Diabetes impairs an interleukin-1β-dependent pathway that enhances neurite outgrowth through JAK/STAT3 modulation of mitochondrial bioenergetics in adult sensory neurons
© Saleh et al.; licensee BioMed Central Ltd. 2013
Received: 29 August 2013
Accepted: 21 October 2013
Published: 24 October 2013
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.
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.
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.
KeywordsNeurotrophic factor Axon regeneration Bioenergetics Neuropathy Dorsal root ganglia
Peripheral nerve injury is associated with an inflammatory response at the site of damage that is associated with enhanced expression of cytokines such as interleukin-1β (IL-1β), TNF-α and IL-6[1–3]. Nerve damage, and associated Wallerian degeneration, causes the secretion of IL-1β by Schwann cells and invading macrophages leading to a cascade of events, in part leading to increased expression of nerve growth factor (NGF) by fibroblasts[4–7]. The induction of NGF provides neurotrophic support for regenerating nerve fibers but this inflammatory process also triggers the development of hyperalgesia[8, 9].
Recent studies reveal that IL-1β may directly target sensory neurons. Adult sensory neurons express receptors for cytokines, such as LIF, IL-6 and IL-1β and these cytokines undergo enhanced expression within the dorsal root ganglia (DRG) upon nerve damage[13, 14]. IL-1β is synthesized by DRG sensory neurons and regulates axonal regeneration following peripheral nerve injury in vivo[7, 15] and in vitro[6, 16].
The JAK/STAT3 pathway is activated after nerve injury in DRG and motor neurons and maintains neuronal survival and drives axon regeneration[17–23]. Phosphorylation on Tyr705 and transcriptional activation of STAT3 is modulated by cytokines, including IL-1β[2, 24, 25] and growth factors[26, 27]. STAT3 phosphorylation on Ser727 regulates its translocation to the mitochondria and modulates the activity of electron transport Complex I[28–30]. In PC12 cells the interaction of STAT3 phosphorylated on Ser727 with mitochondria was associated with NGF induction of neurite outgrowth.
High ATP consumption by growth cones during motility instills a need for optimal mitochondrial function to maintain axon regeneration and plasticity[32, 33]. Mitochondrial dysfunction has been proposed as a central mediator of development of many neurodegenerative diseases[34, 35], including neurological and other diseases associated with diabetes[33, 36, 37]. Recently, we have demonstrated that sensory neurons of diabetic rodents exhibit an abnormal mitochondrial phenotype that contributes to the etiology of diabetic neuropathy[38, 39]. Recent studies reveal reduced cytokine expression in DRG and nerve of diabetic rodents and the ability of neuropoietic cytokines to enhance mitochondrial bioenergetics. We, therefore, tested the hypothesis that impaired IL-1β expression in DRG may contribute to aberrant axon regeneration and plasticity seen in diabetic sensory neuropathy.
IL-1β protein levels are diminished in DRG and nerve tissue under a diabetic state
Effect of 2 or 5 month STZ-diabetes on cytokine levels in rat DRG and sciatic nerve
0.97 ± 0.1*
60.35 ± 15.4*
17.53 ± 6.0
4.53 ± 1.38
0.36 ± 0.05
28.38 ± 2.1
12.65 ± 3.6
2.63 ± 0.57
22.64 ± 7.3*
63.93 ± 18.2*
9.78 ± 3.5
3.09 ± 0.28
9.55 ± 3.7
34.05 ± 6.3
13.87 ± 7.1
4.98 ± 0.90
102.4 ± 17.7*
31.79 ± 17.7
4.64 ± 3.04
45.41 ± 8.5
39.64 ± 3.9
1.99 ± 0.6*
75.7 ± 10.9*
5.99 ± 2.93
0.68 ± 0.1
32.1 ± 5.2
1.93 ± 0.63
15.9 ± 2.5*
64.15 ± 8.1*
2.08 ± 0.77
3.6 ± 1.1
25.68 ± 5.3
2.61 ± 1.15
17.0 ± 2.46*
105.4 ± 14.8*
3.81 ± 1.63
3.4 ± 1.02
30.74 ± 6.6
3.25 ± 0.94
IL-1β elevates neurite outgrowth in adult rat sensory neurons
IL-1 receptor type 1 and P-STAT3-Tyr705 are expressed in control rat DRG neurons
IL-1β activates the JAK/STAT pathway
IL-1β modulates neurite outgrowth via JAK/STAT pathway
Endogenous IL-1β expression modulates neurite outgrowth in cultured adult sensory neurons
STAT3 is localized to mitochondria upon Ser727 phosphorylation triggered by IL-1β
Bioenergetic profile is abnormal in neurons cultured from STZ-diabetic mice and was corrected by IL-1β
We discovered for the first time that IL-1β expression was reduced in sensory neurons and peripheral nerve in diabetes. The generalized down-regulation of cytokine expression seen both in the present study and previously clearly shows that an inflammatory environment is not triggered in the DRG or nerve during early stages of experimental diabetes. Therefore, the primary aim of the present study was to investigate the signal transduction pathways utilized by IL-1β in regulating neurite outgrowth in adult sensory neurons. By understanding these events we hope to identify novel drug targets for therapy in diabetic neuropathy, a severe neurodegenerative disease involving impaired axonal plasticity. The results show that IL-1β augmented neurite outgrowth, in part, through the JAK-STAT3 pathway (the activity of this pathway was impaired in DRG isolated from diabetic animals). Blockade of JAK-STAT3 signaling using pharmacological inhibition or shRNA to STAT3 significantly reduced IL-1β dependent neurite outgrowth. We also uncovered a novel autocrine pathway whereby endogenous neuronal IL-1β enhanced neurite outgrowth. Finally, mitochondrial function was impaired in neurons derived from diabetic mice and could be up-regulated by IL-1β treatment through a JAK-STAT dependent pathway.
