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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell Stem Cell. Author manuscript; available in PMC Jan 7, 2012.
Published in final edited form as:
PMCID: PMC3018289
NIHMSID: NIHMS256441
Proliferative Neural Stem Cells Have High Endogenous ROS Levels that Regulate Self-Renewal and Neurogenesis in a PI3K/Akt-Dependant Manner
Janel E. Le Belle,1 Nicolas M. Orozco,1 Andres A. Paucar,1 Jonathan P. Saxe,5 Jack Mottahedeh,1 April D. Pyle,2,3,4 Hong Wu,3,4,5,6 and Harley I. Kornblum1,3,4,5
1NPI-Semel Institute for Neuroscience & Human Behavior and Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA
2Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA
3Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA
4Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA
5Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA
6Institute for Molecular Medicine, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA
Corresponding Author: Harley I Kornblum, Room 379 Neuroscience Research Building, 635 Charles E. Young Dr. South, Los Angeles, CA 90095, Tel: 310-794-7866, Fax: 310-206-5061, harley/at/ucla.edu
The majority of research on reactive oxygen species (ROS) has focused on their cellular toxicities. Stem cells generally have been thought to maintain low levels of ROS as a protection against these processes. However, recent studies suggest that ROS can also play roles as second messengers, activating normal cellular processes. Here, we investigated ROS function in primary brain-derived neural progenitors. Somewhat surprisingly, we found that proliferative, self-renewing multipotent neural progenitors with the phenotypic characteristics of neural stem cells (NSC) maintained a high ROS status and were highly responsive to ROS stimulation. ROS-mediated enhancements in self-renewal and neurogenesis were dependent on PI3K/Akt signaling. Pharmacological or genetic manipulations that diminished cellular ROS levels also interfered with normal NSC and/or multipotent progenitor function both in vitro and in vivo. This study has identified a redox-mediated regulatory mechanism of NSC function which may have significant implications for brain injury, disease, and repair.
Oxidative stress caused by the cellular accumulation of reactive oxygen species (ROS) is a major contributor to disease and to cell death. In contrast to the damaging effects of ROS, there is evidence that in some systems ROS at lower, non-toxic levels can actually promote cell proliferation and survival (Blanchetot & Boonstra, 2008; Chiarugi & Fiaschi, 2007; Leslie, 2006). These findings suggest a much more complex role for redox balance in cellular biology than was first understood by models of oxidative stress. For example, in the hematopoietic system a low endogenous cellular ROS status has been associated with maintaining the quiescence of hematopoietic stem cells (HSCs) whereas a higher ROS state is associated with a greater proliferation leading to a premature exhaustion of self-renewal in these cells (Jang & Sharkis, 2007). This has led to the hypothesis that keeping ROS levels low within the stem cell niche is an important feature of “stemness” which is directly related to the relatively quiescent state of stem cells in vivo. Although it is thought that the resident neural stem cells (NSCs) within the neurogenic niches of the brain are also relatively quiescent it is not yet known how ROS status or ROS stimuli may affect this population of cells. One might hypothesize that NSCs would utilize and defend against ROS in the same manner as HSCs, maintaining low endogenous levels of ROS. However, despite similarities in the core functions of self-renewal and multipotency, HSCs and NSCs also have many biological differences. For example, the premature replicative senescence observed in HSCs as a result of the hyper-proliferation caused by deletion of the tumor suppressor gene, PTEN, is not observed in PTEN-deleted NSCs (Zhang, et al. 2006; Yilmaz, et al., 2006; Groszer et al., 2006).
Emerging evidence now suggests that in addition to the passive production of ROS by the mitochondria we have evolved a redox mechanism to utilize cellular ROS in a directed manner by NADPH oxidase (NOX) enzymes which are the predominant source of ROS in many cells (Lameth et al., 2008). NOX was originally characterized in phagocytes, which utilize a NOX-generated burst of superoxide to defend against pathogens. It has now become clear that other cell types utilize NOX-generated ROS as second messengers in tightly controlled signal transduction networks. The realization that ROS production is an essential component of cellular signaling has led to the discovery that many ligands essential to normal cell function including peptide and angiogenic growth factors, hormones, and interleukins require the generation of ROS via NOX activation in some non-phagocytotic cells (Kwon et al., 2004; Wang & Lou, 2009; Garrido & Griendling, 2009; Goldstein, et al., 2005; Behrens, et al., 2008). The NOX-stimulated production of ROS, in turn, can activate pathways that have been previously associated with enhanced cell proliferation and survival, including the MAPK and PI3K/Akt pathways (Kwon, et al., 2004; Sundaresan et al., 1995; Figure 1A). NOX isoforms have been identified in a number of different tissues, including the brain, although apart from the deleterious production of high levels of ROS in brain injuries, their function in the CNS is not known (Infanger, et al. 2006; Lambeth et al., 2007; Park, et al., 2008).
