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. 2016 Jul 26;113(30):8514-9.
doi: 10.1073/pnas.1607079113. Epub 2016 Jul 11.

FoxO3 Regulates Neuronal Reprogramming of Cells From Postnatal and Aging Mice

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Free PMC article

FoxO3 Regulates Neuronal Reprogramming of Cells From Postnatal and Aging Mice

Henrik Ahlenius et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.

Keywords: aging; induced neuronal cells; reprogramming.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Fibroblasts from postnatal and adult mice are less susceptible to iN cell reprogramming than MEFs. (A) Experimental approach (Left) and image of iN cells (Right) generated from TTFs of a 17-mo-old (17M) Tau-EGFP animal. Micrograph showing Tau-EGFP epifluorescence (green) and immunofluorescence detection with Tuj1 antibodies (red). (Scale bar, 30 μm.) (B) Sample traces of spontaneous AP firing (i), current-pulse–induced AP (ii), and voltage-gated Na+ and K+ currents (iii) recorded from TTF iN cells reprogrammed from a 20-mo-old animal (20M TTF-iN cell). Insets in red, magnified view of corresponding boxed area. (C) Representative images (Left) for Tuj1 (green) and Map2 (red) immunoreactivity of TTF iN cells generated from embryonic (MEF), postnatal (4 d old; 4D), adult (3 mo old; 3M), middle aged (10 and 15 mo old; 10M and 15M, respectively) and aged (25 mo old; 25M) animals, 3 wk after induction. (Scale bar, 50 μm.) (D) Average fractions of Tuj1+ (Top Right) and Map2+ (Bottom Right) cells 3 wk after infection of initially plated cells. Data are presented as means ± SEM (n = 3–5 experiments with three technical replicates each). Significance was determined by using one-way ANOVA with Bonferroni post hoc test (*P < 0.05; **P < 0.01; ***P < 0.005; ns, not significant).
Fig. S1.
Fig. S1.
Slower and incomplete reprogramming in aging fibroblasts (related to Fig. 1). Example images represent Tuj1 (green) and Map2 (red) immunoreactivity and nuclear stain (DAPI, blue) of iN cells derived from embryonic (MEF; Left), postnatal (4D; Second Left), adult (3M; Second Right) and aged (25M; Right) TTF at 1 wk (1W; Top), 2 wk (2W; Middle), and 3 wk (3W; Bottom) after doxycycline-mediated transgene induction. Note that the iN cells generated from postnatal, adult, and aged fibroblasts show limited neurite arborization at early timepoints, whereas MEF-iN cells display elaborate neuronal morphology already at 1 and 2 wk after doxycycline treatment. (Scale bar, 50 μm.)
Fig. 2.
Fig. 2.
iN cells derived from TTFs are functionally less mature than iN cells derived from MEFs. (A) Example traces of AP generation by TTF iN cells derived from animals of different ages (MEF, 4D, 3M, 10M, 15M, and 25M), as recorded in current-clamp mode, 3 wk after BAM transduction. Step-current (black) induced AP patterns: multiple AP (green), single AP (blue), or failure to generate AP (red). Pie charts indicate fraction of cells with corresponding firing patterns. n = number of cells with qualitatively similar AP firing property/total number of patched cells with elaborate neuronal morphology. (B) Average values of the intrinsic membrane properties of TTF iN cells from different ages (y axis) plotted as mean ± SEM. Parameters (from left to right) include resting membrane-potential (Vrest), membrane resistance (Rm), capacitance (Cm), AP height (APh), and AP threshold (APt). Asterisks indicate significant differences (n = numbers indicated on bar graphs, *P < 0.05, Student’s t test) from corresponding embryonic condition (black dotted line). No significant differences were found between 3M (red dotted line) and older age groups for any parameter tested. (C) Spontaneous AMPAR-mediated