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Neuropharmacology 65 (2013) 65e73

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Ciliary neurotrophic factor activates NF-kB to enhance mitochondrialbioenergetics and prevent neuropathy in sensory neurons ofstreptozotocin-induced diabetic rodents

Ali Saleh a, Subir K. Roy Chowdhury a, Darrell R. Smith a, Savitha Balakrishnan a, Lori Tessler a,Corina Martens a, Dwane Morrowa, Emily Schartner a, Katie E. Frizzi c, Nigel A. Calcutt c,Paul Fernyhough a,b,*

aDivision of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, MB, CanadabDepartment of Pharmacology & Therapeutics, University of Manitoba, Winnipeg, MB, CanadacDepartment of Pathology, UCSD, La Jolla, CA, USA

a r t i c l e i n f o

Article history:Received 23 May 2012Received in revised form12 September 2012Accepted 14 September 2012

Keywords:Axon regenerationBioenergetics profileDorsal root gangliaCNTFNF-kBDiabetesDiabetic neuropathyOxidative phosphorylationRespiration

* Corresponding author. R4046 e 351 Taché Ave., SCentre,Winnipeg, MB R2H 2A6, Canada. Tel.:þ1204 23

E-mail addresses: pfernyhough@sbrc.ca,(P. Fernyhough).

0028-3908/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2012.09.015

a b s t r a c t

Diabetes causes mitochondrial dysfunction in sensory neurons that may contribute to peripheralneuropathy. Ciliary neurotrophic factor (CNTF) promotes sensory neuron survival and axon regenerationand prevents axonal dwindling, nerve conduction deficits and thermal hypoalgesia in diabetic rats. Inthis study, we tested the hypothesis that CNTF protects sensory neuron function during diabetes throughnormalization of impaired mitochondrial bioenergetics. In addition, we investigated whether the NF-kBsignal transduction pathway was mobilized by CNTF. Neurite outgrowth of sensory neurons derived fromstreptozotocin (STZ)-induced diabetic rats was reduced compared to neurons from control rats andexposure to CNTF for 24 h enhanced neurite outgrowth. CNTF also activated NF-kB, as assessed byWestern blotting for the NF-kB p50 subunit and reporter assays for NF-kB promoter activity. Conversely,blockade of NF-kB signaling using SN50 peptide inhibited CNTF-mediated neurite outgrowth. Studies inmice with STZ-induced diabetes demonstrated that systemic therapy with CNTF prevented functionalindices of peripheral neuropathy along with deficiencies in dorsal root ganglion (DRG) NF-kB p50expression and DNA binding activity. DRG neurons derived from STZ-diabetic mice also exhibited defi-ciencies in maximal oxygen consumption rate and associated spare respiratory capacity that were cor-rected by exposure to CNTF for 24 h in an NF-kB-dependent manner. We propose that the ability of CNTFto enhance axon regeneration and protect peripheral nerve from structural and functional indices ofdiabetic peripheral neuropathy is associated with targeting of mitochondrial function, in part via NF-kBactivation, and improvement of cellular bioenergetics.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Diabetic neuropathy is characterized by functional disorders suchas conduction slowing, sensory loss and pain, and the appearanceof structural changes such as segmental demyelination, Walleriandegeneration and microangiopathy that accompany loss of bothmyelinated and unmyelinated fibers (Malik et al., 2005; Said, 2007;Yagihashi, 1996). Rodent models of diabetic neuropathy exhibitsimilar functional disorders, including conduction slowing (Greene

t Boniface Hospital Research5 3692; fax:þ1204 2374092.paulfernyhough@yahoo.com

All rights reserved.

et al., 1975), abnormal thermal nociception (Calcutt et al., 2004)and tactile allodynia (Calcutt et al., 1996). While many mechanismsdownstream of impaired insulin signaling and/or hyperglycemiahave been proposed as contributing to nerve dysfunction (reviewedby Calcutt et al., 2009), how the metabolic disorders of diabetesultimately lead to structural pathology remains unresolved.We haverecently demonstrated that sensory neurons of diabetic rodentsexhibit an abnormal mitochondrial phenotype that contributes tothe etiology of diabetic neuropathy (Chowdhury et al., 2011, inpress). Inner mitochondrial membrane potential is depolarized insensory neuron cultures derived from streptozotocin (STZ)-induceddiabetic rats (Huang et al., 2003; Srinivasan et al., 2000) and lumbardorsal root ganglia (DRG) from diabetic rats exhibit a reduced rateof oxygen consumption and depression of respiratory complexactivity that correlatedwith suppression of expression of proteins of

A. Saleh et al. / Neuropharmacology 65 (2013) 65e7366

respiratory chain complexes (Chowdhury et al., 2010). A quantitativeproteomic study of mitochondrial proteins revealed that proteinsacross a variety of pathways were down-regulated in DRG from STZ-diabetic rats, suggesting impaired mitochondrial biogenesis (Akudeet al., 2011).

Ciliary neurotrophic factor (CNTF) is expressed by Schwann cellsand promotes survival and axonal regeneration of sensory neuronsand motorneurons (Sleeman et al., 2000). Sciatic nerve of STZ-induced diabetic rats exhibits reduced CNTF bioactivity (Calcuttet al., 1992) and systemic delivery of CNTF prevented deficits innerve conduction, axonal caliber, thermal nociception and nerveregeneration following crush injury (Calcutt et al., 2004; Mizisinet al., 2004). The therapeutic potential of CNTF has been restrictedby cachexia. However, there is renewed interest in CNTF due to itsanti-obesity effects mediated, in part, through modulation ofhypothalamic physiology and inhibitory effects on food intake (Allenet al., 2011). Interestingly, CNTF reduced fat deposition in obesemicethrough promotion of mitochondrial biogenesis and enhancedoxidative capacity in adipose tissue (Crowe et al., 2008).

