Chronically increased transforming growth factor-beta1 strongly inhibits hippocampal neurogenesis in aged mice

Abstract

There is increasing evidence that hippocampal learning correlates strongly with neurogenesis in the adult brain. Increases in neurogenesis after brain injury also correlate with improved outcomes. With aging the capacity to generate new neurons decreases dramatically, both under normal conditions and after injury. How this decrease occurs is not fully understood, but we hypothesized that transforming growth factor (TGF)-beta1, a cell cycle regulator that rapidly increases after injury and with age, might play a role. We found that chronic overproduction of TGF-beta1 from astrocytes almost completely blocked the generation of new neurons in aged transgenic mice. Even young adult TGF-beta1 mice had 60% fewer immature, doublecortin-positive, hippocampal neurons than wild-type littermate controls. Bromodeoxyuridine labeling of dividing cells in 2-month-old TGF-beta1 mice confirmed this decrease in neuro-genesis and revealed a similar decrease in astrogenesis. Treatment of early neural progenitor cells with TGF-beta1 inhibited their proliferation. This strongly suggests that TGF-beta1 directly affects these cells before their differentiation into neurons and astrocytes. Together, these data show that TGF-beta1 is a potent inhibitor of hippocampal neural progenitor cell proliferation in adult mice and suggest that it plays a key role in limiting injury and age-related neurogenesis.

Figure 1

TGF-β1 overexpression dramatically decreases the number of immature neurons and nestin-positive processes. A: Light microscopy images of coronal brain sections stained for the immature neuronal marker doublecortin from 9-week-old and 22-month-old female TGF-β1 mice and their wild-type littermates. B: Quantification of doublecortin immunoreactivity in 9-week-old dentate gyrus, n = 3 mice per genotype. C: Quantification of the total number of hippocampal doublecortin cells in 22-month-old mice, n = 6 per genotype. Each circle represents one mouse. D: Nestin immunohistochemistry on a coronal section from a 9-week-old TGF-β1 mouse. Arrows indicate a nestin-positive process; the arrowhead, a blood vessel. E: Quantification of the total number of nestin-positive processes per hippocampal section. n = 5 mice per genotype. Dcx, doublecortin. Bars are mean ± SEM. **P ≤ 0.005, ***P ≤ 0.0005; Student’s t-test. Scale bars = 100 μm (A); 10 μm (D).

Figure 2

TGF-β1 mice have 60% fewer BrdU-positive cells and BrdU-positive neurons in the hippocampus 1 and 28 days after BrdU administration. TGF-β1 transgenic (n = 5 mice per time point) and nontransgenic littermate controls (n = 5 to 8 mice) were injected at 8 weeks of age with BrdU to label dividing cells and analyzed 1 day or 28 days later for the presence of BrdU-positive cells and BrdU-positive neurons. Doublecortin was used to label immature neurons, and NeuN to label mature neurons. A: Quantification of the number of BrdU-positive cells and the number of BrdU-positive cells co-labeling for doublecortin (BrdU⁺/Dcx⁺) 1 day after BrdU administration. Bars are mean ± SEM. B: Quantification of total BrdU-positive cells and BrdU-positive mature neurons (BrdU⁺/NeuN⁺ cells) 28 days after BrdU. Bars are mean ± SEM. C: Example of BrdU-positive cells in the subgranular zone of the dentate gyrus, counterstained with hematoxylin. D: Confocal image of brain section containing BrdU⁺/Dcx⁺ cells. BrdU, magenta; Dcx, green; and NeuN, blue. E: Confocal image of brain section containing BrdU⁺/NeuN⁺ cells. BrdU, green; NeuN, red; and GFAP, blue. F: Example of a cell in the granule cell layer immunostained with caspase-3, counterstained with cresyl violet. G: Quantification of the number of caspase-3-positive cells counted in dentate gyrus subgranular zone and granule cell layer in 13-week-old TGF-β1 mice and their wild-type littermates, n = 5 mice per genotype. H: Relative percentage of surviving neurons calculated as the fraction of new mature neurons (BrdU⁺/NeuN⁺) present at 28 days after BrdU compared with the number of new immature neurons (BrdU⁺/Dcx⁺) present 1 day after BrdU. Bars represent the percent difference between the means in A and B. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005; Student’s t-test. Scale bars = 10 μm.

Figure 3

TGF-β1 mice have decreased astrogenesis, normal microgliogenesis, and prolonged glial survival. Coronal brain sections of TGF-β1 transgenic and nontransgenic littermate controls (n = 5 mice per analysis time point) injected at 8 weeks of age with BrdU to label dividing cells and sacrificed 1 or 28 days later for the presence of BrdU-positive astrocytes and BrdU-positive microglia. GFAP was used to label astrocytes, and Iba1 to label microglia. Bars are mean ± SEM. A: Confocal image of brain section containing a BrdU⁺/GFAP⁺ cell. BrdU, red; GFAP, green; and NeuN, blue. B and C: Quantification of the number of BrdU-positive astrocytes (BrdU⁺/GFAP⁺) 1 and 28 days after BrdU in TGF-β1 and their wild-type littermates. D: Percentage of the BrdU⁺/GFAP⁺ cells that remain 28 days later. E: Confocal image of brain section containing a BrdU⁺/Iba1⁺ cell. Iba1, blue; NeuN, green; and BrdU, magenta. F and G: Quantification of the number of BrdU-positive microglia (BrdU⁺/Iba1⁺) 1 and 28 days after BrdU in TGF-β1 and their wild-type littermates. H: Percent surviving BrdU⁺/Iba1⁺ cells at 28 days. *P ≤ 0.05, Student’s t-test. n.s., not significant. Scale bar = 10 μm.

Figure 4

Cell fates of newly generated cells 1 day after BrdU labeling reveal that excess TGF-β1 does not alter the proportion of cells becoming neurons versus astrocytes. Microglia were excluded from this analysis because they are not derived from neural progenitor cells. Note the area of each pie graph represents the absolute number of new cells; TGF-β1 mice generate only 40% of the normal number of new cells, but the proportion of cells assuming each phenotype in TGF-β1 mice is not altered compared to wild-type mice.

Figure 5

TGF-β1 inhibits proliferation and prolongs the cell cycle of neural progenitor cells. Rat (A, B, D) and mouse (C) neural progenitor cells were cultured in varying amounts of TGF-β1, insulin, FGF-2, and EGF. N2, standard media with N2 supplement. *P ≤ 0.05 compared to no TGF-β1, analysis of variance followed by Dunnett’s posthoc test. Bars are mean ± SEM. Data shown are representative of three to five experiments (rat cells) or two experiments (mouse cells). A: Western blot of lysates from neural progenitor cells grown in either 0 or 1 ng/ml of TGF-β1 and probed for phosphorylated (Smad2-P) and total (Smad2) Smad2. B: MTT assay of rat neural progenitor cells grown in varying amounts of TGF-β1, insulin, and FGF-2. The box labeled N2 on the x axis highlights the concentrations of insulin and FGF-2 present in commercially available N2 neuronal culture supplement. C: MTT assay of mouse neural progenitor cells grown in varying amounts of TGF-β1, EGF, and FGF-2. D: Cell cycle analysis of propidium iodide-stained cells showing the proportion of cells in G₁/G₀, S phase, or G₂/metaphase.