Figure 1
Figure 1
Stimulation of neurosphere cultures by reactive oxygen species promotes proliferation and self-renewal
In this study we sought to determine the role of ROS-mediated signaling in NSCs. we have found a surprising sensitivity to redox regulation in the neural stem cell-enriched pool of cells compared to the more generalized proliferative pool of limited progenitors, as the manipulation of cellular ROS levels predominantly affects self-renewal and neurogenesis. In contrast to what has been previously observed with O-2A progenitors (Smith et al., 2000; Power et al., 2002; Li et al., 2007), we have found that a decrease in normal cellular ROS levels can have an unexpectedly negative impact on self-renewal and neurogenesis both in vitro and in vivo. We observed a higher level of endogenous ROS in NSCs and within the neural stem cell niche, the SVZ, in vivo. We found that the regulation of endogenous ROS levels in NSCs was highly dependent on NADPH oxidase and PI3k/Akt signaling. The prominent effects of cellular ROS levels that we have observed on neural stem and progenitor cell function may be of particular relevance in injury and disease because of the large number of factors which may influence and deregulate ROS-mediated signaling.
The addition of exogenous ROS and a higher endogenous ROS state enriches for self-renewing neural stem and progenitor cells in clonal neurosphere cultures from different species and developmental ages
The addition of low, non-toxic concentrations of hydrogen peroxide (H2O2) to culture media produced a large increase in multipotent--capable of producing neurons, astrocytes and oligodendrocytes--clonal density neurosphere formation over multiple serial passages (Figure 1B; p=0.001). There was a more modest increase in overall cell proliferation (Supplemental Figure 1A). Exogenous ROS had a similar stimulatory effect on clonal neurosphere formation in embryonic and adult mouse and fetal human neurosphere cultures (Figure 1C; p<0.001).
Hematopoietic stem cells have relatively low levels of endogenous ROS (Jang and Sharkis, 2007). To determine whether neural stem cells were also low-ROS cells, we used FACS and the ROS-sensitive dye DCFDA to separate cells into ROShi (top 10%) and ROSlo (bottom 10%) populations and assessed their serial clonal density neurosphere forming capacity. The ROShi population contained almost all of the multipotent sphere-forming cells in primary and secondary clonal cultures (p<0.001; Figure 1D). We replicated this finding using multiple different ROS-sensitive dyes (see Supplemental Figure 1B). In addition, a high-ROS status provided an enrichment in clonal neurosphere formation compared to unselected (US), sorted cells from the same sample. ROSlo cells only formed primary clonal neurospheres and therefore displayed a limited capacity for self-renewal. Culture and resorting of sorted cells demonstrated that ROShi cells gave rise to both ROShi and ROSlo cells in secondary neurospheres but ROSlo cells were not capable of giving rise to ROShi cells (Figure 1E). Consistent with these results in murine cells, we also observed that the ROShi population in human ES-derived neural progenitors had a greater proliferative capacity compared to ROSlo or unselected cells from the same sample (p<0.001; Figure 1F).
Elevated ROS levels and NOX signaling are associated with increased NSC enrichment
Serial clonal density neurosphere formation (self-renewal), sphere diameter (proliferation), and multipotency were assessed in the ROShi cells compared to unselected cells from the same samples over multiple passages. ROShi cells were highly enriched for clonal neurosphere forming cells at all passages although a gradual decrease in this enrichment was observed (p<0.001; Figure 2A). There was also an initial significant increase in neurosphere diameter (p<0.05), but this returned to control levels with successive passages. ROShi spheres maintained a high level of multipotency over serial clonal passages. In agreement with our data utilizing exogenous ROS stimulation, a high endogenous ROS status was also associated with a greater positive effect on self-renewing divisions than on overall proliferation.
Figure 2
Figure 2
Neural stem cells are associated with a high-ROS status and NOX is a significant endogenous source of cellular ROS regulating NSC function in low oxygen conditions
We next sought to identify differences in cellular phenotypes between the ROShi and ROSlo populations since they displayed different capacities for long-term clonal self-renewal. Therefore, we sorted primary adult SVZ cells for 3 different neural stem cell-enriching marker sets: 1. EGFR+GFAP+CD24- cells (Pastrana et al., 2009) 2. ID1+GFAP+ cells (Nam and Nam, 2009), and 3. Lex (SSEA1)+ cells (Capela & Temple et al., 2002) and evaluated their relative endogenous ROS levels. We found that the enriched populations maintained significantly elevated endogenous ROS levels compared to the negative, non-NSC enriched populations from the same samples in each case, indicating that ROShi fraction contains the neural stem cell fraction (p<0.001; Figure 2 C&D). The “stem cell astrocytes” (EGFR+GFAP+CD24- cells) had 48% higher ROS levels than the (EGFR+GFAP-CD24-) transit amplifying cells and approximately 200% more than the EGFR-negative niche astrocyte-containing fraction of cells. The ID1+GFAP+ cells also had 57% higher ROS levels than the GFAP-negative cells.
