miR-34a is a well-known miRNA involved in various cellular functions that acts as a tumor suppressor 
. In this study, we demonstrated a novel function for miR-34a during neuronal differentiation, implicating its expression in the appearance of postmitotic neurons and on the regulation of neurite elongation via SIRT1 downregulation. In addition, its function was also associated with increased levels of acetylated p53 and p53-DNA binding activity, in common with neural differentiation.
We previously determined that miR-34a expression was concomitant with the appearance of postmitotic neurons and astrocytes during mouse NS cell differentiation 
. Here we show that miR-34a promoted the appearance of postmitotic neurons. This is in agreement with previous observations that miR-34a can suppress cell-cycle genes and induce a neural phenotype 
. miR-34a may have a wide range of molecular targets during cell differentiation. Our results showed that the specific effect of miR-34a on the appearance of postmitotic neurons was not mediated by SIRT1. Rather, miR-34a-targets, such as CDK4 and CDK6, as well as MEK1, which regulate cell cycle progression and inhibit cell proliferation, may constitute potential mediators of miR-34a effect on postmitotic neurons. In addition, miR-34a can also regulate Notch-1 and Notch-2 protein expression 
, which could contribute to the observed final outcome.
Here we report an additional effect of miR-34a on neurite elongation. Specifically, overexpression of miR-34a resulted in increased total neurite output. The fact that a similar phenotype was observed after SIRT1 knockdown suggests that this effect might be mediated by SIRT1. However, a recent publication reported that miR-34a expression was not involved in SIRT1 downregulation during human embryonic stem cell differentiation 
. In fact, the authors observed no increase in miR-34a expression after induction of differentiation into embryonic bodies. Differences associated with specific properties of each biological model may explain this discrepancy; in addition to different mechanisms that regulate SIRT1 expression levels. Moreover, the effect of siRNA-mediated silencing of SIRT1 on neurite number/output was more pronounced than the effect of miR-34a overexpression. A possible explanation may rely on the fact that miRNAs usually function as a fine-tuning mechanism, and not as an on/off switch. In this scenario, siRNA transfection would result in increased SIRT1 downregulation when compared with miR-34a overexpression.
SIRT1 expression was shown to decrease during mouse NS cell differentiation. A similar pattern of expression was already reported for the differentiation of human embryonic stem cells 
, suggesting that decreased SIRT1 expression is a prerequisite for successful differentiation. In accordance, SIRT1 downregulation was shown to be necessary for reactivation of key developmental genes, such as the neuroretinal morphogenesis effector delta-like protein 4 (DLL4), the T-box transcription factor (TBX3), and paired box gene 6 (PAX6) 
Our observation that SIRT1 is expressed in β-III Tubulin+
cells at two days of differentiation suggest that SIRT1 is may be necessary in the initial stages of the differentiation process. In agreement, SIRT1 has been shown to positively regulate neuronal differentiation by inhibiting the transactivation of Hes1, a transcription factor that negatively controls cell differentiation 
. In addition, loss of SIRT1 in embryonic stem cells was shown to delay the switch-off of Oct4
expression, thereby reducing the capacity to exit the pluripotent stem cell program 
. This suggests that a tight regulation of SIRT1 expression might exist and that timing and levels of expression may determine commitment toward a certain neural phenotype.
