Functional rejuvenation of aged neural stem cells by Plagl2 and anti-Dyrk1a activity

Abstract

The regenerative potential of neural stem cells (NSCs) declines during aging, leading to cognitive dysfunctions. This decline involves up-regulation of senescence-associated genes, but inactivation of such genes failed to reverse aging of hippocampal NSCs. Because many genes are up-regulated or down-regulated during aging, manipulation of single genes would be insufficient to reverse aging. Here we searched for a gene combination that can rejuvenate NSCs in the aged mouse brain from nuclear factors differentially expressed between embryonic and adult NSCs and their modulators. We found that a combination of inducing the zinc finger transcription factor gene Plagl2 and inhibiting Dyrk1a, a gene associated with Down syndrome (a genetic disorder known to accelerate aging), rejuvenated aged hippocampal NSCs, which already lost proliferative and neurogenic potential. Such rejuvenated NSCs proliferated and produced new neurons continuously at the level observed in juvenile hippocampi, leading to improved cognition. Epigenome, transcriptome, and live-imaging analyses indicated that this gene combination induces up-regulation of embryo-associated genes and down-regulation of age-associated genes by changing their chromatin accessibility, thereby rejuvenating aged dormant NSCs to function like juvenile active NSCs. Thus, aging of NSCs can be reversed to induce functional neurogenesis continuously, offering a way to treat age-related neurological disorders.

Keywords: ATAC-seq; Ascl1; ChIP-seq; Dyrk1a; Plagl2; adult neurogenesis; aged brain; lentivirus; mouse; neural stem cell.

Figure 1.

Screening for NSC-activating genes and analysis of the iPaD lentivirus. (A) Strategy for NSC-activating gene screening. Genes involved in in vitro NSC activation were searched for by lentivirus-mediated overexpression or siRNA knockdown. Top genes were introduced into the hippocampal dentate gyrus of 6- or 18-mo-old mice using lentivirus, and brain sections were examined. (B) Control lentivirus, lentivirus inducing Plagl2 expression, or lentivirus inducing Plagl2 expression and Dyrk1a knockdown (KD) was injected into the hippocampal dentate gyrus of 18-mo-old mice, and at 4 wk postinfection (wpi; at 19 mo of age), the hippocampal dentate gyrus was examined immunohistochemically. Virus-infected cells were mCherry⁺. (C,D) Quantification of DCX⁺dendrite⁺ immature neuron formation in the hippocampal dentate gyrus of 6-mo-old mice at 2wpi (C; n = 3) and 19-mo-old mice at 4wpi (D; n = 4). The upper and lower names along the horizontal axis indicate overexpressed and knockdown genes, respectively. (E,G,H) Control or iPaD lentivirus was injected into the hippocampal dentate gyrus of 18-mo-old mice, and 2 or 4 wk later, the hippocampal dentate gyrus was examined immunohistochemically. Yellow arrowheads indicate virus-infected mitotic cells (E; mCherry⁺BrdU⁺MCM2⁺), active NSCs (G; mCherry⁺BrdU⁺GFAP⁺Sox2⁺), and immature neurons (H; three examples of mCherry⁺BrdU⁺DCX⁺dendrite⁺ cells), while white arrowheads indicate mCherry⁻BrdU⁺ (non-virus-infected) cells. (F) Quantification of quiescent NSCs (MCM2⁻) and active NSCs or IPCs (MCM2⁺ or BrdU-incorporating) in the hippocampal dentate gyrus at 2 wk after control and iPaD lentivirus injection of 18-mo-old mice. BrdU was administered intraperitoneally for 7 d until sacrifice. At least three samples were examined for each condition. Each value represents the mean ± SEM. Scale bars: B, 50 µm; E,G,H, 20 µm.

Figure 2.

Long-term effect of the iPaD lentivirus on neurogenesis. (A) Schematic structures of the control, Plagl2, and iPaD lentiviruses. (B) The control (panel B1) or iPaD (panels B2–B4) lentivirus was injected into the hippocampal dentate gyrus of 18-mo-old mice. Immunohistochemical analysis was performed at 8wpi of the control lentivirus (20 mo of age; panel B1) and at 4wpi (19 mo of age; panel B2), 8wpi (20 mo of age; panel B3), or 12wpi (21 mo of age; panel B4) of iPaD lentivirus. (C,D) Quantification of active NSCs/IPCs (MCM2⁺; C) and immature neurons (DCX2⁺dendrite⁺; D) in the hippocampal dentate gyrus after virus injection at 18 mo of age. Two-way ANOVA with Bonferroni's post hoc test. Non-virus-infected cells were included for quantification, but virtually all MCM2⁺ or DCX⁺dendrite⁺ cells at 20 and 21 mo of age were mCherry⁺. (E) Quantification of MCM2⁺ active NSCs/IPCs and DCX2⁺dendrite⁺ immature neurons in the wild-type hippocampal dentate gyrus at 6, 9, 12, and 18 mo of age. Quantification at 4wpi of iPaD lentivirus injection into 18-mo-old mice (19 mo of age) is shown at the right. At least four samples were examined for each condition. One-way ANOVA with Tukey HSD post hoc test was conducted. Each value represents the mean ± SEM. Scale bar, 50 µm. (F) Schedule of Barnes maze test. Control or iPaD lentivirus was injected into the hippocampal dentate gyrus of 18-mo-old male mice. N = 10 for control, N = 10 for iPaD male mice. (G–I) Quantification of the walking distance (G), latency (H), and errors (I) before entering the target hole. Error bars indicate SEM. Two-way mixed ANOVA. (J) Time spent around the target hole was measured at the probe test. Error bars indicate SEM. (**) P < 0.01, (***) P < 0.001, one-way ANOVA with Tukey's HSD test.

