Reactive oxygen species (ROS) oxidize various molecules, including proteins, lipids, and DNA. When ROS levels are elevated, this activity can lead to oxidative stress, cell death, and aging (Sohal and Orr 2012) and contribute to several chronic and degenerative diseases, including cancer, Alzheimer's, and Parkinson's disease (Hernandes and Britto 2012; Yang et al. 2013). On the other hand, a growing body of literature indicates that ROS also act as important physiological signaling molecules in cell proliferation, differentiation, motility, and apoptosis (Bedard and Krause 2007; Finkel 2011). Accordingly, ROS are not only uncontrolled by-products of aerobic metabolism but are also specifically generated by NADPH oxidases, the mitochondrial respiratory chain, and lipoxygenases (Camello-Almaraz et al. 2006; Bedard and Krause 2007; Taddei et al. 2007). A tight control of cellular ROS concentration is essential to ensure specific signaling. Perturbing this redox balance can result in the aforementioned diseases.
Because of the highly reactive and short-lived nature of ROS (Winterbourn 2008), intracellular ROS signaling likely has to occur within close vicinity of the ROS source. Therefore, localized activation seems essential for ROS signaling. In non-neuronal cells, NADPH oxidases have been localized to distinct subcellular regions involved in cell adhesion and migration, including leading edge, ruffles, and focal adhesions (Ushio-Fukai 2006). Accordingly, ROS derived from NADPH oxidases have been implicated in adhesion of fibroblasts (Chiarugi et al. 2003) and in mig-ration of endothelial cells (Moldovan et al. 2000; Ushio-Fukai et al. 2002; Ikeda et al. 2005), HeLa cells (Nimnual et al. 2003; Kim et al. 2009), smooth muscle cells (Schroder et al. 2007; Lee et al. 2009), and keratinocytes (Kim et al. 2011). Whether ROS produced by NADPH oxidase regulate adhesive and motile processes in neurons such as growth cone protrusion, neurite outgrowth, and axon guidance is not clear.
The family of NADPH oxidases consists of seven members, which all contain a major membrane-bound flavocytochrome b558 enzymatic subunit but differ with respect to the composition of additional membrane-bound and cytoplasmic subunits (Bedard and Krause 2007). The first NADPH oxidase to be characterized was found in phagocytes and contained NOX2/gp91phox (referred to as ‘NOX2’ in the remainder of this article). The fully assembled and active NOX2 complex includes the p22phox, Rac1, p47phox, p67phox, and p40phox subunits, which regulate the enzymatic activity of the NOX2 complex. NADPH oxidase family members NOX1, NOX2, NOX3, and NOX4 are expressed in different portions of the nervous system, particularly in neurons, microglia, and astrocytes (Sorce and Krause 2009; Hernandes and Britto 2012). NADPH oxidase-derived ROS have been implicated in hippocampal synaptic plasticity and memory formation (Kishida et al. 2006), NMDA receptor activation (Brennan et al. 2009), nerve growth factor-induced neuronal differentiation and neurite outgrowth of PC-12 cells (Suzukawa et al. 2000; Ibi et al. 2006), and neuronal apoptosis (Tammariello et al. 2000; Guemez-Gamboa and Moran 2009). On the other hand, microglial cells and proinflammatory cytokine-treated neurons release NADPH oxidase-derived superoxide leading to neuronal toxicity (Barth et al. 2012), as described in Alzheimer's and Parkinson's disease (Sorce and Krause 2009; Gao et al. 2012). We have recently reported that ROS derived from NADPH oxidases regulate F-actin organization, dynamics, and neurite outgrowth (Munnamalai and Suter 2009); however, the exact subcellular localization and interactions of NADPH oxidase with the actin cytoskeleton in neuronal growth cones have not been investigated.
Here, we report on the first localization of a NOX2-type NADPH oxidase in neuronal growth cones. NADPH oxidase inhibition with VAS2870 or celastrol resulted in reduced retrograde F-actin flow and neurite outgrowth, confirming our earlier results. NADPH oxidase activation with a protein kinase C (PKC) activator resulted in increased ROS levels in the growth cone periphery. We found that the regulatory cytosolic subunit p40phox exhibited F-actin association in unstimulated growth cones and little colocalization with plasma membrane-bound NOX2. However, upon growth cone stimulation with the Aplysia cell adhesion molecule (apCAM), p40phox and NOX2 accumulated and colocalized at adhesion sites. In summary, these findings point toward an interesting bidirectional relationship between NADPH oxidase and the actin cytoskeleton in neuronal growth cones.
