Priestley JV. Nerve growth factor modulates the activation status and fast axonal transport of ERK 1/2 in adult nociceptive neurones. Mol Cell Neurosci 2001; 18: 183–96.[CrossRef][ISI][Medline]
Belliveau DJ, Krivko I, Kohn J, Lachance C, Pozniak C, Rusakov D, et al. NGF and neurotrophin-3 both activate TrkA on sympathetic neurons but differentially regulate survival and neuritogenesis. J Cell Biol 1997; 136: 375–88.
Brownlees J, Yates A, Bajaj NP, Davis D, Anderton BH, Leigh PN, et al. Phosphorylation of neurofilament heavy chain side-arms by stress activated protein kinase-1b/Jun N-terminal kinase-3. J Cell Sci 2000; 113: 401–7.[Abstract]
Calcutt NA. Potential mechanisms of neuropathic pain in diabetes. Int Rev Neurobiol 2002; 50: 205–28.[ISI][Medline]
Carletti R, Tacconi S, Bettini E, Ferraguti F. Stress activated protein kinases, a novel family of mitogen-activated protein kinases, are heterogeneously expressed in the adult rat brain and differentially distributed from extracellular- signal-regulated protein kinases. Neuroscience 1995; 69: 1103–10.[CrossRef][ISI][Medline]
Cheng H-L, Feldman EL. Bidirectional regulation of p38 kinase and c-Jun N-terminal protein kinase by insulin-like growth factor-I. J Biol Chem 1998; 273: 14560–5.
Clary DO, Reichardt LF. An alternatively spliced form of the nerve growth factor receptor TrkA confers an enhanced response to neurotrophin 3. Proc Natl Acad Sci USA 1994; 91: 11133–7.
Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 2000; 103: 239–52.[CrossRef][ISI][Medline]
Dechant G, Tsoulfas P, Parada LF, Barde YA. The neurotrophin receptor p75 binds neurotrophin-3 on sympathetic neurons with high affinity and specificity. J Neurosci 1997; 17: 5281–7.
Delcroix J-D, Michael GJ, Priestley JV, Tomlinson DR, Fernyhough P. Effect of nerve growth factor treatment on p75NTR gene expression in lumbar dorsal root ganglia of streptozocin-induced diabetic rats. Diabetes 1998; 47: 1779–85.[Abstract]
Delcroix J-D, Averill S, Fernandes K, Tomlinson DR, Priestley JV, Fernyhough P. Axonal transport of activating transcription factor-2 is modulated by nerve growth factor in nociceptive neurons. J Neurosci 1999; 19: RC24.
Deshmukh M, Johnson EM Jr. Programmed cell death in neurons: focus on the pathway of nerve growth factor deprivation-induced death of sympathetic neurons. Mol Pharmacol 1997; 51: 897–906.
Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993; 329: 977–86.
Fan J, Zhang Q, Tochio H, Li M, Zhang M. Structural basis of diverse sequence-dependent target recognition by the 8 kDa dynein light chain. J Mol Biol 2001; 306: 97–108.[CrossRef][ISI][Medline]
Fernyhough P, Tomlinson DR. The therapeutic potential of neurotrophins for the treatment of diabetic neuropathy. Diabetes Rev 1999; 7: 300–11.
Fernyhough P, Diemel LT, Hardy J, Brewster WJ, Mohiuddin L, Tomlinson DR. Human recombinant nerve growth factor replaces deficient neurotrophic support in the diabetic rat. Eur J Neurosci 1995; 7: 1107–10.[CrossRef][ISI][Medline]
Fernyhough P, Diemel LT, Tomlinson DR. Target tissue production and axonal transport of neurotrophin-3 are reduced in streptozocin-diabetic rats. Diabetologia 1998; 41: 300–6.[CrossRef][ISI][Medline]
Fernyhough P, Gallagher A, Averill SA, Priestley JV, Hounsom L, Patel J, et al. Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes 1999; 48: 881–9.[Abstract]
Giasson BI, Mushynski WE. Study of proline-directed protein kinases involved in phosphorylation of the heavy neurofilament subunit. J Neurosci 1997; 17: 9466–72.