Clonal neurosphere formation was greatly enhanced by the addition of exogenous ROS to the stem cell-enriched fractions derived from mouse SVZ, while the stem cell-negative fractions had limited or no response, an effect that was inhibited by the NOX inhibitor apocynin. (Supplemental Figure 2A).
When cells were sorted directly from the SVZ according to their ROS status and then analyzed for other markers, we found no differences in the expression of Dlx2 in ROShi compared to ROSlo cells, while Mash1 positive cells were enriched in the ROSlo fraction (Figure 2B). These data suggest that transient amplifying cells are not responsible for differences observed in neurosphere formation between the two populations. The ROShi population was significantly enriched for cells expressing nestin and doublecortin (DCX). No significant differences in Sox2 or GFAP expressing populations were observed.
The previous experiments were performed under room oxygen conditions. However, low oxygen conditions are known to stimulate NSC self-renewal (Studer et al., 2000). We found that low, physiological oxygen conditions (4% O2) resulted in elevated endogenous ROS levels (Supplemental Figure 2B), consistent with findings of others in different cell models (Guo et al., 2008), increased clonal neurosphere formation (Figure 2H; p<0.001), and up-regulation of the NOX2 homologue (Figure 2G; p<0.01). Conversely, lowering endogenous ROS levels in the low oxygen cultures through NOX inhibition eliminated the positive effects of hypoxia and resulted in decreased clonal density neurosphere formation (Figure 2H). These data suggest that the enhancement of self-renewal by lower oxygen concentrations is at least partially mediated through enhanced NOX activity which in turn leads to elevated ROS levels.
ROS augments growth and trophic factor stimulation and is required for normal NSC self-renewal
We next wanted to determine if NOX-generated ROS played an important role in facilitating growth factor signal transduction. To do this we placed neurosphere-derived cells in low concentrations of EGF and bFGF which led to a marked reduction in neurosphere formation (Figure 3A). However, clonal neurosphere formation could be restored to levels observed with high growth factor concentrations by supplementing low growth factor conditions with exogenous ROS (p<0.001; Figure 3A). The addition of exogenous ROS to the low-growth factor cultures elevated intracellular ROS levels to those observed in the high growth factor conditions (Figure 3B). No clonal neurospheres were formed in cultures without any growth factors even with the addition of exogenous ROS (data not shown), indicating that ROS on its own is not sufficient to replace growth factor-initiated signaling.
Figure 3
Figure 3
Reactive oxygen species are required for stimulation of normal neural stem cell self-renewal
Since the addition of small amounts of ROS resulted in a gain-of-function we next investigated the effects of a loss-of-function in NOX signaling. In growth factor-supplemented SVZ neurosphere cultures we found that NOX inhibition (DPI) significantly decreases clonal neurosphere formation but this inhibition can be rescued by adding exogenous ROS (H2O2) back to the culture medium. We also observed that cells derived from the SVZ of NOX2 mutant mice had significantly lower endogenous ROS levels (Supplemental Figure 3A) and subsequently displayed a significantly diminished NSC self-renewal and multipotency over serial clonal passages (Figure 3D-E; p<0.01). Mutant neurospheres produce approximately 29% more glial-only spheres (astrocytes and oligodendrocytes) compared to wild-type cultures (Supplemental Figure 3B). Clonal neurosphere formation and multipotency in the NOX2 mutants could also be significantly rescued by the addition of exogenous ROS (H2O2) in the mutant cultures (Figure 3A-B).
