Oxygen Radicals Elicit Paralysis and Collapse of Spinal Cord Neuron Growth Cones upon Exposure to Proinflammatory Cytokines

A persistent inflammatory and oxidative stress is a hallmark of most chronic CNS pathologies (Alzheimer's (ALS)) as well as the aging CNS orchestrated by the proinflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β). Loss of the integrity and plasticity of neuronal morphology and connectivity comprises an early step in neuronal degeneration and ultimate decline of cognitive function. We examined in vitro whether TNFα or IL-1β impaired morphology and motility of growth cones in spinal cord neuron cultures. TNFα and IL-1β paralyzed growth cone motility and induced growth cone collapse in 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. TNFα and IL-1β provoked rapid, NOX-mediated generation of ROS in advancing growth cones, which preceded paralysis of motility and collapse of morphology. Increases in ROS intermediates were accompanied by an aberrant, nonproductive reorganization of actin filaments. These findings suggest that NADPH oxidase serves as a pivotal source of oxidative stress in neurons and together with disruption of actin filament reorganization contributes to the progressive degeneration of neuronal morphology in the diseased or aging CNS.


Introduction
A spreading inflammatory reaction accompanied by oxidative stress is prevalent in most chronic CNS diseases and acute CNS trauma as well as in the aging CNS [1][2][3]. The proinflammatory cytokines TNF (tumor necrosis factor ) and IL-1 (interleukin-1 ) exert pleiotropic functions both in CNS development and CNS pathogenesis [4][5][6]. Persistent high-level expression of TNF and IL-1 is important to the progressive degeneration of neuronal connectivity and loss of neuronal plasticity ultimately leading to cognitive decline. The role of TNF and IL-1 as inducers of apoptotic events is well documented, whereas the recognition of their morphogenetic function is more recent [7]. TNF and IL-1 released from microglia cells inhibited neurite outgrowth, reduced branching, and caused neurite retraction in cultures of Neuro2A cells or primary hippocampal neurons [8,9]. The intricate pattern of neuronal connectivity innate to cognitive function rests as much on the integrity and stability of the axonal and dendritic architecture as on the plasticity of motile structures to maintain, form, or regenerate connections, which is intimately linked to the dynamic reorganization of the actin cytoskeleton [10,11]. ROS intermediates greatly affect both the dynamics and organization of actin filaments during oxidative stress of physiological redox signaling [12,13]. TNF paralyzed actin filament reorganization in neuroblastoma cells due to oxidative damage, whereas physiological levels of ROS intermediates seem to be necessary for proper growth cone motility [14,15]. TNF and IL-1 potently stimulate NADPH oxidase (NOX) activities in neurons and glia cells often localized in coalescing lipid rafts [16,17]. The members of the NADPH oxidase (NOX) family (NOX1-5, DUOX1/2), defined by the large membrane flavoprotein gp91 phox of the phagocyte NAPDH oxidase (NOX2), are ubiquitously expressed in all cell types and have emerged as principal ROS sources both in cellular signaling and disease 2 BioMed Research International progression in response to cytokines, growth factors, and hormones [18,19]. Functional NOX requires an intricate assembly between two membrane proteins (a NOX isoform, gp22 phox ) and several cytosolic factors (p47 phox , p40 phox , p67 phox ) under the regulation of the small GTPases Rac1 or Rac2 [20]. Rho GTPases harbor a dual role in cytokine signaling as regulators of both NOX assembly and the reorganization of actin filament structures [21][22][23]. In light of these reports, we examined whether ROS intermediates generated by NOX activities in neuronal growth cones are implicated in mediating the neurotoxic effects of TNF or IL-1 on neurite outgrowth. A mechanistic understanding of the detrimental consequences of TNF and IL-1 on neuronal connectivity in the CNS neurons is vital to intervene with progressive neurodegeneration in the aging or diseased CNS.

