Research Article Oxygen Radicals Elicit Paralysis and

Research ArticleOxygen Radicals Elicit Paralysis and Collapse ofSpinal Cord Neuron Growth Cones upon Exposure toProinflammatory Cytokines

Thomas B Kuhn

Department of Chemistry and Biochemistry University of Alaska Fairbanks 900 Yukon DriveReichardt Building Room 194 Fairbanks AK 99775-6150 USA

Correspondence should be addressed toThomas B Kuhn tbkuhnalaskaedu

Received 16 October 2013 Revised 25 February 2014 Accepted 11 March 2014 Published 23 June 2014

Academic Editor Tullia Maraldi

Copyright copy 2014 Thomas B Kuhn This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A persistent inflammatory and oxidative stress is a hallmark of most chronic CNS pathologies (Alzheimerrsquos (ALS)) as well as theaging CNS orchestrated by the proinflammatory cytokines tumor necrosis factor alpha (TNF120572) and interleukin-1 beta (IL-1120573)Loss of the integrity and plasticity of neuronal morphology and connectivity comprises an early step in neuronal degenerationand ultimate decline of cognitive function We examined in vitro whether TNF120572 or IL-1120573 impaired morphology and motility ofgrowth cones in spinal cord neuron cultures TNF120572 and IL-1120573 paralyzed growth cone motility and induced growth cone collapsein a dose-dependent manner reflected by complete attenuation of neurite outgrowth Scavenging reactive oxygen species (ROS)or inhibiting NADPH oxidase activity rescued loss of neuronal motility and morphology TNF120572 and IL-1120573 provoked rapid NOX-mediated generation of ROS in advancing growth cones which preceded paralysis ofmotility and collapse ofmorphology Increasesin ROS intermediates were accompanied by an aberrant nonproductive reorganization of actin filaments These findings suggestthat NADPH oxidase serves as a pivotal source of oxidative stress in neurons and together with disruption of actin filamentreorganization contributes to the progressive degeneration of neuronal morphology in the diseased or aging CNS

1 Introduction

A spreading inflammatory reaction accompanied by oxida-tive stress is prevalent in most chronic CNS diseases andacute CNS trauma as well as in the aging CNS [1ndash3] Theproinflammatory cytokines TNF120572 (tumor necrosis factor 120572)and IL-1120573 (interleukin-1120573) exert pleiotropic functions bothin CNS development and CNS pathogenesis [4ndash6] Persistenthigh-level expression of TNF120572 and IL-1120573 is important tothe progressive degeneration of neuronal connectivity andloss of neuronal plasticity ultimately leading to cognitivedecline The role of TNF120572 and IL-1120573 as inducers of apoptoticevents is well documented whereas the recognition of theirmorphogenetic function is more recent [7] TNF120572 and IL-1120573 released from microglia cells inhibited neurite outgrowthreduced branching and caused neurite retraction in culturesof Neuro2A cells or primary hippocampal neurons [8 9]Theintricate pattern of neuronal connectivity innate to cognitive

function rests as much on the integrity and stability of theaxonal and dendritic architecture as on the plasticity ofmotilestructures to maintain form or regenerate connectionswhich is intimately linked to the dynamic reorganizationof the actin cytoskeleton [10 11] ROS intermediates greatlyaffect both the dynamics and organization of actin filamentsduring oxidative stress of physiological redox signaling [1213] TNF120572 paralyzed actin filament reorganization in neurob-lastoma cells due to oxidative damage whereas physiologicallevels of ROS intermediates seem to be necessary for propergrowth cone motility [14 15] TNF120572 and IL-1120573 potentlystimulate NADPH oxidase (NOX) activities in neurons andglia cells often localized in coalescing lipid rafts [16 17]The members of the NADPH oxidase (NOX) family (NOX1ndash5 DUOX12) defined by the large membrane flavoproteingp91phox of the phagocyte NAPDH oxidase (NOX2) areubiquitously expressed in all cell types and have emerged asprincipal ROS sources both in cellular signaling and disease

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 191767 20 pageshttpdxdoiorg1011552014191767

2 BioMed Research International

progression in response to cytokines growth factors andhormones [18 19] Functional NOX requires an intricateassembly between two membrane proteins (a NOX isoformgp22phox) and several cytosolic factors (p47phox p40phoxp67phox) under the regulation of the small GTPases Rac1or Rac2 [20] Rho GTPases harbor a dual role in cytokinesignaling as regulators of both NOX assembly and thereorganization of actin filament structures [21ndash23] In lightof these reports we examined whether ROS intermediatesgenerated by NOX activities in neuronal growth cones areimplicated inmediating the neurotoxic effects of TNF120572 or IL-1120573 on neurite outgrowth Amechanistic understanding of thedetrimental consequences of TNF120572 and IL-1120573 on neuronalconnectivity in the CNS neurons is vital to intervene withprogressive neurodegeneration in the aging or diseased CNS

2 Materials and Methods

21 Reagents Unless state otherwise all reagents were pur-chased from Sigma (St Louis MO) Dulbeccorsquos Modi-fied Eagle Medium (high glucose DMEM) Leibovitzrsquos L-15 and isopropyl 120573-D-thiogalactopyranoside were obtainedfrom Invitrogen Corporation (Carlsbad CA) Character-ized fetal bovine serum (FBS) was from Hyclone (LoganUT) and it was heat-inactivated according to the manu-facturer Laminin was from Roche Diagnostics Corporation(Indianapolis IN) The redox sensitive fluorescent indi-cators carboxymethyl-2101584071015840-dihydrodichlorofluorescein diac-etate 2101584071015840-dihydrodichlorofluorescein diacetate and Cal-cein Blue were obtained from Molecular Probes (EugeneOR) Human recombinant tumor necrosis factor 120572 (TNF120572)and interleukin-1120573 (IL-1120573) Diphenylene iodonium (DPI)N-acetyl-L-cysteine (NAC) and Manganese(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) were purchasedfrom Calbiochem (San Diego CA) Polyclonal rabbit anti-TNF receptor I polyclonal rabbit anti-TNF receptor IIpolyclonal rabbit anti IL-1 receptor type I polyclonal goatanti-NOX2 polyclonal goat anti-p47phox including respectiveblocking peptides were all from St Cruz Biotechnology(St Cruz CA) A monoclonal mouse anti-p4442 MAPKa monoclonal mouse anti-phospho p4442 MAPK and amonoclonal mouse anti-phospho JNKwere fromCell Signal-ing Technologies (Danvers MA) Fluorescein or rhodamine-labeled goat anti-rabbit and goat anti-mouse IgG (resp)were from Chemicon (Temecula CA) whereas rhodamine-conjugated phalloidin was obtained from Cytoskeleton Inc(Denver CO) The polyclonal rabbit anti-p67phox was pur-chased from Abcam (Cambridge MA)

22 Spinal Cord Neuron Cultures Dissociated low-densitycultures of spinal cord (SC) neurons were established asdescribed [24] Briefly spinal cord tissue was dissected from7 day-old chick embryos (E7) and a single cell suspensionobtained after enzymatic digestion (05mgmL trypsin 2mMEDTA 10min 37∘C) followed by trituration After preplating(1 h 37∘C 5 CO

2atmosphere high glucose DMEM pH

7310 FBS) nonadherent cells (predominantly SC neurons)were collected (3min 200timesgmax) and resuspended in SC

medium (high glucose DMEM pH 73 10 FBS 12 nMfluorodeoxyuridine 30 nM uridine and 1 N3 nutrientsupplement) [25] SC explants were prepared by pushingfreshly dissected E7 spinal cord tissue through a wire-mesh(50ndash75120583m) Dissociated SC neurons (75000 cellsmL) or SCexplants (2 per culture) were plated onto glass cover slips (22times 22mm2 number 1 Carolina Biological Supply CompanyBurlington NC) mounted over a 15 cm hole drilled intothe bottom of a 35mm culture dish Glass cover slips weretreated with poly-D-lysine (100 120583gmL borate buffer pH84 30min RT) prior to coating with laminin (5120583gcm230min RT) These SC neuron cultures contained less than5 nonneuronal cells with no immunoreactivity againstmicroglia markers

23 Growth Cone Particle Preparations Preparations ofhighly enriched growth cone particles were obtained fromfreshly dissected whole chick embryo brains (E10-12) [26 27]After homogenizing (Dounce homogenizer ice-cold 5mMHEPES pH 73 1mM MgCl

2 032M Sucrose 6ndash8 volumes

per wet weight) homogenates were centrifuged (15min1600timesgmax 4

∘C) Resulting supernatants were overlaid onto5mM HEPES pH 73 1mM MgCl

2 and 075M sucrose and

then centrifuged (150000timesgmax 1 h 4∘C) and material at

the interface was collected After dilution (6-7 times in lowsucrose buffer) the suspension was overlaid ontoMaxiDense(4-5 sample volumes per volumeMaxidense) and centrifugedat 40000timesgmax (1 h 4

∘C) and growth cone particles (GCPs)were collected on top of the MaxiDense cushion GCPswere resuspended in Krebrsquos buffer (145mMNaCl 5mM KCl12mM NaH

2PO4timesH2O 12mMMgCl

2 and 5mMHEPES

pH 73) and protein concentration determined (BCA assayThermo Scientific Rockford IL USA)

24 Measurement of Neurite Lengths Dissociated SC neu-rons were grown after the onset of neurite outgrowth indi-cated by the majority of cells extending at least one processlonger than 2 cell body diameters Cultures were incubated(1 h) with pharmacological agents followed by bath appli-cation (6ndash8 h) of 100 ngmL TNF120572 100 ngmL IL-1120573 or10 120583gmL ovalbumin After fixation (2 glutaraldehyde) thelength of the longest neurite per neuron was measured ofat least 50 randomly selected SC neurons only consideringprocesses adhering to the following criteria (i) emergingfrom an isolated cell body (ii) longer than two cell diametersand (iii) no contact to other neuronal processes or cell bodiesThe distribution of neurite length in a population of SCneurons was obtained by plotting the percentage of neuronswith neurites longer than a given length against neurite length[28] As a characteristic for neurite outgrowth under a givencondition the neurite length reached by 50 of neurite-bearing SC neurons (NL

50) was calculated as criteria for

statistical significance [29] Purified recombinant Rac1V12-GST Rac1N17-GST or GST were introduced into freshlydissociated SC neurons (7ndash10mgmL 500000 cells 200120583L50mM Tris-Cl pH 75 100mM NaCl and 5mM MgCl

2)

by trituration loading at the time of plating [26] Followingtrituration SC neurons were immediately transferred in SCmedium and plated as described above For each condition

BioMed Research International 3

measurements were performed in duplicate cultures fromtwo independent dissections

25 Measurement of Growth Cone Advance Dissociated SCneurons (12 to 16 h in culture) were transferred into obser-vation medium (LeibovitzrsquoL15 without phenol red pH 745mgmL ovalbumin 1 N3) overlaid with light mineraloil to avoid evaporation and placed onto the microscopestage equilibrated at 37 plusmn 02∘C (Nikon TE2000 U) After15-minute recovery images of advancing growth cones (20xphase contrast) were acquired at 3-minute time intervals for a30min time period (Coolsnapfx Photometrics Tuscon AZ)Cytokines (100 ngmL TNF120572 100 ngmL IL-1120573) or 10 120583gmLOvalbumin were bath-applied at 119905 = 8min The extensionof the growth coneneurite boundary (120583m) was measuredas a function of time (min) (Metamorph Software MeridianInstrument Co Kent WA) At least 20 growth cones weremonitored for each condition in duplicate cultures from atleast two different dissections

26 Recombinant Mutant Rac1 Protein RecombinantRac1V12-GST or Rac1N17-GST protein was induced in E coliDH5120572 with 05mM isopropyl 120573-D-thiogalacto-pyranoside(4 h) [30] After cell lysis (50mM Tris-Cl pH 75 50mMNaCl 5mM MgCl

2 and 1mM dithiothreitol) a soluble

protein fraction was obtained (20000timesgmax 20min 4∘C)and subjected to affinity chromatography on glutathione-conjugated agarose Bound proteins were eluted (5mMglutathione in lysis buffer) concentrated to 5ndash7mgmL in50mM Tris-Cl pH 75 100mM NaCl and 5mM MgCl

2

(Centrifugal Devices Millipore Bedford MA) and storedin 100 120583L aliquots at minus80∘C after snap-freezing Proteinpreparations showed single bands on Coomassie blue-stained 10 SDS polyacrylamide gels

27 Adenoviral Expression of Rac1 Mutants in SC NeuronsRecombinant replication deficient adenovirus carrying genesfor constitutively active Rac1 (Rac1V12 withN-terminal FLAGtag) dominant negative Rac1 (Rac1N17 with N-terminalFLAG tag) or lacZ were expressed in E7 chick SC neurons asdescribed previously [31] At the time of plating dissociatedSC neurons were infected with recombinant adenovirus at200 moi (multiplicity of infection) in 300 120583L SC mediumCultures were replenished with 200 120583L fresh SC mediumafter 12 h and grown for additional 48 h At three days afterinfection yields of neuronal infections generally exceeded70 [31 32] Amplification of viral stocks was performedin 293 HEK cells and titers greater than 5 times 108 plaqueforming units per mL were routinely obtained Viral stockswere stored at ndash80∘C

28 Quantitative ROS Imaging in SC Neuron Cell BodiesDissociated SC neurons were loaded with 10120583M 2101584071015840-dihydrodichlorofluorescein diacetate (DCF) or 5 120583M dihy-roethidium (DMEM10 FBS) for 30min (37∘C 5 CO