Because NOX has been implicated in both growth factor and neurotrophin signaling, we next examined whether it may play a role in the proliferative effects of brain derived neurotrophic factor (BDNF) on neural stem and progenitor cells (Islam et al., 2009). In the presence of standard concentrations of NSC growth factors (EGF and bFGF), we observed that BDNF could significantly increase clonal neurosphere formation. Therefore, we used inhibition of NADPH oxidase or treatment with the antioxidant N-acetyl-cysteine (NAC) in order to determine that NOX signaling played a significant role in the positive effects of BDNF on clonal neurosphere formation (p<0.001; Figure 4A). In addition, we demonstrated that endogenous superoxide (the ROS species produced by NOX) was increased upon BDNF treatment which could be blocked by NOX inhibition (p<0.001; Figure 4B). However, BDNF was not able to stimulate NSC self-renewal in cells derived from NOX2 mutant mice but was stimulatory only to wild-type cells (p<0.01; Supplemental Figure 4A), suggesting that NOX-dependent signaling plays a significant role in the stimulatory effects of BDNF on neural stem and progenitor proliferation.
Figure 4
Figure 4
ROS augment trophic factor signaling and are dependent on the PI3K/Akt signaling pathway for their effects
NOX regulation of neural stem and progenitor cells is dependent on PI3K/Akt/mTOR signaling
Previous studies have suggested that ROS can activate the PI3K/Akt/mTOR pathway through the reversible inactivation of the PTEN protein (Kwon et al., 2004; Leslie, 2006). Consistent with this we found in neurospheres that the addition of stimulatory concentrations of H2O2 induced direct oxidation of the PTEN protein (Figure 4C). To more directly assess the requirement for PTEN expression in the mechanisms underlying the stimulatory effect of ROS, we used cells derived from PTEN- deficient, PTEN heterozygous, and wild-type mice (Groszer et al. 2001), demonstrating that the addition of exogenous ROS is not capable of stimulating the PTEN-deficient cells (P<0.001; Figure 4D). As would be predicted from this model, ROS stimulated clonal neurosphere formation in heterozygous cells to a greater extent than it did wildtype cells, (Figure 4D; p<0.01). Likewise, inhibition of NOX resulted in dramatically reduced clonal neurosphere formation in WT but not in PTEN-deficient cells (Supplemental Figure 4B). Finally, BDNF stimulation of clonal neurosphere formation was also only observed in wild-type but not in PTEN-deficient cells (see Supplemental Figure 4A).
We examined activation status of key downstream nodes of the pathway. Exogenous ROS (H2O2 and Gox) enhanced, while inhibition of endogenous NOX-generated ROS with DPI inhibited the phosphorylation of Akt (Figure 4E). Furthermore we observed increased phospho Akt (pAkt) in the ROShi compared to the ROSlo population of cells, increased pAkt in BDNF-treated neurosphere cultures, and increased pAkt following the addition of H2O2 into low-growth-factor conditions media (Figure 4E). We observed similar results from flow cytometry analysis of S6 phosphorylation (Figure 4F). In addition, the ROShi population from human ES-derived neural cells also had elevated pAkt and pS6 activation (Supplemental Figure 1C).
Pharmacological experiments also support a role for the PI3K pathway. The effects of exogenous ROS on neurosphere formation were abolished by the PI3K inhibitor LY294002 (LY), suggesting that exogenous ROS do not exert their effects by either bypassing the pathway or by providing enough stimulation downstream of PI3K to overcome this inhibition. Since ROS can also mediate effects via activation of the MAPK pathway, we compared the relative effects of LY and the ERK inhibitor U0126 in ROShi and unselected cells (Figure 4G). In both cases, pathway inhibition had a greater effect on the ROShi compared to unselected cells. However, LY had a much greater inhibitory effect on the ROShi cells than the U0126, indicating a greater dependence of these cells on the PI3K pathway, than on the MAPK pathway. Acute LY treatment inhibition significantly decreased endogenous cellular ROS levels (Supplemental Figure 4C) in agreement with our hypothesized pathway for NOX signaling in neural stem cells (Figure 1A).
Cellular ROS levels influence neurogenic potential
Conditional deletion of PTEN results in both enhanced NSC self-renewal and a sustained increase in neurogenesis (Groszer, et al., 2001, 2006; Gregorian et al., 2009). Therefore, we determined whether ROS stimulation of PI3K/Akt signaling had similar effects on neurogenesis. Treatment of clonal density cultures with low, non-toxic levels of exogenous ROS during sphere formation produced significantly higher numbers of neurons as a percentage of total cells when differentiated in the presence of standard conditions (p<0.001; Figure 5A-B). However, treatment of cells with the same exogenous ROS during differentiation resulted in increased cell death and few, if any, neurons were produced (data not shown). Conversely, inhibition of NOX or inhibition of PI3K (LY294002) prior to differentiation significantly reduced neuron numbers (P<0.01; Figure 5A-B). In combination with exogenous ROS stimulation, inhibition of the PI3K pathway (LY294002) eliminated the positive effects of ROS on neurogenesis (p<0.001; Figure 5A-B). In agreement with our data demonstrating that NOX inhibition decreased neurogenesis we found that neurosphere cultures derived from NOX2 mutant mice produced significantly fewer neurons as well (p<0.01; Figure 5C-D).