In the peripheral nervous system other groups have shown that IL-1β can promote neurite outgrowth from DRG neurons. In organ culture of adult DRG IL-1β enhanced axon regeneration. In dissociated rat sensory neurons from postnatal day 9–10 treatment with IL-1β increased neurite outgrowth via p38 MAPK activation. Delivery of IL-1β by miniosmotic pump for 2 weeks to the crushed sciatic of rats was able to accelerate axon regeneration as measured using morphological and functional criteria. IL-1β knockout mice exhibit reduced functional recovery, specifically locomotor function, following nerve crush although IL-1β clearly has several complementary roles at the crush site including mediating neutrophil biology and hyperalgesia. Combined with this previous literature our study reveals that treatment with the cytokines IL-1α and β enhanced neurite outgrowth via direct effects on cultured neurons. Neurons derived from normal or diabetic rats were able to respond to IL-1β with effects of IL-1β more pronounced in the absence of neurotrophic factors. This data provides encouraging evidence that in vivo treatment of damaged nerves in the setting of diabetes can be effective in enhancing axonal plasticity and ideally augmenting levels of distal nerve fibers in the skin (the primary site of fiber loss in diabetes)[43, 44].
Our studies focused on IL-1β dependent signaling via the JAK-STAT3 pathway. During development of the nervous system STAT3 plays an important role in axon pathfinding, neurite outgrowth and glial cell differentiation. In rodents, STAT3 expression is detected in neurons and glia from embryonic day 14 to postnatal in rat brain. STAT3 is an interesting transcription factor in the context of axon regeneration in the peripheral nervous system. STAT3 expression and phosphorylation on Tyr-705 was enhanced in neurons and proximal nerve following crush injury[22, 23]. Intriguingly, STAT3 activation, in part, through the JAK2 signaling pathway occurs in the axons and perikarya of DRG neurons after peripheral, but not central lesion, strongly supporting a role for STAT3 in sensory axon regeneration[18, 19, 23]. cAMP through induction of neuropoietic molecules such as IL-6, LIF and CNTF activates STAT3 (phosphorylated on Tyr-705) as a component of a conditioning injury in the DRG. The appearance of P-STAT3-Tyr705 in the perikarya is a dual leucine zipper kinase (DLK) and JIP3 dependent process involving retrograde transport of activated STAT3 from the site of injury. Activated STAT3 within the perikarya initiates a range of transcriptional changes that drives peripheral axon regeneration (reviewed by[2, 49]). In addition, activation of STAT3 within the axon can afford neuroprotection from axotomy-induced axonal degeneration. Our work reveals that STAT3 can be activated rapidly by IL-1β in vitro and the process of activation involves phosphorylation on Tyr-705. Furthermore, for the first time in adult primary neurons we show that phosphorylation on Ser-727 occurs (which could be linked to IL-1β mediated optimization of mitochondrial bioenergetics – see later).
Sensory neurons of the DRG express IL-1β although ability to secrete the protein remains unclear. We show that cultured sensory neurons expressed IL-1β and exhibited endogenous transcriptional activity for the IL-1β promoter (Figure 5). Blockade of endogenous IL-1β reduced neurite outgrowth thus revealing for the first time an autocrine pathway for local control of axonal plasticity by endogenous IL-1β in adult neurons. Table 1 shows that endogenous IL-1β expression was reduced in DRG and nerve of diabetic animals. In CNS neurons, IL-1β enhances expression and secretion of IL-6 and GDNF[51–54] so that down-regulation of IL-1β expression in diabetes could trigger a generalized sup-optimal neurotrophic environment. The down-regulation of cytokine gene expression and synthesis is an early target of diabetes that is likely to have pathogenic consequences.
The diabetes-induced impairments in IL-1β signaling were linked to aberrant mitochondrial bioenergetics. We used Seahorse Biosciences XF24 analysis to measure cell respiratory control, recording rate of ATP production, proton leak, coupling efficiency, maximum respiratory rate, respiratory control ratio and spare respiratory capacity. Measurements of oxygen consumption rate in the presence of uncoupler revealed that the maximal electron transport capacity was significantly depressed in sensory neurons from STZ-diabetic mice. In our study, phosphorylation of the STAT3 on Ser-727 and the neuronal mitochondrial bioenergetics profile were impaired in DRG of STZ-diabetic mice and rats and this was prevented by IL-1β treatment. We believe these defects in mitochondrial function detected by our bioenergetics analysis were not due to a general loss of mitochondrial number or mass in the cultured cells since our previous work reveals no loss of mitochondrial mass[38, 39, 56]. This data complements our recent findings that diabetes induces mitochondrial abnormalities and dysfunction in sensory neurons in type 1 diabetes[33, 38, 39, 56] and reviewed. Data in Figures 3 and6 show that STAT3 phosphorylation on Ser-727 was impaired by diabetes and enhanced by IL-1β treatment. In Figure 6 we show localization of P-STAT3-Ser727 to the mitochondria. Previous work shows that STAT3 is involved in cellular respiration by regulating the activity of mitochondrial complexes I and II of the electron transport system when phosphorylated on Ser727[28, 30, 31]. STAT3 was observed to localize in mitochondria and interact with complex I components and GRIM 19[57, 58]. The mitochondrial activity of STAT3 was confined to serine phosphorylation at position 727, as overexpression of mutant STAT3-S727A in STAT3 −/− cells did not restore the mitochondrial complex I activity. Thus, activated STAT3 targets multiple sites within the axon, including the microtubule network and mitochondria, to optimize function and enhance axonal plasticity subsequent to damage.