Measurement of Neurite
Lengths. Dissociated SC neurons were grown after the onset of neurite outgrowth indicated by the majority of cells extending at least one process longer than 2 cell body diameters. Cultures were incubated (1 h) with pharmacological agents, followed by bath application (6-8 h) of 100 ng/mL TNF , 100 ng/mL IL-1 , or 10 g/mL ovalbumin. After fixation (2% glutaraldehyde), the length of the longest neurite per neuron was measured of at least 50 randomly selected SC neurons only considering processes adhering to the following criteria: (i) emerging from an isolated cell body, (ii) longer than two cell diameters, and (iii) no contact to other neuronal processes or cell bodies. The distribution of neurite length in a population of SC neurons was obtained by plotting the percentage of neurons with neurites longer than a given length against neurite length [28]. As a characteristic for neurite outgrowth under a given condition, the neurite length reached by 50% of neuritebearing SC neurons (NL 50 ) was calculated as criteria for statistical significance [29]. Purified, recombinant Rac1 V12 -GST, Rac1 N17 -GST, or GST were introduced into freshly dissociated SC neurons (7-10 mg/mL, 500,000 cells, 200 L 50 mM Tris-Cl pH 7.5, 100 mM NaCl, and 5 mM MgCl 2 ) by trituration loading at the time of plating [26]. Following trituration, SC neurons were immediately transferred in SC medium and plated as described above. For each condition, measurements were performed in duplicate cultures from two independent dissections.

Measurement of Growth Cone
Advance. Dissociated SC neurons (12 to 16 h in culture) were transferred into observation medium (Leibovitz'L15 without phenol red, pH 7.4, 5 mg/mL ovalbumin, 1% N3), overlaid with light mineral oil to avoid evaporation, and placed onto the microscope stage equilibrated at 37 ± 0.2 ∘ C (Nikon TE2000 U). After 15-minute recovery, images of advancing growth cones (20x, phase contrast) were acquired at 3-minute time intervals for a 30 min time period (Coolsnapfx, Photometrics, Tuscon, AZ). Cytokines (100 ng/mL TNF , 100 ng/mL IL-1 ) or 10 g/mL Ovalbumin were bath-applied at = 8 min. The extension of the growth cone/neurite boundary ( m) was measured as a function of time (min) (Metamorph Software, Meridian Instrument Co, Kent, WA). At least 20 growth cones were monitored for each condition in duplicate cultures from at least two different dissections.

Adenoviral Expression of Rac1 Mutants in SC Neurons.
Recombinant, replication deficient adenovirus carrying genes for constitutively active Rac1 (Rac1 V12 with N-terminal FLAG tag), dominant negative Rac1 (Rac1 N17 with N-terminal FLAG tag), or lacZ were expressed in E7 chick SC neurons as described previously [31]. At the time of plating, dissociated SC neurons were infected with recombinant adenovirus at 200 moi (multiplicity of infection) in 300 L SC medium. Cultures were replenished with 200 L fresh SC medium after 12 h and grown for additional 48 h. At three days after infection, yields of neuronal infections generally exceeded 70% [31,32]. Amplification of viral stocks was performed in 293 HEK cells and titers greater than 5 × 10 8 plaque forming units per mL were routinely obtained. Viral stocks were stored at -80 ∘ C.

Quantitative ROS Imaging in SC Neuron Cell Bodies.