Figure 5
Figure 5
ROS stimulation during mitogenic expansion enhances neurogenesis in a PI3K-dependent manner
NOX-generated ROS regulates SVZ proliferation and neurogenesis in vivo
We next tested whether our ex vivo findings extend to an in vivo stem cell system. To this end, we tested the effects of the NOX inhibitor apocynin (Apo) on SVZ proliferation. We first assessed the effects of Apo treatment on endogenous ROS levels using the in vivo ROS-sensitive dye, hydroethidine (HEt). Even in control (vehicle-treated) animals, the SVZ had significantly higher ROS levels than surrounding brain tissues such as the striatum and cortex (p<0.01; Figure 6A-C). The SVZ also had approximately 8-fold enriched expression for the NOX2 homologue compared to neighboring cortical tissue (p<0.001; Figure 6B). The 3 week Apo treatment resulted in a significant reduction in SVZ ROS levels (p<0.01; Figure 6A & D) and in the number of Ki67 (proliferative) cells within the SVZ (p<0.02; Figure 6E). Cells acutely dissociated from the SVZ of mice similarly treated with Apo in vivo produced significantly fewer clonal neurospheres in primary cultures compared to vehicle-treated mice (p<0.01; Figure 6F), indicating decreased neural stem or progenitor cell numbers. However, this deficit recovered in subsequent serial clonal passages, demonstrating that although APO administration acutely inhibited proliferation in vivo, the competency for self-renewal in the SVZ-derived cells was not affected.
Figure 6
Figure 6
In vivo inhibition of NADPH oxidase by Apocynin decreases SVZ proliferation, endogenous ROS levels, and NSC self-renewal
Consistent with our observations on apocynin-treated animals, we found that NOX2 mutant mice also had diminished numbers of Ki67 (proliferating) cells within the SVZ compared to wildtype mice (p<0.03; Figure 7A). NOX2 mutant and wild-type mice were pulsed with BrdU followed by a 4 week wash-out period during which time labeled SVZ cells would be expected to leave the SVZ and migrate through the rostral migratory stream to the olfactory bulb where they normally differentiate into post-mitotic neurons. We found that a larger number of BrdU positive cells remained within the SVZ of mutant mice, whilst there were also fewer BrdU+ cells in the olfactory bulb of mutant mice and fewer new neurons (BrdU+/NeuN+) produced there (p<0.01; Figure 7A-B). As a result of this defect in cell proliferation and possibly also in migration we observed that the granule cell layer of the olfactory bulb in mutant mice was smaller than those of wildtype mice (p<0.05; Figure 7C-D).
Figure 7
Figure 7
In vivo SVZ proliferation and neurogenesis are significantly impacted by changes in cellular ROS
Using flow cytometry analysis of acutely dissociated SVZ, we found that the NOX2 mutants have more immature progenitor cells (nestin+ and Sox2+) and fewer cells expressing markers for neuroblasts (DCX) or transit amplifying cells (Mash1 and Dlx2; Figure 7G). Although these data suggest an increase in some progenitor cells, our in vitro findings indicate a diminished capacity for the generation of clonal, serially passagable neurospheres, suggesting a diminished number of neural stem cells in NOX2 mutants. Therefore, the ex vivo cell phenotypes we have observed indicate that there may also be defects in cell maturation and differentiation.
In addition to the negative effects on NSCs caused by decreased NOX activity, we have also conversely demonstrated that increased NOX activity in vivo can have stimulatory effects. Systemic administration of a low, non-toxic dose of the neuroinflammatory stimulus, lipopolysaccharide (LPS), resulted in a significant enhancement in SVZ proliferation (p<0.001; Figure 7E-F) whilst inhibition of NOX activity by Apo co-treatment eliminated the stimulatory effects of LPS on SVZ proliferation (p<0.03; Figure 7E-F). Although neuro-inflammatory cells are likely play a role in this effect in vivo, low dose LPS stimulates NSC self-renewal in vitro which is also blocked by NOX inhibition and antioxidant treatment (Supplemental Figure 5).