This study demonstrates a novel role for of IL-1β in preventing the diabetic phenotype of sensory neurons. This work confirms previous studies demonstrating diabetes-induced reductions in peripheral nerve tissue of cytokines such as IL-6, TNFα and CNTF[40, 59, 60]. The mechanism of action of IL-1β was mediated, in part, via optimization of mitochondrial bioenergetics through the JAK/STAT pathway, possibly encompassing a novel pathway linked to STAT3 modulation of mitochondrial function. We propose that a generalized down-regulation of cytokine expression leading to sub-optimal axonal plasticity at distal nerve sites contributes to development of sensory neuropathy in early stages of experimental diabetes.
Materials and methods
Induction of type 1 diabetes in rodents
Male Sprague Dawley rats were made diabetic with a single intraperitoneal injection of 75 mg/kg streptozotocin (STZ; Sigma, St Louis, MO, USA) and male outbred Swiss Webster mice were made diabetic by injection of 90 mg/kg STZ on two consecutive days, with each injection preceded by a 12 h fast. Only animals with blood glucose levels of > 19 mM at the start and end of the study were retained as diabetic. Sensory neuropathy was confirmed in rodents at 2 months using loss of thermal sensitivity as a marker (data not shown). Tissue was collected from rodents after 2–5 months of diabetes. Animal procedures followed guidelines of University of Manitoba Animal Care Committee using Canadian Council of Animal Care rules.
Sensory neuron cultures and treatments
DRG from adult male rats or mice were dissociated using previously described methods[61–63]. Neurons were cultured in defined Hams F12 media in the presence of modified Bottensteins N2 supplement without insulin (0.1 mg/ml transferrin, 20 nM progesterone, 100 mM putrescine, 30 nM sodium selenite 1.0 mg/ml BSA; all additives were from Sigma, St Louis, MO, USA; culture medium was from Life Technologies, Grand Island, NY, USA). In some experiments the media was also supplemented with a low dose cocktail of neurotrophic factors (0.1 ng/ml NGF, 1.0 ng/ml GDNF, 1.0 ng/ml NT-3, and 0.1 nM insulin – all from Promega, Madison, WI, USA). This treatment improved viability of cultures and attempted to mimic the levels of neurotrophic support experienced in vivo by sensory neurons. Normal neurons were cultured in the presence of 10 mM glucose and 0.1 nM insulin and diabetic neurons with 25 mM glucose and zero insulin. Recombinant human IL-1β and recombinant human IL-1α were obtained from Peprotech (Cedarlane, USA). Rabbit anti-rat IL-1β antibody was from Millipore (CA, USA). AG490 was purchased from Millipore (Canada).
Measurement of cytokine protein levels using Luminex system
Levels of cytokines (IL-1α, IL-1β, IL-10 and IL-2) were measured using a Bio-Plex Rat 9-Plex kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Studies on rats were performed to ensure enough protein for detection. Lumbar DRG, mid sciatic nerve and tibial nerve tissue samples from control and STZ-induced diabetic rats were homogenized on ice with a polytron in the presence of Tissue Extraction Reagent I (Invitrogen, #FNN0071A). Multiplex beads were added to each well of a 96-well plate and 100 μl of either the standards or homogenized samples were added, agitated for 30 min then washed 3 times. Beads were incubated with the detection antibodies for 30 min and data were quantified on a Bio-Rad Bioplex 200 Luminex system and analyzed with associated software (Bio-Rad, Hercules, CA, USA). Samples were assayed in triplicate, while cytokine standards were in duplicate. A minimum of 100 beads per analyte was used and the levels (pg/mg) of the cytokines were calculated from the standard curve and corrected to the protein concentration.
shRNA knockdown of gene expression in DRG neurons
For shRNA-based gene silencing, GFP expressing clones specific for IL-1β and STAT3 (clone Id: V3LMM_471275 and V3LMM_424801, respectively) were obtained from OpenBiosystems (Lafayatte, CO, USA). The pGIPz lentiviral system from the OpenBiosystems database is held at University of Manitoba, Winnipeg, Canada. A control scrambled shRNA unrelated to IL-1β and STAT3 sequence was used as a negative control for lentiviral transduction and plasmid transfection. For gene silencing studies that were focused on determination of gene expression rat DRG cells were transduced with lentivirus at 20X infectious unit in the presence of polybrene (8 μg/ml) for 2 h at 37°C, complete medium was added and neurons cultured for additional 48 h. For neurite outgrowth studies neurons were transduced with lentivirus or transfected with plasmid (as described in next section). Fluorescence images derived from GFP-expressing virally transduced or plasmid transfected neurons were acquired by using a LSM510 confocal microscope (Carl Zeiss) with 20X air objective.