- Top of page
- Materials and methods
- Acknowledgements and conflict of interests disclosure
- Supporting Information
This is the first report on subcellular localization of NADPH oxidase in neuronal growth cones. Expression of NOX1-NOX4 family members at the RNA and protein levels has been shown for various brain regions including cortex, cerebellum, hippocampus, as well as for peripheral ganglia (Sorce and Krause 2009; Hernandes and Britto 2012). NOX2 subunits have been localized in cultured neurons, such as hippocampal (Tejada-Simon et al. 2005; Park and Jin 2008), cerebellar granule (Coyoy et al. 2008), sympathetic (Tammariello et al. 2000; Hilburger et al. 2005), and dorsal root ganglion neurons (Cao et al. 2009). However, these studies did not investigate the detailed subcellular distribution of NADPH oxidase proteins. Here, we have shown that a NOX2-type NADPH oxidase is localized to the plasma membrane of Aplysia neuronal growth cones, exhibiting a similar but less homogenous plasma membrane distribution compared to the cell adhesion protein apCAM. Whether another type of NADPH oxidase is also present in Aplysia growth cones, remains to be investigated. NADPH oxidase inhibition with VAS2870 or celastrol impaired actin organization and dynamics as well as neurite outgrowth, confirming our previous findings (this study; Munnamalai and Suter 2009), while NADPH oxidase activation increased H2O2 levels in the growth cone P domain (Figs. 1, 2; Figure S1). The cytosolic subunit p40phox exhibited relatively little colocalization with NOX2 in unstimulated growth cones, but had significant colocalization with F-actin bundles (Fig. 3). We observed variations in p40phox levels between growth cones, which correlated with F-actin content (Fig. 4). Cytochalasin B significantly reduced the amount of p40phox in the growth cone periphery (Fig. 5), while cell fractionation and actin coimmunoprecipitation experiments confirmed a partial association of p40phox with F-actin (Fig. 6). Cell fractionation studies were performed on total CNS lysates suggesting that the differential association of NOX2 and p40phox not only occurs in the growth cone but also in other regions of the cell. Differential localization of NOX2 and p40phox in other compartments of unstimulated neurons is further supported by confocal imaging of the neuronal cell body (Figure S4). Finally, we have found increased p40phox/NOX2 colocalization at adhesion sites during apCAM-evoked neuronal growth (Fig. 7). In summary, these findings provide evidence for a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which may contribute to the control of neurite outgrowth.
Very little is known about NADPH oxidase signaling in neuronal development, particularly in axonal growth and guidance. Previous work from our laboratory and others suggested a role for NADPH oxidase-derived ROS in regulating neurite outgrowth (Suzukawa et al. 2000; Ibi et al. 2006; Munnamalai and Suter 2009). Tail wound-derived H2O2 can promote regeneration of sensory axons in Zebrafish embryos (Rieger and Sagasti 2011). Furthermore, we have shown that NADPH oxidase inhibition in Aplysia growth cones results in reduced F-actin content (this study; Munnamalai and Suter 2009). ROS have also been implicated in Rac1-dependent Ca2 + release in Aplysia growth cones (Zhang and Forscher 2009). Because of the short half-life and high reactivity of ROS, target proteins need to be close to the ROS source to establish signaling specificity (Ushio-Fukai 2006). Consistent with this idea, we found NADPH oxidase localized in the growth cone periphery at sites where actin assembly occurs. This study is also interesting from an evolutionary point of view. While ancestral NOX2, p22phox , p47phox , and p67phox homologs have been identified for several invertebrate species including Monosiga brevicollis, Nematostella vectensis, Strongylocentrotus purpuratus, as well as the snail Lottia gigantea, no p40phox homolog has been identified in these species thus far, which has led to the conclusion that p40phox may have evolved later with the chordata (Kawahara et al. 2007; Sumimoto 2008). Very recently several predicted Aplysia NADPH oxidase-like sequences have been released to the NCBI database, including NOX2-like (XP_005090645), NOX5-like XP_005101964), dual oxidase 1 (DUOX1)-like (XP_005108327), and DUOX2-like (XP_005106629). Our results suggest that a NOX2-type NADPH complex with all membrane and cytosolic subunits including the regulatory subunit p40phox already evolved with the appearance of the molluscan clade. Other available antibodies against NOX2-type subunits did not detect proteins with high enough specificity (data not shown); thus, because of this limitation we were unable to detect other subunits of the NOX2-type complex in Aplysia growth cones thus far.
Our quantification of H2O2 levels following PKC activation revealed that the NADPH oxidase is functionally active in growth cones. VAS2870 abolished the PDBu-stimulated increase in H2O2 levels in the growth cone periphery within 30 s (Fig. 2i). Treatment of growth cones with VAS2870 alone for 15 min resulted only in a modest decrease of H2O2 levels (Fig. 2h). These findings are in line with another study demonstrating that VAS2870 blocked the increase of ROS levels in endothelial cells activated by oxidized low-density lipoprotein, but had minimal effect on the basal ROS levels without stimulation (Stielow et al. 2006). There are several possible reasons why VAS2870 treatment alone did not lower basal H2O2 levels more. First, NADPH oxidases are not the only source for superoxide/hydrogen peroxide. Second, we are measuring hydrogen peroxide in this assay and not superoxide, which is the primary product of NADPH oxidase. Third, ROS are highly reactive, act locally, and are tightly regulated. Finally, the dye used to detect hydrogen peroxide may not be able to detect small ROS changes. Thus, it might be difficult to optically detect local changes in ROS levels induced by NADPH oxidase inhibition with sufficient temporal resolution, despite the fact that such changes can affect cellular physiology and morphology.
