Cell-density-dependent regulation of neural precursor cell function

Abstract

Stress-induced reductions of neural precursor cells from the subgranular zone of the hippocampal dentate gyrus have been linked to impaired neurogenesis and cognitive dysfunction. Given the importance of redox state in regulating multiple damage-responsive pathways in the CNS, we hypothesize that oxidative stress plays a major role in affecting neurogenesis and subsequent cognitive function after cell injury/depletion. Using an in vitro system, we showed that the level of reactive oxygen species (ROS), which depend critically on changes in cell density, were significantly higher in neural precursor cells when compared with other primary and transformed cell lines. ROS were significantly elevated ( approximately 4-fold) under low- (<1 x 10(4) cells per cm(2)) versus high-density (>1 x 10(5) cells per cm(2)) conditions. Higher ROS levels found at lower cell densities were associated with elevated proliferation and increased metabolic activity. These ROS were likely a result of altered mitochondrial function that ultimately compromised the growth rate of cells. At high cell densities, intracellular ROS and oxidative damage were reduced in concert with an increased expression of mitochondrial superoxide dismutase 2. Our finding that DNA-damage-induced depletion of neural precursor cells in the subgranular zone of mice also led to increased ROS and altered proliferation validated our in vitro system. Increased ROS and proliferation associated with the reduction of precursor cell numbers both in vitro and in vivo could be reversed with the antioxidant alpha-lipoic acid. These data showed that neural precursor cells were predisposed to microenvironmental cues that regulate redox-sensitive pathways to control cellular proliferation after CNS damage.

Fig. 1.

Cell-density-dependent production of ROS. Cells grown at different densities were treated while anchored with the fluorogenic dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate and assessed for intracellular ROS by FACS analysis. FACS histograms (A) show that neural precursor cells are not autofluorescent and have elevated ROS at LD versus HD. ROS levels drop rapidly as neural precursors grow to HD (B) but are significantly higher at all densities when compared with other cells (C–G). All data in B–G are averaged from four or more experiments (±SEM).

Fig. 2.

Cell growth depends on ROS levels. Cells grown at varying densities were assessed for population doubling, cell cycle distribution, and ROS levels in the presence (5–100 μM) and absence of lipoic acid over 2–3 days. In the presence of lipoic acid, LD and HD cultures showed concentration-dependent decreases in population doubling (A) and S-phase fractions (B). Increasing concentrations of LA were also found to progressively reduce the LD/HD ratio of ROS found at different densities (C). All data are averaged from three or more experiments (±SEM).

Fig. 3.

Cells cultured under LD conditions exhibit a persistent inhibition of growth. Cells were cultured at LD or HD for 2 days before reseeding at equal densities and subsequent analysis over the course of 10 days. Cultures derived from LD conditions (A, solid lines) exhibited reduced XTT absorbance compared with cultures derived from HD (A, dashed lines), even after cells were reseeded again at equal densities on day 6. Cultures grown from LD conditions had significantly fewer cells than those grown from HD conditions, as indicated by a ratio (LD/HD) of total cell counts that was always less than unity (B). The slower growth of cells from LD conditions persisted over 10 days and was observed at two different densities (i.e., 1.2 × 10⁴/cm² solid line, or 4.0 × 10⁴/cm² dashed line, B). All data averaged from three experiments (±SEM).

Fig. 4.

mt alterations correspond to density-dependent changes in oxidative stress. FACS analysis of anchored cells treated with the mt probe NAO indicated little density-dependent fluctuation in mt content (A, dashed lines), yielding a LD/HD ratio of NAO fluorescence near unity (B). This result was confirmed by Western analysis of mt porin levels (that also served as loading controls for these blots) and showed that mt content varied little with cell density (C). However, FACS analysis of LD anchored cells treated with the mt function probe R123 showed a dramatic drop in fluorescence (A, solid lines) that resulted in a considerable decline in the fluorescent ratio (LD/HD) of R123 (B). Qualitatively similar results (B) were obtained with the dye tetramethylrhodamine methyl ester, suggesting that reduced cell densities lead to physiologic changes in mt ψ_m. Further analysis of mt proteins showed that aconitase was significantly lower (10-fold) in LD cultures (C), and that MnSOD was higher (3- to 4-fold) in HD cultures. All data in B are averaged from four or more experiments (±SEM).

Fig. 5.

Relationship between density-dependent ROS and growth factor. Differing densities of neural precursor cells were cultured in serum-free media supplemented with varying levels of FGF2 (0–100 ng/ml). FACS analysis of cells cultured in the absence of FGF2 showed higher levels of ROS at all cell densities tested. Addition of FGF2 lowered ROS levels but to a much greater extent in HD cultures, but the presence of varying FGF2 did not account for the cell-density-dependent differences in ROS reported in this study. All data are averaged from three or more experiments (±SEM).

Fig. 6.

Proliferation and oxidative stress within the hippocampus. Brain tissues from irradiated (5–10 Gy) and nonirradiated mice with or without treatment with LA (100 mg/kg) were processed for immunohistochemical analysis or MDA assays. Numbers of proliferating cells within the SGZ of the dentate gyrus were quantified as described. Unirradiated controls showed a typical age-dependent decline in the number of proliferating precursors (A, filled circles) that was markedly enhanced by LA treatment (A, open circles). Analysis of tissues from irradiated mice demonstrated the effectiveness of X-irradiation to reduce the numbers of proliferating neural precursors in the SGZ (A, filled squares). Proliferating cells then recover over the next 2 weeks; this was not affected by the presence of LA (A, open squares). During this increased proliferation, oxidative stress increased as measured by MDA levels of hippocampal tissue (10 Gy, 2 weeks), an effect that could be reversed by LA (B). Compared with unirradiated controls (C), tissue sections from these irradiated mice showed that MDA staining (purple/brown cells) was concentrated in both blades of the SGZ (D). All data in A are averaged from five or more experiments (±SEM).