Luciferase reporter constructs for IL-1β and cell transfection
Reporter plasmid with the IL-1β promoter upstream from luciferase was kindly donated by Dr. Jian Fei (Shanghai Research Center for Model Organisms, Shanghai, China). Rat DRG cells (30×103) were transfected in triplicate with 1.8 μg of IL-1β Luc-promoter plasmid DNA and 0.2 μg of pCMV-Renilla (Promega, Madison, WI, USA) using the Amaxa Nucleofector electroporation kit for low numbers of cells according to the manufacturer’s instructions (ESBE Scientific, Toronto, ON, Canada). In some experiments co-transfection was performed with plasmid carrying shRNA to STAT3 (clone ID: V3LMM_424801) or shRNA to IL-1β (clone ID: V3LMM_471275) obtained from OpenBiosystems (Lafayatte, CO, USA). Cells were lysed using passive lysis buffer provided with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The luciferase activity was measured using a luminometer (model LMAXII; Molecular Devices, Sunnyvale, CA, USA). 20 μl of each sample was loaded in a 96-well plate and was mixed with 100 μl of Luciferase Assay Reagent II and firefly luciferase activity was first recorded. Then, 100 μl of Stop-and-Glo Reagent was added, and Renilla luciferase activity was measured. All values are normalized to Renilla luciferase activity. For lentiviral infection or plasmid transfection cultures of rat cells were preferred due to high cell yields.
Quantification of neurite outgrowth
GFP-based or immunostaining-based fluorescent images were captured and the mean pixel area determined using ImageJ software (adjusted for the cell body signal). All values were normalized for neuronal number. In this culture system the level of total neurite outgrowth has been previously validated to be directly related to an arborizing form of axonal plasticity and homologous with in vivo collateral sprouting. Studies were performed with rat cultures due to higher cell yields.
Cultured DRG neurons from adult rats were harvested after 24 h or intact lumbar DRG from adult mice were rapidly isolated and then homogenized in ice-cold stabilization buffer containing: 0.1 M Pipes, 5 mM MgCl2, 5 mM EGTA, 0.5% Triton X-100, 20% glycerol, 10 mM NaF, 1 mM PMSF, and protease inhibitor cocktail. Proteins were assayed using DC protein assay (BioRad; Hercules, CA, USA) and Western blot analysis performed as previously described[39, 67]. The samples (5 μg total protein/lane) were resolved on a 10% SDS-PAGE gel, and electroblotted (100 V, 1 h) onto a nitrocellulose membrane. Blots were then blocked in 5% nonfat milk containing 0.05% Tween overnight at 4°C, rinsed in TBS-T and then incubated with the primary antibodies for phospho-STAT3 (Tyr705) or phospho-STAT3 (Ser727) or T-STAT3 antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA) or IL-1RAcP (1:500; Santa Cruz Biotechnologies, CA). Total ERK (1: 1500; Santa Cruz Biotechnologies, CA) was used as a loading control. After four washes of 10 min in TBS-T, secondary antibody was applied for 1 h at room temperature. The blots were rinsed, incubated in Western Blotting Luminol Reagent (Santa Cruz Biotechnologies, CA, USA), and imaged using the BioRad Fluor-S Max image analyzer. For culture work source material was from rats to ensure high enough protein yields. DRG samples were collected from control vs diabetic mice since the Swiss Webster mice were cheaper to maintain, reveal less inter-animal variability and onset of neuropathy was quicker compared with rats.
Co-localization studies and immunocytochemistry for detection of tubulin and phospho-STAT3 (Tyr705 or Ser727)
For co-localization experiments DRG neuron cultures from adult rats were incubated with 500 nM Mitotracker deep red TM (Molecular Probes, Invitrogen, USA) at 37°C for 30 min. Cultures were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) for 15 min at room temperature then permeabilized with 0.3% Triton X-100 in PBS for 5 min. Cells were then incubated in blocking buffer (Roche, Indianapolis, IN, USA) diluted with FBS and 1.0 mM PBS (1:1:3) for 1 h then rinsed three times with PBS. Primary antibodies used were: β-tubulin isotype III (1:1000) neuron specific from Sigma Aldrich, Oakville, ON, Canada; phospho-STAT3 (Tyr705 or Ser727 (1:300) Cell Signaling Technology, Danvers, MA, USA). Antibodies were added to all wells and plates were incubated at 4°C overnight. The following day, the coverslips were incubated with FITC- and CY3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature, mounted and imaged using a Carl Zeiss Axioscope-2 fluorescence microscope equipped with an AxioCam camera. Images were captured using AxioVision3 software. Co-localization coefficients between P-STAT3-Ser727 and MitoTracker deep red were analyzed using confocal imaging with Zeiss LSM510 software and followed procedures previously described.