Reactive oxygen species regulate neural stem cell function
In the current manuscript we have demonstrated that both exogenous and endogenous ROS can have a significant impact on neural stem and progenitor cell proliferation, self-renewal and neurogenesis. Our observations of the effects of ROS on these cells are surprising for the fact that the neural stem cell compartment appears to be disproportionately dependent on ROS-mediated signaling in the brain. This is not inconsistent with observations by others that embryonic and neural stem cells have enhanced antioxidant capacity compared to more differentiated progeny (Madhavan et al., 2006) as this activity may be a protective mechanism in stem cell populations with active oxidant-mediated signaling to prevent excessive or toxic levels of ROS from being generated. Stem cell populations have been observed to possess an enhanced resistance to oxidative stress-mediated cell death (Madhavan et al., 2006, 2008; Romanko et al., 2004). One such mechanism important for cellular redox regulation could be FOXO proteins. When FOXO genes are deleted from neural stem and progenitor cells, antioxidant defenses are significantly depleted and endogenous ROS levels undergo large increases (Renault et al., 2009; Paik et al., 2009). As a result of this elevated cellular ROS there is an initial hyper-proliferation of NSCs leading to brain overgrowth on par with what has been observed with PTEN deletion in the developing brain. However, toxic levels of ROS build up over time leading to a premature senescence in the cells, suggesting that control of endogenous ROS levels may play a significant role in the regulation of self-renewal and proliferation in neural stem and progenitor cells. Accordingly, Yoneyama et al. (2010) have recently observed that NOX inhibition and antioxidant treatments significantly inhibit hippocampal progenitor proliferation. On the other hand, another recent study has identified a novel ROS-regulating gene, Prdm16, which results in brain undergrowth when deleted (Chuikov, et al., 2010). Prdm16 was identified by the authors as a result of BMI-1 inhibition, which has also been shown to regulate cellular ROS levels in hematopoetic stem cells by specifically altering mitochondrial ROS and not NADPH oxidase-generated ROS (Liu et al., 2009). Thus, the contradictory inhibitory effects of Prdm16-mediated ROS regulation on NSCs may be related to the endogenous source of the ROS and the cellular compartment in which they act.
Previous studies have disagreed on whether stem cells generally have lower or higher endogenous ROS levels than their differentiated progeny (Madhavan et al., 2006; Tsatmali et al, 2005; Limoli et al., 2004; Jang & Sharkis, 2007; Diehn et al., 2009). Definitive NSCs might be expected to have a lower endogenous ROS status than that of the highly proliferative, transit amplifying progenitors because the adult neural stem cell in vivo is thought to be a relatively quiescent cell under normal circumstances (Doetsch, et al., 1997). Thus, the higher ROS status of the SVZ that we observed may play a role in maintaining the proliferation of progenitor cells within this neurogenic niche. However, in order for the stem cell population to maintain a more quiescent state in this environment it would necessitate that they are able to maintain a lower endogenous ROS level when not dividing, suggesting a robust antioxidant regulation in a subset of specialized cells in vivo. Our ex vivo and in vitro data are consistent with high endogenous ROS levels in neural stem cells but could be reflective of an “activated” state in the cells as a result of removal from their normal in vivo environment. In vivo we observed significantly reduced SVZ proliferation and neurogenesis when endogenous ROS levels are reduced in the NOX2 mutants and APO-treated mice. This suggests that in order to maintain normal levels of neurogenesis, the neural stem cells must need to be able to increase ROS levels when required for cell division but does not rule out the possibility that NSCs maintain a low ROS state in vivo when they are in a quiescent state.
The effects of ROS on NSC function are dependent on PI3k/Akt signaling
The most often cited mechanism by which ROS contribute to cellular signaling is by modifying the actions of proteins through the reversible oxidation of essential cysteine residues (Ross et al., 2007; Leslie et al., 2003; Kwon et al., 2004), although other mechanisms have been proposed such as cell cycle targets (cyclin D1 and forkhead proteins) (Abid, et al., 2004; Burch and Heintz, 2005; Blanchetot and Boonstra, 2008). Our data are consistent with a model of post-translational oxidative inactivation of the tumor suppressor PTEN, a negative regulator of PI3K signaling. Although the involvement of other pathways, such as MAPK signaling has not been ruled out, our data suggest a critical role for the PI3K/Akt pathway and are similar to the phenotype observed following genetic deletion of PTEN (Groszer et al., 2001, 2006; Gregorian et al., 2009).
Perhaps more surprising than the stimulatory effects of exogenous ROS, we have found that the inhibition of normal endogenous ROS production by NOX inhibition or mutation negatively regulated the PI3K/Akt pathway and NSC function. Thus, the high ROS status of NSCs appears to be required to maintain their self-renewal and neurogenesis by maintaining adequate levels of PI3K signaling.