Gold BG, Austin DR, Storm-Dickerson T. Multiple signals underlie the axotomy-induced up-regulation of c-JUN in adult sensory neurons. Neurosci Lett 1994; 176: 123–7.[CrossRef][ISI][Medline]
Gupta S, Barrett T, Whitmarsh AJ, Cavanagh J, Sluss HK, Dérijard B, et al. Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J 1996; 15: 2760–70.[ISI][Medline]
Harper SJ, LoGrasso P. Signalling for survival and death in neurones: the role of stress-activated kinases, JNK and p38. Cell Signal 2001; 13: 299–310.[CrossRef][ISI][Medline]
Harrington AW, Kim JY, Yoon SO. Activation of Rac GTPase by p75 is necessary for c-jun N-terminal kinase-mediated apoptosis. J Neurosci 2002; 22: 156–66.
Howe CL, Valletta JS, Rusnak AS, Mobley WC. NGF signaling from clathrin-coated vesicles: evidence that signaling endosomes serve as a platform for the Ras-MAPK pathway. Neuron 2001; 32: 801–14.[CrossRef][ISI][Medline]
Jenkins R, McMahon SB, Bond AB, Hunt SP. Expression of c-Jun as a response to dorsal root and peripheral nerve section in damaged and adjacent intact primary sensory neurons in the rat. Eur J Neurosci 1993; 5: 751–9.[CrossRef][ISI][Medline]
Ji R, Samad T, Jin S, Schmoll R, Woolf C. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 2002; 36: 57–68.[CrossRef][ISI][Medline]
Kaplan DR, Miller FD. Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 2000; 10: 381–91.[CrossRef][ISI][Medline]
Kenney AM, Kocsis JD. Peripheral axotomy induces long-term c-Jun amino-terminal-kinase-1 activation and activator protein-1 binding activity by c-Jun and junD in adult rat dorsal root ganglia in vivo. J Neurosci 1998; 18: 1318–28.
Kim AH, Yano H, Cho H, Meyer D, Monks B, Margolis B, et al. Akt1 regulates a JNK scaffold during excitotoxic apoptosis. Neuron 2002; 35: 697–709.[CrossRef][ISI][Medline]
Kuan C-Y, Yang DD, Roy DRS, Davis RJ, Rakic P, Flavell RA. The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development. Neuron 1999; 22: 667–76.[CrossRef][ISI][Medline]
Makkinje A, Quinn DA, Chen A, Cadilla CL, Force T, Bonventre JV, et al. Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. A potential marker transcript for chronic pathologic conditions, such as diabetic nephropathy. Possible role in the response to persistent stress. J Biol Chem 2000; 275: 17838–47.
Michael GJ, Averill S, Nitkunan A, Rattray M, Bennett DLH, Yan Q, et al. Nerve growth factor treatment increases brain-derived neurotrophic factor selectively in TrkA-expressing dorsal root ganglion cells and in their central terminations within the spinal cord. J Neurosci 1997; 17: 8476–90.
Michael GJ, Averill S, Shortland PJ, Yan Q, Priestley JV. Axotomy results in major changes in BDNF expression by dorsal root ganglion cells: BDNF expression in large trkB and trkC cells, in pericellular baskets, and in projections to deep dorsal horn and dorsal column nuclei. Eur J Neurosci 1999; 11: 3539–51.[CrossRef][ISI][Medline]
Mischel PS, Smith SG, Vining ER, Valletta JS, Mobley WC, Reichardt LF. The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA. J Biol Chem 2001; 276: 11294–301.
Mizisin AP, Kalichman MW, Bache M, Dines KC, DiStefano PS. NT-3 attenuates functional and structural disorders in sensory nerves of galactose-fed rats. J Neuropathol Exp Neurol 1998; 57: 803–13.[ISI][Medline]
Mizisin AP, Calcutt NA, Tomlinson DR, Gallagher A, Fernyhough P. Neurotrophin-3 reverses nerve conduction velocity deficits in streptozotocin-diabetic rats. J Periph Nerv Syst 1999; 4: 211–21.[ISI][Medline]
Murphy PG, Grondin J, Altares M, Richardson PM. Induction of interleukin-6 in axotomized sensory neurons. J Neurosci 1995; 15: 5130–8.[Abstract]
Nagata K, Puls A, Futter G, Aspenstrom P, Schaefer E, Nakata T, et al. The MAP kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3. EMBO J 1998; 17: 149–58.[CrossRef][ISI][Medline]
Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000; 404: 787–90.[CrossRef][Medline]
Priestley JV. Immunocytochemical techniques for the study of the nervous system. In: Bachelard H, Turner A, editors. Neuro chemistry: a practical approach. Oxford: Oxford University Press; 1997. p. 71–120.