Measurement of mitochondrial respiration in cultured DRG neurons from mice
An XF24 Analyzer (Seahorse Biosciences, Billerica, MA, USA) was used to measure neuronal bioenergetic function. The XF24 creates a transient 7 μl chamber in specialized 24-well microplates that allows for oxygen consumption rate (OCR) to be monitored in real time. Culture medium was changed 1 h before the assay to unbuffered DMEM (Dulbecco’s modified Eagle’s medium, pH 7.4) supplemented with 1 mM pyruvate, and 10 mM D-glucose. Neuron density in the range of 2,500-5,000 cells per well gave linear OCR. Oligomycin (1 μM), carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 1.0 μM) and rotenone (1 μM) + antimycin A (1 μM) were injected sequentially through ports in the Seahorse Flux Pak cartridges. Each loop was started with mixing for 3 min, then delayed for 2 min and OCR measured for 3 min. This allowed determination of the basal level of oxygen consumption, the amount of oxygen consumption linked to ATP production, the level of non-ATP-linked oxygen consumption (proton leak), the maximal respiration capacity and the non-mitochondrial oxygen consumption[41, 69]. Oligomycin inhibits the ATP synthase leading to a build-up of the proton gradient that inhibits electron flux and reveals the state of coupling efficiency. Uncoupling of the respiratory chain by FCCP injection reveals the maximal capacity to reduce oxygen. Finally, rotenone + antimycin A were injected to inhibit the flux of electrons through complexes I and III, and thus no oxygen was further consumed at cytochrome c oxidase. The remaining OCR determined after this intervention is primarily non-mitochondrial. Following OCR measurement the cells were immediately fixed and stained for β-tubulin III as described above. The plates were then inserted into a Cellomics Arrayscan-VTI HCS Reader (Thermo Scientific, Pittsburgh, PA, USA) equipped with Cellomics Arrayscan-VTI software to determine total neuronal number in each well. Data are expressed as OCR in pmoles/min for 1,000 cells. Cultures from control vs diabetic mice were used since the Swiss Webster mice were cheaper to maintain, reveal less inter-animal variability and onset of neuropathy was quicker compared with rats.
Where appropriate, data (presented as mean ± SEM) were subjected to one-way ANOVA with post-hoc comparison using Dunnett’s t test (for dose response studies) or Tukey’s tests or regression analysis with a one-phase exponential decay parametric test with Fisher’s parameter (GraphPad Prism 4, GraphPad Software Inc., San Diego, CA). Where appropriate two-way ANOVA was performed with Bonferronis post hoc test. In all other cases two-tailed Student’s t-Tests were performed.
Dorsal root ganglia
Nerve growth factor
Oxygen consumption rate.
These studies were supported by grants to P.F. from Canadian Institutes for Health Research (CIHR; grant # MOP-84214) and Juvenile Diabetes Research Foundation (grant # 1-2008-193). The authors thank St. Boniface Research for funding support for Dr. Darrell Smith. We thank Dr. Jian Fei (Shanghai Research Center for Model Organisms, Shanghai, China) for the IL-1β plasmid reporter gift. We also thank Dr. Gordon Glazner, University of Manitoba and St. Boniface Hospital Research Centre, for permitting access to the Carl Zeiss LSM 510 microscope.
- Gaudet AD, Popovich PG, Ramer MS: Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. J Neuroinflammation. 2011, 8: 110-PubMed CentralView ArticlePubMedGoogle Scholar
- Patodia S, Raivich G: Downstream effector molecules in successful peripheral nerve regeneration. Cell Tissue Res. 2012, 349: 15-26.View ArticlePubMedGoogle Scholar
- Zigmond RE: gp130 cytokines are positive signals triggering changes in gene expression and axon outgrowth in peripheral neurons following injury. Front Mol Neurosci. 2011, 4: 62-PubMed CentralPubMedGoogle Scholar
- Lindholm D, Heumann R, Hengerer B, Thoenen H: Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J Biol Chem. 1988, 263: 16348-16351.PubMedGoogle Scholar
- Lindholm D, Heumann R, Meyer M, Thoenen H: Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature. 1987, 330: 658-659.View ArticlePubMedGoogle Scholar
- Temporin K, Tanaka H, Kuroda Y, Okada K, Yachi K, Moritomo H, Murase T, Yoshikawa H: IL-1beta promotes neurite outgrowth by deactivating RhoA via p38 MAPK pathway. Biochem Biophys Res Commun. 2008, 365: 375-380.View ArticlePubMedGoogle Scholar
- Nadeau S, Filali M, Zhang J, Kerr BJ, Rivest S, Soulet D, Iwakura Y, de Rivero Vaccari JP, Keane RW, Lacroix S: Functional recovery after peripheral nerve injury is dependent on the pro-inflammatory cytokines IL-1beta and TNF: implications for neuropathic pain. J Neurosci. 2011, 31: 12533-12542.View ArticlePubMedGoogle Scholar