Dissociated SC neurons were loaded with 10 M 2 ,7dihydrodichlorofluorescein diacetate (DCF) or 5 M dihyroethidium (DMEM/10% FBS) for 30 min (37 ∘ C, 5% CO 2 atmosphere), washed, and allowed to recover (15 min, DMEM/10% FBS). Cultures were switched to observation medium, overlaid with light mineral oil, transferred to the heated microscope stage (Zeiss Axiovert125S), and allowed to adapt for 15 min. Images of dissociated SC neurons (random fields of view) were acquired under phase contrast (20x Plan-Apo objective) and FITC illumination (Ex 465-495 nm, DM 505, Em 515-555) using a Peltier cooled CCD camera (Sensys, Photometrics, Tuscon, AZ) after the recovery phase (defined as basal condition), after pharmacological treatments (30 min), and after cytokine exposure (100 ng/mL TNF or 100 ng/mL IL-1 ). Hydrogen peroxide was added to all cultures following treatments to ensure proper loading. As our criteria for SC neurons, we analyzed only cells displaying a large round cell body and one neuronal process at least longer than three cell diameters. Maximum DCF fluorescence intensity per neuronal cell body was determined on a pixelby-pixel basis following background subtraction (average background of all images at = 0 min for each condition) followed by erosion (2 pixels) and overlay with the original image (Zeiss imaging analysis software KS 300). All values were normalized to the average DCF fluorescence intensity in control (initial conditions). No morphological changes of neuronal cell bodies were detectable in our assay conditions indicated by constant cell body areas. ROS measurements were performed in duplicate cultures obtained from 3 to 5 independent dissections. At least 50 SC neurons were measured in duplicate cultures of three independent dissections. Images of advancing growth cones were acquired under phase contrast (40x Plan-Apo), FITC illumination (DCF fluorescence), and DAPI illumination (CB fluorescence) (Coolsnapfx) before (pre-stimulus images at = 0, 2, 4 min) and after (after stimulus images, 2 min time intervals, 16 min time period) bath application of 100 ng/mL TNF , 100 ng/mL IL-1 , or 10 g/mL ovalbumin. As our positive loading control, following the observation period, all growth cones were exposed to 100 M hydrogen peroxide. For image analysis, DCF and CB fluorescence intensities ( DCF and CB ) were integrated (pixel-by-pixel basis) over the growth cone area for each growth cone observed at each time point ( Int DCF, and Int CalcB, with = time interval) after background subtraction ( = 0 image) and the ratio of integrated DCF and CB fluorescence intensities for each growth cone at each time point was calculated ( = Int DCF, / Int with = time interval). Next, the average ratio of integrated DCF and CB fluorescence intensities of all growth cones observed at = 0 min (pre-stimulus) was calculated ( av 0 ) followed by normalization of all ratios for at = 0 min ( / av 0 , = growth cone 1, 2, to for each condition). At least 15 growth cones were analyzed per condition from three different dissections to provide statistical significance ( * < 0.05).

ROS Quantification in Growth Cone Particle Preparations.
Freshly prepared GCPs (100 g in Kreb's buffer) were loaded with 20 M DCF (30 min, 4 ∘ C) in the presence of pharmacological reagents (10 M DPI, 500 M NAC), washed (14,000 ×g max ), allowed to recover (10 min, 4 ∘ C) with pharmacological reagents present, and then exposed to 100 or 200 ng/mL TNF (45 min, 4 ∘ C). For lysis, GCPs were resuspended in 2% SDS, 10 mM Tris-Cl pH 7.5, 10 mM NaF, 5 mM dithiothreitol, and 2 mM EGTA; sonicated; and cleared by centrifugation (14,000 ×g max , 5 min). Total DCF fluorescence intensity was measured (100 L aliquots, black 96 well plates) using a Beckman Coulter Multimode DTX 880 microplate reader (495 nm excitation filter, 525 emission filter). All data were adjusted to total soluble protein concentration (BCA assay) and normalized to control condition to account for unspecific fluorescence and/or autofluorescence artifact. For all conditions, measurements were obtained in duplicates from three different GCP preparations.

Actin Filament Quantification.