EPSCs, as probed by voltage-clamp recordings from MEF (black), 4D (green), 3M (red), and 25M (blue) TTF iN cells. Pie charts represent ratio of cells with (gray fraction) or without (white fraction) synaptic response (i). Sample traces of spontaneous EPSCs recorded (ii), and average values of EPSC amplitude (iii), and frequency (iv) presented as mean ± SEM (n = numbers associated with bar graphs). Asterisks, significant difference (*P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test), compared with the MEF condition (dotted line). (D) Evoked AMPAR-mediated EPSCs recorded from MEF iN cells (black), and iN cells derived from 4D (green), 3M (red), and 25M (blue) animals. Example traces (i) and average EPSC amplitudes (mean ± SEM) (ii) of each condition are plotted. Asterisks indicate significant difference (n = indicated on bar graphs, **P < 0.01, ***P < 0.005, Student’s t test) between MEF and aging conditions.
Fig. S2.
Fig. S2.
Patch-clamp configuration for postsynaptic recording (related to Fig. 2). TTF-derived iN cells were additionally infected with lentivirus expressing GFP and cocultured with low-density mouse primary hippocampal neurons for 3 wk to allow them to form synaptic connections. Left, GFP fluorescence view; Center, bright-field; and Right, as both views merged. Rec, recording electrode, placed on a GFP+ TTF-iN cell. Black arrowheads (Center), non-GFP primary neurons. (Scale bars, 10 μm.)
Fig. S3.
Fig. S3.
Similar morphological maturation of iN cells derived from aging fibroblasts (related to Fig. 2). Example images of Tuj1-immunopositive iN cells (Upper Left) overlaid with trace (Lower Left) and quantification (Right) of total neurite length (using NeuronJ) of iN cells derived from differently aged fibroblasts, 3W after BAM transduction. (Scale bar, 50 μm.) Average values represented as means ± SEM, (n ≥ 10 cells, three independent experiments per condition). Significance was determined by using one-way ANOVA with Bonnferoni post hoc test. ns, not significant.
Fig. 3.
Fig. 3.
Aging-associated features in donor fibroblasts. (A) Representative images indicating age-dependent decrease of EdU incorporation (EdU staining in red, Top) and increase in senescence associated β-Gal activity (black staining, Bottom) in fibroblasts from different age groups (MEFs, 4D, 3M, and 25M, Left to Right) counterstained with DAPI (blue). (B) Average bar graphs indicate means ± SEM of percentages of EdU+ (i), SA-β-Gal+ (ii) fibroblasts and average relative mRNA levels for senescence markers p16 (iii) and p19 (iv) measured by qRT-PCR. Asterisks indicate significant difference (n = 3 independent batches; *P < 0.05; **P < 0.01; ***P < 0.005; ns, not significant; ANOVA with Bonferroni post hoc test). (C) Representative images showing lentivirus-mediated EGFP expression (green) and DAPI (blue) staining in MEF, 4D, 3M, and 25M TTF. (D) Average plating efficiency (DAPI staining/field of view, normalized to MEF condition; i) and infection efficieny (percentage of EGFP+/DAPI-stained cells; ii) calculated for MEF, 4D, 3M, and 25M TTF (left to right). Bar graphs represent mean ± SEM, and statistical significance were calculated by using ANOVA with Bonferroni post hoc test (n = 3 batches, *P < 0.05, **P < 0.01; ns, not significant). (Scale bars, 20 μm.)
Fig. 4.
Fig. 4.