The effects of ROS-mediated PI3K pathway signaling are context dependent
Despite the broad influence of ROS-mediated signaling indicated by the stimulatory effects of exogenous ROS and the negative effects of NOX inhibition in neural stem cell-enriched populations, there are many cases in which cellular response to ROS is highly dependent on other factors such as cell phenotype, cell differentiation state, or other signaling co-factors. For example, conditional deletion of PTEN in nestin-expressing neural stem and progenitors in the developing brain and in GFAP-expressing stem cells in the SVZ of the adult brain leads to an enhanced and sustained neural stem cell self-renewal and neurogenesis, contributing to brain overgrowth (Groszner et al. 2001, 2006; Gregorian et al., 2009). However, studies in the haematopoeitc system indicate that although PTEN deletion results in a similar enhancement in self-renewal in haematopoietic stem cells (HSCs), it also results in a premature senescence in these cells (Zhang, et al., 2006; Yilmaz, et al., 2006; Chen et al., 2008). The effects of cellular ROS levels may also be similarly cell type-dependent. For example, HSCs have been shown to have lower endogenous ROS levels than their more differentiated haematopoietic cell counterparts (Jang & Sharkis, 2007).
Previous work has established that O2-A progenitor cells are modulated by changes in cellular redox status, namely that they maintain a low ROS status which promotes cell division and maintains an undifferentiated state (Li et al., 2007; Power et al., 2002; Smith et al., 2000). Li et al., (2007) determined that one mechanism by which higher ROS inhibit O-2A progenitors is through c-Cbl mediated receptor tyrosine kinase (RTK) ubiquitination and breakdown. On the other hand it has recently been shown in other cell types that PTEN deletion prevents c-Cbl mediated RTK breakdown (Vivanco et al., 2010). Therefore, NOX-mediated oxidative inactivation of PTEN should have similar RTK-stabilizing effects. Additionally, EGF signaling activates NOX and is required for aSVZ neurosphere cultures, whereas EGF is not utilized by O2-A progenitors (Kondo and Raff, 2000). Thus phenotypic differences in cell NOX activity and EGF signaling could be important factors in the functional differences we have observed between NSCs and O-2A progenitors.
The effects of ROS are also dependent on the differentiation state of the cells. For example, the neurotrophic factor BDNF promotes differentiation of postmitotic neurons, but we found that in undifferentiated cells in the presence of growth factors that it will promote NSC self-renewal in a NOX- and ROS-dependent manner. Similarly, we found that the effects of exogenous ROS stimulation are dependent on the differentiation state of cells. ROS stimulation of undifferentiated cells in the presence of growth factors promotes both NSC self-renewal and neurogenic potential but, on the other hand, the same levels of ROS which were stimulatory to proliferative cells were found to be toxic to the same cells when present during differentiation following growth factor withdrawal. Consistently, the effect of PTEN deletion is also dependent on the differentiation state of the cells. For example, while PTEN deletion in undifferentiated, mitotic cells produced enhanced NSC proliferation and neurogenesis (Groszer et al., 2001), PTEN deletion in post-mitotic neurons does not influence cell phenotypes or cause cells to re-enter the cell cycle and divide (Kwon et al., 2006). Rather, enhanced PI3K pathway signaling in differentiated neurons results in cellular hypertrophy which can also contribute to a macrocephalic phenotype in vivo (Zhou, et al., 2009).
In conclusion, we have identified a novel redox-mediated regulatory mechanism of self-renewal and differentiation potential which is required for normal neural stem cell function and to support normal ontogeny. However, a large number of environmental factors and genetic mutations can potentially influence and de-regulate ROS-mediated signaling which may contribute to abnormal brain development or transformation and tumorigenesis. Thus, understanding how normal and transformed cells utilize ROS may play an important role in identifying new targets for anti-cancer treatments or points of vulnerability in brain development.
Animals
Unless otherwise specified all experiments were carried out on adult CD1 mice from Charles River, USA. PTEN mutants were generated as described in Groszer et al., 2001 and Gregorian et al. 2009. NADPH oxidase (gp91phox) mutant mice and wild-type controls were obtained from Jackson Labs (USA) and backcrossed onto a CD-1 background. In vivo administration of apocynin (5mg/kg/day; Sigma) was performed by daily intraperitoneal injections for 3 weeks. Lipopolysaccharide (LPS E. Coli serotype 0111:B4; 0.1 mg/kg, Sigma) was administered in vivo via I.P. injection 48 hours prior to perfusion-fixation. In vivo administration of BrdU (50mg/kg/injection) was performed every two hours for 8 hours. BrdU-injected mice were perfused 4 weeks later.