2

atmosphere) washed and allowed to recover (15minDMEM10 FBS) Cultures were switched to observationmedium overlaid with light mineral oil transferred to the

heated microscope stage (Zeiss Axiovert125S) and allowedto adapt for 15min Images of dissociated SC neurons(random fields of view) were acquired under phase contrast(20x Plan-Apo objective) and FITC illumination (Ex 465ndash495 nm DM 505 Em 515ndash555) using a Peltier cooled CCDcamera (Sensys Photometrics TusconAZ) after the recoveryphase (defined as basal condition) after pharmacologicaltreatments (30min) and after cytokine exposure (100 ngmLTNF120572 or 100 ngmL IL-1120573) Hydrogen peroxide was added toall cultures following treatments to ensure proper loading Asour criteria for SC neurons we analyzed only cells displayinga large round cell body and one neuronal process at leastlonger than three cell diametersMaximumDCFfluorescenceintensity per neuronal cell body was determined on a pixel-by-pixel basis following background subtraction (averagebackground of all images at 119905 = 0min for each condition)followed by erosion (2 pixels) and overlay with the originalimage (Zeiss imaging analysis software KS 300) All valueswere normalized to the average DCF fluorescence intensityin control (initial conditions) No morphological changes ofneuronal cell bodies were detectable in our assay conditionsindicated by constant cell body areas ROS measurementswere performed in duplicate cultures obtained from 3 to 5independent dissections At least 50 SC neurons were mea-sured in duplicate cultures of three independent dissections

29 Quantitative ROS Imaging in Advancing SC Neu-ron Growth Cones SC explants were incubated (30minDMEM10 FBS) with 20120583MDCF (51015840-(and 61015840)chloromethyl-dichlorodihydrofluorescein diacetate) and 4 120583M CalceinBlue (CB) an oxidation-inert fluorescence indicator Afterrecovery (15min DMEM10 FBS) cultures were switchedto observation medium and overlaid with light mineral oilImages of advancing growth coneswere acquired under phasecontrast (40x Plan-Apo) FITC illumination (DCF fluores-cence) and DAPI illumination (CB fluorescence) (Cool-snapfx) before (pre-stimulus images at 119905 = 0 2 4min) andafter (after stimulus images 2min time intervals 16min timeperiod) bath application of 100 ngmL TNF120572 100 ngmL IL-1120573 or 10 120583gmL ovalbumin As our positive loading controlfollowing the observation period all growth cones wereexposed to 100 120583M hydrogen peroxide For image analysisDCF and CB fluorescence intensities (119865DCF and 119865CB) wereintegrated (pixel-by-pixel basis) over the growth cone area foreach growth cone observed at each time point ( Int119865DCF119905 andInt119865CalcB119905with 119905= time interval) after background subtraction

(119905 = 0 image) and the ratio of integrated DCF and CBfluorescence intensities for each growth cone at each timepoint was calculated (119877

119905=

Int119865DCF119905

Int119865 with 119905 = time

interval) Next the average ratio of integrated DCF andCB fluorescence intensities of all growth cones observedat 119905 = 0min (pre-stimulus) was calculated (av119877

0) followed

by normalization of all ratios for at 119905 = 0min (119877119905

119899av1198770

119899 = growth cone 1 2 to 119899 for each condition) At least15 growth cones were analyzed per condition from threedifferent dissections to provide statistical significance (lowast119875 lt005)


210 ROS Quantification in Growth Cone Particle Prepa-rations Freshly prepared GCPs (100120583g in Krebrsquos buffer)were loaded with 20 120583M DCF (30min 4∘C) in the presenceof pharmacological reagents (10 120583M DPI 500120583M NAC)washed (14000timesgmax) allowed to recover (10min 4∘C) withpharmacological reagents present and then exposed to 100 or200 ngmL TNF120572 (45min 4∘C) For lysis GCPs were resus-pended in 2 SDS 10mM Tris-Cl pH 75 10mM NaF 5mMdithiothreitol and 2mM EGTA sonicated and cleared bycentrifugation (14000timesgmax 5min) Total DCF fluorescenceintensity was measured (100120583L aliquots black 96 well plates)using a Beckman Coulter Multimode DTX 880 microplatereader (495 nm excitation filter 525 emission filter) All datawere adjusted to total soluble protein concentration (BCAassay) and normalized to control condition to account forunspecific fluorescence andor autofluorescence artifact Forall conditions measurements were obtained in duplicatesfrom three different GCP preparations

211 Indirect Immunocytochemistry Dissociated SC neuronswere fixed (4 paraformaldehyde 10mM MES pH 61138mM KCl 3mM MgCl

2 2mM EGTA 15min RT) fol-

lowed by three washes with TBS (20mM Tris-Cl pH 75150mM NaCl) After blocking (30min) cultures were incu-bated (2 h RT) with primary antibodies (4120583gmL 2mgmLBSA in TBS) against cytokine receptors or MAP kinasesrinsed with TBS (3 times 15min each) and incubated withrespective secondary antibodies Cultures were transferredto 60 glycerolPBS Images were acquired under FITCillumination on a Radiance 2000 confocal microscope (BioRad)

212 Actin Filament Quantification To visualize actin fil-aments SC neurons were fixed and permeabilized (05Triton X-100 15min) After rinsing (01 Triton X-100 inTBS) cultures were incubated (20min) with rhodamine-conjugated phalloidin (1 10 in 1 Triton X-100 in TBSCytoskeleton Inc Denver CO) washed and stored in 60glycerol (4∘C) until inspection Images were acquired (40xoil Plan Fluor) using a Zeiss LSM 510 confocal microscopeequipped with a HeNe laser and an Argon laser For eachcondition 60 randomly selected SC neuron growth coneswere scored for the presence of at least one large lamellipodia-like structure (three dissections 119899 = 180) and the percentageof responding growth cones determined

213 Plasma Membrane Translocation of 11990167phox FreshlypreparedGCPs (100 120583g per sample) were treatedwithmethyl-120573-cyclodextrin (01) or buffer (30min 4∘C) and thenexposed to 200 ngmL TNF120572 (1 h 4∘C) whereas cultures ofE10 forebrain neurons were subjected to 200 nM PMA (1 h37∘C) GCPs (centrifugation) or forebrain neurons (scraping)were transferred into a 033M sucrose buffer (20mM Tris-HCL pH 80 2mM EDTA 05 EGTA 2mM AEBSF and25 120583gmL Leupeptin) lysed by sonication and centrifuged(25000timesgmax 15min) to obtain an enriched plasma mem-brane fraction (pellet) Pellets were resuspended in sucrosebuffer containing 1 Triton X-100 and 001 saponin (4∘C

15min) or in 20mM Tris-HCL pH 80 2mM EDTA 05mMEGTA and 2mM AEBSF with regard to gel electrophoresisor ELISA analysis respectively Suspensions were centrifuged(25000timesgmax 15min 4∘C) membrane protein was recov-ered as the supernatant and total protein concentrationwas determined For ELISA aliquots of plasma membraneprotein (20120583gmL TBS1 Triton-X-100) were incubated(12 h 25∘C) in 96-well high protein absorbent plates (Falcon)After blockingwith BSA (5wv TBS 1Trition-X-100 1 h)wells were incubated (overnight 4∘C)with a polyclonal rabbitanti-p67phox (3 120583gmL) rinsed (TBS1 Triton-X-100) andincubated with a goat anti-rabbit IgG antibody conjugated toHRP (1 2000 45min 25∘C) After three consecutive washeswells were supplemented with 100 120583L TMB (tetramethylben-zidine) and absorbance at 620 nm was measured 10min later(Beckman Coulter Multimode DTX 880 microplate readerAll values were adjusted to total membrane protein and nor-malized to control conditions (duplicates three independentexperiments)

214 Gel Electrophoresis and Western Blotting SC tissue SCneuron cultures or GDPs were solubilized in 2 SDS 10mMTris-Cl pH 75 10mM NaF 5mM dithiothreitol and 2mMEGTA and sonicated and a total soluble protein fractionwas obtained by centrifugation (supernatant 14000timesgmax5min) Fractions of total plasma membrane proteins wereobtained as described above Samples containing 20120583g oftotal soluble protein in Laemmli buffer were subjected to10 SDS gel electrophoresis followed by western blottingonto PVDF membranes (Bio-Rad Hercules CA) [33 34]Membranes were blocked (2 nonfat dry milk TBS pH 85overnight 4∘C) washed (TBS005 Tween 202 BSA)and incubated with polyclonal antibodies against cytokinereceptor MAP kinase phosphoMAP kinase NOX2 p47phoxor p67phox (1 h 4 120583gmL in TBS005 Tween 202 BSA)Membranes were washed (TBS005 Tween 202 BSA)and incubated (1 h RT) with the respective secondary alka-line phosphatase-conjugated IgG (1 10000 Sigma St LouisMO) followed by colorimetric development (NBTBCIP onestep Thermo Scientific Rockford IL) The specificity ofantibodies was verified in lysates ofHeLa cells RAW264 cellsand SH-SY5Y cells (including blocking peptides)

215 Statistical Analysis One-way ANOVA analysis and aKruskal-Wallis test were employed for comparisons amongmultiple conditions Dunnettrsquos 119905-test was used when compar-ing the means of multiple conditions with a single controlAll statistical values are given as SEM with a significance oflowast119875 lt 005 unless indicated otherwise Measurements were

obtained in duplicates from3 to 5 separate experiments unlessstated otherwise

3 Results

31 TNF120572 and IL-1120573 Paralyze Growth Cone Motility andInduce Growth Cone Collapse Recent reports detailed thatTNF120572 exerts morphogenetic functions (rearrangements ofthe actin cytoskeleton) without induction of apoptosis [7]

BioMed Research International 5IL

-1120573

Ov

TNF120572

(a)

0min

0min

0min

9min 16min 27min

85min 12min 16min

3min 7min 11min

IL-1120573

Ov

TNF120572

(b)

90

50

(kD

a)

IL-1RTNF-R2TNF-R1

(c)

Figure 1 TNF120572 and IL-1120573 impair motility and morphology of neuronal growth cones (a) Acute exposure of dissociated E7 SC neurons(laminin) to 100 ngmL TNF120572 (top panel) or IL-1120573 (middle panel) provoked paralysis of growth cone advance and rapid degeneration ofthe morphology of growth cones and neurites as opposed to 10120583gmL ovalbumin (bottom panel) Stars indicate location of the growthconeneurite border with respect to first image in each panel respectively (scale bar = 10120583m) (b) To restrict cytokine exposure exclusivelyto advancing growth cones polystyrene beads (25 times 105 beadsmL 4120583m in diameter) coated with TNF120572 or IL-1120573 were applied to SC neuroncultures and growth cone-bead encounters observed under phase contrast (63x oil phase contrast) Following physical contact of growth conesto cytokine-coated beads (TNF120572mdashtop panel IL-1120573mdashmiddle panel) growth cone motility ceased followed by the progressive degenerationof growth cone morphology upon reaching complete collapse Contact with ovalbumin-coated beads had no influence on growth conemorphology and advance (bottom panel) (c) E7 SC neurons grown on laminin for 2 days were fixed in paraformaldehyde Cytokine receptorswere revealed by indirect immunocytochemistry and analyzed by confocal microscopy (Zeiss LSM510 40x oil NA 130) TNF120572 receptor 1(TNF-R1) and IL-1120573 receptor (IL-1R) were expressed on cell bodies (arrows) and neurites were expressed as well as on growth cones andfilopodia (arrowheads) In contrast TNF120572 receptor 2 (TNF-R2) expression was restricted to cell bodies (Scale bars upper panel 20120583mlower panel 10 120583m)Western blots of whole spinal cord (E7 chick) extracts revealed immunoreactivity (stars) against avian cytokine receptorsTNF-R1 (48 kDa) TNF-R2 (70 kDa) and IL-1R (76 kDa) as determined in spinal cord lysates (50120583g total protein per lane)

We examined whether TNF120572 or IL-1120573 has the potency toaltermotility of neuronal growth cones an actin-cytoskeletondriven mechanism Live-video phase contrast microscopy ofadvancing SC neuron growth cones revealed that an acuteexposure to TNF120572 or IL-1120573 paralyzed growth cone motilityand also caused the collapse of growth cone morphology

accompanied by neurite beading and retraction compared tocontrol (ovalbumin) (Figure 1(a)) To demonstrate a directeffect of cytokines on growth cones we utilized polystyrenebeads (4 120583m in diameter) covalently coated with TNF120572IL-1120573 or ovalbumin (control) to restrict contact betweenneurons and cytokines exclusively to advancing growth cones


10 20Time (min)

60

40

20

0

minus20

ControlTNF 50ngmL

TNF 100ngmLH2O2 100 120583m

Gro

wth

cone

adva

nce (120583

m)

(a)

10 20 30Time (min)

40

20

0

minus20

minus40

Gro

wth

cone

adva

nce (120583

m)

IL-1120573 50ngmLIL-1120573 100ngmL Control

(b)

10 20 30Time (min)

60

40

20

0

minus20Gro

wth

cone

adva

nce (120583

m)

ControlTNF

TNF + MnTNF + DPI

(c)

10 20 30Time (min)

0

40

20

minus20

minus40Gro

wth

cone

adva

nce (120583

m)

ControlIL-1120573 IL-1120573 + Mn

IL-1120573 + DPI

(d)

60

80

40

20

0

lowast

lowast

lowast

Col

lapse

d gr

owth

cone

s (

)

25

TNF50

TNF100

TNFCon

(e)

lowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowast

60

80

40

20

0Col

lapse

d gr

owth

cone

s (

)

TNFMn TNFMn

Con DPI TNFDPI DPI

ILMnILIL

(f)

Figure 2 TNF120572 and IL-1120573 provoke a redox-sensitive collapse of growth cone motility and morphology Advancing growth cones in SCneurons cultures (laminin) were randomly selected and images were acquired at 3min time intervals (20x magnification phase contrast) inthe presence of 10120583MMnTBAP 2120583MDPI or PBS (equal volume) before and after acute exposure (119905 = 6min) to TNF120572 (50 or 100 ngmL) IL-1120573 (50 or 100 ngmL) or ovalbumin (10 120583gmL) Growth cone advance was measured as the extension of the growth coneneurite boundary(120583m) per time interval and plotted against time (min) with slopes indicating growth rates (a and b) Growth cones ceased motility andadvance within minutes upon exposure to cytokines (open diamonds open circles) compared to a steady growth cone advance under control(ovalbumin closed squares) Growth cones exposed to 100 120583MH