CNTF and other cytokines signal, in part, through the multi-component transcription factor, NF-kB (Vallabhapurapu and Karin,2009). Decreased expression and activity of NF-kB in DRG accom-panies early signs of sensory neuropathy in STZ-diabetic rodents(Berti-Mattera et al., 2008, 2011; Purves and Tomlinson, 2002). TheNF-kB transcriptional complex includes p50/p65 (NF-kB1) and p50/c-Rel (NF-kB2) heterodimers that are retained in the cytoplasm bythe inhibitory protein IkBa. Various stimuli induce IkBa phosphor-ylation and subsequent degradation through a canonical pathwaycomprising Ik kinase (IkK), which unmasks the nuclear localizationsignal of the NF-kB heterodimer. This dimer then translocates intothe nucleus, where it binds to its cognate DNA sequence and regu-lates gene transcription (Baeuerle and Baltimore, 1996; Rothwarfand Karin, 1999). Recent studies in fibroblasts also describe a keyrole for NF-kB in augmenting mitochondrial function (Mauroet al., 2011).

Adult sensory neurons exhibit rapidly elevated NF-kB followingeither axotomy in vivo or dissection and culture and subsequentlysurvive without the need for exogenous neurotrophic factors(Fernyhough et al., 2005; Gushchina et al., 2010). Conversely, severeinhibition of NF-kB results in death of neurons when maintainedin vitro (Fernyhough et al., 2005), although the response to axot-omy is more complex with no direct evidence that NF-kB activationalone is required for survival (Gushchina et al., 2009). In embryonicsensory neurons CNTF enhances neurite outgrowth and neuronalsurvival through an NF-kB dependent pathway (Gallagher et al.,2007; Middleton et al., 2000). Adult sensory neurons expressreceptors for CNTF and other cytokines (Cafferty et al., 2004; Coprayet al., 2001; Gardiner et al., 2002; Sango et al., 2008; Wang et al.,2009) and these cytokines undergo enhanced expression withinthe DRG and nerve upon nerve damage (Murphy et al., 1995;Subang and Richardson, 2001). The role of CNTF and other cyto-kines during steady state maintenance of neuronal phenotype isless well understood andmay involve signal transduction pathwaysin addition to NF-kB, for example the JAK-STAT pathway.

Given the positive effect of CNTF on neurite outgrowth andmitochondrial biogenesis and physiology we tested the hypothesisthat CNTF could reverse deficits in mitochondrial bioenergetics andnerve physiology, putatively via activation of NF-kB, in sensoryneurons derived from diabetic rodents.

2. Methods

2.1. Induction, treatment and confirmation of type 1 diabetes

Male Sprague Dawley rats were made diabetic with a single intraperitonealinjection of 75 mg/kg STZ (Sigma, St Louis, MO, USA) and female C57Bl/6J mice were

made diabetic by injection of 90 mg/kg STZ on two consecutive days, with eachinjection preceded by a 12 h fast. Only animals with blood glucose levels of >15 mMat the start and end of the study were retained as diabetic. HbA1c was measuredusing the A1CNow kit (Bayer Healthcare, Toronto, Ontario, Canada). A sub-group ofSTZ-diabetic C57Bl/6J mice was treated with human recombinant CNTF (2 mg/kg s.c.thrice weekly; a gift from Regeneron Inc.) for the last 12 weeks of diabetes. The doseand frequency of CNTF were chosen based on efficacy in STZ-diabetic rats (Calcuttet al., 2004; Mizisin et al., 2004), with the dose adjusted to reflect scaling effectsbetween rats and mice (Freireich et al., 1966). Tissue was collected from rats after3e5 months of diabetes and from mice after 30 weeks of diabetes. Animal proce-dures followed guidelines of University of Manitoba Animal Care Committee usingCanadian Council of Animal Care rules or of the Institutional Animal Care and UseCommittee at UCSD.

2.2. Determination of thermal sensitivity and nerve conduction velocity

Hind paw thermal response latencies were measured as previously described(Beiswenger et al., 2008). In short, rodents were placed in plexiglass cubicles on topof the modified Hargreaves device (UARD, La Jolla, CA, USA). The heat source wasplaced directly below the middle of one of the hind paws and the time until pawwithdrawal measured. The heating rate of 1 �C/s selectively activates C fibers(Yeomans and Proudfit, 1996). Response latency of each paw was measured 4 timesat 5 min intervals and the median value of trials 2e4 used to represent the responselatency for that paw. Motor and sensory nerve conduction velocities (MNCV andSNCV) were measured in mice anesthetized with a mixture of pentobarbital anddiazepam (12.5 and 1.25 mg/kg respectively in sterile 0.9% saline) using needleelectrodes to stimulate (0.05 ms, 1e5 V) the sciatic nerve at the sciatic notch andAchilles tendon. Evoked electromyograms were recorded using needle electrodesplaced in the ipsilateral interosseus muscles and MNCV and SNCV calculated usingthe peakepeak latency between pairs of M or Hwaves and the distance between thetwo stimulation sites. Nerve and rectal temperatures were maintained at 37 �C andall measurements were made in triplicate, with the median of the three used torepresent values for that animal.