To visualize actin filaments, SC neurons were fixed and permeabilized (0.5% Triton X-100, 15 min). After rinsing (0.1% Triton X-100 in TBS), cultures were incubated (20 min) with rhodamineconjugated phalloidin (1 : 10 in 1% Triton X-100 in TBS, Cytoskeleton Inc., Denver, CO), washed, and stored in 60% glycerol (4 ∘ C) until inspection. Images were acquired (40x oil, Plan Fluor) using a Zeiss LSM 510 confocal microscope equipped with a HeNe laser and an Argon laser. For each condition, 60 randomly selected SC neuron growth cones were scored for the presence of at least one large lamellipodialike structure (three dissections, = 180) and the percentage of responding growth cones determined.

Statistical Analysis.
One-way ANOVA analysis and a Kruskal-Wallis test were employed for comparisons among multiple conditions. Dunnett's -test was used when comparing the means of multiple conditions with a single control.
All statistical values are given as SEM with a significance of * < 0.05 unless indicated otherwise. Measurements were obtained in duplicates from 3 to 5 separate experiments unless stated otherwise.

TNF and IL-1 Paralyze Growth Cone Motility and
Induce Growth Cone Collapse. Recent reports detailed that TNF exerts morphogenetic functions (rearrangements of the actin cytoskeleton) without induction of apoptosis [7]. To restrict cytokine exposure exclusively to advancing growth cones, polystyrene beads (2.5 × 10 5 beads/mL, 4 m in diameter) coated with TNF or IL-1 were applied to SC neuron cultures and growth cone-bead encounters observed under phase contrast (63x oil, phase contrast). Following physical contact of growth cones to cytokine-coated beads (TNF -top panel, IL-1 -middle panel), growth cone motility ceased followed by the progressive degeneration of growth cone morphology upon reaching complete collapse. Contact with ovalbumin-coated beads had no influence on growth cone morphology and advance (bottom panel). (c) E7 SC neurons grown on laminin for 2 days were fixed in paraformaldehyde. Cytokine receptors were revealed by indirect immunocytochemistry and analyzed by confocal microscopy (Zeiss LSM510, 40x oil, NA 1.30). TNF receptor 1 (TNF-R1) and IL-1 receptor (IL-1R) were expressed on cell bodies (arrows) and neurites were expressed as well as on growth cones and filopodia (arrowheads). In contrast, TNF receptor 2 (TNF-R2) expression was restricted to cell bodies. We examined whether TNF or IL-1 has the potency to alter motility of neuronal growth cones, an actin-cytoskeleton driven mechanism. Live-video, phase contrast microscopy of advancing SC neuron growth cones revealed that an acute exposure to TNF or IL-1 paralyzed growth cone motility and also caused the collapse of growth cone morphology accompanied by neurite beading and retraction compared to control (ovalbumin) (Figure 1(a)). To demonstrate a direct effect of cytokines on growth cones, we utilized polystyrene beads (4 m in diameter) covalently coated with TNF , IL-1 , or ovalbumin (control) to restrict contact between neurons and cytokines exclusively to advancing growth cones 10 20 Time ( , respectively (open circles). All data were obtained from at least three different dissections (duplicate cultures each, >30 growth cones total) with error bars representing SEM. (e) TNF elicited a dose-dependent growth cone collapse at concentrations higher than 50 ng/mL. Growth cones with collapsed morphology were quantified (random fields of view) 30 min after application to allow for possible recovery of morphology. (f) Preincubation of SC neuron culture either with 10 M MnTBAP or with 2 M DPI provided significant protection against growth cone collapse in the presence of 100 ng/mL TNF or 100 ng/mL IL-1 (dark grey bars; * * < 0.05) as opposed to cytokines alone (black bars), which caused substantial growth cone collapse (dark bar, * < 0.05) compared to control (open bar). A presence of 10 M MnTBAP (light grey bar, Mn) had no effect on basal levels of collapsed growth cones, whereas 2 M DPI increased the percentage of collapse growth cones. All data (e and f) were obtained from at least three different dissections (duplicate cultures each). Error bars represent ±SEM. [35]. Growth cones encountering cytokine-coated beads displayed a series of stereotypic changes in behavior ultimately resulting in growth cone collapse (Figure 1(b)). After establishing long-lasting adhesion contacts with filopodia, growth cones entered a phase characterized by highly mobile, lamellipodia-like structures, which were nonproductive for advance, followed by a collapse of morphology (TNF : 78 ± 4%, = 22 and IL-1 : 83 ± 7%, = 18 of observed growth cones, resp.). None of these growth cone responses occurred upon contact with ovalbumin-coated beads. Cytokine receptors were expressed on SC neurons and their processes (Figure 1(c)). Whole SC tissue extracts (western blotting) exhibited immunoreactivity against TNF receptor 1 (TNF-R1, apparent MW = 48 kDa), TNF receptor 2 (TNF-R2, apparent MW = 70 kDa), and IL-1 receptor type 1 (IL-1R, apparent MW = 76 kDa) in accordance with previous reports [36]. Expression of TNF-R1 and IL-1R was found on neuronal cell bodies, neurites, as well as growth cones; however, TNF-R2 expression was predominantly localized to neuronal cell bodies with virtually no expression on neurites or growth cones.