- Stemkowski PL, Smith PA: Sensory neurons, ion channels, inflammation and the onset of neuropathic pain. Can J Neurol Sci. 2012, 39: 416-435.View ArticlePubMedGoogle Scholar
- Watkins LR, Maier SF: Beyond neurons: evidence that immune and glial cells contribute to pathological pain states. Physiol Rev. 2002, 82: 981-1011.View ArticlePubMedGoogle Scholar
- Gardiner NJ, Cafferty WB, Slack SE, Thompson SW: Expression of gp130 and leukaemia inhibitory factor receptor subunits in adult rat sensory neurones: regulation by nerve injury. J Neurochem. 2002, 83: 100-109.View ArticlePubMedGoogle Scholar
- Cafferty WB, Gardiner NJ, Das P, Qiu J, McMahon SB, Thompson SW: Conditioning injury-induced spinal axon regeneration fails in interleukin-6 knock-out mice. J Neurosci. 2004, 24: 4432-4443.View ArticlePubMedGoogle Scholar
- Copray JC, Mantingh I, Brouwer N, Biber K, Kust BM, Liem RS, Huitinga I, Tilders FJ, Van Dam AM, Boddeke HW: Expression of interleukin-1 beta in rat dorsal root ganglia. J Neuroimmunol. 2001, 118: 203-211.View ArticlePubMedGoogle Scholar
- Murphy PG, Grondin J, Altares M, Richardson PM: Induction of interleukin-6 in axotomized sensory neurons. J Neurosci. 1995, 15: 5130-5138.PubMedGoogle Scholar
- Subang MC, Richardson PM: Influence of injury and cytokines on synthesis of monocyte chemoattractant protein-1 mRNA in peripheral nervous tissue. Eur J Neurosci. 2001, 13: 521-528.View ArticlePubMedGoogle Scholar
- Temporin K, Tanaka H, Kuroda Y, Okada K, Yachi K, Moritomo H, Murase T, Yoshikawa H: Interleukin-1 beta promotes sensory nerve regeneration after sciatic nerve injury. Neurosci Lett. 2008, 440: 130-133.View ArticlePubMedGoogle Scholar
- Edoff K, Jerregard H: Effects of IL-1beta, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells. J Neurosci Res. 2002, 67: 255-263.View ArticlePubMedGoogle Scholar
- Lee N, Neitzel KL, Devlin BK, MacLennan AJ: STAT3 phosphorylation in injured axons before sensory and motor neuron nuclei: potential role for STAT3 as a retrograde signaling transcription factor. J Comp Neurol. 2004, 474: 535-545.View ArticlePubMedGoogle Scholar
- Qiu J, Cafferty WB, McMahon SB, Thompson SW: Conditioning injury-induced spinal axon regeneration requires signal transducer and activator of transcription 3 activation. J Neurosci. 2005, 25: 1645-1653.View ArticlePubMedGoogle Scholar
- Ben-Yaakov K, Dagan SY, Segal-Ruder Y, Shalem O, Vuppalanchi D, Willis DE, Yudin D, Rishal I, Rother F, Bader M, et al: Axonal transcription factors signal retrogradely in lesioned peripheral nerve. EMBO J. 2012, 31: 1350-1363.PubMed CentralView ArticlePubMedGoogle Scholar
- Schweizer U, Gunnersen J, Karch C, Wiese S, Holtmann B, Takeda K, Akira S, Sendtner M: Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult. J Cell Biol. 2002, 156: 287-297.PubMed CentralView ArticlePubMedGoogle Scholar
- Bareyre FM, Garzorz N, Lang C, Misgeld T, Buning H, Kerschensteiner M: In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration. Proc Natl Acad Sci USA. 2011, 108: 6282-6287.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwaiger FW, Hager G, Schmitt AB, Horvat A, Hager G, Streif R, Spitzer C, Gamal S, Breuer S, Brook GA, et al: Peripheral but not central axotomy induces changes in Janus kinases (JAK) and signal transducers and activators of transcription (STAT). Eur J Neurosci. 2000, 12: 1165-1176.View ArticlePubMedGoogle Scholar
- Sheu JY, Kulhanek DJ, Eckenstein FP: Differential patterns of ERK and STAT3 phosphorylation after sciatic nerve transection in the rat. Exp Neurol. 2000, 166: 392-402.View ArticlePubMedGoogle Scholar
- Wu YY, Bradshaw RA: Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12-E2 cells. J Biol Chem. 2000, 275: 2147-2156.View ArticlePubMedGoogle Scholar
- Zorina Y, Iyengar R, Bromberg KD: Cannabinoid 1 receptor and interleukin-6 receptor together induce integration of protein kinase and transcription factor signaling to trigger neurite outgrowth. J Biol Chem. 2010, 285: 1358-1370.PubMed CentralView ArticlePubMedGoogle Scholar
- Quesnelle KM, Boehm AL, Grandis JR: STAT-mediated EGFR signaling in cancer. J Cell Biochem. 2007, 102: 311-319.View ArticlePubMedGoogle Scholar
- Haas CA, Hofmann HD, Kirsch M: Expression of CNTF/LIF-receptor components and activation of STAT3 signaling in axotomized facial motoneurons: evidence for a sequential postlesional function of the cytokines. J Neurobiol. 1999, 41: 559-571.View ArticlePubMedGoogle Scholar