2.3. Sensory neuron cultures and treatments

DRG from adult male age matched control or diabetic (3e5 months of disease)Sprague Dawley rats or mice were dissociated using previously described methods(Gardiner et al., 2005; Huang et al., 2005). Neurons were cultured in defined HamsF12 media in the presence of modified Bottensteins N2 supplement without insulin(0.1 mg/ml transferrin, 20 nM progesterone, 100 mM putrescine, 30 nM sodiumselenite, 1 mg/ml BSA; all additives were from Sigma, St Louis, MO, USA; culturemedium was from Life Technologies, Grand Island, NY, USA). In experimentsinvolving plasmid transfection, treatment with SN50 and mitochondrial functionalanalysis the media was also supplemented with a cocktail of neurotrophic factors(0.1 ng/ml NGF, 1 ng/ml GDNF, 0.1 ng/ml NT-3, and 0.1 nM insulin: all from Promega,Madison, WI, USA) to improve neuronal viability. Neurons from control animalswere cultured in the presence of 10 mM glucose and 1 nM insulin. Neurons derivedfrom STZ-induced diabetic rodentswere exposed to 25mMglucose in the absence ofinsulin to mimic physiological conditions. All cultures were maintained for 1 or 2days. Recombinant human CNTF was from R&D (Minneapolis, MN, USA). SN50peptide (Calbiochem, La Jolla, CA, USA) was used to prevent NF-kB nuclear trans-location, thus preventing its transcriptional activity.

2.4. Luciferase reporter constructs for NF-kB and rat neuron transfection

Reporter plasmid with the wild-type NF-kB promoter upstream from luciferasewas kindly donated by Dr. M. Bouchard (Drexel University College of Medicine,Philadelphia, PA). Rat DRG cells (30 � 103) were transfected in triplicate with 1.8 mgof NF-kB promoter/luciferase plasmid DNA and 0.2 mg of PRLTK-Renilla (Promega,Madison, WI, USA) using the Amaxa electroporation kit for low numbers of cellsaccording to the manufacturer’s instructions (ESBE Scientific, Toronto, ON, Canada).Cells were lysed using passive lysis buffer provided with the Dual-LuciferaseReporter Assay System (Promega, Madison, WI, USA). The luciferase activity wasmeasured using a luminometer (model LMAXII; Molecular Devices, Sunnyvale, CA,USA). 20 ml of each sample was loaded in a 96-well plate and was mixed with 100 mlof Luciferase Assay Reagent II and firefly luciferase activity was first recorded. Then,100 ml of Stop-and-Glo Reagent was added, and Renilla luciferase activity wasmeasured. All values are normalized to Renilla luciferase activity.

2.5. Western blotting for NF-kB subunit and CNTF receptor a protein expression

Rat DRG neurons were harvested after 24 h of culture while intact lumbar DRGfrom adult mice were rapidly isolated, frozen and then homogenized in ice-coldstabilization buffer containing: 0.1 M Pipes, 5 mM MgCl2, 5 mM EGTA, 0.5% TritonX-100, 20% glycerol, 10 mM NaF, 1 mM PMSF, and protease inhibitor cocktail(Fernyhough et al., 1999). Proteins were assayed using DC protein assay (BioRad;Hercules, CA, USA) and Western blot analysis performed as previously described(Smith et al., 2009). The samples (5 mg total protein/lane) were resolved on a 10%

A. Saleh et al. / Neuropharmacology 65 (2013) 65e73 67

SDS-PAGE gel, and electroblotted (100 V, 1 h) onto a nitrocellulose membrane. Blotswere then blocked in 5% nonfat milk containing 0.05% Tween overnight at 4 �C,rinsed in PBS-T and then incubatedwith the primary antibody for p50 subunit of NF-kB (1:1000; Epitomics, CA, USA) or anti-rat CNTF receptor a (1:500) antibody fromR&D Systems, Minneapolis, MN, USA. Total ERK (1:1500; Santa Cruz Biotechnologies,CA) was used as a loading control (previous studies reveal this protein to not changeexpression in DRG under diabetic conditions: Fernyhough et al., 1999). After fourwashes of 10 min in PBS-T, secondary antibody was applied for 1 h at roomtemperature. The blots were rinsed, incubated in Western Blotting Luminol Reagent(Santa Cruz Biotechnologies, CA, USA), and imaged using the BioRad Fluor-S imageanalyzer.

2.6. Immunocytochemistry for detection of tubulin and CNTF receptor in rat cultures

Rat neurons grown on glass coverslips were fixed with 4% paraformaldehyde inphosphate buffered saline (PBS, pH 7.4) for 15 min at room temperature then per-meabilized with 0.3% Triton X-100 in PBS for 5 min. Cells were then incubated inblocking 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:b-tubulin isotype III (1:1000) neuron specific from Sigma Aldrich, Oakville, ON,Canada and anti-rat CNTF receptor a (1:500) antibody from R&D Systems, Minne-apolis, MN, USA (also used for Western blotting studies). Antibodies were added toall wells and plates were incubated at 4 �C overnight. The following day, thecoverslips were incubated with FITC- and CY3-conjugated secondary antibodies(Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at roomtemperature, mounted and imaged using a Carl Zeiss Axioscope-2 fluorescencemicroscope equipped with an AxioCam camera. Images were captured using Axio-Vision4.8 software.