Purves T, Middlemas A, Agthong S, Jude EB, Boulton AJM, Fernyhough P, et al. A role for mitogen-activated protein kinases in the aetiology of diabetic neuropathy. FASEB J 2001; 15: 2508–14.
Raivich G, Hellweg R, Kreutzberg GW. NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration. Neuron 1991; 7: 151–64.[CrossRef][ISI][Medline]
Reynolds AJ, Hendry IA, Bartlett SE. Anterograde and retrograde transport of active extracellular signal-related kinase 1 (ERK1) in the ligated rat sciatic nerve. Neuroscience 2001; 105: 761–71.[CrossRef][ISI][Medline]
Richardson PM. Neurotrophic factors in regeneration. Curr Opin Neurobiol 1991; 1: 401–6.[CrossRef][Medline]
Rotshenker S, Aamar S, Barak V. Interleukin-1 activity in lesioned peripheral nerve. J Neuroimmunol 1992; 39: 75–80.[CrossRef][ISI][Medline]
Sanna MG, da Silva Correia J, Ducrey O, Lee J, Nomoto K, Schrantz N, et al. IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition. Mol Cell Biol 2002; 22: 1754–66.
Shin H, Hsueh YP, Yang FC, Kim E, Sheng M. An intramolecular interaction between Src homology 3 domain and guanylate kinase-like domain required for channel clustering by postsynaptic density-95/SAP90. J Neurosci 2000; 20: 3580–7.
Stockinger W, Brandes C, Fasching D, Hermann M, Gotthardt M, Herz J, et al. The reelin receptor ApoER2 recruits JNK-interacting proteins-1 and -2. J Biol Chem 2000; 275: 25625–32.
Thomas PK, Tomlinson DR. Diabetic and hypoglycemic neuropathy. In: Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo JF, editors. Peripheral neuropathy. Philadelphia: W.B. Saunders; 1993. p. 1219–50.
Tokuoka H, Saito T, Yorifuji H, Wei F-Y, Kishimoto T, Hisanaga S. Brain-derived neurotrophic factor-induced phosphorylation of neurofilament H subunit in primary cultures of embryo rat cortical neurons. J Cell Sci 2000; 113: 1059–68.[Abstract]
Tomlinson DR. Mitogen-activated protein kinases as glucose transducers for diabetic complications. Diabetologia 1999; 42: 1271–81.[CrossRef][ISI][Medline]
Tomlinson DR, Fernyhough P, Diemel LT. Neurotrophins and peripheral neuropathy. Philos Trans R Soc Lond B Biol Sci 1996; 351: 455–62.[ISI][Medline]
Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, et al. Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules. J Cell Biol 2001; 152: 959–70.
Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Genet Dev 2002; 12: 14–21.[CrossRef][ISI][Medline]
Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 1996; 74: 589–607.[CrossRef][ISI][Medline]
Whitmarsh AJ, Kuan CY, Kennedy NJ, Kelkar N, Haydar TF, Mordes JP, et al. Requirement of the JIP1 scaffold protein for stress-induced JNK activation. Genes Dev 2001; 15: 2421–32.
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995; 270: 1326–31.
Xu X, Raber J, Yang D, Su B, Mucke L. Dynamic regulation of c-Jun N-terminal kinase activity in mouse brain by environmental stimuli. Proc Natl Acad Sci USA 1997; 94: 12655–60.
Yagihashi S. Nerve structural defects in diabetic neuropathy: do animals exhibit similar changes? Neurosci Res Commun 1997; 21: 25–32.
Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 1997; 389: 865–70.[CrossRef][Medline]
Yano H, Lee FS, Kong H, Chuang JZ, Arevalo JC, Perez P, et al. Association of Trk neurotrophin receptors with components of the cytoplasmic dynein motor. J Neurosci 2001; 21: RC125.
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