2O2(open triangles) mostly responded with paralysis yet slow recovery was

measured In the presence of 50 ngmL cytokines (open diamonds) growth cones resumed advance after a lag phase however at much slowergrowth rates whereas no recovery was detected at concentrations of 100 ngmL TNF120572 or IL-1120573 (open circles) (c and d) ROS scavengingwith 5 120583MMnTBAP (open triangles) or NOX inhibition with 2 120583MDPI (open diamonds) rescued growth cone advance upon acute exposureto 100 ngmL TNF120572 (c) or 100 ngmL IL-1120573 (d) respectively (open circles) All data were obtained from at least three different dissections(duplicate cultures each gt30 growth cones total) with error bars representing SEM (e) TNF120572 elicited a dose-dependent growth cone collapseat concentrations higher than 50 ngmL Growth cones with collapsed morphology were quantified (random fields of view) 30min afterapplication to allow for possible recovery of morphology (f) Preincubation of SC neuron culture either with 10 120583M MnTBAP or with 2120583MDPI provided significant protection against growth cone collapse in the presence of 100 ngmL TNF120572 or 100 ngmL IL-1120573 (dark grey barslowastlowast

119875 lt 005) as opposed to cytokines alone (black bars) which caused substantial growth cone collapse (dark bar lowast119875 lt 005) compared tocontrol (open bar) A presence of 10 120583MMnTBAP (light grey bar Mn) had no effect on basal levels of collapsed growth cones whereas 2120583MDPI increased the percentage of collapse growth cones All data (e and f) were obtained from at least three different dissections (duplicatecultures each) Error bars represent plusmnSEM


[35] Growth cones encountering cytokine-coated beadsdisplayed a series of stereotypic changes in behavior ulti-mately resulting in growth cone collapse (Figure 1(b)) Afterestablishing long-lasting adhesion contacts with filopodiagrowth cones entered a phase characterized by highlymobilelamellipodia-like structures which were nonproductive foradvance followed by a collapse of morphology (TNF12057278 plusmn 4 119899 = 22 and IL-1120573 83 plusmn 7 119899 = 18 ofobserved growth cones resp) None of these growth coneresponses occurred upon contact with ovalbumin-coatedbeads Cytokine receptors were expressed on SC neuronsand their processes (Figure 1(c)) Whole SC tissue extracts(western blotting) exhibited immunoreactivity against TNFreceptor 1 (TNF-R1 apparent MW = 48 kDa) TNF receptor2 (TNF-R2 apparentMW= 70 kDa) and IL-1120573 receptor type1 (IL-1R apparentMW=76 kDa) in accordancewith previousreports [36] Expression of TNF-R1 and IL-1R was foundon neuronal cell bodies neurites as well as growth coneshowever TNF-R2 expression was predominantly localized toneuronal cell bodies with virtually no expression on neuritesor growth cones

Next we quantified growth cones advance (the spatialdisplacement of the growth coneneurite boundary overtime) in SC explant cultures upon acute exposure to TNF120572IL-1120573 or ovalbumin (Ov control) (Figure 2) Both TNF120572and IL-1120573 caused a rapid dose-dependent paralysis of growthcone advance and even neurite retraction within less than 10minutes of addition compared to Ov (Figures 2(a) and 2(b))Acute bath application of 100 ngmL TNF120572 or IL-1120573 reducedthe percentage of advancing growth cones by 83plusmn6 (119899 = 48lowast119875 lt 005) compared to bath application of 10 120583gmL Ov In

contrast a considerable fraction of growth cones resumedadvance following bath application of 50 ngmL TNF120572 or IL-1120573 (TNF120572 42 plusmn 14 119899 = 24 lowast119875 lt 005 and IL-1120573 30 plusmn8 119899 = 20 lowast119875 lt 005) however at much slower growthrates (31 plusmn 6 120583mh and 21 plusmn 5 120583mh resp) compared togrowth rates prior to cytokines application (TNF120572 79 plusmn8 120583mh 119899 = 22 and IL-1120573 89 plusmn 9 120583mh 119899 = 26) Asexpected application of 10 120583gmLOv had no effect on growthcone advance (before application 108 plusmn 10 120583mh and afterapplication 113 plusmn 6 120583mh) According to reports and ourown findings we tested whether ROS intermediates wereimplicated in cytokine-mediated growth cone paralysis andcollapse Scavenging ROS with 10 120583MMnTBAP or inhibitionof NADPH oxidase (NOX) activities with 2 120583M DPI bothprotected growth cone advance upon exposure to 100 ngmLTNF120572 (69 plusmn 1 119899 = 26 and 71 plusmn 10 119899 = 38 of advancinggrowth cones resp) albeit with decreased growth rates (96 plusmn9 120583mh 17 reduction and 82 plusmn 9 120583mh 26 reductionresp) compared to 10 120583gmL Ov (Figure 2(c)) Growth coneadvance was not affected by an addition of MnTBAP (124 plusmn10 120583mh) or DPI (98plusmn13 120583mh) in the presence of ovalbuminduring the period of observation Growth cone advance inthe presence of 100 ngmL IL-1120573 was also rescued by 10 120583MMnTBAP or 2 120583M DPI (77 plusmn 12 119899 = 38 and 69 plusmn 6119899 = 50 resp) (Figure 2(d)) Exogenous addition of ROS togrowth cones (100 120583M hydrogen peroxide) also reduced thepercentage of advancing growth cones (44 plusmn 2 119899 = 42lowast119875 lt 005)

Both cytokines also elicited a dose-dependent growthcone collapse quantified 30min after addition as shown forTNF120572 (25 ngmL 16 plusmn 3 119899 = 135 50 ngmL 59 plusmn 4lowast119875 lt 005 119899 = 125 100 ngmL 71 plusmn 8 lowast119875 lt 005 119899 = 122)compared to 10 120583gmL Ov (13 plusmn 3 119899 = 69) (Figure 2(e))Moreover scavenging ROS (10 120583M MnTBAP) or inhibitingNOX (2 120583M DPI) (32 plusmn 4 119899 = 100 and 34 plusmn 4 119899 =99 resp lowastlowast119875 lt 005) protected growth cone morphologyfrom the presence of 100 ngmL TNF120572 as opposed to theabsence of pharmacological agents (63 plusmn 7 119899 = 87 lowast119875 lt005) (Figure 2(f)) Similarly MnTBAP (31 plusmn 3 119899 = 156)and DPI (44 plusmn 1 119899 = 186) also negated IL-1120573-inducedgrowth cone collapse (69 plusmn 8 119899 = 196 lowast119875 lt 005) Thepercentage of collapsed growth cone remained unaltered bythe presence of 10 120583MMnTBAP (11 plusmn 6 119899 = 99) yet it didsignificantly increase with 2120583MDPI (27plusmn1 119899 = 63) Takentogether these data revealed that TNF120572 and IL-1120573 causedrapid degeneration of growth conemorphology and completeloss of motility through a redox-dependent mechanism Therapid response of growth cones to cytokines together with theexpression of cytokines receptors on growth cones stronglysuggested a direct mechanism as opposed to indirect effectsthrough neurodegenerative processes originating in neuronalcell bodies

32 A Cytokine-Activated NADPH Oxidase Generates ROS inGrowth Cones and Cell Bodies of SC Neurons To determinethe generation of intracellular ROS in advancing growthcones exposed to cytokines we employed quantitative ratio-metric fluorescence analysis utilizing the oxidation-sensitivefluorescent indicator 2101584071015840-dihydrodichlorofluorescein (DCF10 120583M) and the redox inert fluorescence indicator CalceinBlue (CB 4 120583M) Ratiometric analysis distinguishes changesin DCF fluorescence due to ROS formation from dilu-tionconcentration effects simply due to rapid changes ingrowth cone morphology Bath application of 100 ngmLTNF120572 or 100 ngmL IL-1120573 elicited a rise in the relative maxi-mum DCFCB fluorescence ratio indicative of the formationof ROS (Figure 3) As shown in Figure 3(a) (false coloredDCFCB ratio images) ROS formation was sustained andpreceded loss of morphology of the advancing growth conereflected by atrophy and beading of filopodia condensationof growth cone body and retraction Quantitative analysisdemonstrated a significant and sustained ROS formation inadvancing growth cones upon exposure to TNF120572 (100 ngmL119899 = 14) IL-1120573 (100 ngmL 119899 = 10) or 100 120583M hydrogenperoxide (positive control 119899 = 11) compared to 10 120583gmLovalbumin (negative control 119899 = 22) (Figure 3(b)) Neitherloading with DCF (131 plusmn 18 120583mh 119899 gt 15) nor loadingwith CB (118 plusmn 14 120583mh 119899 gt 15) at the concentrations usedaffected growth cone advance per se compared to unloadedcontrol (113 plusmn 9 120583mh)

To examine NOX-dependent ROS production in neu-ronal growth cones we utilized freshly isolated growthcone particle preparations (GCPs) obtained from chick E10forebrain neurons which are capable of showing complexresponses to extrinsic stimuli [37ndash39] As shown in Fig-ure 4(a) addition of 100 ngmL TNF120572 to laminin-adherent


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Figure 3 TNF120572 stimulates ROS formation in advancing growth cones ROS formation in advancing SC neuron growth cones was revealed byratiometric fluorescence imaging Dissociated E7 chick SC neurons (laminin) were loaded (30min) with the oxidation-sensitive fluorescentindicator 2101584071015840-dihydrodichlorofluorescein (10120583M DCF) and the oxidation-inert fluorescent indicator Calcein blue (4120583M CB) Images ofrandomly selected growth cones were acquired under FITC fluorescence illumination (DCF) DAPI fluorescence illumination (CB) andphase contrast at short time intervals before and after addition of stimuli (40x oil identical parameters) (a) Intracellular ROS formationis represented by a heat spectrum ranging from blue (basal ROS levels) to red (increased ROS levels) Growth cone morphology (arrow)disintegrated over a time period of 11 minutes upon acute exposure to 100 ngmL TNF120572 (phase images upper panel) Ratio imaging (middlepanel) revealed a sharp increase in ROS production (119905 = +3min) shortly after TNF120572 exposure that persisted (119905 = +8min) compared to basallevels (119905 = minus3min) further illustrated by profile images of ratiometric images (lower panel) (Scale bar = 5120583m) Note the loss of filopodiaand lamellipodia and the contraction of the growth cone body (last panel) were preceded by an increase in ROS (b) ROS formation inadvancing SC neuron growth cones was quantified by ratiometric fluorescence imaging Maximum DCF and CB fluorescence intensities pergrowth cone area were determined on a pixel-by-pixel basis and DCFCB ratios calculated for each condition and time point All DCFCBratios were normalized (average DCFCB ratio at 119905 = 0min) and plotted against time Exposure to 100 ngmL TNF120572 (filled circles 119899 = 14)100 ngmL IL-1120573 (filled squares 119899 = 10) or 100120583M hydrogen peroxide (positive control open circles 119899 = 11) at 119905 = 3min (arrow) elicited asignificant ROS formation (stippled line = significance threshold) in advancing growth cones within less than 5min upon addition comparedto 10 120583gmL ovalbumin (negative control open triangles 119899 = 22)


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Figure 4 NADPH oxidase mediates ROS formation in growth cones exposed to TNF120572 (a) Exposure of freshly isolated growth coneparticles (GCPs) to TNF120572 activates MAP kinase GCPs were plated on laminin in Krebs buffer (2 h) washed and incubated with theMAP kinase inhibitors PD98059 (25 120583M 20min) or PBS (equivalent volume) prior to an addition of 100 ngmL TNF120572 Attached GCPswere lysed and equal amounts of total protein were subjected to SDS gel electrophoresis (10) followed by western blotting and detection(chemiluminescence) of MAP kinase (top panel) and phospho-MAP kinase immunoreactivity (bottom panel) Actin served as a loadingcontrol (middle panel) Exposure of GCPs to TNF120572 stimulated MAP kinase activity (increased phospho-MAP kinase) which was negatedby PD98059 Also TNF120572 treatment enhanced expression of MAP kinase (increases in total MAP kinase) a response apparently diminishedby a presence of PD98059 Confocal images acquired (63x oil) of cultured chick forebrain neurons (laminin 24 h) exposed to 100 ngmL ofTNF120572 (TNF120572) revealed phospho-JNK immunoreactivity (white signal) as a discrete punctate pattern in cell bodies and neuronal processin contrast to the homogenous appearance in neuronal soma under control conditions (PBS equivalent volume) Cultures were fixed with4 paraformaldehyde and immunostained against phospho-JNK (b) Neuronal growth cones contain a functional NADPH oxidase activityLysates obtained from SC neuron cultures revealed immunoreactivity against the large membrane subunit NOX2 and the cytosolic subunitsp67phox and p47phox Incubation of chick (E7) forebrain neurons with the NOX activator PMA (400 ngmL) increased plasma membraneassociation of the cytosolic subunit p67phox (lowast) compared to controls (Con) indicative for the formation of the NOX multiprotein complexNative gel electrophoresis combined with in-gel NBT staining of freshly isolated growth cone particles (GCPs) obtained from E10ndash12 chickforebrain demonstrated one NADPH oxidoreductases activity () colocalizing with NOX2 immunoreactivity (c) GCPs were loaded with10120583M DCF (30min) in the presence of 2120583M DPI (NOX inhibitor) 500 120583MNAC (antioxidant) or PBS (mock equivalent volume) prior toaddition of TNF120572 (45min) DCF fluorescence was determined in GCP lysates adjusted to total soluble protein and normalized to control(Con open bar) TNF120572 stimulated a significant dose-dependent ROS formation in GCPs (T