2.7. Quantification of neurite outgrowth in rat cultures

Rat cultures were fixed as described above and immunostained using an anti-body to neuron-specific b-tubulin III. Fluorescent images were captured and themean pixel area determined using ImageJ software, adjusted for the cell body signal.In both cases all values were adjusted for neuronal number. In this culture systemthe level of total neurite outgrowth has been previously validated to be directlyrelated to an arborizing form of axonal plasticity and homologous to collateralsprouting in vivo (Smith and Skene, 1997).

2.8. Measurement of mitochondrial respiration in cultured DRG neurons from mice

An XF24 Analyzer (Seahorse Biosciences, Billerica, MA, USA) was used tomeasure neuronal bioenergetics function of mouse cultures. The XF24 createsa transient 7 ml chamber in specialized 24-well microplates that allows for oxygenconsumption rate (OCR) to be monitored in real time (Hill et al., 2009). Culturemedium was changed 1 h before the assay to unbuffered DMEM (Dulbecco’smodified Eagle’s medium, pH 7.4) supplemented with 1 mM pyruvate, and 10 mMD-glucose. Neuron density in the range of 2500e5000 cells per well gave linear OCR.Oligomycin (1 mM), carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP;1.0 mM) and rotenone (1 mM) þ antimycin A (1 mM) were injected sequentiallythrough ports in the Seahorse Flux Pak cartridges. Each loopwas startedwithmixingfor 3 min, then delayed for 2 min and OCR measured for 3 min. This alloweddetermination of the basal level of oxygen consumption, the amount of oxygenconsumption linked to ATP production, the level of non-ATP-linked oxygenconsumption (proton leak), the maximal respiration capacity and the non-mitochondrial oxygen consumption (Brand and Nicholls, 2011; Hill et al., 2009).Oligomycin inhibits the ATP synthase leading to a build-up of the proton gradientthat inhibits electron flux and reveals the state of coupling efficiency. Uncoupling ofthe respiratory chain by FCCP injection reveals the maximal capacity to reduceoxygen. Finally, rotenoneþ antimycin Awere injected to inhibit the flux of electronsthrough complexes I and III, and thus no oxygen was further consumed at cyto-chrome c oxidase. The remaining OCR determined after this intervention is primarilynon-mitochondrial. Following OCR measurement the cells were immediately fixedand stained for b-tubulin III as described above. The plates were then inserted intoa Cellomics Arrayscan-VTI HCS Reader (Thermo Scientific, Pittsburgh, PA, USA)equipped with Cellomics Arrayscan-VTI software to determine total neuronalnumber in each well. Data are expressed as OCR in pmol/min for 1000 cells.

2.9. ELISA-based NF-kB p50 DNA binding assay

Transcriptional activation of NF-kB, assessed by protein binding to transcriptionfactor consensus sequences, was measured using a transcription factor ELISA. Mousep50 subunit of NF-kB DNA binding activity was quantified using a Trans-AM NF-kBp50 Kit (Active Motif, Carlsbad, CA, USA) following the instructions of the manu-facturer. Briefly, mouse DRG were homogenized in 50 ml lysis buffer and 20 ml wasused in the p50 DNA binding assay (at a concentration of 3 mg protein). The samples(in duplicate) were shaken for 1 h at RT in 30 ml binding buffer. After washing, a kitantibody to p50 was applied for 1 h at RT. Specific binding of p50 to DNA wasestimated by spectrophotometry at a wavelength of 450 nm.

2.10. Statistical analysis

Where appropriate, data (presented as mean� SEM)were subjected to one-wayANOVA with post-hoc comparison using Dunnett’s (for dose response studies) orTukey’s tests (GraphPad Prism 4, GraphPad Software Inc., San Diego, CA). In all othercases unpaired two-tailed Student’s t-Tests were performed. For the in vitrowork, inmost part, single rats were used for each experiment with cultures performed inreplicate (n ¼ 3 or 4). For each study at least 3 independent experiments wereperformed. In each case the data shown is a representative sample unless statedotherwise.

3. Results

3.1. CNTF enhances neurite outgrowth in cultured adult ratsensory neurons

Adult rat DRG sensory neurons were cultured for 24 h underdefined conditions in the presence of a range of concentrations ofCNTF with no other neurotrophic factors present. Cultures werefixed, stained for neuron-specific b-tubulin III and then levels oftotal neurite outgrowth quantified. CNTF significantly elevatedneurite outgrowth in a dose-dependent manner, with concentra-tions as lowas 1 ng/ml being effective (Fig.1AeC). Themajor sourceof neurites was the medium to large sub-population of neurons(approximately 51 � 11.7% of this group of neurons respondedto CNTF). However, the small diameter neurons also exhibitedincreased outgrowth (Table 1). Cultures were also prepared on thesame day from age matched control and 3e5 month STZ-induceddiabetic rats. CNTF significantly enhanced neurite outgrowth incultures from control and diabetic rats, although levels of growthin control cultures were significantly higher under all conditions(Fig. 1D). Immunocytochemical staining for the CNTF receptora revealed that all neuronal sizes expressed the receptor with noevidence of non-neuronal expression (Fig. 1EeH). A similar patternof staining for the receptor was seen in diabetic cultures (data notshown). Expression of the CNTF receptor a subunit by culturedneurons from normal vs diabetic rats was not significantly different(Fig.1I) and treatment with CNTF did not effect the expression (datanot shown). Similar effects of CNTF on morphology were observed,except total levels of growth were higher, when neurons werecultured with a low dose cocktail of neurotrophic factors (data notshown).