Human and mouse cell culture
Standard high-density neurosphere cultures and clonal density neurosphere assays were established for mouse and human cells according to the methods of Groszer et al., 2006 and Gregorian et al., 2009. See supplemental experimental methods for more detailed information. Exogenous ROS used were hydrogen peroxide (2-4uM H2O2) and glucose oxidase (2uU; GOx). NOX inhibitors used were apocynin (100uM; APO) and diphenylene iodonium (1nM; DPI). The antioxidant N-acetyl-cysteine (1mM; NAC) was also used.
Flow cytometry
The isolation of Lex-, EGFR-positive, and negative cells for clonal analysis was performed using Fluorescent Activated Cell Sorting (FACS) to place one cell per well in 96-well plates. FACS was performed with a FACSDiVa cell sorter (BD Biosciences) using a purification-mode algorithm. Sort gates were set by side and forward scatter to eliminate dead and aggregated cells and by Alexa secondary fluorophores to define positive cells. Purity of the sorted cells was confirmed by flow cytometric re-analysis of positive and negative cell samples.
Stem cell sorting
A combination of live cell sorting for extracellular EGFR and CD24 was performed with a FACSDiVa cell sorter followed by DCFDA dye-labeling, fixation, permeablization, intracellular staining for GFAP, ID1, and flow analysis.
Western Blotting
All primary antibodies (total Akt and phospho-specific Akt) and positive and negative controls were purchased from Cell Signaling Technologies. Neurospheres from each condition were lysed in buffer containing 0.1% triton X-100 in 50 mM Tris-HCl and 150 mM NaCl and Protease Inhibitor Cocktail (Sigma). Samples were prepared according to standard western blot protocol. See supplemental methods for details. Oxidized PTEN was visualized according to the methods of Delgado-Esteban, 2007.
Quantitative real time-PCR (qRT-PCR)
RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's protocol. 1μg of total RNA was treated with 1 Unit of Amplification Grade DNAse I (Sigma-Aldrich) at room temperature for 15 min followed by inactivation at 70 °C for 10 min as described by the manufacturer. See supplemental experimental methods.
Measuring endogenous ROS levels
In cell culture the ROS-sensitive dye DCFDA (5 uM; Molecular Probes), (Hydroethidine, 2uM, Sigma), and (HPF-APF, 5uM, Invitrogen) was used to measure endogenous cellular ROS levels in control and treated cultures as well as in cells from mutant and wild-type animals. In vivo ROS levels were determined using the ROS-sensitive dye, hydroethidine (10 mg/kg; Invitrogen, Kunz et al., 2007). See supplemental experimental methods.
Immunohistochemistry
Perfused-fixed mouse brains were stabilized by incubation in 10% sucrose for 48 hours. Brains were cryo-sectioned at 20uM. Brain sections were immunostained for Ki67 and BrdU according to the methods of Tang et al., 2007. Double-labeling with the neuronal marker NeuN (Abcam 1:200) were carried out on sections. Ten serial sections, spaced 120 μm apart, through the SVZ and olfactory bulb (OB) were quantified with the unbiased optical fractionator approach (Tsai et al., 2006) (StereoInvestigator; MicroBrightField, Colchester, VT). Hoescht counter-stain was used to measure olfactory bulb granule cell layer area using image analysis software (MCID, Imaging Research, St. Catherines, ON, Canada).
Statistical Analysis
All data are expressed as mean ± SEM, unless otherwise indicated. t-tests were performed using Microsoft Excel to determine statistical significance of treatment sets. For multiple comparisons, one- or two-way ANOVA was performed, as appropriate, and Bonferroni post-hoc t-tests were done to determine significance. Alpha values were 0.05 except when adjusted by the post-hoc tests.
Supplementary Material
01
Acknowledgments
This work is supported by the following grants and awards: Cure Autism Now Fellowship (to J.E.L.), Autism Speaks Basic and Clinical grant (to H.K.), Autism Speaks Environmental Sciences grant (to J.E.L.), Center for Autism Research and Treatment (CART) Pilot Grant Award #06LEB2008 which is supported by NIH/NICHD grant # P50-HD-055784 (to J.E.L.), NIH MH65756 (to H.I.K. and H.W.), Henry Singleton Brain Cancer Research Program and James S. McDonnell Foundation Award (to H.W.), Miriam and Sheldon Adelson Program in Neural Repair and Rehabilitation (to H.W. and H.I.K.), University of California, Cancer Research Coordinating Committee grant (to A.D.P.), and the Jonsson Comprehensive Cancer Center grant (to A.D.P.). Flow cytometry and cell sorting was performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility which is supported by National Institutes of Health awards CA-16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, the David Geffen School of Medicine at UCLA, and the UCLA Chancellor's Office. The authors declare no financial conflict of interest that might be construed to influence the results or interpretation of this manuscript.
Footnotes
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