In this study, we performed gene expression profiling of primary astrocytes that exhibited differential effects on the neuronal differentiation of adult NSPCs (13
). In conjunction with our previous study analyzing genes differentially expressed in neurogenic and non-neurogenic regions (17
), we identified candidate genes that potentially affect NSPC lineage determination. Using luciferase reporter assays, we tested the functional effects of a large number of the candidate genes encoding secreted and membrane proteins on adult NSPC differentiation, followed by confirmation using more precise immunofluorescent staining and quantification. We demonstrated that two interleukins, IL-1β and IL-6, and a combination of a group of factors that included these two interleukins could promote NSPC neuronal differentiation, whereas IGFBP6, decorin, and enkephalin inhibited neuronal differentiation of cultured adult NSPCs. These results provide molecular evidence that astrocytes play critical roles in modulating adult NSPC fate choices. The finding that cytokines and chemokines could promote adult NSPC neuronal differentiation might help us to understand how injuries induce neurogenesis in adult brains.
The lack of neurogenesis in adult spinal cord could be due to both the lack of positive regulators and the presence of inhibitory factors. Our findings indicate that at least 3 molecules that were expressed at higher levels in ADS astrocytes or spinal cord tissue had negative effects on the neuronal differentiation of NSPCs: IGFBP-6, decorin and enkephalin. IGF-1 has been shown to promote neurogenesis both in vivo and in cultured NSCs (41
). IGFBPs are a family of proteins that can regulate IGF function both positively and negatively (31
). While IGFBP5, a positive regulator of IGFs, was highly expressed in the DG as opposed to non-neurogenic tissues (17
), IGFBP6 is a negative regulator of IGF signaling with highest specificity to IGF-II (44
). Overexpression of IGFBP6 in astrocytes of transgenic mice (GFAP promoter-IGFBP6 transgenic mice) leads to decreased IGF levels and brain size (44
). The function of IGFBP6 in adult neurogenesis has not been shown previously. In our functional assays, neither IGFBP5 nor IGFs had significant effects on NSPC neuronal differentiation, probably due to the fact that our NSPC culture medium contained a high concentration of insulin. By contrast, IGFBP6 did inhibit neuronal differentiation of adult NSPCs co-cultured with NBH astrocytes in our study, possibly by inhibiting the effect of insulin expressed by NBH astrocytes. Our finding suggests that the high levels of IGFBP6 expressed by ADS astrocytes may contribute, at least partially, to the inhibitory effect of ADS astrocytes on NSPC neuronal differentiation in the adult spinal cord. It will be interesting to know whether GFAP promoter-IGFBP6 transgenic animals have reduced adult hippocampal neurogenesis. In addition, TGFβs have been shown to inhibit cell proliferation but promote differentiation (45
). We previously found that TGFβ2 was expressed at higher levels in neurogenic tissues than in non-neurogenic tissues (17
). However, when we applied TGFβ2 alone to NSPCs, we did not observe any effect on either NSPC proliferation or differentiation, but TGFβ2 was a necessary component of the combined factors (Combo) that exhibited the strongest effects on promoting NSPC neuronal differentiation in our assay. Interestingly, in this study we found that decorin, an extracellular proteoglycan that can bind and inhibit several cytokines including TGFβ2 (33
), was expressed at higher levels in ADS astrocytes. We then confirmed that decorin could significantly inhibit NSPC neuronal differentiation promoted by NBH astrocytes (). Our data suggest that IGF/IGFBPs and TGFβ2/decorin pathways may be involved in regulating NSPC neuronal differentiation in the adult stem cell niche. Additional studies at molecular and cell signaling levels and further in vivo assays will unveil how these two pathways regulate adult neurogenesis.