miR-34a appears to be a key player parting the p53 regulatory network 
. miR-34a transcription is directly activated by p53, and in turn miR-34a regulates the expression of some p53 target genes 
. Nevertheless, transfection with p53 siRNA did not affect miR-34a expression, suggesting that induction of miR-34a during mouse NS cell differentiation is p53 independent (data not shown). These results are in accordance with previous data showing a p53-independent role for miR-34a during megakaryocytic differentiation of K562 cells 
. Instead, p53 seems to act downstream of miR-34a in this cellular context. Our results suggested that miR-34a indirectly regulates p53, possibly through a SIRT1-dependent mechanism. Numerous studies indicated that SIRT1 plays a crucial role in p53-mediating responses by deacetylating human p53 at lysine 382 (K379 in mouse p53) in the C-terminal domain, which attenuates its transcriptional activity 
. Based on this, downregulation of SIRT1 mediated by miR-34a would result in increased levels of acetylated p53. The acetylated form has been reported to have increased transcriptional activity, and promote coactivator recruitment and site-specific DNA binding 
In fact, p53 was previously shown to be necessary for neurite outgrowth and axonal regeneration, which required specific acetylation of p53 
. Further, SIRT1-mediated regulation of p53 might also contribute to the induction of differentiation, since SIRT1 was shown to inhibit p53-mediated suppression of Nanog
expression, which is required to maintain cells in an undifferentiated status 
. Several reports support a role for p53 in neural differentiation 
, although the precise mechanism(s) remain unclear. It may involve the ability of p53 to promote cell cycle arrest by inducing p21
. Alternatively, p53 could directly regulate the transcription of neuronal genes. By genome-wide chromatin immunoprecipitation (ChIP), new putative p53 target genes have been identified during NGF-mediated PC12 neuronal differentiation. These include wnt7b
, which is involved in dendritic development, and the tfcp214
/grhl3 grainyhead homolog, implicated in ectodermal development 
. Further, p53 regulates the expression of both actin-binding protein Coronin 1b and the GTPase Rab13, which are required for physiological neurite outgrowth in PC12 cells and dorsal root ganglion neurons 
. Nevertheless, while the present study provides a foundation for the role of miR-34a in neural stem cell differentiation, the role of p53 in this pathway remains unclear.
Our results also demonstrate that SIRT1 expression positively regulates astrogliogenesis. In fact, it has been previously shown that increased SIRT1 activity causes differentiation of neural progenitor cells into astrocytes at the expense of neurons 
. SIRT1 represses the pro-neuronal basic helix-loop-helix (bHLH) transcription factor Mash1 and influences cell-fate decision. This indicates that SIRT1 might play a distinct role on differentiation under specific conditions, according to substrate and co-factor availability, as well as redox state of the cell.
We found that transient transfection of miR-34a into mouse NS cells had almost no effect on the astrocytic subpopulation. This is in accordance with previous studies showing that miR-34a overexpression did not affect human astrocytes 
. Curiously, miR-34a was shown to positively regulate the proportion of GFAP+
cells when cells were pre-treated with resveratrol. Given that miR-34a negatively regulates SIRT1 expression and SIRT1 induces the astroglial lineage, we would have expected an opposite effect of miR-34a in GFAP+
cells. This suggests that under certain conditions, miR-34a may have the capacity to influence commitment toward astrogliogenesis by a SIRT1-independent mechanism. This data might be particularly important in the specific case of gliomas, since miR-34a has been extensively studied as a therapeutic target for tumor suppression.
It is thought that miRNAs may act as a buffering system, modulating molecules whenever cell balance is lost. Recent findings indicate that simple inactivation of miRNAs is not sufficient to collapse the signaling networks in which miRNAs are involved, and suggest that the use of sensitized backgrounds provides an efficient approach to study miRNA function analysis 
. Therefore, it is possible that the particular environment generated by resveratrol treatment would render the cells sensitive to the modulation of miR-34a, revealing an additional miR-34a function, i.e. the capacity to regulate the glial lineage. Resveratrol is a naturally occurring polyphenol with antioxidant, anti-inflammatory, anti-aging, cardioprotective, and neuroprotective activities 
. Multiple biochemical and molecular actions seem to contribute to its effects including inhibition of cyclooxygenase (COX) 
, cytochromes P450, cell invasion, transformation, and angiogenesis 
Resveratrol has been shown to up-regulate antioxidant enzymes such as glutathione peroxidase, catalase, and quinone reductase. It inhibits lipid peroxidation, ornithine decarboxylase (ODC), protein kinases, and cellular, proliferation 
and was shown to inhibit inflammatory processes, including NF-κB activation and inflammatory gene expression 
. Resveratrol effectively induces apoptosis modulated through multiple pathways including up-regulation of p53, activation of caspases, modulation of Bcl-2 family proteins, inhibition of D-type cyclins, and interference with NF-κB and AP-1 mediated cascades 
. Therefore, multiple targets could contribute to its effects in astrogliogenesis induced by miR-34a. Additional studies, possibly using genetic backgrounds with reduced activity of regulatory pathways modulated by resveratrol would be necessary to confirm the capacity and the mechanism of miR-34a to regulate astrogliogenesis.
In conclusion, our results support a role for miR-34a in the regulation of neural differentiation. In addition, miR-34a-mediated silencing of SIRT1 may be necessary for correct establishment of specific differentiation programs during neural stem cell differentiation.