100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)

which was abolished by DPI or NAC Peroxide (H2O2) served as a positive control (d) Plasma membrane association of p67phox normalized

to control conditions was quantified (western blotting) using plasma membrane-enriched fractions Exposure of GCPs to 200 ngmL TNF120572significantly increased the relative plasma membrane association of p67phox (TNF lowast119875 lt 005) compared to control (Con) indicating theformation of a functional NOX complex Preincubation of GCPs withmethyl-120573-cyclodextrin (01) negated plasmamembrane translocationof p67phox in the presence of TNF120572 (mCdx-TNF) All data represent SEM from at least two experiments (triplicate conditions each) withlowast

119875 lt 005


GCPs resulted in MAP kinase and JNK activation whichwas corroborated in cultured forebrain neurons No basalMAP kinase activity (phosphor MAP kinase) was detectablein GCPs under control conditions possibly due a lack ofgrowth factors (serum free plating conditions) Indicative forthe formation of a functional NOX multiprotein complexthe NOX activator PMA (200 ngmL) significantly increasedthe relative plasma membrane association of p67phox in chickforebrain neurons (Figure 4(b)) Moreover native gel elec-trophoresis and NBT staining revealed several NADPH oxi-doreductase activities in GCPs one colocalizing with NOX2immunoreactivity The large membrane subunit NOX2 andthe cytosolic subunits p67phox and p47phox were also detectedin lysates of SC neurons (Figure 4(b)) Addition of TNF120572(100 ngmL) to DCF-loaded GCPs stimulated a significantdose-dependent increase in ROS formation which wasnegated by the NOX inhibitor DPI (5 120583M) or the ROSscavenger NAC (1mM) (Figure 4(c)) The small spatialdimensions of plated GCPs prevented ROS quantificationutilizing microscopic imaging Therefore we resorted toa protocol involving lysis as previously established [14]Moreover plasma membrane association of p67phox in GCPssignificantly increased in response to 100 ngmL TNF120572which was abolished by the lipid raft disrupter methyl-120573-cyclodextrin suggesting that the assembly of NOX occurredpredominantly in lipid rafts (Figure 4(d)) The insignificantincrease of p67phox in plasma membrane fractions frommethyl-120573-cyclodextrin-treated cells could result from unspe-cific adsorption due to the dramatic change in membranelipid composition TNF120572-coated polystyrene beads or bathapplication of 100 ngmL TNF120572 to DCF-loaded SC neuronsstimulated a transient increase in DCF fluorescence intensityin neuronal cell bodies (Figures 5(a) and 5(b)) Cytokine-stimulated ROS formation was dose-dependent with signif-icant stimulation at concentrations higher than 50 ngmLand saturation at 100 ngmL (data not shown) MaximumROS formation (162 plusmn 01 lowast119875 lt 001) in SC neuron cellbodies occurred 53 plusmn 08min following bath application of100 ngmL TNF120572 lasting for 64 plusmn 06min (16 plusmn 2 cells pertime interval) (Figure 5(c)) 100 ngmL IL-1120573 also increasedROS formation (139 plusmn 002 lowast119875 lt 001) within 5 plusmn 07minupon application and lasted for 78 plusmn 14min (119899 = 20plusmn 2 cellsper time interval) Scavenging ROSwith 2mMNACor 10 120583MMnTBAP both suppressed TNF120572-stimulated ROS formation(075 plusmn 011 119899 = 29 and 099 plusmn 005 119899 = 65 resp) tolevels indistinguishable from control (10 120583gmL ovalbumin099 plusmn 005 119899 = 123) in contrast to TNF120572 alone (153 plusmn005 lowast119875 lt 001 119899 = 184) (Figure 5(d)) Inhibiting NOXactivity (5 120583MDPI) also negated ROS formation (103plusmn 007119899 = 70 lowast119875 lt 001) upon exposure to TNF120572 Similar datawere obtained upon acute addition of 100 ngmL IL-1120573 to SCneurons (data not shown) As our positive control 200120583Mhydrogen peroxide greatly increased relative maximum DCFfluorescence intensity (175plusmn002 119899 = 109 lowast119875 lt 001) whichwas abolished in the presence of 10120583M MnTBAP (097 plusmn002 119899 = 109) (Figure 5(d)) Depleting Rac1 activity usingadenoviral-mediated expression of FLAG-tagged dominant-negative Rac1N17 completely suppressed TNF120572 or IL-1120573

-stimulatedROS formation (113plusmn005 119899 = 93 and 084plusmn005119899 = 71 resp) as opposed to lacZ expression (TNF120572 153 plusmn005 lowast119875 lt 005 119899 = 184 and IL-1120573 130 plusmn 005 lowast119875 lt 005119899 = 117 resp) (Figure 6(a)) [31] In contrast expression ofconstitutively active Rac1V12 in SC neurons was sufficient toincrease ROS formation (165 plusmn 004 119899 = 450 lowast119875 lt 005)compared to 200120583M hydrogen peroxide (198 plusmn 016 119899 =60 lowast119875 lt 005) (Figures 6(b) and 6(c)) ROS scavenging with2mMNACor 10 120583MMnTBAPaswell asNOX inhibitionwith10 120583M DPI abolished Rac1V12-stimulated ROS formation(111 plusmn 009 119899 = 62 096 plusmn 009 119899 = 109 and 105 plusmn007 119899 = 138 resp) Similar results were obtained usingpurified recombinant GST chimeras of Rac1V12 and Rac1N17transfected into E7 SC neurons by trituration loading (datanot shown) Expressing Rac1N17 alone (103 plusmn 004 119899 = 247)or lacZ (099 plusmn 003 119899 = 105) our control had no effecton basal levels of ROS (Figure 6(c)) However addition of2mMNAC or 10 120583MDPI to Rac1N17-expressing SC neuronsboth reduced ROS formation significantly below basal levels(076plusmn004 119899 = 94 and 055plusmn002 119899 = 106 resp lowast119875 lt 005)These findings revealed that TNF120572 and IL-1120573 stimulate afunctional assembly of a Rac1-regulated NOX2 complex inthe plasma membrane of neuronal growth cones resulting ina transient increase in ROS formation

33 TNF120572 and IL-1120573 Attenuate Neurite Outgrowth of SCNeurons in a Redox-Dependent Manner Next we examinedwhether the redox-dependent impairment of growth coneadvance is of consequence for neurite outgrowth Bathapplication of 100 ngmL TNF120572 or IL-1120573 attenuated neu-rite outgrowth in SC neuron cultures in a dose-dependentmanner indicated by a shift in the distribution to shorterneurite lengths compared to 10 120583gmL ovalbumin (Ov) ourcontrol (Figures 7(a) and 7(b) Table 1) Scavenging ROS(10 120583MMnTBAP) rescued neurite outgrowth in the presenceof TNF120572 and IL-1120573 whereas the NOX inhibitor (2120583MDPI) provided only partial protection of neurite outgrowth(Figures 7(c) and 7(d)) Notably neurite outgrowth of SCneurons on laminin exhibited inherent redox dependenceeven in the absence of TNF120572 or IL-1120573 (Figures 7(e) and7(f)) In accordance with previous reports increasing con-centrations of the ROS scavenger N-acetyl-L-cysteine (2mMNAC 50 reduction) or 20120583M MnTBAP (10 reduction)inhibited neurite outgrowth compared to control whereas10 120583MMnTBAPwas ineffective [15] However wemeasured asignificant increase in neurite outgrowthwith 5120583MMnTBAP(25 increase)

To assess the role of Rac1 on neurite outgrowth Rac1V12

(constitutively active Rac1) or Rac1N17 (dominant negativeRac1) was introduced as purified recombinant GST chimerainto freshly dissected SC neurons by trituration loading[26] (Figure 8) Depletion of Rac1 activity (Rac1N17-GST) inSC neurons protected neurite outgrowth upon addition of100 ngmL TNF120572 (NL

50= 81 plusmn 5 120583m 119899 = 85 lowastlowast119875 lt 005)

or 100 ngmL IL-1120573 (NL50= 79 plusmn 6 120583m 119899 = 68 lowastlowast119875 lt 005)

compared to GST-loaded SC neurons (NL50= 64 plusmn 3 120583m 119899 =

75 and NL50= 69 plusmn 2 120583m 119899 = 51) respectively (Figure 8(a))

However introduction of Rac1N17-GST diminished neurite


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Figure 5 Cytokines elicit ROS formation in cell bodies of SC neurons Dissociated E7 SC neurons were loaded with 10120583M DCF (30min)allowed to recover for 15min and then incubated (45min) with NAC MnTBAP DPI or PBS (equal volume) prior to cytokines exposureImages were acquired at identical parameters (20x) (a) DCF-loaded SC neurons exhibited a distinct neuronal morphology (arrowheads)extending one long and several shorter neuronal processes (left panel phase contrast image) Note cultures are virtually free of nonneuronalcells Under control conditions SC neuron cell bodies displayed basal levels of ROS formation (middle panel arrowheads) Degeneratingcells (star) exhibit very high fluorescence intensity in the absence of any stimuli Addition of 100 ngmL TNF120572 stimulated a robust increase inintracellular ROS (right panel arrowheads 8min after addition) Fluorescence images were false colored (heat spectrum) with fluorescenceintensities increasing from blue (basal ROS levels) to red (high ROS levels) (b) TNF120572-coated polystyrene beads increased DCF fluorescenceintensity in SC neurons indicative of ROS formation Although qualitative in nature SC neurons with multiple bead contacts (arrow head)as opposed to single bead contact (asterisk) displayed more intense DCF fluorescence (c) Maximum DCF fluorescence intensities perneuronal cell body were determined on a pixel-by-pixel basis after background subtraction over time and all values normalized to the averagemaximum DCF fluorescence intensity under control conditions at t = 0min (relative maximum DCF fluorescence intensity) SC neuronsresponded with a transient increase in ROS formation in cell bodies (16 plusmn 2 cells per time interval) upon exposure to 100 ngmL TNF120572(25)min (threshold of statistical significance lowast119875 lt 001 shown as open triangles) (d) 100 ngmL TNF120572 stimulated a significant increase inROS formation ( lowast119875 lt 001) which was negated by the presence of ROS scavengers (TNF120572-NAC 2mM or TNF120572-MnTBAP 10120583M resp)or NOX inhibitor (TNF120572-DPI 5 120583M) to levels indistinguishable from 10 120583gmL ovalbumin (Ov) our control As a control for DCF loading200 120583Mhydrogen peroxide (H

2O2) greatly increased relativemaximumDCF fluorescence ( lowast119875 lt 001) which was suppressed in the presence

of 10 120583MMnTBAP (H2O2-Mn) All measurements were obtained from at least three independent dissections (duplicate cultures each)


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Figure 6 TNF120572 and IL-1120573 stimulate a Rac1-mediated ROS formation in SC neurons Dissociated SC neurons grown on laminin were infectedwith recombinant replication deficient adenovirus (200 moi infection at the time of plating) carrying FLAG-tagged constitutively activeRac1V12 FLAG-tagged dominant negative Rac1N17 or lacZ Three days after infection SC neurons were loaded with 10120583M DCF (30min)incubated with NACMnTBAP or DPI and exposed to cytokines (100 ngmL) Random images were acquired (20x) under FITC fluorescenceillumination relativemaximumDCF fluorescence intensity was quantified and all values normalized to lacZ-expressing SC neurons exposedto 10 120583gmL Ovalbumin (lacZ) our control (a) Expression of RacN17 completely abolished ROS formation in SC neurons upon exposure to100 ngmL TNF120572 or IL-1120573 (TNF120572-N17 and IL-1120573-N17 resp) compared to lacZ-expressing SC neurons (TNF120572 and IL-1120573 resp lowast119875 lt 001)without altering basal levels of ROS formation (N17) (b) Expression of Rac1V12 was sufficient to induce ROS formation in SC neurons whichwas negated by 2mM NAC 10 120583MMnTBAP or 10120583MDPI Peroxide (200120583MolL H

2O2) served as a DCF loading control (c) DCF-loaded

SC neurons expressing RacV12 (V12) revealed increases in ROS formation both in cell bodies (arrowhead) as well as in neuronal growthcones (arrow) and distal neurites (left panel) In contrast SC neurons expressing RacN17 (N17) exhibited basal DCF fluorescence intensitycompared to lacZ (Con) expressing SC neurons (right panel) Note the intense DCF fluorescence in a degenerating cell (asterisk) A heatspectrum ranging from blue (basal ROS levels) to red (increased ROS levels) indicates ROS production (scale bar = 40120583m)

outgrowth even in the absence of adverse stimuli (NL50

=84 plusmn 4 120583m 119899 = 132 lowast119875 lt 005) compared to GST-loadedSC neurons (NL

50= 91 plusmn 3 120583m 119899 = 99) in accordance with

previous findings [26 40] Constitutive activation of Rac1(Rac1V12-GST) in SC neurons also resulted in reduction ofneurite length (NL

50= 83 plusmn 5 120583m 119899 = 195 lowast119875 lt 005)

compared to GST-loaded SC neurons (NL50

= 98 plusmn 5 120583m119899 = 112) (Figure 8(b)) Interestingly addition of 5 120583MMnTBAP restored neurite outgrowth of RacV12-GST-loadedSC neurons (NL

50= 108 plusmn 6 119899 = 136 lowastlowast119875 lt 005) to

levels indistinguishable from GST-loaded SC neurons (NL50

= 103plusmn7 120583m 119899 = 103 lowastlowast119875 lt 005)This finding implied a rolefor Rac1 as a regulator of TNF120572 or IL-1120573-stimulated oxidativestress These findings provided evidence that attenuation ofneurite outgrowth in the presence of TNF120572 or IL-1120573 requireda persistent Rac1-regulated formation of ROS Moreoverneurite outgrowth on laminin exhibited an innate redox-sensitivity

