FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis

Abstract

The PI3K-AKT-FoxO pathway is integral to lifespan regulation in lower organisms and essential for the stability of long-lived cells in mammals. Here, we report the impact of combined FoxO1, 3, and 4 deficiencies on mammalian brain physiology with a particular emphasis on the study of the neural stem/progenitor cell (NSC) pool. We show that the FoxO family plays a prominent role in NSC proliferation and renewal. FoxO-deficient mice show initial increased brain size and proliferation of neural progenitor cells during early postnatal life, followed by precocious significant decline in the NSC pool and accompanying neurogenesis in adult brains. Mechanistically, integrated transcriptomic, promoter, and functional analyses of FoxO-deficient NSC cultures identified direct gene targets with known links to the regulation of human brain size and the control of cellular proliferation, differentiation, and oxidative defense. Thus, the FoxO family coordinately regulates diverse genes and pathways to govern key aspects of NSC homeostasis in the mammalian brain.

Fig. 1

Increased brain size and cycling of NSC in FoxO null mice and their derived culture. (A) Representative photograph of brains from 20 week old FoxO WT (left) and null (right) mice. (B) The number of glia and neorons was estimated by counting S100 or NeuN-positive cells in the cerebral cortex of 10 day old FoxO WT (■) and null () pups. There are more S100 or NeuN-positive cells in FoxO null brain compared with the control (*, p<0.1). ** indicates the region where S100+ and NeuN+ cells were scored. hGFAP-cre mice were crossed with ROSA26R mice, where Cre-mediated recombination drives the constitutive expression of the ß-galactosidase. X-gal staining positice cells are shown in blue. (C) Increase in cell cycle re-entry of FoxO null SVZ cells. Young mice (P8) were single-pulse labeled with BrdU 24hrs prior to the sacrifice and brain sections were stained for BrdU (red), Ki67 (green), and Sox2 (cyan). The fraction of Sox2 positive cells re-entered cell cycle (open arrow, BrdU+/Ki67+/Sox2+ triple positive) increased in FoxO null SVZ and that of no longer dividing (filled arrow, BrdU+/Ki67-/Sox2+) is higher in WT SVZ. Percent mean ±s.d. of Ki67+ cells from BrdU+/Sox2+ cells from 10 WT and 6 FoxO null mice is shown ( *, p<0.001 by two tail t-test). Bar=40μm (D) Ki67 positive NSC of FoxO WT and null cultures (n=5, P2). Bar=20μm.

Fig. 2

FoxO deficient brain exhibits decrease in NSC reserve and neurogenesis. (A) Decreased Sox2 positive NSC in 32 week old FoxO null (right) brain. Bar=50μm. (B) Decline in neural progenitor proliferation and neurogenesis measured by Ki67 and DCX expression in SVZ of lateral wall from 32 week old FoxO null mice (right). 200x microscopic field of comparable region from WT littermate control mouse is shown (left). Insets are higher magnification view of boxed regions. Bar=50μm. Sox2 (A), DCX (B), Ki67(B) expression levels in young (8 week old, (■)) and relatively old (15-32 week old, ()) mice SVZ were measured by laser scanning and plotted (4 pairs, *, p=0.004, **, p=0.0039, ‡, p=0.026, by two tail paired t-test). Decreased self-renewal capacity of NSC derived from 18-22 week old FoxO null mice (C) and acutely deleted for FoxOs in vitro (D). For conditional deletion NSC were isolated from SVZ of 4 week old Rosa26-CreERT2⁺ (null) or ^-(WT) FoxO1/3/4^L/L mice and treated with 400nM 4OHT prior to the assay. Results are representative of three independent experiments with two primary cultures. Both average diameters for all the neurospheres and for multipotential ones are shown as mean±s.d. Proliferation index is shown as fraction of BrdU incorporation from triplicate cultures in two independent experiments. §, p=0.0043, #, p<0.0001, *, p=0.01, **, p=0.03, †, p=0.06, ‡, p=0.096 by two tail t-test. Bar= 100 μm.

Fig. 3

Regulation of NSC self-renewal by FoxO through controlling intracellular ROS level. (A) Accumulation of intracellular ROS in FoxO null NSC (P7) is measured by DCF-DA staining and flowcytometry. Numbers indicate mean intensity level (A) and % of total event above the threshold (C). Both NAC treatment (1mM) and enforced expression of SESN3 normalized and suppressed ROS level (A, C) and partially rescued self renewal in FoxO null NSC (B, D). Expression of SESN3-v5 in NSC is shown below by immunobloting of v5 epitope (C). (B, D) Representative images of cultured neurospheres from two independent experiments are shown. Both average diameters for all the neurospheres and for multipotential ones are shown as mean±s.d. Proliferation index is shown as fraction of BrdU incorporation from triplicate cultures in two independent experiments. *, p<0.01, †, p< 0.05 by ANOVA with Bonferroni's post test. Bar= 100 μm