Loss of FoxO3 improves reprogramming efficiency and functional maturation of iN cells derived from TTFs but not from MEFs. (A) Tuj1-immunoreactive, traced MEF iN cells derived in control condition (infected with BAM factors only, BAM; Upper) or in the presence of FoxO3 transcription factor (infected with BAM factors + FoxO3, BAMF; Lower). Bar graphs represent average values (means ± SEM, n = 3 batches, *P < 0.05, ANOVA with Bonferroni post hoc test) of cell number and relative neurite length for each condition, as indicated. (B) Map2-immunoreactive MEF iN cells derived from WT (Upper) and FoxO3−/− (KO; Lower) littermate animals. Average percentages are means ± SEM, n = 3 experiments (*P < 0.05, ANOVA with Bonferoni post hoc test). (C) Same as B, except for TTF iN cells derived from 3M (Left) and 20M (Right) old WT (Upper) or FoxO3 KO (Lower) animals. (D) Representative images (Left) of Tuj1+ iN cells derived from 3M TTF, in the presence of shRNA hairpins for scrambled control (Sh Ctrl; Upper) or FoxO3 (Sh FoxO3; Lower). Average numbers of Tuj1+ cells are plotted as means ± SEM (Right) for each condition, n = 3 technical replicates (*P < 0.05, ANOVA with Bonferroni post hoc test). (E) Example traces of APs recorded from iN cells derived from MEF (Left), 3M (Center), and 20–27 M (Right) TTF of WT (Upper, black) or KO (Lower, red) mice. (F) Percentages (means ± SEM) of iN cells with no (Failure), single, or multiple APs recorded from MEF (Left), 3M (Center), and 20–27 M TTFs (Right). Asterisks indicate significant difference (n = 3 independent batches, *P < 0.05, Student’s t test) for pairwise comparison (dotted lines). ns, not significant. (G) Average values of the Vrest, AP height, and AP threshold of iN cells derived from MEF, 3M, and 20–27M TTFs (n = 21, 21, and 43, respectively, for each genotype). Asterisks, significant difference (*P < 0.05, ***P < 0.005), ns, not significant, Student’s t test. (Scale bars: 50 μm.)
Fig. S4.
Fig. S4.
Relative expression of FoxO genes in fibroblasts (related to Fig. 4). (A) Table includes primer sequences designed for qRT-PCR assays of corresponding FoxO genes and their predicted product sizes. Gapdh was used as loading control. (B) Relative expression (normalized to Gapdh) values are plotted as means ± SEM (n = 3 independent batches) for FoxO1, FoxO3, FoxO4, and FoxO6 (left to right, respectively), from WT (black) and FoxO3 KO (red) fibroblasts of embryonic (MEF), 4-d-old (4D) and 3-mo-old (3M) animals. Statistical significance (*P < 0.05; ***P < 0.005; ns; P > 0.05) between WT and KO conditions (dotted lines, black) were performed by using unpaired, one-tailed, Student’s t test. (C) The qRT-PCR products of WT and FoxO3 KO MEFs from B were run on 1% agarose gel, which generated single bands for each primer pair (Gapdh, FoxO1, FoxO3, FoxO4, and FoxO6; from left to right, respectively) comparable with their sizes predicted in A. Asterisk (red) in FoxO3 KO condition indicates loss of FoxO3 transcript. L, 1Kb+ ladder (Life Technologies).
Fig. S5.
Fig. S5.
Germ-line loss of FoxO3 does not affect infection efficiency, proliferation, or senescence (related to Fig. 4). (A) Representative images (Left) of infection efficiency (as quantified with lentivirus expressing GFP, 24 h postinduction) for TTFs derived from embryonic (MEF; Upper) or 3-mo-old (3M; Lower) WT (Left) and FoxO3−/− (KO, Right) littermate animals. Average percentages of GFP-expressing cells (Right) were calculated with respected to total DAPI staining (blue). (B) Sample images of 3M TTFs stained with EdU (Upper Left) and SA-β-Gal (Lower Left), respectively, used as proliferation and senescence markers. Average quantifications for EdU/SA-β-Gal staining (Upper Right), as well as p16/p19 mRNA expression levels (as measured by qRT-PCR; Lower Right) for WT and KO conditions are indicated. For all graphs, average values represent means ± SEM, n = 3 independent batches. No significant differences were detected in either experimental conditions (ns, P < 0.05, ANOVA with Bonferroni post hoc test) for either parameter tested. (Scale bars, 20 μm.)

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