- Wegrzyn J, Potla R, Chwae YJ, Sepuri NB, Zhang Q, Koeck T, Derecka M, Szczepanek K, Szelag M, Gornicka A, et al: Function of mitochondrial Stat3 in cellular respiration. Science. 2009, 323: 793-797.PubMed CentralView ArticlePubMedGoogle Scholar
- Boengler K, Hilfiker-Kleiner D, Heusch G, Schulz R: Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion. Basic Res Cardiol. 2010, 105: 771-785.PubMed CentralView ArticlePubMedGoogle Scholar
- Qiu H, Lizano P, Laure L, Sui X, Rashed E, Park JY, Hong C, Gao S, Holle E, Morin D, et al: H11 kinase/heat shock protein 22 deletion impairs both nuclear and mitochondrial functions of STAT3 and accelerates the transition into heart failure on cardiac overload. Circulation. 2011, 124: 406-415.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou L, Too HP: Mitochondrial localized STAT3 is involved in NGF induced neurite outgrowth. PLoS One. 2011, 6: e21680-PubMed CentralView ArticlePubMedGoogle Scholar
- Bernstein BW, Bamburg JR: Actin-ATP hydrolysis is a major energy drain for neurons. J Neurosci. 2003, 23: 1-6.PubMedGoogle Scholar
- Saleh A, Roy Chowdhury SK, Smith DR, Balakrishnan S, Tessler L, Martens C, Morrow D, Schartner E, Frizzi KE, Calcutt NA, Fernyhough P: Ciliary neurotrophic factor activates NF-kappaB to enhance mitochondrial bioenergetics and prevent neuropathy in sensory neurons of streptozotocin-induced diabetic rodents. Neuropharmacology. 2013, 65: 65-73.PubMed CentralView ArticlePubMedGoogle Scholar
- Ashrafi G, Schwarz TL: The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ. 2013, 20: 31-42.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsunemi T, La Spada AR: PGC-1alpha at the intersection of bioenergetics regulation and neuron function: from Huntington’s disease to Parkinson’s disease and beyond. Prog Neurobiol. 2012, 97: 142-151.PubMed CentralView ArticlePubMedGoogle Scholar
- Chowdhury SK, Dobrowsky RT, Fernyhough P: Nutrient excess and altered mitochondrial proteome and function contribute to neurodegeneration in diabetes. Mitochondrion. 2011, 11: 845-854.View ArticlePubMedGoogle Scholar
- Sivitz WI, Yorek MA: Mitochondrial dysfunction in diabetes: from molecular mechanisms to functional significance and therapeutic opportunities. Antioxid Redox Signal. 2010, 12: 537-577.PubMed CentralView ArticlePubMedGoogle Scholar
- Chowdhury SK, Zherebitskaya E, Smith DR, Akude E, Chattopadhyay S, Jolivalt CG, Calcutt NA, Fernyhough P: Mitochondrial respiratory chain dysfunction in dorsal root ganglia of streptozotocin-induced diabetic rats and its correction by insulin treatment. Diabetes. 2010, 59: 1082-1091.PubMed CentralView ArticlePubMedGoogle Scholar
- Roy Chowdhury SK, Smith DR, Saleh A, Schapansky J, Marquez A, Gomes S, Akude E, Morrow D, Calcutt NA, Fernyhough P: Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes. Brain. 2012, 135: 1751-1766.PubMed CentralView ArticlePubMedGoogle Scholar
- Saleh A, Smith DR, Balakrishnan S, Dunn L, Martens C, Tweed CW, Fernyhough P: Tumor necrosis factor-alpha elevates neurite outgrowth through an NF-kappaB-dependent pathway in cultured adult sensory neurons: diminished expression in diabetes may contribute to sensory neuropathy. Brain Res. 2011, 1423: 87-95.View ArticlePubMedGoogle Scholar
- Brand MD, Nicholls DG: Assessing mitochondrial dysfunction in cells. Biochem J. 2011, 435: 297-312.PubMed CentralView ArticlePubMedGoogle Scholar
- Horie H, Sakai I, Akahori Y, Kadoya T: IL-1 beta enhances neurite regeneration from transected-nerve terminals of adult rat DRG. Neuroreport. 1997, 8: 1955-1959.View ArticlePubMedGoogle Scholar
- Kennedy WR, Wendelschafer-Crabb G, Johnson T: Quantitation of epidermal nerves in diabetic neuropathy. Neurology. 1996, 47: 1042-1048.View ArticlePubMedGoogle Scholar
- Quattrini C, Tavakoli M, Jeziorska M, Kallinikos P, Tesfaye S, Finnigan J, Marshall A, Boulton AJ, Efron N, Malik RA: Surrogate markers of small fiber damage in human diabetic neuropathy. Diabetes. 2007, 56: 2148-2154.View ArticlePubMedGoogle Scholar
- Dziennis S, Alkayed NJ: Role of signal transducer and activator of transcription 3 in neuronal survival and regeneration. Rev Neurosci. 2008, 19: 341-361.PubMed CentralView ArticlePubMedGoogle Scholar
- Gautron L, De Smedt-Peyrusse V, Laye S: Characterization of STAT3-expressing cells in the postnatal rat brain. Brain Res. 2006, 1098: 26-32.View ArticlePubMedGoogle Scholar
- Wu D, Zhang Y, Bo X, Huang W, Xiao F, Zhang X, Miao T, Magoulas C, Subang MC, Richardson PM: Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons. Exp Neurol. 2007, 204: 66-76.View ArticlePubMedGoogle Scholar
