BMP4-SMAD1/5/9-RUNX2 pathway activation inhibits neurogenesis and oligodendrogenesis in Alzheimer's patients' iPSCs in senescence-related conditions

Abstract

In addition to increasing β-amyloid plaque deposition and tau tangle formation, inhibition of neurogenesis has recently been observed in Alzheimer's disease (AD). This study generated a cellular model that recapitulated neurogenesis defects observed in patients with AD, using induced pluripotent stem cell lines derived from sporadic and familial AD (AD iPSCs). AD iPSCs exhibited impaired neuron and oligodendrocyte generation when expression of several senescence markers was induced. Compound screening using these cellular models identified three drugs able to restore neurogenesis, and extensive morphological quantification revealed cell-line- and drug-type-dependent neuronal generation. We also found involvement of elevated Sma- and Mad-related protein 1/5/9 (SMAD1/5/9) phosphorylation and greater Runt-related transcription factor 2 (RUNX2) expression in neurogenesis defects in AD. Moreover, BMP4 was elevated in AD iPSC medium during neural differentiation and cerebrospinal fluid of patients with AD, suggesting a BMP4-SMAD1/5/9-RUNX2 signaling pathway contribution to neurogenesis defects in AD under senescence-related conditions.

Keywords: Alzheimer’s disease; BMP4; RUNX2; SMAD1/5/9; compound screening; induced pluripotent stem cell (iPSC); morphological analysis; neurogenesis.

Graphical abstract

Figure 1

Neuronal, oligodendrocyte, and astrocyte differentiation potentials of iPSCs from HCs and patients with sAD under low-density culture conditions (A) Schematic of neuronal differentiation of iPSCs under low-density culture conditions. Control (HC1–HC8) and sAD (sAD1–sAD8) iPSCs were analyzed on day 28. (B and C) Immunofluorescence of MAP2, TUJ1, and PAX6. Mean positive area was quantified in (C) Scale bar, 50 μm. ^∗p < 0.05, n = 13 images taken from the same well per group. (D) Quantitative real-time PCR analysis of MAP2, TUJ1, and PAX6 mRNA expression. Mean mRNA levels in HC1 were set to 1. n = 4–6 independent experiments. ^∗p < 0.05. (E and F) Immunofluorescence and quantification of OLIG2 and S100β. ^∗p < 0.05, n = 15 images taken from the same well per group. Scale bar, 50 μm.

Figure 2

Senescence-related propensities of HC and sAD cells under low- and high-density culture conditions Control (HC1–HC3) and sAD (sAD1–sAD3) iPSCs were analyzed after neuronal differentiation for 28 days. (A and B) Neuronal differentiation potential under high-density culture conditions was analyzed by immunofluorescence (A) and quantitative real-time PCR analysis (B) of MAP2, TUJ1, and PAX6 (n = 4 independent experiments). (C–E) Immunofluorescence of SA-β-gal (C) and O-GlcNAc (D) on day 28 under low- or high-density culture conditions, quantified in (E). n = 10–15 images taken from the same well per group. (F and G) The mRNA levels of IL-6 were quantified by quantitative real-time PCR analysis (F), and the IL-6 protein levels in the medium were quantified by ELISA (G). Mean mRNA or protein levels in HC1 were set to 1. ^∗p < 0.05. n = 4–6 independent experiments. Scale bars, 50 μm.

Figure 3

Image-based screening of small chemical compounds for improving NG of sAD iPSCs (A) Outline of the compound screening experiments. Cells were treated with the compounds during stages II and III or only during stage III. Cells were fixed on day 28, followed by immunofluorescence and image analysis to detect MAP2- and/or TUJ1-positive neurites (detailed in Figure S5). (B) Representative images of MAP2 and TUJ1 staining in LDN-, ISO-, and SAG-treated sAD1 and sAD3 cells (excerpt from Figure S5B). Scale bars, 50 μm. (C and D) Total length of MAP2- and/or TUJ1-positive neurites. n = 10–15 images taken from the same well per group. (E) Ratio of the total length, total area, and branchpoints of MAP2-positive neurons to TUJ1-positive neurons. (F) PCA using morphological features (total length, total area, and number of branchpoints).

Figure 4

Effect of LDN on neural differentiation of HC and sAD iPSCs Control (HC1) and sAD iPSCs (sAD1 and sAD3) were cultured under low-density conditions and treated with or without 250 nM LDN during stage I or stage II of neuronal differentiation (+first LDN or +second LDN, respectively). (A) Immunofluorescence of MAP2 and TUJ1. Scale bar, 50 μm. (B) Quantification of MAP2- and/or TUJ1-positive neurites in (A) (detailed in Figure S5). n = 15 images taken from the same well per group. (C) Quantitative real-time PCR analysis of mRNA expression levels of MAP2 and TUJ1. Mean mRNA levels in HC1 were set to 1. n = 4 independent experiments, ^∗p < 0.05 compared with non-treated HC1 cells.

