Research teams from Harvard Medical School in Boston and Stanford University in Palo Alto, California, found that deleting FoxO genes in mice resulted in NSCs that initially proliferated overtime, but ultimately were unable to provide normal levels of new cells to the aging brain. “They just exhaust the reservoir of cells too early, and later on they don’t have any more,” said Anne Brunet, principal investigator on the Stanford study. The work suggests that FoxOs normally act to promote NSC quiescence. “We think that this quiescence is actually characteristic of stemness,” Brunet said. The research also demonstrates a role for FoxOs in mammals parallel to the longevity function they have in invertebrates.
The FoxO transcription factor family includes FoxO1, FoxO3, and FoxO4, which act similarly, plus the more distantly related FoxO6. FoxO1, FoxO3 and FoxO4 are activated by stresses, such as free radicals, to move to the nucleus and turn on genes such as cell cycle inhibitors, DNA repair mediators, differentiation controllers, and apoptotis promoters (reviewed in Salih and Brunet, 2008: PMID18394876).
In invertebrates, FoxOs are involved in lifespan and quiescence. The Caenorhabditis elegans homolog, DAF-16, is required for the nematodes to enter the dormant dauer phase, and, in conjunction with mutant daf-2, results in longer-lived worms (Kenyon et al., 1993: PMID8247153). Similarly, over-expression of Drosophila dFOXO increases longevity (Giannakou et al., 2004: PMID15192154).
The exact role of FoxOs in mammals is unclear, although certain FoxO3 gene variants are associated with increased lifespan in people (Willcox et al., 2008: PMID18765803). These data led the researchers to hypothesize a cellular longevity role for FoxOs in mammals, where, unlike in nematodes that only live for a few weeks, a constant supply of new cells is required. “We thought, maybe this gene is important for preserving long-lived cells like stem cells,” said Ji-Hye Paik, first author on the Harvard report.
In hematopoietic stem cells (HSCs), knockout of FoxO genes leads to defective quiescence and decreased self-renewal, leading to a depleted stem cell population (Miyamoto et al., 2007: PMID18371339; Tothova et al., 2007: PMID17254970). The new papers show that the same is true in NSCs, which normally give rise to progenitors of neurons, astrocytes and oligodendrocytes necessary for functions such as learning and memory in adults.
Paik led one research effort in the laboratory of Ronald DePinho at Harvard Medical School and the Dana Farber Cancer Institute in Boston (Paik et al., 2009: PMID19896444). The researchers engineered mice with Lox sites flanking their FoxO1, FoxO3 and FoxO4 genes, plus the Cre recombinase under control of the human GFAP promoter, which is expressed in neural stem and progenitor cells. The resulting mice expressed FoxOs normally, except in the neural stem cells and their descendants, where the genes were deleted by Cre’s activity.
Early in life, the animals showed increased proliferation of neural progenitors, compared to control mice carrying the floxed FoxOs but not the Cre recombinase. To examine the cell division rates in the neural stem cell niche, the researchers treated 8-day-old animals with BrdU, which labels actively dividing DNA. There were more dividing NSCs, as evidenced by positive BrdU staining combined with expression of the pluripotency marker Sox2, in the FoxO knockout mice than in control animals. At 12 weeks of age, the FoxO knockout brains were heavier, packed with the additional cells. (No word on whether the mice were smarter, although FoxO knockouts do have “emotional problems,” such as anxiety, Paik said (Polter et al., 2009: PMID18823877)).
However, older mice told a different story, with a decline in the neural stem cell pool and in neurogenesis. By 15 weeks, the FoxO knockout mice had 25 percent fewer Sox2-positive cells in the neural stem cell niche than control animals.
The Stanford research was led by first author Valérie Renault (Renault et al., 2009: PMID19896443). These researchers focused on FoxO3, the most abundant member of the family. When they knocked out the FoxO3 gene in all cells, or in only the brain via floxed FoxO3 and Cre controlled by the nestin promoter, they also saw heavier brains in young animals. One month after injecting BrdU into 5-month-old FoxO3 knockout animals, the mice had fewer BrdU-positive stem cells than mice expressing FoxO3, indicating that the NSCs had divided repeatedly and diluted the marker.
Both teams also analyzed the proliferation of NSCs in vitro. While young cells showed increased division, cells from adults or those that underwent repeated passages evinced reduced self-renewal and differentiation.
The researchers delved into the possible mechanism for FoxO’s regulation of homeostasis with gene expression analyses. Both groups found changes in expression of cell cycle genes such as cyclins and neural differentiation markers such as myelin basic protein, as well as novel targets. The work suggested several potential reasons for the FoxO knockout’s effects, including diminished quiescence, reduced response to oxidative stress, upregulation of the Wnt pathway, altered cellular response to hypoxia, and increased expression of the cell cycle gene Aspm.
“Seeing two different papers coming at it slightly differently, but coming up with the same result, is really nice,” said Karen Arden of the University of California, San Diego, who was not involved in the current research. Combined with the HSC reports and other work, there is a “common thread,” she said, that “FoxOs keep cells in check.” Might FoxOs do the same in other tissues as well? “It wouldn’t surprise me,” Arden said, but she noted that FoxO roles vary between tissues, so it is not certain that they will do the same job throughout the body.
The current work supports the hypothesis that invertebrate longevity genes regulate stem cell niche longevity in mammals. “I think this is not unique to FoxO,” Brunet said. “Several longevity genes are likely to also affect the pool of stem cells.”
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