34 Cytokines Elicit a Redox-Dependent Reorganization ofActin Filaments in Neuronal Growth Cones Earlier studies inneuroblastoma cells revealed oxidative damage to actin due toTNF120572 exposure [14] We found that growth cones respondedrapidly to cytokine exposure with changes in morphologyprior to collapse suggesting reorganization of actin filamentsWe quantified the percentage of growth cones respondingto cytokines with at least one distinct actin filament-richlamellipodial structure as a function of both time and phar-macological treatment (Figure 9) Growth cones exhibited arapid yet transient increase in actin filament-rich structureswithin 15min upon exposure to 100 ngmL TNF120572 (65 plusmn 5119899 = 180 lowast119875 lt 005) or 100 ngmL IL-1120573 (84 plusmn 5 119899 =180 lowast119875 lt 005) compared to control (23 plusmn 5 119899 = 180lowast119875 lt 005) and persisted up to 30min (TNF120572 67 plusmn 5119899 = 180 lowast119875 lt 005 and IL-1120573 58 plusmn 5 119899 = 180 lowast119875 lt 005)(Figures 9(a) and 9(d)) However the percentage of growthcones with actin filament-rich structures greatly subsided


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Figure 7 TNF120572 and IL-1120573 impede neurite outgrowth in a redox-sensitive manner Dissociated SC neurons were grown on laminin past theonset of neurite initiation and then incubated with 10120583MMnTBAP 2 120583MDPI or PBS (10120583L) for 1 h prior to bath application of cytokines orovalbumin (6 to 8 h) followed by fixation (2 glutaraldehyde) The longest neurite per neuron was measured of randomly selected neuronsand the percentage of neuronswith a given neurite lengthwas plotted against neurite length (a and b) Persistent presence of TNF120572 (a) or IL-1120573(b) significantly reduced neurite outgrowth in a dose-dependent manner (40 ngmL open triangles and 100 ngmL open squares) indicatedby the shift of the neurite length distribution to shorter neurites compared to control (10 120583gmL ovalbumin filled circles) (c and d) Despite acontinuous presence of 100 ngmL TNF120572 ((c) open squares) or 100 ngmL IL-1120573 ((d) open squares) scavenging ROS with 10120583MMnTBAP(open triangles) rescued neurite outgrowth compared to controls (10120583gmL ovalbumin filled circles) whereas inhibiting NOX activity with2 120583MDPI (open diamonds) was only partially protective (Neurite numbermeasured ge 60 for each condition two experiments and duplicatecultures) (e) In the presence of 20 120583M MnTBAP (open diamonds) neurite outgrowth was significantly decreased compared to control(PBS filled circles) However concentrations of 10120583M MnTBAP (open triangles) did not significantly alter neurite outgrowth and 5120583MMnTBAP (open circles) on the contrary causes a significant increase in neurite length (Neurite number measured gt 75 for each conditiontwo experiments and duplicate cultures) (f) Lastly an overabundance of the radical scavenger NAC (2mM open squares) dramaticallyreduced neurite outgrowth compared to control (PBS filled circles) indicated by the shift towards shorter neurites


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rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0

V12GST V12 MnTBAPMnTBAP

lowast


Neu

rite l

engt

h (120583

m)

(b)

Figure 8 The small GTPase Rac1 mediates redox-dependent neurite outgrowth Freshly dissected SC neurons were trituration-loaded withpurified recombinant Rac1V12-GST Rac1N17-GST or GST only (7mgmL each) and grown (laminin) after the onset of neurite initiationCultures were supplemented with 100 ngmL TNF120572 100 ngmL IL-1120573 or 10120583gmL ovalbumin (8 h) and the average neurite length of thelongest neurite reached by 50 of SC neurons (NL

50) for each condition quantified (119899 gt 50) (a) Introduction of Rac1N17-GST (dominant

negativemutation black bars) significantly protected neurite outgrowth in the presence of TNF120572 (TNF120572-N17 lowastlowast119875 lt 005) or IL-1120573 (IL-1120573-N17lowastlowast

119875 lt 005) compared to SC neurons loaded with GST which exhibited a great reduction in neurite length in the presence of cytokines (greybars lowast119875 gt 005) compared to control (ovalbumin open bar) Not unexpected Rac1N17-GST even in the absence of cytokines reduced neuriteoutgrowth (N17 lowast119875 lt 005) compared to control (b) Similarly introduction of Rac1V12-GST (constitutively active mutation) significantlyreduced neurite lengths (V12 lowast119875 lt 005) compared to GST-loaded SC neurons (GST) Notably the presence of 5 120583M MnTBAP increasedneurite outgrowth of Rac1V12-GST-loaded SC neurons (V12-MnTBAP lowastlowast119875 lt 005) to levels indistinguishable from neurite outgrowth ofGST-loaded SC neurons treated with 5120583MMnTBAP (MnTBAP lowastlowast119875 lt 005) compared to control All data were obtained from at least threedifferent dissections (duplicate cultures each) with error bars representing

Table 1 Redox-sensitive inhibition of neurite outgrowth inresponse to TNF120572 and IL-1120573

Condition NL50plusmn SEM 119899

Control (10mgmL Ov) 147 plusmn 16 120583m 104TNF120572 40 ngmL 118 plusmn 6 120583mlowast 74TNF120572 100 ngmL 86 plusmn 13 120583mlowast 173TNF120572 100 ngmL + 10mMMnTBAP 145 plusmn 13 120583m 62TNF120572 100 ngmL + 2mMDPI 113 plusmn 12 120583mlowastlowast 56Control (10mgmL Ov) 128 plusmn 7 120583m 61IL-1120573 40 ngmL 109 plusmn 4 120583mlowast 92IL-1120573 100 ngmL 80 plusmn 3 120583mlowast 107IL-1120573 100 ngmL + 10mMMnTBAP 112 plusmn 9 120583mlowastlowast 113IL-1120573 100 ngmL + 2mMDPI 87 plusmn 8 120583mlowast 78PBS 128 plusmn 12 120583m 156MnTABP 5mM 161 plusmn 10 120583mlowast 82MnTABP 10mM 133 plusmn 7 120583m 75MnTABP 20mM 115 plusmn 7 120583mlowast 56PBS 179 plusmn 7 120583m 103NAC 2mM 89 plusmn 7 120583mlowast 102SC neuron cultures were incubated with pharmacological inhibitors (MnT-BAP DPI) or PBS prior to addition of cytokines for 6ndash8 hours After fixationthe average neurite length of the longest neurite per neuron reached by 50of neurons (NL50) was quantified for each condition

lowastSignificant differencesfrom controls lowastlowastSignificant difference from cytokine only at 119875 lt 005 byone-way ANOVA and Dunnettrsquos 119905-test

45min after addition (TNF120572 40 plusmn 4 119899 = 180 lowastlowast119875 lt 005and IL-1120573 37 plusmn 5 119899 = 180 lowastlowast119875 lt 005) compared to theinitial response Inhibiting NOX activity (5120583M DPI) greatly

diminished the percentage of responding growth cones uponbath application of cytokines (DPI + TNF120572 32 plusmn 6 119899 = 180and DPI + IL-1120573 49 plusmn 6 119899 = 180 lowastlowast119875 lt 005) comparedto absence of DPI (TNF120572 60 plusmn 6 119899 = 180 lowast119875 lt 005 andIL-1120573 76 plusmn 6 119899 = 180 lowast119875 lt 005) measured 15min afteraddition of cytokines (Figures 9(b) and 9(e)) The percentageof responding growth cones was not altered by DPI treatmentalone (18 plusmn 6 119899 = 180) compared to control (23 plusmn 6119899 = 180) Similarly scavenging ROS with 20120583M MnTBAPalso negated the formation of actin filament-rich structuresin growth cones exposed to cytokines (Mn + TNF120572 24 plusmn8 119899 = 180 and Mn + IL-1120573 18 plusmn 8 119899 = 180) to levelsindistinguishable from control (18 plusmn 4 119899 = 180) comparedto cytokines alone (TNF120572 66 plusmn 8 119899 = 180 lowast119875 lt 005 andIL-1120573 84plusmn 8 119899 = 180 lowast119875 lt 005) (Figures 9(c) and 9(f)) Apresence of MnTBAP did not alter the percentage of growthcones with actin filament-rich structures (18 plusmn 3 119899 = 180)Taken together these data suggested that TNF120572 and IL-1120573stimulated a redox-dependent yet transient reorganization ofactin filaments in neuronal growth cones prior to collapse ofmorphology

4 Discussion

We demonstrated that long-term exposure of SC neuronsto the proinflammatory cytokines TNF120572 and IL-1120573 pro-voked the loss of growth cone motility and the subsequentdegeneration of growth cone morphology (collapse) Thesechanges in growth cone advance and behavior translatedinto the impairment of neurite outgrowth and disruption ofprocess architecture of SC neurons which was rescued either


lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)

Con TNF TNF TNF IL IL IL

Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILILDPIDPI DPI

Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn

Figure 9 Cytokines elicit redox-dependent reorganization of actin filaments in neuronal growth cones SC neurons grown on laminin (2days) were incubated with 40 120583MMnTBAP 5120583MDPI or left untreated prior to addition of TNF120572 or IL-1120573 (200 ngmL) for increasing timeperiods (15min 30min and 45min) Cultures were fixed permeabilized (Triton-X-100) and stained with rhodamine phalloidin to revealfilamentous actin Random images were acquired (40x confocal microscope) growth cones were scored for the presence of at least onedistinct actin filament-rich structure and all values normalized to control conditions ( responding growth cones) (a) Both TNF120572 and IL-1120573 significantly increased the percentage of growth cones with actin-filament rich structures ( lowast119875 lt 001) at 15min and 30min after exposurefollowed by a significant decrease at 45min after exposure ( lowastlowast119875 lt 001 compared to 119905 = 15min) as opposed to control (b) Inhibition ofNOX activity with 2 120583M DPI largely negated the formation of actin-filament rich structures upon exposure to TNF120572 (TNF-DPI) or IL-1120573(IL-DPI lowastlowast119875 lt 001) as opposed to TNF120572 only (TNF120572 lowast119875 lt 001) or IL-1120573 only (IL lowast119875 lt 001) respectively Large actin filament-richstructures were not affected by DPI in the absence of cytokines (DPI) compared to control (Con) (c) Scavenging ROS with 10120583MMnTBAPabolished the formation of actin filament-rich structures in response to TNF120572 (TNF120572-Mn) or IL-1120573 (IL-Mn) when compared to TNF120572 only(TNF lowast119875 lt 001) or IL-1120573 only (IL lowast119875 lt 001) The degree of actin filament-rich structures in the presence of MnTBAP alone (Mn) wasindistinguishable from control (Con) lowastSignificant difference from control and lowastlowastsignificant difference from respective cytokine treatmentat 119875 lt 005 by Kruskal Wallis test and Dunnettrsquos 119905-test (dndashf) Cultured SC neurons were treated with pharmacological inhibitors prior tobath application of cytokines fixed with 4 paraformaldehyde and stained for actin filaments with rhodamine phalloidin (red signal) (d)Representative images of SC neurons with increasing exposure time to TNF120572 Actin filament-rich structures in growth cones (lamellipodia)are indicated with arrowheads (e) Incubation with DPI largely abolished the formation of actin filament-rich structures in SC neuronscultures (f) Scavenging ROS with MnTBAP effectively negated appearance of actin filament-rich structures in growth cones of SC neurons


Extrinsic stimuli (TNF120572)

NOX activity

N17Rac1 V12Rac1

Low HighIntracellular [ROS]

Gro

wth

cone

mot

ility

Figure 10 A Rac1-dependent redox rheostat regulates growth conemotility in response to extrinsic stimuli Exposure of neuronalgrowth cones either to TNF120572 or to IL-1120573 rapidly stimulates ROSgeneration through NOX activity under the regulation of the smallGTPase Rac1 and concomitant paralysis and degeneration of mor-phology of growth conesMoreover growth conemotility is not onlysensitive to the overabundance of ROS either via NOX activationor via constitutive Rac1 activity (Rac1V12) but is also impaired bya substantial depletion of ROS (antioxidants) or inhibition of Rac1activity (Rac1N17) Consequently productive growth cone motilityrequires an optimal concentration of intracellular ROS generated bya Rac1-regulated NOX activity It is plausible that this Rac1NOX-redox rheostat is responsive to many extrinsic stimuli includingcytokines growth factors hormones and cell adhesion moleculesfor which redox signaling mechanism has been demonstrated

by scavenging ROS inhibiting NOX activity or depletingRac1 activity despite a presence of TNF120572 or IL-1120573 Bothcytokines stimulated the formation of ROS intermediates anda transient phase of actin filament organization in advancinggrowth cones Importantly ROS intermediates and actinfilament reorganization preceded paralysis of growth conemotility and collapse of morphology implying a causativeaction of this signaling mechanism Taking into accountthat exhaustive ROS scavenging (excess of antioxidants) alsoimpaired neurite outgrowth it is feasible that productivegrowth cone motility could demand an optimal level of ROSintermediates We propose that a redox rheostat under theregulation of the Rac1 shapes growth conemotility and henceneurite outgrowth in response to many extrinsic stimuliother than cytokines (Figure 10)

Prolonged bath application of TNF120572 or IL-1120573 (6ndash8 h)to dissociated SC neurons or SC explants stunted neuriteoutgrowth in a dose-dependent manner (Figure 7 Table 1) inaccordance with previous findings in hippocampal neuronsor neuroblastoma cells [8 9] In lieu of the well-documentedapoptotic potency of TNF120572 and IL-1120573 it was imperativeto determine whether inhibition of neurite outgrowth hadits origin in neuronal cell bodies (apoptosis necrosis) ordirectly in the distal compartment of growth cones Foremostcytokines were applied to SC neurons after the onset of

neurite outgrowth with the majority of processes longer thantwo cell diameters Since the number of neurites per neuron(a measure of neurite initiation) remained unaltered andno measureable neuronal cell death occurred over the timeperiod of cytokine exposure (6ndash8 h) as determined by alivedead fluorescence assay (calcein greenpropidium iodidecounterstaining) neither a decrease in neurite initiationnor a decrease in neuronal cell death could account forthe observed reduction in neurite outgrowth Significantneuronal cell death (gt15) was however apparent 24 hoursafter cytokine addition and increased to over 40 after 2days Evidence for direct action of cytokines on growthcone motility and morphology was obtained by live-videophase microscopy (Figures 1 and 2) Acute exposure to TNF120572or IL-1120573 (bath applied) impaired growth cone motility ina dose-dependent manner within 10 to 15 minutes uponapplication Initial paralysis of growth cone advance wasfollowed by degeneration ofmorphology and subsequent col-lapse Restricting cytokine exposure exclusively to advancinggrowth cones in our bead assay corroborated these findingsIn addition bead assays revealed a transient increase inlamellipodia-like structures or in the vicinity of neuron-bead contacts which were nonproductive for growth coneadvance This transient increase in actin filaments (Figure 9)was reminiscent of our findings in SH-SY5Y neuroblastomayet in the case of primary neurons it revealed a physiologicalconsequence [14] Direct effects of cytokines on growth coneswere further supported by the expression of IL-R1 (IL-1120573receptor) and TNF-R1 (high affinity p55 TNF120572 receptor) onthe entire neuronal surface including cell bodies and growthcones (Figure 1) Together these findings provided strongevidence that TNF120572 and IL-1120573 directly impaired growth conemotility and morphology as the underlying cause for theattenuation of neurite outgrowth over longer time periods ofexposure