Figure 5

Increased phosphorylation of SMAD1/5/9 in sAD cells (A) HC1, sAD1, and sAD3 cells were immunostained with phosphorylated SMAD1/5/9 antibody (P-SMAD1/5/9) after induction of neuronal differentiation for 28 days with or without 250 nM LDN treatment during stage II. n = 10–15 images taken from the same well per group. Scale bar, 50 μm. (B) Immunofluorescence of P-SMAD1/5/9 in HC1-8 and sAD1-8 on day 28. Scale bar, 50 μm. (C) Protein expression of P-SMAD1/5/9 in HC1 and sAD3 cells 0, 5, 10, 15, 20, 25, and 28 days after neuronal differentiation were analyzed by western blot. The mean protein levels on day 0 of HC1 were set to 1. n = 3 independent experiments. (D) Immunofluorescence of OLIG2 and S100β and its quantification. Scale bar, 50 μm n = 10–15 images taken from the same well per group. ^∗p < 0.05.

Figure 6

RUNX2 inhibition restored NG and oligodendrogenesis of sAD and fAD cells (A) Quantitative real-time PCR analysis of RUNX2 mRNA expression 0, 5, 10, 15, 20, 25, and 28 days after neuronal differentiation. The mean mRNA levels on day 0 of HC1 were set to 1. n = 4–6 independent experiments. (B) The mRNA expression of RUNX2 in cells treated with or without 250 nM LDN during stage II (left) and HC1–HC8 and sAD1–sAD8 iPSCs (right), analyzed by quantitative real-time PCR on day 28. The mean mRNA levels of HC1 were set to 1. n = 4–6 independent experiments. ^∗p < 0.05. (C) iPSCs from sAD and fAD patients were immunostained with MAP2 antibody (green) and TUJ1 antibody (yellow) after induction of neuronal differentiation with DMSO (0.5% [vol/vol], days 15–28) or CADD522 (50 μM, days 15–28, +CADD) for 28 days. Scale bar, 50 μm. (D) Quantitative real-time PCR analysis of mRNA expression of MAP2 and TUJ1 under the same conditions as in (C). The mean mRNA levels in DMSO-treated sAD1 or fAD1 cells were set to 1. ^∗p < 0.05 compared with DMSO-treated cells. n = 6 independent experiments. (E) Immunofluorescence of OLIG2 and S100β under the same conditions as in (C). (F) Immunofluorescence of cleaved caspase-3 (cCASP3) under the same conditions as in (C). Scale bar, 50 μm. (G) Western blot of P-Smad1/5/9 and Runx2 in the hippocampus of 8-week SAMR1 or 8-week SAMP8 mice. n = 3 mice per group. The mean protein level of SAMR1 was set to 1. ^∗p < 0.05.

Figure 7

Elevated BMP4 level in AD cells and in cerebrospinal fluid samples from patients with AD (A) After 28 days of neuronal differentiation, mRNA levels of BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, and BMP10 in control (HC1–HC4), sAD (sAD1–sAD3), and fAD (fAD1 and fAD2) cells were analyzed by quantitative real-time PCR. n = 4–6 independent experiments. (B and C) mRNA expression and secretion of BMP4 and BMP7 on day 28. n = 6 independent experiments. (D) Quantitative real-time PCR analysis of BMP4 mRNA expression on days 0–28. n = 4 independent experiments. The mean mRNA levels of HC1 and sAD1 (A and B) and on day 0 of HC1 (C) were set to 1. ^∗p < 0.05. N.D., not detected. (E) Methylation levels of 6 CpG sites in the promoter region of the BMP4 gene were analyzed by unmethylated (U) and methylated (M) CpG-specific PCR in undifferentiated HC1 and sAD3 iPSCs. (F) Methylation levels of cg245268999 of undifferentiated control (HC) and sAD iPSCs by EpiTaq HS. n = 5–7 independent experiments. (G) mRNA expression of the BMP4 gene and methylation levels of the CpG site (cg24526899) in the promoter region of control iPSCs (HC1) after treatment for 48 h with DMSO or 1 μM 5-aza-2′ deoxycytidine (5-Aza-dC). The mean mRNA levels of DMSO-treated HC1 were set to 1. n = 3 independent experiments. (H) Concentrations of BMP4 in cerebrospinal fluid obtained from 20 HCs and 20 patients with AD. (I) A model of NG inhibition in AD iPSCs. BRII, BMP receptor type 2.