3.2. Blockade of NF-kB signaling inhibits CNTF-directed neuriteoutgrowth

The importance of NF-kB in cytokine-induced neurite outgrowthwas evaluated using the NF-kB inhibitor SN50 which blocks nuclearentry of activated NF-kB dimers (Fernyhough et al., 2005). Culturedrat sensory neurons were treated for 24 h with CNTF in the presenceor absence of SN50. Cultures were fixed and stained for b-tubulinand neurite outgrowth quantified (Fig. 2). SN50, but not inactiveSN50M, significantly blocked CNTF-directed neurite outgrowth(Fig. 2).

3.3. CNTF up-regulates NF-kB promoter activity in cultured ratsensory neurons

A luciferase reporter assay was used to confirm CNTF-dependentactivation of NF-kB in DRG neurons. CNTF treatment of normalcultured neurons for 24 h significantly elevated NF-kB promoteractivity in a dose-dependent manner (Fig. 3A). Cultures derivedfrom age-matched control and 3e5 month STZ-induced diabeticrats were prepared on the same day and transfected with wild-typeNF-kB reporter plasmids. Control and diabetic neurons bothexhibited CNTF dependent induction of NF-kB promoter activity

Table 1CNTF induces neurite outgrowth in small and medium/large populations of DRGsensory neurons.

Neuron size # of neuronsanalyzed/study

% of totalpopulation

% of neuronswith neurites

ControlMedium/large (>25 mm) 46e100 21.3 � 3.3 32.7 � 6.2#Small (>25 mm) 233e286 78.7 � 3.3* 10.5 � 3.1xDCNTFMedium/large (>25 mm) 52e130 23.3 � 4.5 51.0 � 11.7Small (<25 mm) 223e310 76.7 � 4.5* 34.7 � 7.0

Incidence of neurons with neurites in control versus CNTF (10 ng/ml) treatedcultures at 1 day. The values in brackets indicate the neuron diameter of eachpopulation. Values are mean � SEM (n ¼ 3 independent experiments). *p < 0.05 vsmedium/large neuron size group in control or CNTF groups (oneway ANOVA withTukey’s posthoc). #p < 0.05 vs CNTF treated medium/large neuron size group andxp < 0.05 vs CNTF treated small neuron size group (both by paired Student t-Test).

Fig. 1. CNTF significantly increased axonal outgrowth in normal and, to a lesser extent,in diabetic neurons. Cultured adult DRG sensory neurons from normal rats were grownfor 1 day in the absence of neurotrophic factors with a range of cytokines underdefined conditions and then fixed and stained for neuron-specific b-tubulin III. In (A) isuntreated, or (B) treated with CNTF (10 ng/ml). In (C) is shown total axon outgrowth ofneurons in response to a range of CNTF concentrations. Values are means � SEM (n ¼ 3replicates). *p < 0.05 vs control (oneway ANOVA). In (D) is shown total axon outgrowthfor sensory neurons isolated from control (white column) or 3e5 month STZ-diabeticrats (black column) cultured for 1 day � CNTF (at 10 ng/ml). Values are the means �SEM (n ¼ 3 replicates). For control, *p < 0.05 vs CNTF; for diabetic, #p < 0.05 vs CNTF,xp < 0.05 vs diabetic. DRG sensory neurons from normal rats were cultured for 1 day inthe absence of neurotrophic factors and were fixed and immunostained with (E) noantibody, (F) stained with CNTF receptor a antibody, (G) with b-tubulin III antibody,and (H) merge of F and G. The white arrows indicate co-localization of receptor andneuron-specific staining. The bar ¼ 100 mm. (I) Western blot for CNTF receptora comparing control and diabetic cultures at 24 h in vitro with quantification.Expression levels are indicated below the blots and are expressed relative to T-ERK andare means � SEM (n ¼ 4 replicates); no significant difference between groups.

A. Saleh et al. / Neuropharmacology 65 (2013) 65e7368

(Fig. 3B). The degree of induction was the same in control and dia-betic neurons, however, the basal activity of NF-kB was reduced indiabetic neurons (Fig. 3C). Treatment of control cultures for up to24 h with 10 ng/ml CNTF caused increased expression of NF-kB p50relative to T-ERK, peaking around 30e60 min and falling back tobasal levels by 24 h (Fig. 3D; T-ERK protein is used as a reference

Fig. 2. Inhibition of NF-kB by SN50 impairs cytokine-mediated axon outgrowth. DRGsensory neurons from normal rats were cultured for 1 day in the presence of low doseneurotrophic factors (to improve viability in presence of SN50) and were exposed to theNF-kB peptide inhibitor SN50 (10 mg/ml), or its control peptide SN50M (10 mg/ml) �CNTF (10 ng/ml). Neurons were fixed and stained for neuron-specific b-tubulin III.Shown is (A) no treatment, (B) þSN50, (C) þCNTF, and (D) þCNTF þ SN50. In (E) isshown the impact on total axon outgrowth. Data are mean � SEM (n ¼ 3 replicates).*p < 0.05 vs control, or SN50, or SN50M; **p < 0.05 vs CNTF (by one way ANOVA).