Initial evidence for a role of ROS intermediates wassuggested by studies demonstrating that antioxidants (ROSscavenging) or DPI (inhibiting NOX-like activities) largelyrescued neurite outgrowth growth cone motility growthcone morphology and lastly actin filament organization inthe presence of cytokines Interestingly neurite outgrowth ofSC neurons on laminin exhibited innate redox dependenceWhereas moderate MnTBAP concentrations (5 120583M) signif-icantly increased neurite outgrowth concentration higherthan 10 120583M stunted neurite outgrowth corroborating studiesinAplysia neurons [15] It is plausible thatmoderateMnTBAPconcentration (5120583M) scavenges excessive amounts of super-oxide derived frommitochondrial respiration and as byprod-uct of several enzymatic reactions Under basal culture con-ditions this excessive superoxide might compromise optimalgrowth cone motility (actin dynamics tubulin dynamics)and hence the observed enhancement of neurite outgrowthby moderate concentrations of scavenger [41ndash43] In con-trast higher MnTBAP concentrations could impair vitalredox-dependent mechanism necessary for proper neuriteoutgrowth [44] Using the oxidation-sensitive fluorescenceindicator DCF we demonstrated substantial yet transient


generation of ROS (likely superoxide) in growth cones andcell bodies of SC neurons upon exposure to TNF120572 and IL-1120573 (Figures 3 and 5) Similar results were obtained using thesuperoxide-specific fluorescence indicator dihydroethidiumyet investigations suffered from considerable neurotoxicity ofdihydroethidium (data not shown) Previous studies in chickcortical neurons and human SH-SY5Y neuroblastoma cellsdemonstrated superoxide production in response to TNF120572utilizing dihydroethidium oxidation or a SOD-inhibitablecytochrome C oxidation as demonstrated [14 45] A sig-nificant contribution from nonneuronal cells (microglia)was unlikely since (i) SC neuron cultures contained lessthan 3 nonneuronal cells (ii) analyzed cells all exhibiteddistinct neuronal characteristics (ie round cell body oneprocess longer than 2 cell diameters) and (iii) microgliainvade and populate the developing CNS at late embryonicor even postnatal stages [46] Ratiometric imaging demon-strated significant ROS production in advancing growthcones within 3 to 5min upon exposure to inflammatorystress as opposed to changes in motility and morphologyobserved 10 to 15min following exposure Several findingsstrongly suggested a NOX2-like activity as the source ofROS in SC neurons in particular superoxide [14 47] SCneurons exhibited immunoreactivity against NOX2 and thecytosolic subunits p47phox and p67phox A presence of otherNOX isoforms can however not be excluded yet antibodyquality against NOX1 or NOX3 was insufficient to producea conclusive finding TNF120572 stimulated a dose-dependentincrease in ROS intermediates in freshly isolated growthcone particle preparations in conjunction with a presenceof NADPH oxidoreductases activity The phorbol ester PMAas well as TNF120572 induced translocation of the cytosolicsubunit p67phox to plasma membranes Parallel studies onthe translocation of the cytosolic subunit p47phox to plasmamembranes were hampered by the considerable variabilityof immunoreactivity Expression of NOX-like activities wasreported in both primary PNS and CNS neurons [48ndash51]Lastly the antioxidant MnTBAP inhibits the TNF120572 or IL-1120573-stimulated ROS formation Also pharmacological inhibitionwith DPI was effective in blocking TNF120572 or IL-1120573-mediatedresponses of SCneurons includingROS formation andpartialrescue of growth motility neurite outgrowth growth conecollapse and actin filament reorganization (Figures 2 4 5 7and 9) Notably all these experiments revealed considerabletoxicity of DPI and thus depending on the duration of DPIapplication put restrictions on the usable concentrations ofDPI Mechanistically DPI blocks single electron transportreactions such as NOX-mediated generation of superoxideNevertheless numerous other enzymatic reactions are alsocompromised foremost electron transport of complex I inmitochondria which could account for the observed toxicityLastly depleting Rac1 activity negated cytokine-mediatedROS formation in SC neurons whereas Rac1 overexpressionalone was sufficient to increase ROS formation The smallGTPase Rac1 emerged as a pivotal regulator of cytokine-stimulated ROS formation in SC neurons in accordance withprevious reports [22 52 53] In our study depleting Rac1activity abolished ROS formation in the presence of TNF120572

or IL-1120573 yet without affecting basal ROS levels (Figures 6(b)ndash6(d)) In contrast overexpression of constitutively active Rac1alone was sufficient to stimulated ROS generation in SCneurons and also reduced neurite outgrowth which wasnot further exacerbated upon addition of TNF120572 or IL-1120573(data not shown) Effects of Rac1 depletion or overexpressionwere addressed by trituration loading of purified recombi-nant Rac1-GST mutant chimeras since adenoviral-mediatedexpression of Rac1 mutants required a 3-day expressionperiod incompatible with neurite outgrowth analysis [2631] Not unexpectedly introduction of Rac1N17 or Rac1V12resulted in a measurable reduction in neurite outgrowthon laminin even in the absence of cytokines [11 26 5455] In support of our findings numerous reports linkRac1 as a regulator of ROS intermediates in cellular signaltransduction Neurite extension in PC12 cells upon NGFstimulation encompasses increases in Rac1 activity andH

2O2

formation [56] A Rac1 mutant lacking residues 124ndash135of the insert region blocking ROS generation disruptedmembrane ruffling and mitogenesis in fibroblasts and causeda downregulation of RhoA [44 57ndash59] With respect toactin cytoskeleton dynamics TNF120572 and IL-1120573 induced atransient phase of actin filament reorganization (increasesin actin filament density) in neuronal growth cones whichwas however nonproductive for motility Interestingly actinfilament reorganization was not repeatable with anotheraddition of cytokines even after wash-out and extensiverecover time suggesting permanent or persistent damageto actin filament reorganization by cytokines Irreversibleoxidative damage (carbonylation) to actin was detectable inneuroblastoma cells exposed to TNF120572 [14] These findingsprovided evidence that NOX activation in neuronal growthcones serves as source of ROS intermediates in response tocytokines and that ROS intermediates are likely causative forcytokine-mediated degeneration of neuronal growth cones

Inflammatory and oxidative stress is a hallmark ofneurodegeneration in most chronic acute and even somepsychiatric CNS pathologies [3 60 61]The proinflammatorycytokines TNF120572 and IL-1120573 are important to orchestrateinflammatory and oxidative stress in the disease and agingCNS although pleiotropic actions in the adult and developingCNS are known hence establishing a complex and delicatebalance between neuroprotection and neurotoxicity Mem-bers of the NoxDuox family have emerged as [46 62] keysources of oxidative stress in aging and the progression ofmany pathologies beyond the CNS and have been recognizedas key therapeutic targets [63ndash66] Our findings providesupport for a role of NOX activity in primary neurons asa principal source of oxidative stress triggered by TNF120572 orIL-1120573 [16] Overabundance of ROS intermediates is likely todisrupt proper actin filament dynamics which is vital forthe plasticity and morphology of distal neuronal processesincluding neurite outgrowth sprouting and spine dynamicsStrategies to block NOX activities could thus proof beneficialto blunt inflammatory stress in the diseased and aging CNSand to halt cognitive decline [66 67]


Abbreviations

DCF 2101584071015840-dichlorofluoresceinDMEM Dulbeccorsquos Modified Eagle MediumDPI Diphenylene iodoniumFBS Fetal bovine serumGST Glutathione-S-transferaseIL-1120573 Interleukin-1 betaMnTBAP Manganese(III) tetrakis(4-benzoic acid)

porphyrin chlorideNAC N-acetyl-L-cysteineROS Reactive oxygen speciesSC Spinal cordTBS Tris-buffered salineTNF120572 Tumor necrosis factor alpha

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

I would like to extend my appreciation to Drs RichardBridges and Mike Kavanaugh (University of Montana) andDr Paul Letourneau (University of Minnesota Minneapolis)for many helpful discussions that were vital in shapingthis research Thanks to Shellie Stewart-Smeets for hercontribution on actin filament experiments and DianneCabrie for technical assistance I am indebted to Dr MarkWright (University of Alaska Fairbanks) for his support withnative gel electrophoresis and Dr Kelly Auer (Universityof Alaska Fairbanks) for her support with MAP kinaseanalysis Support for this work was provided by the Christo-pherReeve Paralysis Foundation (KA1-0004-2)NIHNINDSgrant 1U54NS41069 andCOBRETheUniversity ofMontana

References

[1] S Amor F Puentes D Baker and P van der Valk ldquoInflamma-tion in neurodegenerative diseasesrdquo Immunology vol 129 no 2pp 154ndash169 2010

[2] M Cacquevel N Lebeurrier S Cheenne and D VivienldquoCytokines in neuroinflammation and Alzheimers diseaserdquoCurrent Drug Targets vol 5 no 6 pp 529ndash534 2004

[3] S-M Lucas N J Rothwell and R M Gibson ldquoThe role ofinflammation in CNS injury and diseaserdquo British Journal ofPharmacology vol 147 supplement 1 pp S232ndashS240 2006

[4] M K McCoy and M G Tansey ldquoTNF signaling inhibition inthe CNS implications for normal brain function and neurode-generative diseaserdquo Journal of Neuroinflammation vol 5 article45 2008

[5] R E Mrak and W S T Griffin ldquoGlia and their cytokines inprogression of neurodegenerationrdquo Neurobiology of Aging vol26 no 3 pp 349ndash354 2005

[6] B Viviani S Bartesaghi E Corsini C L Galli and MMarinovich ldquoCytokines role in neurodegenerative eventsrdquo Tox-icology Letters vol 149 no 1ndash3 pp 85ndash89 2004

[7] S J Mathew D Haubert M Kronke and M Leptin ldquoLookingbeyond death a morphogenetic role for the TNF signalling

pathwayrdquo Journal of Cell Science vol 122 no 12 pp 1939ndash19462009

[8] G Munch J Gasic-Milenkovic S Dukic-Stefanovic et alldquoMicroglial activation induces cell death inhibits neurite out-growth and causes neurite retraction of differentiated neurob-lastoma cellsrdquo Experimental Brain Research vol 150 no 1 pp1ndash8 2003

[9] H Neumann R Schweigreiter T Yamashita K RosenkranzH Wekerle and Y-A Barde ldquoTumor necrosis factor inhibitsneurite outgrowth and branching of hippocampal neurons by aRho-dependent mechanismrdquo The Journal of Neuroscience vol22 no 3 pp 854ndash862 2002

[10] R A Gungabissoon and J R Bamburg ldquoRegulation of growthcone actin dynamics by ADFcofilinrdquo Journal of Histochemistryand Cytochemistry vol 51 no 4 pp 411ndash420 2003

[11] L Luo ldquoRho GTPases in neuronal morphogenesisrdquo NatureReviews Neuroscience vol 1 no 3 pp 173ndash180 2000

[12] L Moldovan K Irani N I Moldovan T Finkel and P JGoldschmidt-Clermont ldquoThe actin cytoskeleton reorganiza-tion induced by Rac1 requires the production of superoxiderdquoAntioxidants and Redox Signaling vol 1 no 1 pp 29ndash43 1999

[13] V J Thannickal and B L Fanburg ldquoReactive oxygen speciesin cell signalingrdquo American Journal of Physiology Lung CellularandMolecular Physiology vol 279 no 6 pp L1005ndashL1028 2000

[14] B M Barth S Stewart-Smeets and T B Kuhn ldquoProinflamma-tory cytokines provoke oxidative damage to actin in neuronalcells mediated by Rac1 and NADPH oxidaserdquo Molecular andCellular Neuroscience vol 41 no 2 pp 274ndash285 2009

[15] V Munnamalai and D M Suter ldquoReactive oxygen speciesregulate F-actin dynamics in neuronal growth cones andneuriteoutgrowthrdquo Journal of Neurochemistry vol 108 no 3 pp 644ndash661 2009

[16] S Chakraborty D K Kaushik M Gupta and A BasuldquoInflammasome signaling at the heart of central nervous systempathologyrdquo Journal of Neuroscience Research vol 88 no 8 pp1615ndash1631 2010

[17] M J Morgan Y-S Kim and Z Liu ldquoLipid rafts and oxidativestress-induced cell deathrdquo Antioxidants and Redox Signalingvol 9 no 9 pp 1471ndash1483 2007

[18] B T Kawahara M T Quinn and J D Lambeth ldquoMolecularevolution of the reactive oxygen-generating NADPH oxidase(NoxDuox) family of enzymesrdquoBMCEvolutionary Biology vol7 article 109 2007

[19] J D Lambeth ldquoNOX enzymes and the biology of reactiveoxygenrdquo Nature Reviews Immunology vol 4 no 3 pp 181ndash1892004

[20] Y Groemping and K Rittinger ldquoActivation and assembly of theNADPH oxidase a structural perspectiverdquo Biochemical Journalvol 386 no 3 pp 401ndash416 2005