Cytoskeletal association of regulatory NOX subunits has been reported for p40phox, p47phox, and p67phox in non-neuronal cells (Nauseef et al. 1991; El Benna et al. 1994, 1999; Li and Shah 2002). Furthermore, p40phox and p47phox associate with the neutrophil cytoskeleton through a PX-domain-dependent interaction with the actin-associated protein moesin (Wientjes et al. 2001; Zhan et al. 2004), while p40phox also interacts with the actin-binding protein coronin (Grogan et al. 1997), as well as directly with the actin cytoskeleton (Shao et al. 2010). These findings suggested that actin association of cytosolic NADPH oxidase subunits may have a regulatory function by keeping the complex in an inactive state until a stimulus releases the subunits from the cytoskeleton during the activation process. In agreement with this idea, COS-7 expression experiments revealed that p40phox is associated with actin and moesin, and that a mutated form of p40phox shows higher actin association and inhibits NOX2 activity (Chen et al. 2007). Furthermore, the actin-binding protein cortactin has been suggested to regulate NOX2 activation in lung endothelial cells trough an interaction with p47phox (Usatyuk et al. 2007). Thus, actin rearrangements could also deliver cytosolic NADPH oxidase subunits to the plasma membrane. Does NOX2 localization show actin dependence? In migrating endothelial cells, NOX2 colocalizes with F-actin along the leading edge in an IQ motif containing GTPase activating protein 1 (IQGAP1)-dependent manner, while manipulating the actin cytoskeleton with latrunculin and jasplakinolide alters NOX2 localization (Ikeda et al. 2005). Our growth cone studies also suggest a certain actin dependence of NOX2 localization as indicated by the reduction of NOX2 following cytochalasin treatment and the colocalization of NOX2 with actin along the leading edge and in transition zone ruffle/intrapodia (Fig. 5g and h). Thus, a fraction of NOX2 along with p40phox appears to undergo retrograde flow together with F-actin. In summary, our present work in neuronal growth cones is consistent with previous findings made with leukocytes and endothelial cells indicating that the actin cytoskeleton may play a role in NADPH oxidase activation through localization of its subunits.
On the other hand, NADPH oxidase-derived ROS regulate actin organization and dynamics, growth cone motility, and neurite growth (this study; Munnamalai and Suter 2009) and actin organization and migration of non-neuronal cells (Nimnual et al. 2003; Wojciak-Stothard et al. 2005; Schroder et al. 2007; Kim et al. 2009, 2011; Kuiper et al. 2011). Thus, together with these findings, our current study suggests an interesting bidirectional relationship between NADPH oxidase and F-actin. We propose that the cytosolic NADPH oxidase subunits such as p40 are associated with actin structures in unstimulated growth cones. Upon growth cone stimulation by guidance cues, cytosolic subunits p47phox, p67phox, p40phox, and Rac1 translocate to the plasma membrane (either with or without F-actin) and activate the membrane-bound subunits NOX2/p22phox. NADPH oxidase-produced superoxide (or most likely hydrogen peroxide) diffuses back into the growth cone for local regulation of the cytoskeleton and related directional growth (see graphical abstract of the online version). In agreement with this hypothesis, we found increased p40phox/NOX2 levels and colocalization at growth cone contact sites with apCAM beads (Fig. 7) and interacting growth cones (arrow in Fig. 3e), where apCAM exhibits increased density as well (Thompson et al. 1996; Suter et al. 1998). These results indicate that apCAM clustering can trigger actin remodeling as well as NADPH oxidase activation related to neurite outgrowth. The details of the functional relationship and order of events of apCAM clustering, actin reorganization, and NADPH oxidase activation remain unclear at this point. apCAM clustering could first activate NADPH oxidase followed by ROS-mediated actin reorganization; or apCAM-mediated actin remodeling could activate NOX2 by translocating cytosolic subunits close to plasma membrane. Thus, additional studies are needed to address the important question whether NADPH oxidase activation plays a functional role in axonal growth triggered by apCAM or other guidance cues.
Acknowledgements and conflict of interests disclosure
- Top of page
- Materials and methods
- Acknowledgements and conflict of interests disclosure
- Supporting Information
We thank members of the Suter laboratory for their comments on this manuscript. PF-6 AM was kindly provided to us by Dr. Christopher Chang and Vivian Lin, University of California, Berkeley CA, USA. We also thank Kelsey Martin, Samuel Schacher, and Eric Kandel for providing 4E8 hybridoma cells to produce 4E8 antibody. This work was supported by grants from the NIH (R01 NS049233 to D.M.S. and P20 GM103500 to M. Q.), NSF (1146944-IOS to D.M.S.), and the Bindley Bioscience Center at Purdue University (D.M.S.). V. M. was partially supported by fellowships through the PULSe and PUN Integrative Neuroscience graduate programs at Purdue University. The authors have no conflict of interest to declare.
All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.