Fig 4

Suppression of ROS does not inhibit hyper-proliferation of FoxO null NSC. (A) Proliferation of FoxO WT (blue line) and null (red line) NSC (Rosa26-CreERT2⁺ and ^-:FoxO1/3/4^L/L respectively) over the 8 passages after 4OHT treatment is plotted. Y axis indicates log₂ (cells counted after 5 days in culture/cells seeded). Result is representative of three independent cultures (*, p=0.0047; **, p=0.015). (B) NAC treatment does not attenuate proliferation of FoxO null NSC. P2 NSC were treated with up to 5mM NAC for 24hr and fraction of BrdU+ cells was plotted. Bar=30μm (C) NAC treatment does not inhibit increased cycling of Sox2+ cells in 8 day old FoxO null brain. 3 day old FoxO null mice were treated with NAC for 5 days before sacrificed. Percent mean ±s.d. of Ki67+ cells from BrdU+/Sox2+ cells from NAC (n=6) or water (n=6) treated FoxO null mice is shown (p=0.08 by two tail t-test). (D) Increased apoptosis in late passage (P8) FoxO null NSC was reversed by 1mM NAC treatment. No apparent changes in apoptosis were observed in early passage (P2). Cleaved caspase3 staining positive cells were scored and normalized by total number of DAPI positive cells. Fractions of apoptotic cells are plotted. *, p=0.017, **, p=0.021.

Fig.5

FoxO-dependent expression of ASPM regulates NSC proliferation. Differential expression of ASPM mRNA in FoxO WT and null embryonic NSC measured by rtqPCR (A) and protein by IHC analysis (B) on FoxO WT and null E15.5 embryonic brain. Bar=50μm. (C) Number of mitotic spindle-localized ASPM positive NSC in FoxO WT and null cultures. Representative images are shown. Bar=10μm. (D) The ASPM promoter is occupied by both FoxO1 and FoxO3 in NSC. On the schematic representation of the ASPM promoter FoxO binding sites are depicted relative to the +1 transcriptional start site. ChIP analysis of NSC with FoxO1, FoxO3, IgG, or PolII antibodies. Primers were designed to encompass the FoxO binding sites. (E) Knockdown of ASPM by shRNAs in FoxO null NSC. ASPM mRNA level was measured by rtqPCR after 48hr of shASPM lentiviral transduction. Decreased proliferative potential in ASPM knockdown NSC was measured by BrdU incorporation assay (F) and neurosphere formation (G) *, p<0.01, #, p<0.05 by ANOVA with Bonferroni's post test. NA, not analyzed, NT, non targeting control shRNA infected culture. Bar=100μm.

Fig. 6

FoxOs negatively regulate Wnt signaling in NSC. (A) Expression of soluble antagonists of Wnt. Differential expression of sFRP1/2 and SOST mRNA in FoxO WT and null embryonic NSC measured by rtqPCR. (B) Promoters for sFRP1/2 and SOST are occupied by both FoxO1 and FoxO3 in NSC. On the schematic representation of the promoter FoxO binding sites are depicted relative to the +1 transcriptional start site. ChIP analysis of NSC with FoxO1, FoxO3, IgG, or PolII antibodies. Quantification of ChIP analysis on each promoter was assessed by rtqPCR analysis. (C) Wnt3a-induced canonical Wnt signaling is measured TOFlash reporter assay. Note enhanced signal in FoxO null () NSC compared to WT (■) . *, p=0.08053. (D) Attenuated Wnt3a-dependent canonical signaling by constitutively active FoxOs. CA1, CA3; FoxO1-ADA and FoxO3-AAA mutant respectively. *, p<0.1. (E) Enhanced canonical signaling in sFRP1/2 and SOST knockdown NSC (■) compared to WT () is reversed by the addition of soluble sFRP1/2 and SOST (500ng/ml). *, p=0.068; **, p<0.005 (F) Exogenously added sFRP1/2 and SOST (500ng/ml) reversed increased proliferation of FoxO null NSC. Wnt3a, 50ng/ml of recombinant wnt3a was added for 24hrs. KD, 72 hrs after knockdown by siRNAs for sFRP1/2 and SOST. Fractions of Ki67 positive nuclei are plotted. (G) Decreased long term proliferation potential of NSC by Wnt3a stimulation (100ng/ml). Y axis indicates log₂ (cells counted after 5 days in culture/cells seeded). Lines in red, FoxO null; blue, WT; yellow, Wnt3a treated. *, p=0.0041; **, p<0.01; #, p=0.0169.

Fig. 7

Model for FoxO-dependent regulation of NSC. FoxOs control NSC biology at multiple levels. FoxOs (A) negatively regulate G0 exit of NSC and keep their quiescence within the niche, (B) support their long-term self-renewal activity, (C) constrain proliferation of lineage-committed neural progenitors, and (D) contribute to continued neurogenesis.