[21] A Leonardi H Ellinger-Ziegelbauer G Franzoso K Brownand U Siebenlist ldquoPhysical and functional interaction of fil-amin (actin-binding protein-280) and tumor necrosis factorreceptor-associated factor 2rdquo The Journal of Biological Chem-istry vol 275 no 1 pp 271ndash278 2000

[22] M Peppelenbosch E Boone G E Jones et al ldquoMultiple signaltransduction pathways regulate TNF-induced actin reorganiza-tion inmacrophages inhibition of Cdc42-mediated filopodiumformation by TNFrdquo Journal of Immunology vol 162 no 2 pp837ndash845 1999

[23] R Singh B Wang A Shirvaikar et al ldquoThe IL-1 receptor andrho directly associate to drive cell activation in inflammationrdquo


Journal of Clinical Investigation vol 103 no 11 pp 1561ndash15701999

[24] T B Kuhn ldquoGrowing and working with spinal motor neuronsrdquoMethods in Cell Biology vol 71 pp 67ndash87 2003

[25] J E Bottenstein and G H Sato ldquoGrowth of a rat neuroblastomacell line in serum free supplemented mediumrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 76 no 1 pp 514ndash517 1979

[26] T B Kuhn M D Brown and J R Bamburg ldquoRac1-dependentactin filament organization in growth cones is necessary forb1-integrin-mediated advance but not for growth on poly-D-lysinerdquo Journal of Neurobiology vol 37 pp 524ndash540 1998

[27] R Owen Lockerbie V E Miller and K H Pfenninger ldquoReg-ulated plasmalemmal expansion in nerve growth conesrdquo TheJournal of Cell Biology vol 112 no 6 pp 1215ndash1227 1991

[28] S Chang F G Rathjen and J A Raper ldquoExtension of neuriteson axons is impaired by antibodies against specific neural cellsurface glycoproteinsrdquo The Journal of Cell Biology vol 104 no2 pp 355ndash362 1987

[29] E T Stoeckli T B Kuhn C O Duc M A Ruegg and PSonderegger ldquoThe axonally secreted protein axonin-1 is a potentsubstratum for neurite growthrdquoThe Journal of Cell Biology vol112 no 3 pp 449ndash455 1991

[30] A Hall ldquoRho GTpases and the actin cytoskeletonrdquo Science vol279 no 5350 pp 509ndash514 1998

[31] T B Kuhn M D Brown C L Wilcox J A Raper and J RBamburg ldquoMyelin and collapsin-1 inducemotor neuron growthcone collapse through different pathways inhibition of collapseby opposing mutants of rac1rdquo The Journal of Neuroscience vol19 no 6 pp 1965ndash1975 1999

[32] M Brown B Cornejo T Kuhn and J Bamburg ldquoCdc42stimulates neurite outgrowth and formation of growth conefilopdia and lamellipodiardquo Journal of Neurobiology vol 43 pp352ndash364 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] H Towbin T Staehelin and J Gordon ldquoElectrophoretic trans-fer of proteins frompolyacrylamide gels to nitrocellulose sheetsprocedure and some applicationsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 76 no9 pp 4350ndash4354 1979

[35] T B Kuhn M F Schmidt and S B Kater ldquoLaminin andfibronectin guideposts signal sustained but opposite effects topassing growth conesrdquoNeuron vol 14 no 2 pp 275ndash285 1995

[36] M A Wride and E J Sanders ldquoExpression of tumor necrosisfactor-120572 (TNF120572)-cross-reactive proteins during early chickembryo developmentrdquoDevelopmental Dynamics vol 198 no 3pp 225ndash239 1993

[37] C Cypher and P C Letourneau ldquoIdentification of cytoskeletalfocal adhesion and cell adhesion proteins in growth coneparticles isolated from developing chick brainrdquo Journal ofNeuroscience Research vol 30 no 1 pp 259ndash265 1991

[38] S Helmke and K H Pfenninger ldquoGrowth cone enrichment andcytoskeletal association of non-receptor tyrosine kinasesrdquo CellMotility and the Cytoskeleton vol 30 no 3 pp 194ndash207 1995

[39] P Negre-Aminou and K H Pfenninger ldquoArachidonic acidturnover and phospholipase A2 activity in neuronal growthconesrdquo Journal of Neurochemistry vol 60 no 3 pp 1126ndash11361993

[40] Z Jin and SM Strittmatter ldquoRac1mediates collapsin-1-inducedgrowth cone collapserdquoThe Journal ofNeuroscience vol 17 no 16pp 6256ndash6263 1997

[41] C E Cooper R P Patel P S Brookes and VM Darley-UsmarldquoNanotransducers in cellular redox signaling modification ofthiols by reactive oxygen and nitrogen speciesrdquo Trends inBiochemical Sciences vol 27 no 10 pp 489ndash492 2002

[42] I Dalle-Donne R Rossi A Milzani P di Simplicio and RColombo ldquoThe actin cytoskeleton response to oxidants fromsmall heat shock protein phosphorylation to changes in theredox state of actin itselfrdquo Free Radical Biology and Medicinevol 31 no 12 pp 1624ndash1632 2001

[43] I Lassing F Schmitzberger M Bjornstedt et al ldquoMolecularand structural basis for redox regulation of 120573-actinrdquo Journal ofMolecular Biology vol 370 no 2 pp 331ndash348 2007

[44] A S Nimnual L J Taylor and D Bar-Sagi ldquoRedox-dependentdownregulation of Rho by Racrdquo Nature Cell Biology vol 5 no3 pp 236ndash241 2003

[45] S J Gustafson K L Dunlap C M McGill and T B Kuhn ldquoAnonpolar blueberry fraction blunts NADPH oxidase activationin neuronal cells exposed to tumor necrosis factor-120572rdquoOxidativeMedicine and Cellular Longevity vol 2012 Article ID 768101 12pages 2012

[46] J E Merrill ldquoTumor necrosis factor alpha interleukin 1 andrelated cytokines in brain development normal and pathologi-calrdquo Developmental Neuroscience vol 14 no 1 pp 1ndash10 1992

[47] P L Hordijk ldquoRegulation of NADPH oxidases the role of RacproteinsrdquoCirculation Research vol 98 no 4 pp 453ndash462 2006

[48] K M Noh and J Y Koh ldquoInduction and activation by zinc ofNADPH oxidase in cultured cortical neurons and astrocytesrdquoThe Journal of Neuroscience vol 20 no 23 article RC111 2000

[49] P Vallet Y Charnay K Steger et al ldquoNeuronal expressionof the NADPH oxidase NOX4 and its regulation in mouseexperimental brain ischemiardquo Neuroscience vol 132 no 2 pp233ndash238 2005

[50] M Dvorakova B Hohler E Richter J B Burritt and WKummer ldquoRat sensory neurons contain cytochrome b558 largesubunit immunoreactivityrdquo NeuroReport vol 10 no 12 pp2615ndash2617 1999

[51] S P Tammariello M T Quinn and S Estus ldquoNADPH oxidasecontributes directly to oxidative stress and apoptosis in nervegrowth factor-deprived sympathetic neuronsrdquo The Journal ofNeuroscience vol 20 no 1 article RC53 2000

[52] A Puls A G Eliopoulos C D Nobes T Bridges L S Youngand A Hall ldquoActivation of the small GTPase Cdc42 by theinflammatory cytokines TNF120572 and IL-1 and by the Epstein-Barr virus transforming protein LMP1rdquo Journal of Cell Sciencevol 112 no 17 pp 2983ndash2992 1999

[53] B Wojciak-Stothard A Entwistle R Garg and A RidleyldquoRegulation of TNF alpha-induced reorganization of the actincytoskeleton and cell-cell junctions by rho rac and cdc42 inhuman endothelial cellsrdquo Journal of Cell Physiology vol 176 pp150ndash165 1998

[54] AHall andCDNobes ldquoRhoGTPasesmolecular switches thatcontrol the organization and dynamics of the actin cytoskele-tonrdquo Philosophical Transactions of the Royal Society B BiologicalSciences vol 355 no 1399 pp 965ndash970 2000

[55] G Meyer and E L Feldman ldquoSignaling mechanisms thatregulate actin-based motility processes in the nervous systemrdquoJournal of Neurochemistry vol 83 no 3 pp 490ndash503 2002


[56] K Suzukawa K Miura J Mitsushita et al ldquoNerve growthfactor-induced neuronal differentiation requires generation ofrac1-regulated reactive oxygen speciesrdquoThe Journal of BiologicalChemistry vol 275 no 18 pp 13175ndash13178 2000

[57] J L Freeman A Abo and J D Lambeth ldquoRac ldquoinsert regionrdquois a novel effector region that is implicated in the activationof NADPH oxidase but not PAK65rdquo The Journal of BiologicalChemistry vol 271 no 33 pp 19794ndash19801 1996

[58] T Joneson and D Bar-Sagi ldquoA Rac1 effector site controllingmitogenesis through superoxide productionrdquo The Journal ofBiological Chemistry vol 273 no 29 pp 17991ndash17994 1998

[59] A E Karnoub C J Der and S L Campbell ldquoThe insertregion of Rac1 is essential formembrane ruffling but not cellulartransformationrdquo Molecular and Cellular Biology vol 21 no 8pp 2847ndash2857 2001

[60] S M Allan and N J Rothwell ldquoCytokines and acute neurode-generationrdquoNature ReviewsNeuroscience vol 2 no 10 pp 734ndash744 2001

[61] Y Sei L Vitkovic and M M Yokoyama ldquoCytokines in thecentral nervous system regulatory roles in neuronal functioncell death and repairrdquo Neuroimmunomodulation vol 2 no 3pp 121ndash133 1995

[62] L Vitkovic J Bockaert and C Jacque ldquoldquoInflammatoryrdquocytokines neuromodulators in normal brainrdquo Journal of Neu-rochemistry vol 74 no 2 pp 457ndash471 2000

[63] K Bedard and K-H Krause ldquoThe NOX family of ROS-generatingNADPHoxidases physiology and pathophysiologyrdquoPhysiological Reviews vol 87 no 1 pp 245ndash313 2007

[64] T Kawahara and J D Lambeth ldquoMolecular evolution of Phox-related regulatory subunits forNADPHoxidase enzymesrdquoBMCEvolutionary Biology vol 7 article 178 2007

[65] K-H Krause D Lambeth and M Kronke ldquoNOX enzymes asdrug targetsrdquo Cellular and Molecular Life Sciences vol 69 no14 pp 2279ndash2282 2012

[66] J D Lambeth K-H Krause and R A Clark ldquoNOXenzymes as novel targets for drug developmentrdquo Seminars inImmunopathology vol 30 no 3 pp 339ndash363 2008

[67] H-M Gao H Zhou and J-S Hong ldquoNADPH oxidases noveltherapeutic targets for neurodegenerative diseasesrdquo Trends inPharmacological Sciences vol 33 no 6 pp 295ndash303 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


progression in response to cytokines growth factors andhormones [18 19] Functional NOX requires an intricateassembly between two membrane proteins (a NOX isoformgp22phox) and several cytosolic factors (p47phox p40phoxp67phox) under the regulation of the small GTPases Rac1or Rac2 [20] Rho GTPases harbor a dual role in cytokinesignaling as regulators of both NOX assembly and thereorganization of actin filament structures [21ndash23] In lightof these reports we examined whether ROS intermediatesgenerated by NOX activities in neuronal growth cones areimplicated inmediating the neurotoxic effects of TNF120572 or IL-1120573 on neurite outgrowth Amechanistic understanding of thedetrimental consequences of TNF120572 and IL-1120573 on neuronalconnectivity in the CNS neurons is vital to intervene withprogressive neurodegeneration in the aging or diseased CNS

2 Materials and Methods

21 Reagents Unless state otherwise all reagents were pur-chased from Sigma (St Louis MO) Dulbeccorsquos Modi-fied Eagle Medium (high glucose DMEM) Leibovitzrsquos L-15 and isopropyl 120573-D-thiogalactopyranoside were obtainedfrom Invitrogen Corporation (Carlsbad CA) Character-ized fetal bovine serum (FBS) was from Hyclone (LoganUT) and it was heat-inactivated according to the manu-facturer Laminin was from Roche Diagnostics Corporation(Indianapolis IN) The redox sensitive fluorescent indi-cators carboxymethyl-2101584071015840-dihydrodichlorofluorescein diac-etate 2101584071015840-dihydrodichlorofluorescein diacetate and Cal-cein Blue were obtained from Molecular Probes (EugeneOR) Human recombinant tumor necrosis factor 120572 (TNF120572)and interleukin-1120573 (IL-1120573) Diphenylene iodonium (DPI)N-acetyl-L-cysteine (NAC) and Manganese(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) were purchasedfrom Calbiochem (San Diego CA) Polyclonal rabbit anti-TNF receptor I polyclonal rabbit anti-TNF receptor IIpolyclonal rabbit anti IL-1 receptor type I polyclonal goatanti-NOX2 polyclonal goat anti-p47phox including respectiveblocking peptides were all from St Cruz Biotechnology(St Cruz CA) A monoclonal mouse anti-p4442 MAPKa monoclonal mouse anti-phospho p4442 MAPK and amonoclonal mouse anti-phospho JNKwere fromCell Signal-ing Technologies (Danvers MA) Fluorescein or rhodamine-labeled goat anti-rabbit and goat anti-mouse IgG (resp)were from Chemicon (Temecula CA) whereas rhodamine-conjugated phalloidin was obtained from Cytoskeleton Inc(Denver CO) The polyclonal rabbit anti-p67phox was pur-chased from Abcam (Cambridge MA)

22 Spinal Cord Neuron Cultures Dissociated low-densitycultures of spinal cord (SC) neurons were established asdescribed [24] Briefly spinal cord tissue was dissected from7 day-old chick embryos (E7) and a single cell suspensionobtained after enzymatic digestion (05mgmL trypsin 2mMEDTA 10min 37∘C) followed by trituration After preplating(1 h 37∘C 5 CO

