Thursday, 17 October 2019

Stem Cell Self Renewal

Modulating Adult Neural Stem Cell Self Renewal

Stem Cell

The vascular niche-derived factor PEDF enhances Notch signaling in adult neural stem cells via an unexpected mechanism involving nuclear export of a transcriptional repressor, to promote both proliferation and multipotentiality.

The potential to use neural stem cells (NSCs) for brain repair ‘stems’ from their capacity for self-renewal. According to conventional wisdom, NSCs are restricted to three distinct methods of dividing. They can divide asymmetrically to generate another NSC and a committed cell, in a process that renews the stem cell population, symmetrically to expand the population, or symmetrically to extinguish the population. These modes of division must be balanced to ensure the maintenance of the NSC population, prevent their unrestricted proliferation and limit the premature or inappropriate differentiation of progeny. 

Recent evidence for the close apposition of adult periventricular NSCs and blood vessels 1,2 have confirmed previous findings 3,4 that factors derived from the vasculature contribute to the regulation of the adult NSC pool. Vascular cues must cross-talk and collaborate with other important stem cells regulatory pathways such as Notch signaling 5,6 to determine adult NSC self-renewal and expansion. A new study by Andreu-Agulló et al. The vasculature reveals that derived PEDF 3,4 promotes the Notch signaling dependent renewal of adult periventricular NSCs through an unconventional mechanism.

Specifically, PEDF increased Notch signaling in dividing NSCs in vitro. However, it did not do this by promoting the cleavage of Notch to its transcriptionally active intracellular domain (NICD), but it instead enhanced the ability of NICD, in complex with its partner C promoter–binding factor 1 (CBF-1), to initiate transcription. Here, they found that PEDF activated a Hes1-luciferase reporter without increasing NICD concentration, but this still required the presence and activity of both NICD and CBF1.

Andreu-Agulló et al. next sought to identify precisely which cells in the adult periventricular area in vivo were responsive to PEDF. Because of their quiescent nature, adult NSCs retain labels incorporated during division (such as the thymidine analog BrdU) for extended periods of time, and these label-retaining cells have been commonly equated with adult NSCs 8. In mice engineered to express an enhanced green fluorescent protein (EGFP) under the control of four CBF1- responsive elements found that proliferating label-retaining cells could be neatly classified into EGFP-high (high Notch signaling) and EGFP-low (low Notch signaling) subpopulations of approximately equal size. Administration of PEDF into the lateral ventricle markedly increased the proportion of EGFP-high label-retaining cells at the expense of the EGFP-low population.

If one were to stop here and assume that all label-retaining cells are NSCs, one might conclude that PEDF promotes the symmetrical division of NSCs with high levels of Notch signaling, generating more of these cells at the expense of NSCs with low levels of Notch signaling. However, subsequent experiments suggested a more interesting conclusion.

Sorting adult periventricular cells for high or low EGFP expression revealed that EGFP-high cells generated larger neurospheres than EGFP-low cells. Neurospheres are clusters of undifferentiated cells that are the products of division commonly used to retrospectively identify NSCs. EGFP- high–derived neurospheres maintained higher subcloning efficiency over multiple passages and contained a higher percentage of multipotent cells than EGFP-low–derived neurospheres. PEDF treatment of EGFP- low cells increased their capacity to generate multipotent primary neurospheres and these neurospheres were able to generate more secondary or tertiary neurospheres even in the absence of PEDF, suggesting that PEDF increased their self-renewal capacity. PEDF did not increase the size of primary EGFP- high–derived neurospheres, but it enhanced the proportion of progeny with high CBF1 activity and their ability to generate secondary or tertiary neurospheres.

These data suggest that PEDF enhances Notch signaling by increasing CBF1 activity. How does PEDF promote CBF1 activity? The answer turned out to be complicated. Immunostaining dissociated cultures of adult periventricular cells for NICD, Andreu-Agulló et al. 7 found that 24 h after plating, 45% of cell pairs (generated by a single-cell division) consisted of one cell expressing high levels of NICD and one with low levels of NICD. PEDF did not increase the proportion of NICD-high cell pairs, indicating that it does not increase self-renewal by promoting symmetric cell divisions in vitro. 