A Cytokine-Activated NADPH Oxidase Generates ROS in Growth Cones and Cell Bodies of SC Neurons.
To determine the generation of intracellular ROS in advancing growth cones exposed to cytokines, we employed quantitative ratiometric fluorescence analysis utilizing the oxidation-sensitive fluorescent indicator 2 ,7 -dihydrodichlorofluorescein (DCF, 10 M) and the redox inert fluorescence indicator Calcein Blue (CB, 4 M). Ratiometric analysis distinguishes changes in DCF fluorescence due to ROS formation from dilution/concentration effects simply due to rapid changes in growth cone morphology. Bath application of 100 ng/mL TNF or 100 ng/mL IL-1 elicited a rise in the relative maximum DCF/CB fluorescence ratio indicative of the formation of ROS ( Figure 3). As shown in Figure 3(a) (false colored DCF/CB ratio images), ROS formation was sustained and preceded loss of morphology of the advancing growth cone reflected by atrophy and beading of filopodia, condensation of growth cone body, and retraction. Quantitative analysis demonstrated a significant and sustained ROS formation in advancing growth cones upon exposure to TNF (100 ng/mL, = 14), IL-1 (100 ng/mL, = 10), or 100 M hydrogen peroxide (positive control, = 11) compared to 10 g/mL ovalbumin (negative control, = 22) (Figure 3(b)). Neither loading with DCF (131 ± 18 m/h, > 15) nor loading with CB (118 ± 14 m/h, > 15) at the concentrations used affected growth cone advance per se compared to unloaded control (113 ± 9 m/h).

TNF and IL-1 Attenuate Neurite Outgrowth of SC Neurons in a Redox-Dependent
Manner. Next, we examined whether the redox-dependent impairment of growth cone advance is of consequence for neurite outgrowth. Bath application of 100 ng/mL TNF or IL-1 attenuated neurite outgrowth in SC neuron cultures in a dose-dependent manner indicated by a shift in the distribution to shorter neurite lengths compared to 10 g/mL ovalbumin (Ov), our control (Figures 7(a) and 7(b), Table 1). Scavenging ROS (10 M MnTBAP) rescued neurite outgrowth in the presence of TNF and IL-1 , whereas the NOX inhibitor (2 M DPI) provided only partial protection of neurite outgrowth (Figures 7(c) and 7(d)). Notably, neurite outgrowth of SC neurons on laminin exhibited inherent redox dependence even in the absence of TNF or IL-1 (Figures 7(e) and 7(f)). In accordance with previous reports, increasing concentrations of the ROS scavenger N-acetyl-L-cysteine (2 mM NAC, 50% reduction) or 20 M MnTBAP (10% reduction) inhibited neurite outgrowth compared to control, whereas 10 M MnTBAP was ineffective [15]. However, we measured a significant increase in neurite outgrowth with 5 M MnTBAP (25% increase).