2atmosphere high glucose DMEM pH

7310 FBS) nonadherent cells (predominantly SC neurons)were collected (3min 200timesgmax) and resuspended in SC

medium (high glucose DMEM pH 73 10 FBS 12 nMfluorodeoxyuridine 30 nM uridine and 1 N3 nutrientsupplement) [25] SC explants were prepared by pushingfreshly dissected E7 spinal cord tissue through a wire-mesh(50ndash75120583m) Dissociated SC neurons (75000 cellsmL) or SCexplants (2 per culture) were plated onto glass cover slips (22times 22mm2 number 1 Carolina Biological Supply CompanyBurlington NC) mounted over a 15 cm hole drilled intothe bottom of a 35mm culture dish Glass cover slips weretreated with poly-D-lysine (100 120583gmL borate buffer pH84 30min RT) prior to coating with laminin (5120583gcm230min RT) These SC neuron cultures contained less than5 nonneuronal cells with no immunoreactivity againstmicroglia markers

23 Growth Cone Particle Preparations Preparations ofhighly enriched growth cone particles were obtained fromfreshly dissected whole chick embryo brains (E10-12) [26 27]After homogenizing (Dounce homogenizer ice-cold 5mMHEPES pH 73 1mM MgCl

2 032M Sucrose 6ndash8 volumes

per wet weight) homogenates were centrifuged (15min1600timesgmax 4

∘C) Resulting supernatants were overlaid onto5mM HEPES pH 73 1mM MgCl

2 and 075M sucrose and

then centrifuged (150000timesgmax 1 h 4∘C) and material at

the interface was collected After dilution (6-7 times in lowsucrose buffer) the suspension was overlaid ontoMaxiDense(4-5 sample volumes per volumeMaxidense) and centrifugedat 40000timesgmax (1 h 4

∘C) and growth cone particles (GCPs)were collected on top of the MaxiDense cushion GCPswere resuspended in Krebrsquos buffer (145mMNaCl 5mM KCl12mM NaH

2PO4timesH2O 12mMMgCl

2 and 5mMHEPES

pH 73) and protein concentration determined (BCA assayThermo Scientific Rockford IL USA)

24 Measurement of Neurite Lengths Dissociated SC neu-rons were grown after the onset of neurite outgrowth indi-cated by the majority of cells extending at least one processlonger than 2 cell body diameters Cultures were incubated(1 h) with pharmacological agents followed by bath appli-cation (6ndash8 h) of 100 ngmL TNF120572 100 ngmL IL-1120573 or10 120583gmL ovalbumin After fixation (2 glutaraldehyde) thelength of the longest neurite per neuron was measured ofat least 50 randomly selected SC neurons only consideringprocesses adhering to the following criteria (i) emergingfrom an isolated cell body (ii) longer than two cell diametersand (iii) no contact to other neuronal processes or cell bodiesThe distribution of neurite length in a population of SCneurons was obtained by plotting the percentage of neuronswith neurites longer than a given length against neurite length[28] As a characteristic for neurite outgrowth under a givencondition the neurite length reached by 50 of neurite-bearing SC neurons (NL

50) was calculated as criteria for

statistical significance [29] Purified recombinant Rac1V12-GST Rac1N17-GST or GST were introduced into freshlydissociated SC neurons (7ndash10mgmL 500000 cells 200120583L50mM Tris-Cl pH 75 100mM NaCl and 5mM MgCl

2)

by trituration loading at the time of plating [26] Followingtrituration SC neurons were immediately transferred in SCmedium and plated as described above For each condition







2




2





119905=

Int119865DCF119905

Int119865 with 119905 = time


0) followed


119899av1198770













3 Results



-1120573

Ov

TNF120572

(a)

0min

0min

0min

9min 16min 27min

85min 12min 16min

3min 7min 11min

IL-1120573

Ov

TNF120572

(b)

90

50

(kD

a)

IL-1RTNF-R2TNF-R1

(c)





10 20Time (min)

60

40

20

0

minus20

ControlTNF 50ngmL

TNF 100ngmLH2O2 100 120583m

Gro

wth

cone

adva

nce (120583

m)

(a)

10 20 30Time (min)

40

20

0

minus20

minus40

Gro

wth

cone

adva

nce (120583

m)


(b)

10 20 30Time (min)

60

40

20

0

minus20Gro

wth

cone

adva

nce (120583

m)

ControlTNF

TNF + MnTNF + DPI

(c)

10 20 30Time (min)

0

40

20

minus20

minus40Gro

wth

cone

adva

nce (120583

m)

ControlIL-1120573 IL-1120573 + Mn

IL-1120573 + DPI

(d)

60

80

40

20

0

lowast

lowast

lowast

Col

lapse

d gr

owth

cone

s (

)

25

TNF50

TNF100

TNFCon

(e)

lowast



lowast

60

80

40

20

0Col

lapse

d gr

owth

cone

s (

)

TNFMn TNFMn

Con DPI TNFDPI DPI

ILMnILIL

(f)













(a)

Rel

max

DCF

CB

fluor

esce

nce r

atio


Time (min)0 2 4 6 8 10 12

H2O2

5

4

3

2

1

0

(b)



Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







2




2





119905=

Int119865DCF119905

Int119865 with 119905 = time


0) followed


119899av1198770













3 Results



-1120573

Ov

TNF120572

(a)

0min

0min

0min

9min 16min 27min

85min 12min 16min

3min 7min 11min

IL-1120573

Ov

TNF120572

(b)

90

50

(kD

a)

IL-1RTNF-R2TNF-R1

(c)





10 20Time (min)

60

40

20

0

minus20

ControlTNF 50ngmL

TNF 100ngmLH2O2 100 120583m

Gro

wth

cone

adva

nce (120583

m)

(a)

10 20 30Time (min)

40

20

0

minus20

minus40

Gro

wth

cone

adva

nce (120583

m)


(b)

10 20 30Time (min)

60

40

20

0

minus20Gro

wth

cone

adva

nce (120583

m)

ControlTNF

TNF + MnTNF + DPI

(c)

10 20 30Time (min)

0

40

20

minus20

minus40Gro

wth

cone

adva

nce (120583

m)

ControlIL-1120573 IL-1120573 + Mn

IL-1120573 + DPI

(d)

60

80

40

20

0

lowast

lowast

lowast

Col

lapse

d gr

owth

cone

s (

)

25

TNF50

TNF100

TNFCon

(e)

lowast



lowast

60

80

40

20

0Col

lapse

d gr

owth

cone

s (

)

TNFMn TNFMn

Con DPI TNFDPI DPI

ILMnILIL

(f)













(a)

Rel

max

DCF

CB

fluor

esce

nce r

atio


Time (min)0 2 4 6 8 10 12

H2O2

5

4

3

2

1

0

(b)



Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












3 Results



-1120573

Ov

TNF120572

(a)

0min

0min

0min

9min 16min 27min

85min 12min 16min

3min 7min 11min

IL-1120573

Ov

TNF120572

(b)

90

50

(kD

a)

IL-1RTNF-R2TNF-R1

(c)





10 20Time (min)

60

40

20

0

minus20

ControlTNF 50ngmL

TNF 100ngmLH2O2 100 120583m

Gro

wth

cone

adva

nce (120583

m)

(a)

10 20 30Time (min)

40

20

0

minus20

minus40

Gro

wth

cone

adva

nce (120583

m)


(b)

10 20 30Time (min)

60

40

20

0

minus20Gro

wth

cone

adva

nce (120583

m)

ControlTNF

TNF + MnTNF + DPI

(c)

10 20 30Time (min)

0

40

20

minus20

minus40Gro

wth

cone

adva

nce (120583

m)

ControlIL-1120573 IL-1120573 + Mn

IL-1120573 + DPI

(d)

60

80

40

20

0

lowast

lowast

lowast

Col

lapse

d gr

owth

cone

s (

)

25

TNF50

TNF100

TNFCon

(e)

lowast



lowast

60

80

40

20

0Col

lapse

d gr

owth

cone

s (

)

TNFMn TNFMn

Con DPI TNFDPI DPI

ILMnILIL

(f)













(a)

Rel

max

DCF

CB

fluor

esce

nce r

atio


Time (min)0 2 4 6 8 10 12

H2O2

5

4

3

2

1

0

(b)



Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


-1120573

Ov

TNF120572

(a)

0min

0min

0min

9min 16min 27min

85min 12min 16min

3min 7min 11min

IL-1120573

Ov

TNF120572

(b)

90

50

(kD

a)

IL-1RTNF-R2TNF-R1

(c)





10 20Time (min)

60

40

20

0

minus20

ControlTNF 50ngmL

TNF 100ngmLH2O2 100 120583m

Gro

wth

cone

adva

nce (120583

m)

(a)

10 20 30Time (min)

40

20

0

minus20

minus40

Gro

wth

cone

adva

nce (120583

m)


(b)

10 20 30Time (min)

60

40

20

0

minus20Gro

wth

cone

adva

nce (120583

m)

ControlTNF

TNF + MnTNF + DPI

(c)

10 20 30Time (min)

0

40

20

minus20

minus40Gro

wth

cone

adva

nce (120583

m)

ControlIL-1120573 IL-1120573 + Mn

IL-1120573 + DPI

(d)

60

80

40

20

0

lowast

lowast

lowast

Col

lapse

d gr

owth

cone

s (

)

25

TNF50

TNF100

TNFCon

(e)

lowast



lowast

60

80

40

20

0Col

lapse

d gr

owth

cone

s (

)

TNFMn TNFMn

Con DPI TNFDPI DPI

ILMnILIL

(f)













(a)

Rel

max

DCF

CB

fluor

esce

nce r

atio


Time (min)0 2 4 6 8 10 12

H2O2

5

4

3

2

1

0

(b)



Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10 20Time (min)

60

40

20

0

minus20

ControlTNF 50ngmL

TNF 100ngmLH2O2 100 120583m

Gro

wth

cone

adva

nce (120583

m)

(a)

10 20 30Time (min)

40

20

0

minus20

minus40

Gro

wth

cone

adva

nce (120583

m)


(b)

10 20 30Time (min)

60

40

20

0

minus20Gro

wth

cone

adva

nce (120583

m)

ControlTNF

TNF + MnTNF + DPI

(c)

10 20 30Time (min)

0

40

20

minus20

minus40Gro

wth

cone

adva

nce (120583

m)

ControlIL-1120573 IL-1120573 + Mn

IL-1120573 + DPI

(d)

60

80

40

20

0

lowast

lowast

lowast

Col

lapse

d gr

owth

cone

s (

)

25

TNF50

TNF100

TNFCon

(e)

lowast



lowast

60

80

40

20

0Col

lapse

d gr

owth

cone

s (

)

TNFMn TNFMn

Con DPI TNFDPI DPI

ILMnILIL

(f)













(a)

Rel

max

DCF

CB

fluor

esce

nce r

atio


Time (min)0 2 4 6 8 10 12

H2O2

5

4

3

2

1

0

(b)



Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









(a)

Rel

max

DCF

CB

fluor

esce

nce r

atio


Time (min)0 2 4 6 8 10 12

H2O2

5

4

3

2

1

0

(b)



Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

Rel

max

DCF

CB

fluor

esce

nce r

atio


Time (min)0 2 4 6 8 10 12

H2O2

5

4

3

2

1

0

(b)



Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Con TNFTNFPD PD

Anti-MAPK

Anti-actin

Anti-phosphoMAPK

(a)

NOX2 p67 p47

CON PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

NADH NADPH

NOX2 p67 p47

PMA

MW

(kD

)

130

94

49

lowast

GCPAnti

NOX2

(b)

lowast

lowast

lowast


7

6

5

4

3

2

1

0

Rel

DCF

fluo

resc

ence

per120583

g to

tal p

rote

in

H2O2

(c)

3

2

1

0

Rel

mem

bran

e p67

phox

mCdx mCdx

lowast

TNFTNF

Con

(d)


100= 100 ngmL T

200= 200 ngmL lowast119875 lt 005)



119875 lt 005













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b)

lowast

lowast

lowast

Time (min)

20

16

12

00 2 4 6 8 10 12

Rel

max

DCF

fluo

resc

ence

lowastP lt 005

(c)

lowast

lowast


OvDPI

H2O2 H2O2

2

1

0

Rel

max

DCF

fluo

resc

ence

(d)





lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast

lowast

2

1

0


IL-1 IL-1

Rel

max

DCF

fluo

resc

ence

(a)

lowastlowast


H2O2H2O2

2

15

1

05

0

Rel

max

DCF

fluo

resc

ence

(b)

(c)

















100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



100

80

60

40

20

00 100 200 300

ConTNF 40

TNF 100

neur

ite le

ngthgtX

()

Neu

rons

with

(a)


IL-1 40IL-1 100Con

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(b)


ConTNF

TNF + MnTNF + DPI

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(c)


ConIL-1

IL-1 + Mn

100

80

60

40

20

0

+ DPIIL-1

neur

ite le

ngthgtX

()

Neu

rons

with

(d)


ConMn 5

Mn 10

Mn 20

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(e)


ConNAC

100

80

60

40

20

0

neur

ite le

ngthgtX

()

Neu

rons

with

(f)



100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100

80

60

40

20

0

Con TNF N17N17N17

TNF IL-1 IL-1

lowast

lowastlowast


Neu

rite l

engt

h (120583

m)

(a)

100

80

60

40

20

0


lowast


Neu

rite l

engt

h (120583

m)

(b)











4 Discussion



lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast lowast

lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

(a)

(b)

(c)

(d)

(e)

(f)

15998400 30998400 45998400 15998400 30998400 45998400


lowast

lowast

100

80

60

40

20

0Resp

ondi

ng g

row

th co

nes (

)


Treatments

lowastlowast

lowast

lowast

100

80

60

40

20

0

Resp

ondi

ng g

row

th co

nes (

)

Con TNFTNF ILIL

Treatments

MnMn Mn




NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



NOX activity

N17Rac1 V12Rac1


Gro

wth

cone

mot

ility









2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2O2




Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abbreviations





Acknowledgments


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Oxygen Radicals Elicit Paralysis and