Fig. 3. CNTF activates NF-kB in adult DRG sensory neuron culture. Cultured DRGsensory neurons from normal rats, in the presence of low dose neurotrophic factors,were transfected with WT-NF-kB promoter. One day after transfection with functionalWT-NF-kB promoter the neurons were stimulated for 12 h with a range of doses ofCNTF (A). NF-kB-induced luciferase activity was normalized to Renilla luciferaseactivity. Fold induction represents luciferase activity in CNTF-treated cells comparedwith untreated cells and is the mean of three independent experiments. Values aremeans � SEM, n ¼ 3; *p < 0.05 vs control. In (B) neurons from control or STZ-induceddiabetic rats were transfected with WT-NF-kB promoter and were then treated for 12 hwith CNTF (10 ng/ml). Values are mean � SEM, n ¼ 3; *p < 0.05 vs control. (C) showsbasal NF-kB reporter activity in cultures from age matched control rats vs STZ-diabetic.

A. Saleh et al. / Neuropharmacology 65 (2013) 65e73 69

protein in sensory neurons and expression did not change withCNTF treatment). These studies could have been further strength-ened by performing ChiP analyses to reveal endogenous binding ofNF-kB dimers to specific gene promoters, however, the low cellnumbers (<200,000 neurons total per culture) precluded such anapproach.

3.4. Peripheral neuropathy and impaired NF-kB p50 expression/activity in DRG from STZ-diabetic mice were normalized by systemicCNTF treatment

After 18 weeks of diabetes mice exhibited a mild decrease inweight gain and significant thermal hypoalgesia compared tocontrol mice (Table 2). CNTF treatment (2 mg/kg thrice weekly) wasbegun in a sub-group of diabetic mice at week 18 and by week 30had reversed thermal hypoalgesia and normalized both MNCV andSNCV (Table 2). CNTF treatment was associated with mild cachexiathat exaggerated the diabetes-induced weight loss but no otherside effects were noted. DRG removed from mice after 30 weeks ofdiabetes showed deficits in NF-kB p50 expression and DNA bindingactivity that were prevented by CNTF treatment between weeks 18and 30 of diabetes (Fig. 4).

3.5. Bioenergetic profile is abnormal in neurons cultured fromSTZ-diabetic mice and is corrected by CNTF

Optimal conditions for neurite outgrowth demand efficientmitochondrial function and supply of ATP to the motile growthcone (Bernstein and Bamburg, 2003). To assess the mitochondrialbioenergetics profile in sensory neurons derived from DRG of agematched control and 3e5 month STZ-diabetic mice, the oxygenconsumption rate (OCR) was measured in neurons cultured for 24 husing the Seahorse Biosciences XF24 analyzer. OCR induced by theuncoupler FCCP (1 mM) in neurons cultured from diabetic mice wassignificantly decreased compared to neurons cultured from age-matched control mice (Fig. 5A). This significant impairment ofmaximal electron transport activity is indicative of suboptimalspare respiratory capacity (Fig. 5D). Neurons from diabetic micethat were cultured for 24 h with CNTF (10 ng/ml) showed signifi-cantly improved basal respiration, maximal OCR (Fig. 5B) and sparerespiratory capacity (Fig. 5E). Neurons from diabetic mice weretreated with CNTF and co-treated with SN50M or SN50. Inhibitionof NF-kB by SN50 induced an approximate 2-fold suppressionof maximal OCR and spare respiratory capacity (Fig. 5C and F).Respiratory control ratio and coupling efficiency were not signifi-cantly impacted by diabetes or CNTF treatment (data not shown).

4. Discussion

Our previouswork demonstrated that CNTF replacement therapyprevented the development of conduction slowing, axonal dwin-dling, thermal hypoalgesia and impaired axon regeneration indiabetic rats (Calcutt et al., 2004; Mizisin et al., 2004) but nomechanism of action was identified. The present study demon-strates that the aberrant mitochondrial bioenergetic profile ofsensory neurons from diabetic rodents is a direct target of CNTF.Additionally, the work demonstrates for the first time that CNTFsignals via the NF-kB transcription factor to modulate mitochondrialbioenergetics. We propose this mitochondrial-dependent process,

Values are means � SEM, n ¼ 3; *p < 0.05 vs control. In (D) are shown representativeWestern blots and quantification for NF-kB p50 and T-ERK showing effect of CNTF(10 ng/ml) treatment for up to 24 h on expression. Values are mean � SEM, n ¼ 3,*p < 0.05 vs control (by one way ANOVA).

Table 2Physiological parameters in control and STZ-diabetic C57Bl/6 mice treated with vehicle or CNTF (2 mg/kg thrice weekly) betweenweeks 18 and 30 of diabetes. Data are groupmean� SEM. Statistical analysis by one-way ANOVA followed by Dunnett’s post-hoc test to compare both diabetic groups against the control group. *p< 0.05 and **p< 0.01 vscontrol.

Body weight (g) Blood glucose (mM) Thermal latency (s) MNCV (m/s) SNCV (m/s)

0 wk 18 wk 30 wk 30 wk 18 wk 30 wk 30 wk 30 wk

Control 19.0 � 0.4 23.1 � 0.5 24.3 � 0.5 8.8 � 0.6 5.1 � 0.3 5.4 � 0.1 51.4 � 3.5 52.4 � 3.3STZ 18.3 � 0.5 21.9 � 0.3* 22.3 � 0.4** 22.6 � 1.8** 8.5 � 0.4** 8.5 � 0.5** 38.0 � 2.4** 45.6 � 2.1STZ þ CNTF 18.7 � 0.5 21.3 � 0.2** 19.9 � 0.5** 20.9 � 1.4** 8.7 � 0.4** 6.3 � 0.5 48.1 � 4.0 53.4 � 3.3

A. Saleh et al. / Neuropharmacology 65 (2013) 65e7370

in part, underlies the ability of CNTF to protect peripheral nervesfrom functional and structural indices of diabetic neuropathy and toenhance axon regeneration.