- Shin JE, Cho Y, Beirowski B, Milbrandt J, Cavalli V, DiAntonio A: Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration. Neuron. 2012, 74: 1015-1022.PubMed CentralView ArticlePubMedGoogle Scholar
- Patodia S, Raivich G: Role of transcription factors in peripheral nerve regeneration. Front Mol Neurosci. 2012, 5: 8-PubMed CentralView ArticlePubMedGoogle Scholar
- Selvaraj BT, Frank N, Bender FL, Asan E, Sendtner M: Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease. J Cell Biol. 2012, 199: 437-451.PubMed CentralView ArticlePubMedGoogle Scholar
- Saavedra A, Baltazar G, Duarte EP: Interleukin-1beta mediates GDNF up-regulation upon dopaminergic injury in ventral midbrain cell cultures. Neurobiol Dis. 2007, 25: 92-104.View ArticlePubMedGoogle Scholar
- Tsakiri N, Kimber I, Rothwell NJ, Pinteaux E: Differential effects of interleukin-1 alpha and beta on interleukin-6 and chemokine synthesis in neurones. Mol Cell Neurosci. 2008, 38: 259-265.View ArticlePubMedGoogle Scholar
- Tsakiri N, Kimber I, Rothwell NJ, Pinteaux E: Interleukin-1-induced interleukin-6 synthesis is mediated by the neutral sphingomyelinase/Src kinase pathway in neurones. Br J Pharmacol. 2008, 153: 775-783.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsakiri N, Kimber I, Rothwell NJ, Pinteaux E: Mechanisms of interleukin-6 synthesis and release induced by interleukin-1 and cell depolarisation in neurones. Mol Cell Neurosci. 2008, 37: 110-118.View ArticlePubMedGoogle Scholar
- Calcutt NA: Future treatments for diabetic neuropathy: clues from experimental neuropathy. Curr Diab Rep. 2002, 2: 482-488.View ArticlePubMedGoogle Scholar
- Akude E, Zherebitskaya E, Chowdhury SK, Smith DR, Dobrowsky RT, Fernyhough P: Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats. Diabetes. 2011, 60: 288-297.PubMed CentralView ArticlePubMedGoogle Scholar
- Tammineni P, Anugula C, Mohammed F, Anjaneyulu M, Larner AC, Sepuri NB: The import of the transcription factor STAT3 into mitochondria depends on GRIM-19, a component of the electron transport chain. J Biol Chem. 2013, 288: 4723-4732.PubMed CentralView ArticlePubMedGoogle Scholar
- Shulga N, Pastorino JG: GRIM-19-mediated translocation of STAT3 to mitochondria is necessary for TNF-induced necroptosis. J Cell Sci. 2012, 125: 2995-3003.PubMed CentralView ArticlePubMedGoogle Scholar
- Mizisin AP, Vu Y, Shuff M, Calcutt NA: Ciliary neurotrophic factor improves nerve conduction and ameliorates regeneration deficits in diabetic rats. Diabetes. 2004, 53: 1807-1812.View ArticlePubMedGoogle Scholar
- Calcutt NA, Muir D, Powell HC, Mizisin AP: Reduced ciliary neuronotrophic factor-like activity in nerves from diabetic or galactose-fed rats. Brain Res. 1992, 575: 320-324.View ArticlePubMedGoogle Scholar
- Huang TJ, Sayers NM, Verkhratsky A, Fernyhough P: Neurotrophin-3 prevents mitochondrial dysfunction in sensory neurons of streptozotocin-diabetic rats. Exp Neurol. 2005, 194: 279-283.View ArticlePubMedGoogle Scholar
- Gardiner NJ, Fernyhough P, Tomlinson DR, Mayer U, von der Mark H, Streuli CH: Alpha7 integrin mediates neurite outgrowth of distinct populations of adult sensory neurons. Mol Cell Neurosci. 2005, 28: 229-240.View ArticlePubMedGoogle Scholar
- Urban MJ, Pan P, Farmer KL, Zhao H, Blagg BS, Dobrowsky RT: Modulating molecular chaperones improves sensory fiber recovery and mitochondrial function in diabetic peripheral neuropathy. Exp Neurol. 2012, 235: 388-396.PubMed CentralView ArticlePubMedGoogle Scholar
- Saleh A, Shan L, Halayko AJ, Kung S, Gounni AS: Critical role for STAT3 in IL-17A-mediated CCL11 expression in human airway smooth muscle cells. J Immunol. 2009, 182: 3357-3365.View ArticlePubMedGoogle Scholar
- Li L, Fei Z, Ren J, Sun R, Liu Z, Sheng Z, Wang L, Sun X, Yu J, Wang Z, Fei J: Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice. BMC Immunol. 2008, 9: 49-PubMed CentralView ArticlePubMedGoogle Scholar
- Smith DS, Skene JH: A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth. J Neurosci. 1997, 17: 646-658.PubMedGoogle Scholar
- Fernyhough P, Gallagher A, Averill SA, Priestley JV, Hounsom L, Patel J, Tomlinson DR: Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes. 1999, 48: 881-889.View ArticlePubMedGoogle Scholar
- Dunn KW, Kamocka MM, McDonald JH: A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol. 2011, 300: C723-742.PubMed CentralView ArticlePubMedGoogle Scholar
- Hill BG, Dranka BP, Zou L, Chatham JC, Darley-Usmar VM: Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal. Biochem J. 2009, 424: 99-107.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.