Instead, PEDF increased the proportion of NICD-high/low cell pairs in which both cells expressed high levels of epidermal growth factor receptor (EGFR); EGF is a mitogen for adult periventricular NSCs 9. Chromatin immunoprecipitation and luciferase assays identified the Egfr promoter as a target of Notch signaling. Thus, PEDF promotes the renewal of adult NSCs expressing low levels of NICD by increasing the proportion that is responsive to mitogenic EGF signaling.

Stem Cell Self Renewal

The nuclear receptor co-repressor (N-CoR) suppresses Notch-induced astrogliogenesis in the developing brain 10. Andreu-Agulló et al. 7 asked whether N-CoR was involved in PEDF- induced maintenance of CBF-1 activity and the subsequent upregulation of EGFR. Chromatin immunoprecipitation revealed that N-CoR bound to a CBF1-binding element in the Egfr promoter and was displaced from this element by PEDF treatment. Furthermore, overexpression of full-length N-CoR abrogated PEDF-induced activation of Hes-1 or EGFR- luciferase reporters, whereas a repression-dead N-CoR had no effect. 

It also observed that PEDF treatment decreased nuclear N-CoR and increased its cytoplasmic localization. The PEDF-induced nuclear export of N-CoR and subsequent increase in neurosphere formation were blocked by overexpression of p65–nuclear factor κB (NF-κB) lacking the nuclear export signal and transactivation domain. Overexpression of a p65 subunit lacking only the transactivation domain did not reduce PEDF increases in neurosphere formation. Together, these in vitro data suggest that p65-mediated export of N-CoR from the nucleus is responsible for the increase in CBF1 activity in NSCs after PEDF treatment.

Notably, Andreu-Agulló et al. 7 confirmed these findings in vivo. PEDF reduced nuclear expression of N-CoR, but increased EGFR expression in label-retaining cells; a C-terminal PEDF fragment that antagonized PEDF signaling did the reverse. Thus, PEDF activates NF-κB signaling in adult periventricular NSCs, which results in p65-mediated nuclear export of N-CoR away from CBF1 binding sites, allowing the CBF1-NICD complex to initiate transcription of Hes1 and EGFR. Ultimately, this increases the capacity of PEDF-stimulated NSCs with low levels of Notch signaling to proliferate and generate multiple cell types.

Stem Cell Self Renewal

Andreu-Agulló et al. 7 have thus elucidated the pathways used by PEDF to regulate adult periventricular NSC function. The identity of the receptor that initiates PEDF signaling, however, remains unknown. Pharmacological inhibition of the only known putative PEDF receptor, adipose triglyceride lipase 11, did not affect PEDF actions on adult periventricular NSCs. If an expression of the PEDF receptor is restricted to adult periventricular NSCs, its use as a prospective marker would substantially aid in their study.

The interpretation of PEDF’s role in regulating adult NSC function, however, could depend on which population of label-retaining cells one selects as the ‘true’ stem cell. Presuming that NSCs with high levels of Notch signaling and EGFR are at the top of the hierarchy might mean that PEDF functions to extend the cell- generating capacity of NSCs with low levels of Notch signaling. 

Alternatively, one could assume that label-retaining cells with low Notch signaling and EGFR expression are the quiescent NSCs at the top of the hierarchy; in this case, PEDF would function to activate them. Indeed, intraventricular infusion of PEDF promotes NSC division, whereas inhibition of PEDF signaling decreases it. Furthermore, injury-mediated activation of adult periventricular NSCs increases their expression of EGFR 12 and Notch signaling.

It may also be more reasonable for a quiescent non-EGFR expressing NSC to progress to an active EGFR-expressing NSC, initiated in part by PEDF, to generate transit-amplifying cells known to express EGFR 12. The inability of NSCs with low levels of NICD to generate NSCs with high levels of NICD in vitro could simply be a cell culture limitation and their decreased capacity to generate neurospheres could be a reflection of their quiescent state.

Tracing the fate of NSCs expressing high or low levels of NICD/ EGFR in vivo may provide some insight into the future. Because EGFR is a direct target of Notch signaling, quiescent NSCs may need shielding from excessive Notch signaling, as increasing levels of EGFR and EGF signaling have been demonstrated to inhibit neural precursor proliferation and promote astrogliogenesis.

In this regard, it is worth noting that Numb physically interacts with NICD to inhibit Notch signaling, but, paradoxically, it has been found to maintain the neural precursor pool during development 15 .Whether Numb and PEDF signaling interact and have opposing roles in regulating adult NSC quiescence or self-renewal would also be an interesting avenue for future investigations.

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