CNTF enhanced neurite outgrowth and activated NF-kB incultured sensory neurons, while long term CNTF therapy in STZ-diabetic mice was effective in normalizing NF-kB subunit expres-sion and activity. In the present work gene reporter technology,DNA binding (ELISA-based) and Western blotting was used toassess NF-kB activity. Future studies using CHiP analysis andnuclear translocation of subunits by immunocytochemistry wouldimprove the robustness of this component of the work by clearlyrevealing the identity of NF-kB dimers binding to endogenousgene promoters. However, these findings suggest that down-regulation of cytokine gene expression and synthesis is an earlytarget of diabetes that is likely to have pathogenic consequences.If diabetes directly impairs the NF-kB pathway, for example bydegrading p50 subunits, then cytokine therapy would not be soefficacious. In peripheral nerve CNTF is synthesized by Schwanncells and its down-regulation during diabetes is a consequence ofglucose metabolism by the Schwann cell enzyme aldose reductase(Mizisin et al., 1997). Possible downstream targets of the diabeticstate include the Wnt/b-catenin pathway modulated by Norrinthat has been shown to up-regulate CNTF expression in retinalMüller cells (Seitz et al., 2010). The transcription factor, SRY-boxcontaining gene 10 (Sox10), is also strongly implicated in posi-tive regulation of CNTF expression in Schwann cells (Ito et al.,2006).

The basal neurite outgrowth levels of cultured diabeticneurons were lower than age matched controls. However, theresponse to CNTF was relatively similar in the two cell types. In

Fig. 4. Decreased NF-kB subunit p50 expression is normalized in DRG from STZ-diabetic mice treated with CNTF. Age matched control and STZ-diabetic C57Bl6/Jmice were maintained for 18 wk and then a sub-group of diabetic mice treatedsystemically with 2 mg/kg CNTF thrice weekly for the final 12 wk. In (A) are shownWestern blots of DRG homogenates probed for NF-kB p50 and total ERK (loadingcontrol; each lane represents an individual mouse) and (B) bar chart of quantificationof p50 expression levels. In (C) is presented DNA binding activity of DRG homogenates.Values are means � SEM, n ¼ 5; *p < 0.05 vs control or Db þ CNTF (one way ANOVA).

addition, CNTF receptor alpha expression was not altered in dia-betic neurons and the NF-kB reporter assay revealed equallyrobust induction of transcriptional activity in neurons from age-matched control versus diabetic animals. This suggests no loss ofCNTF sensitivity, and hence the lowered neurite outgrowth indiabetic neurons, maybe due to factors downstream of NF-kBsignaling, for example reduced expression and/or abnormalphosphorylation of structural proteins such as neurofilaments(Fernyhough et al., 1999; Mohiuddin et al., 1995; Sayers et al.,2003). The ability of CNTF to promote neurite outgrowth incultured neurons does not necessarily imply that loss of CNTFdrives diabetes-induced distal axonal degeneration in vivo. Themeasure of neurite outgrowth is more relevant to the ability ofCNTF to trigger repair via axon regeneration and/or collateralsprouting. Several recent studies reveal multiple pathways thatmay contribute to axonal degeneration linked to mitochondrialdysfunction in the axon. For example, diabetes-induced abnor-malities in function of mitofusin2, slow Wallerian degeneration(WLDS) protein and extranuclear lamin B may instigate axonaldegeneration via a mitochondrial-dependent pathway (Averyet al., 2012; Misko et al., 2012; Yoon et al., 2012).

The diabetes-induced impairments in CNTF and NF-kB signalingwere mechanistically linked to aberrant mitochondrial bioener-getics. Diabetes blunted themaximal OCR and significantly loweredspare respiratory capacity, suggesting that the neurons wereenergetically stressed and mitochondrial workload was increased.Reduced spare respiratory capacity, especially in neurons that havea wide variation of ATP demands, limits their ability to meetenergetic needs and renders the cells more susceptible tosecondary stressors (Brand and Nicholls, 2011). CNTF correctedthese abnormalities in an NF-kB-dependent manner supportingstudies in fibroblasts that describe a key role for NF-kB in aug-menting mitochondrial function (Mauro et al., 2011). Enhancementof mitochondrial performance by CNTF will therefore allowincreased ATP synthesis under conditions of stress or increasedenergy demand. This CNTF-driven enhanced mitochondrial oxida-tive capacity can also increase axonal plasticity, as growth conemotility increased local ATP demands (Bernstein and Bamburg,2003). We propose that this mitochondrial-dependent processunderlies the ability of CNTF to prevent functional indices of dia-betic neuropathy in STZ-diabetic mice (present study) and rats(Calcutt et al., 2004; Mizisin et al., 2004). CNTF can also activateSTAT3 andMAP kinase pathways that may augment axon extension(Liu and Snider, 2001; Qiu et al., 2005). Neurite outgrowth ispotentiated, in part, by nuclear translocation of STAT3 uponinduction by CNTF and blocked by suppressor of cytokine signaling(SOCS3) (Miao et al., 2006). Recent studies reveal that the JAK-STAT3 pathway-dependent augmentation of neurite outgrowth isassociated with modulation of mitochondrial function with a noveldirect interaction of phosphorylated STAT3 (on serine) with theelectron transport system (Wegrzyn et al., 2009; Zhou and Too,2011), but this aspect is beyond the scope of the current work. Inaddition, in retinal ganglion neurons the combined deletion ofphosphatase and tensin homolog (PTEN) and SOCS3 generated