Discussion
We demonstrated that long-term exposure of SC neurons to the proinflammatory cytokines TNF and IL-1 provoked the loss of growth cone motility and the subsequent degeneration of growth cone morphology (collapse). These changes in growth cone advance and behavior translated into the impairment of neurite outgrowth and disruption of process architecture of SC neurons, which was rescued either   Figure 10: A Rac1-dependent redox rheostat regulates growth cone motility in response to extrinsic stimuli. Exposure of neuronal growth cones either to TNF or to IL-1 rapidly stimulates ROS generation through NOX activity under the regulation of the small GTPase Rac1 and concomitant paralysis and degeneration of morphology of growth cones. Moreover, growth cone motility is not only sensitive to the overabundance of ROS either via NOX activation or via constitutive Rac1 activity (Rac1 V12 ) but is also impaired by a substantial depletion of ROS (antioxidants) or inhibition of Rac1 activity (Rac1 N17 ). Consequently, productive growth cone motility requires an optimal concentration of intracellular ROS generated by a Rac1-regulated NOX activity. It is plausible that this Rac1/NOXredox rheostat is responsive to many extrinsic stimuli including cytokines, growth factors, hormones, and cell adhesion molecules for which redox signaling mechanism has been demonstrated. by scavenging ROS, inhibiting NOX activity, or depleting Rac1 activity despite a presence of TNF or IL-1 . Both cytokines stimulated the formation of ROS intermediates and a transient phase of actin filament organization in advancing growth cones. Importantly, ROS intermediates and actin filament reorganization preceded paralysis of growth cone motility and collapse of morphology implying a causative action of this signaling mechanism. Taking into account that exhaustive ROS scavenging (excess of antioxidants) also impaired neurite outgrowth, it is feasible that productive growth cone motility could demand an optimal level of ROS intermediates. We propose that a redox rheostat under the regulation of the Rac1 shapes growth cone motility and hence neurite outgrowth, in response to many extrinsic stimuli other than cytokines ( Figure 10).
Prolonged bath application of TNF or IL-1 (6-8 h) to dissociated SC neurons or SC explants stunted neurite outgrowth in a dose-dependent manner (Figure 7, Table 1) in accordance with previous findings in hippocampal neurons or neuroblastoma cells [8,9]. In lieu of the well-documented apoptotic potency of TNF and IL-1 , it was imperative to determine whether inhibition of neurite outgrowth had its origin in neuronal cell bodies (apoptosis, necrosis) or directly in the distal compartment of growth cones. Foremost, cytokines were applied to SC neurons after the onset of neurite outgrowth with the majority of processes longer than two cell diameters. Since the number of neurites per neuron (a measure of neurite initiation) remained unaltered and no measureable neuronal cell death occurred over the time period of cytokine exposure (6-8 h) as determined by a live/dead fluorescence assay (calcein green/propidium iodide counterstaining), neither a decrease in neurite initiation nor a decrease in neuronal cell death could account for the observed reduction in neurite outgrowth. Significant neuronal cell death (>15%) was however apparent 24 hours after cytokine addition and increased to over 40% after 2 days. Evidence for direct action of cytokines on growth cone motility and morphology was obtained by live-video phase microscopy (Figures 1 and 2). Acute exposure to TNF or IL-1 (bath applied) impaired growth cone motility in a dose-dependent manner within 10 to 15 minutes upon application. Initial paralysis of growth cone advance was followed by degeneration of morphology and subsequent collapse. Restricting cytokine exposure exclusively to advancing growth cones in our bead assay corroborated these findings. In addition, bead assays revealed a transient increase in lamellipodia-like structures or in the vicinity of neuronbead contacts, which were nonproductive for growth cone advance. This transient increase in actin filaments (Figure 9) was reminiscent of our findings in SH-SY5Y neuroblastoma, yet in the case of primary neurons it revealed a physiological consequence [14]. Direct effects of cytokines on growth cones were further supported by the expression of IL-R1 (IL-1 receptor) and TNF-R1 (high affinity p55 TNF receptor) on the entire neuronal surface including cell bodies and growth cones ( Figure 1). Together these findings provided strong evidence that TNF and IL-1 directly impaired growth cone motility and morphology as the underlying cause for the attenuation of neurite outgrowth over longer time periods of exposure.