Figure 3.

Clonal analysis of iPaD lentivirus-infected NSCs. (A) Strategy for clonal analysis using Ai14 mice. Schematic structures of the control-CreER^T2 and iPaD-CreER^T2 lentiviruses and the Rosa26 locus of Ai14 mice (bottom left), and the schedule of experiments for sparse labeling of NSCs (bottom right). (B) Quantification of clones containing RGL cells. (C) Immunohistological and morphological analyses of sparsely labeled cells (tdTomato⁺). (D) Immunohistological and morphological analyses of sparsely labeled cells (tdTomato⁺) from control-CreER^T2 (top two panels) and iPaD-CreER^T2 (bottom three panels) lentivirus-infected hippocampal dentate gyrus. Cell types were determined by morphology and the following markers (indicated by yellow arrowheads): RGL cells (GFAP⁺Nestin⁺), astrocytes (GFAP⁺S100β⁺), IPCs (MCM2⁺DCX⁺dendrite^–), immature neurons (MCM2^–DCX⁺dendrite⁺), and mature neurons (DCX^–Prox1⁺). (E–H) Quantification of the clonal composition of sparsely labeled NSCs with the control-CreER^T2 (E,G) and iPaD-CreER^T2 (F,H) lentiviruses in 9-mo-old (E,F) and 19-mo-old (G,H) Ai14 mice. The number in parentheses indicates the number of each clone. (R) RGL cell, (P) IPC, (N) immature and mature neuron, (A) astrocyte. The number of clones per hemisphere was 38.3 ± 2.7 at 9 mo of age and 43.5 ± 3.6 at 19 mo of age. (I) Clonal sizes of sparsely labeled NSCs at 9 mo of age (left) and 19 mo of age (right). One-way ANOVA with Tukey HSD post hoc test was conducted. (J) Quantification of DCX⁺ IPCs/neurons and astrocytes per activated clone. Six samples were examined for each condition. (N) IPCs and immature and mature neurons, (A) astrocytes. Two-way ANOVA with Bonferroni's post hoc test was conducted. Each value represents the mean ± SEM. Scale bars: C (top), 100 µm C (bottom),D, 20 µm.

Figure 4.

The mechanism of iPaD-dependent rejuvenation of aged NSCs. (A–D) β-Galactosidase staining (A), quantification of its positive cells (B) and p16^INK4a and p19^Arf expression (C), and quantification (D) in 1-mo-old NSCs infected with the control virus and 20-mo-old NSCs infected with the control or iPaD virus. Intensities of p16^Ink4a and p19^ARF bands were normalized with the intensity of α-Actin. (E) E13, young (1-mo-old), adult (9-mo-old), and aged (19- to 22-mo-old) NSCs were infected with the control virus. Aged NSCs were also infected with iPaD virus. (F) Principal component analysis (PCA) of the transcriptomes shown in E. (G–I) Analysis by c-means clustering revealed eight clusters of transcriptomic changes in NSCs from embryonic to aged stages. Transcriptomic changes (G), gene ontology analyses (H), and enrichment of aged genes (I) of clusters 2, 3, and 6 are shown. (J) Fold changes of RNA levels of representative genes in clusters 2, 3, and 6.

Figure 5.

Induction of Ascl1 expression by the iPaD lentivirus. (A) In situ hybridization of Ascl1 in the hippocampal DG-SGZ and the LV-SVZ of 1.5- and 17-mo-old mice. (B) The iPaD or control lentivirus was injected into the hippocampal dentate gyrus of 18-mo-old mice, and 4 wk later, the infected regions were examined immunohistochemically. Subsets of iPaD lentivirus-infected cells expressed Ascl1 (arrowhead), whereas none of the control lentivirus-infected cells did. (C) Bioluminescence imaging and quantification of Luc2-Ascl1 levels in iPaD lentivirus-infected cells. The iPaD lentivirus was injected into the hippocampal dentate gyrus of 10-mo-old Ascl1 reporter mice, in which Luc2-Ascl1 fusion protein was expressed from the endogenous Ascl1 locus. At 4 wk after injection (11 mo of age), time-lapse imaging of brain slices was performed. Luc2-Ascl1 expression occurred in iPaD virus-infected cells (arrowheads). Three representative cells were quantified. Scale bars: A, 200 µm; B, 30 µm; C, 100 µm.

Figure 6.

The epigenetic mechanism of iPaD-dependent rejuvenation of aged NSCs. (A) Motif enrichment analysis of Plagl2 ChIP-seq. (B) GO analysis of genes up-regulated by iPaD and containing Plagl2-binding sites. (C,D) Changes of chromatin/DNA modification gene expression. (E) Analysis by k-means clustering of ATAC-seq signals around promoters of embryonic, adult, and aged NSCs revealed six clusters. (F) Proportions of Plagl2 and Ascl1 target genes in each cluster. (G) Changes of chromatin accessibility in aged NSCs by iPaD. In aged NSCs, closed genes containing Plagl2-binding sites exhibited higher accessibility by iPaD, while opened genes containing Plagl2-binding sites exhibited lower accessibility by iPaD. (H,I) RNA-seq, ATAC-seq, and Plagl2 ChIP-seq enrichment profiles of Nestin (H) and Gfap (I).