Fig. 5. Mitochondrial bioenergetics is abnormal in cultured neurons from diabetic mice and is corrected by CNTF. OCR was measured at basal level with subsequent and sequentialaddition of oligomycin (1 mM), FCCP (1 mM), and rotenone (1 mM) þ antimycin A (AA; 1 mM) to DRG neurons cultured from age-matched control (blue) and 3e5 month STZ-induceddiabetic mice (black) in the presence of low dose neurotrophic factors. Oligomycin inhibits the ATP synthase leading to a build-up of the proton gradient and dearth of ATP thatinhibits 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 þ antimycin Awere injected to inhibit the flux of electrons through complexes I and III, and thus no oxygenwas further consumed at cytochrome c oxidase. The remainingOCR determined after this intervention is primarily non-mitochondrial. Data are expressed as OCR in pmol/min for 1000 cells (there were approximately 2500e5000 cells per well).Dotted lines, aee in (A) have been used later for quantification of bioenergetics parameters. The OCR measurements in (A) control (blue) and diabetic (black), (B) diabetic (black) ortreated with CNTF (red), and (C) diabetic treated with CNTF and SN50M (red) or CNTF and SN50 (green) at the 1 mM concentration of FCCP were plotted. Spare respiratory capacity(dea) in (DeF) is presented for control (blue), diabetic (black), diabetic treated with CNTF (red) and diabetic treated with CNTF and SN50 (green) and was calculated after sub-tracting the non-mitochondrial respiration (e) as described (Brand and Nicholls, 2011). Values are mean � SEM of n ¼ 5 replicate cultures; *p < 0.05 and **p < 0.01 by Student’s t-Test.

A. Saleh et al. / Neuropharmacology 65 (2013) 65e73 71

a robust and remarkable enhancement of axon regeneration (Sunet al., 2011). The role of SOCS3 in axon regeneration is complex,however, since under cytokine stimulation SOCS3 can enhanceretrograde axonal transport of c-jun N-terminal kinase interactingprotein-1 (JIP1), and reveals a beneficial effect of the combination ofSOCS3 and JIP1 on axonal growth in vitro (Miao et al., 2011). Cyto-kinesmay therefore also enhance axonal regeneration by optimizingretrograde axonal transport of signaling molecules that, in turn,control the cell body response to stress and/or axon damage.

Exploration of the therapeutic potential of CNTF has beenlimited by cachexia-related side effects in both animals andhumans (Henderson et al., 1994). There is now renewed interest inthe therapeutic potential of CNTF due to its anti-obesity effectsmediated, in part, through modulation of hypothalamic physiologyand inhibitory effects on food intake (Allen et al., 2011). In addition,

CNTF has been shown to reduce fat deposition in obese micethrough promotion of mitochondrial biogenesis and enhancedoxidative capacity in adipose tissue (Crowe et al., 2008). Further-more, recent studies reveal CNTF to have therapeutic potential innumerous neurological disorders ranging across Parkinson’sdisease (via enhanced neurogenesis) (Yang et al., 2008), optic nervetrauma (Hellstrom et al., 2011), spinal muscular atrophy (Simonet al., 2010), CharcoteMarieeTooth type 1A (Nobbio et al., 2009)and spinal cord injury (Cao et al., 2010). Signal transduction path-ways utilized by CNTF in mediating anti-obesity effects includeactivation of the AMP-activated protein kinase (AMPK)/peroxisomeproliferator-activated receptor g coactivator-1a (PGC-1a) pathwayin muscle and adipocytes and triggering enhanced mitochondrialoxidative capacity and biogenesis (Crowe et al., 2008; Steinberget al., 2009). Pertinently, AMPK activation and PGC-1a expression

A. Saleh et al. / Neuropharmacology 65 (2013) 65e7372

are impaired in sensory neurons in diabetes and this signaling axismay be an additional target for CNTF action (Chowdhury et al.,2011; Roy Chowdhury et al., 2012). Future studies, possibly usingmodified forms of CNTF that lack cachexic side-effects (Blanchardet al., 2010; Rezende et al., 2009) may be warranted to furtherstudy the impact of CNTF on neuropathy in animal models of type 1and type 2 diabetes. Additionally, investigations will be needed todetermine if the AMPK pathway is targeted in conjunctionwith NF-kB signaling and linked to improved mitochondrial activity andprotection from sensory neuron dysfunction.

Acknowledgments

Dr. Ali Saleh and Dr. Subir Roy Chowdhury were supported bygrants to P.F. from Canadian Institutes for Health Research (CIHR;grant # MOP-84214) and from Juvenile Diabetes Research Foun-dation (grant # 1-2008-193). This work was also funded and sup-ported by St Boniface Hospital Research and the National Institutesfor Health grant DK57629 (N.A.C). We thank Mr. Randy Van DerPloeg and Ms. Emily Mutch for expert technical assistance.

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