Initial evidence for a role of ROS intermediates was suggested by studies demonstrating that antioxidants (ROS scavenging) or DPI (inhibiting NOX-like activities) largely rescued neurite outgrowth, growth cone motility, growth cone morphology, and lastly actin filament organization in the presence of cytokines. Interestingly, neurite outgrowth of SC neurons on laminin exhibited innate redox dependence. Whereas moderate MnTBAP concentrations (5 M) significantly increased neurite outgrowth, concentration higher than 10 M stunted neurite outgrowth corroborating studies in Aplysia neurons [15]. It is plausible that moderate MnTBAP concentration (5 M) scavenges excessive amounts of superoxide derived from mitochondrial respiration and as byproduct of several enzymatic reactions. Under basal culture conditions, this excessive superoxide might compromise optimal growth cone motility (actin dynamics, tubulin dynamics) and hence the observed enhancement of neurite outgrowth by moderate concentrations of scavenger [41][42][43]. In contrast, higher MnTBAP concentrations could impair vital redox-dependent mechanism necessary for proper neurite outgrowth [44]. Using the oxidation-sensitive fluorescence indicator DCF, we demonstrated substantial yet transient generation of ROS (likely superoxide) in growth cones and cell bodies of SC neurons upon exposure to TNF and IL-1 (Figures 3 and 5). Similar results were obtained using the superoxide-specific fluorescence indicator dihydroethidium, yet investigations suffered from considerable neurotoxicity of dihydroethidium (data not shown). Previous studies in chick cortical neurons and human SH-SY5Y neuroblastoma cells demonstrated superoxide production in response to TNF utilizing dihydroethidium oxidation or a SOD-inhibitable cytochrome C oxidation as demonstrated [14,45]. A significant contribution from nonneuronal cells (microglia) was unlikely since (i) SC neuron cultures contained less than 3% nonneuronal cells, (ii) analyzed cells all exhibited distinct neuronal characteristics (i.e., round cell body, one process longer than 2 cell diameters), and (iii) microglia invade and populate the developing CNS at late embryonic or even postnatal stages [46]. Ratiometric imaging demonstrated significant ROS production in advancing growth cones within 3 to 5 min upon exposure to inflammatory stress as opposed to changes in motility and morphology observed 10 to 15 min following exposure. Several findings strongly suggested a NOX2-like activity as the source of ROS in SC neurons, in particular superoxide [14,47]. SC neurons exhibited immunoreactivity against NOX2 and the cytosolic subunits p47 phox and p67 phox . A presence of other NOX isoforms can however not be excluded, yet antibody quality against NOX1 or NOX3 was insufficient to produce a conclusive finding. TNF stimulated a dose-dependent increase in ROS intermediates in freshly isolated growth cone particle preparations in conjunction with a presence of NADPH oxidoreductases activity. The phorbol ester PMA as well as TNF induced translocation of the cytosolic subunit p67 phox to plasma membranes. Parallel studies on the translocation of the cytosolic subunit p47 phox to plasma membranes were hampered by the considerable variability of immunoreactivity. Expression of NOX-like activities was reported in both primary PNS and CNS neurons [48][49][50][51]. Lastly, the antioxidant MnTBAP inhibits the TNF or IL-1stimulated ROS formation. Also, pharmacological inhibition with DPI was effective in blocking TNF or IL-1 -mediated responses of SC neurons including ROS formation and partial rescue of growth motility, neurite outgrowth, growth cone collapse, and actin filament reorganization (Figures 2, 4, 5, 7, and 9). Notably, all these experiments revealed considerable toxicity of DPI and thus, depending on the duration of DPI application, put restrictions on the usable