Traditionally, cytokines and chemokines have been shown to be responsible for damaging neuroinflammation during diseases and CNS injuries (46
). Transgenic mice that chronically overexpress IL-6 under the GFAP promoter (GFAP promoter-IL-6 mice) exhibit CNS damage, with the severity of the damage correlating with levels of IL-6 expression (46
). Cytokines and chemokines can have distinct biological effects when present at different concentrations and in various combinations (47
). On the other hand, recent findings indicate cytokines can also have neuroprotective and regenerative effects (7
). The effects of cytokines on adult neurogenesis have recently triggered great attention because of their potential roles in regenerating damaged adult CNS. To date, experimental evidence supports the hypothesis that cytokines play inhibitory roles in adult neurogenesis. For example, both IL-6 and LIF instruct embryonic NSCs into the astrocyte lineage (49
). GFAP promoter-IL-6 transgenic mice exhibit a 63% reduction in adult hippocampal neurogenesis and significantly reduced neuronal differentiation of NSCs in vivo (14
). IL-6 inhibits RA-initiated neuronal differentiation of adult NSPCs and inflammation blockade restores hippocampal neurogenesis in irradiated adult brains (15
). The role of cytokines in promoting neuronal differentiation of adult CNS stem cells or progenitor cells have not been shown previously. The fact that cultured adult NSPCs express receptors for both IL-1 and IL-6 indicates that these cells are ready to respond to these stimulations. To our surprise, our gene expression analysis indicated that neurogenesis-promoting astrocytes express many cytokines, chemokines and inflammation-related proteins at higher levels than did neurogenesis-inhibitory cells. Specifically, we found that IL-1β and IL-6 promoted NSPC neuronal differentiation, contradicting the current theory that inflammatory cytokines, such as IL-6, inhibit adult neurogenesis and NSPC neuronal differentiation (15
). To address this apparent inconsistency, we performed additional parallel experiments using both our condition and published conditions, and found that in the presence of RA, high levels of IL-6 [50 ng/ml used by Monje et al (15
)] inhibited NSPC neuronal differentiation. However, in the absence of RA, relatively low levels of IL-6 (20 ng/ml, used in this study) promoted neuronal differentiation of adult NSPCs ( and ). Since IL-6, like many cytokines, can have distinct physiological effects at different concentrations and in different biological contexts (47
), the differences between our results and published work could be due to the differences in the amounts and conditions that we used in the experiments. It is also possible that IL-6 modulates adult NSPC fate differently depending on the context. When other neurogenic cues (such as RA) are present, IL-6 inhibits neuronal differentiation of adult NSPCs (15
), but in the absence of other neurogenic cues, IL-6 promotes neuronal differentiation of adult NSPCs (this study). The results of our present study suggest that the effects of cytokines on adult NSPCs are complex and are likely context- and concentration-dependent.
Interestingly, we found that even though TGFβ2, IP-10, Cathepsin S, and VCAM1 did not display any effect on NSPC neuronal differentiation when applied alone, the combination of these factors and IL-1β and IL-6 could significantly promote neuronal differentiation of NSPCs. This is consistent with our finding that IL-1β and IL-6 antibodies partially blocked the effects of co-cultured NBH astrocytes on NSPCs. IP-10 is a chemokine induced by several cytokines and has chemoattractant effects on immune cells such as monocytes and lymphocytes (50
). VCAM-1 is an extracellular matrix adhesion protein that regulates cell-cell interaction and can be induced by both IL-1β and IL-6 during neural inflammation (51
). Cathepsin S belongs to a group of Cysteine proteases of the papain family and is involved in MHC presentation during immune responses. Cathepsin S degrades proteins in the extracellular matrix (52
) and may modulate cell surface receptors and hence cellular functions. The role of these factors that are normally involved in inflammatory or immune responses, to promote neuronal differentiation of NSPCs is unclear. Extensive studies have demonstrated that injuries can induce neurogenesis in adult brain regions that are normally non-neurogenic; however, the underlying mechanism is not clear. Our current findings provide a new perspective on understanding injury-induced neurogenesis by identifying the molecular cues, which will facilitate the understanding of adult neurogenesis and the development of NSC-based therapies. The fact that we did not observe a synergistic effect between these factors and RA suggests that these factors may act through the same pathways as RA to promote NSPC neuronal differentiation. The signaling mechanisms underlying the neuronal differentiation effect of these factors are currently under investigation.
In summary, we have performed the initial identification and functional analysis of a group of positive and negative regulators expressed by astrocytes that can modulate adult NSPC differentiation, and we have provided molecular evidence that astrocytes play critical roles in modulating adult NSPC fate determination. Further mechanistic studies using antibodies and signaling pathway inhibitors will unveil the molecular mechanisms underlying these cytokines regulate NSPC proliferation and fate choice both in normal brains and after injuries.