concentrations of DPI. Mechanistically, DPI blocks single electron transport reactions such as NOX-mediated generation of superoxide. Nevertheless, numerous other enzymatic reactions are also compromised foremost electron transport of complex I in mitochondria, which could account for the observed toxicity. Lastly, depleting Rac1 activity negated cytokine-mediated ROS formation in SC neurons, whereas Rac1 overexpression alone was sufficient to increase ROS formation. The small GTPase Rac1 emerged as a pivotal regulator of cytokinestimulated ROS formation in SC neurons in accordance with previous reports [22,52,53]. In our study, depleting Rac1 activity abolished ROS formation in the presence of TNF or IL-1 yet without affecting basal ROS levels (Figures 6(b)-6(d)). In contrast, overexpression of constitutively active Rac1 alone was sufficient to stimulated ROS generation in SC neurons and also reduced neurite outgrowth, which was not further exacerbated upon addition of TNF or IL-1 (data not shown). Effects of Rac1 depletion or overexpression were addressed by trituration loading of purified recombinant Rac1-GST mutant chimeras since adenoviral-mediated expression of Rac1 mutants required a 3-day expression period incompatible with neurite outgrowth analysis [26,31]. Not unexpectedly, introduction of Rac1 N17 or Rac1 V12 resulted in a measurable reduction in neurite outgrowth on laminin even in the absence of cytokines [11,26,54,55]. In support of our findings, numerous reports link Rac1 as a regulator of ROS intermediates in cellular signal transduction. Neurite extension in PC12 cells upon NGF stimulation encompasses increases in Rac1 activity and H 2 O 2 formation [56]. A Rac1 mutant lacking residues 124-135 of the insert region blocking ROS generation disrupted membrane ruffling and mitogenesis in fibroblasts and caused a downregulation of RhoA [44,[57][58][59]. With respect to actin cytoskeleton dynamics, TNF and IL-1 induced a transient phase of actin filament reorganization (increases in actin filament density) in neuronal growth cones, which was however nonproductive for motility. Interestingly, actin filament reorganization was not repeatable with another addition of cytokines even after wash-out and extensive recover time suggesting permanent or persistent damage to actin filament reorganization by cytokines. Irreversible oxidative damage (carbonylation) to actin was detectable in neuroblastoma cells exposed to TNF [14]. These findings provided evidence that NOX activation in neuronal growth cones serves as source of ROS intermediates in response to cytokines and that ROS intermediates are likely causative for cytokine-mediated degeneration of neuronal growth cones.
Inflammatory and oxidative stress is a hallmark of neurodegeneration in most chronic, acute, and even some psychiatric CNS pathologies [3,60,61]. The proinflammatory cytokines TNF and IL-1 are important to orchestrate inflammatory and oxidative stress in the disease and aging CNS although pleiotropic actions in the adult and developing CNS are known, hence establishing a complex and delicate balance between neuroprotection and neurotoxicity. Members of the Nox/Duox family have emerged as [46,62] key sources of oxidative stress in aging and the progression of many pathologies beyond the CNS and have been recognized as key therapeutic targets [63][64][65][66]. Our findings provide support for a role of NOX activity in primary neurons as a principal source of oxidative stress triggered by TNF or IL-1 [16]. Overabundance of ROS intermediates is likely to disrupt proper actin filament dynamics, which is vital for the plasticity and morphology of distal neuronal processes including neurite outgrowth, sprouting, and spine dynamics. Strategies to block NOX activities could thus proof beneficial to blunt inflammatory stress in the diseased and aging CNS and to halt cognitive decline [66,67].