Research by a Stanford University team of scientists has shed new light on how and why neural stem cells (NSCs), the cells behind the generation of new neurons in the adult brain, become less active as brains age. Anne Brunet, PhD, professor of genetics, and her team developed in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms and carried out a genome-wide search for genes that, when knocked out, increase the activation of neural stem cells in old mice, but not young animals. Of the top gene knockouts identified, the team found that knocking out Slc2a4—which encodes the insulin-dependent GLUT4 glucose transporter—notably improved the function of old NSCs.
“We first found 300 genes that had this ability—which is a lot,” emphasized Brunet, the Michele and Timothy Barakett Endowed Professor. After narrowing the candidates down to 10, “one in particular caught our attention,” Brunet said. “It was the gene for the glucose transporter known as the GLUT4 protein, suggesting that elevated glucose levels in and around old neural stem cells could be keeping those cells inactive.”
The study results could point to some intriguing next steps in addressing this old neural stem cell passivity—or even stimulating neurogenesis in younger brains in need of repair— by targeting newly identified pathways that could reactivate the stem cells.
Brunet and colleagues described their findings in Nature, in a paper titled “CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells,” in which they stated “Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during aging.”
Most neurons in the human brain last a lifetime, and for good reason. Intricate, long-term information is preserved in the complex structural relationships between their synapses. To lose the neurons would be to lose that critical information—that is, to forget. Some new neurons are still produced in the adult brain by a population of neural stem cells. “The adult mammalian brain contains several NSC regions that give rise to newborn neurons and can repair tissue damaged by stroke or brain injuries,” the investigators stated.
There are parts of the brain, such as the hippocampus and the olfactory bulb, where many neurons have shorter lives, where they regularly expire and may be replaced by new ones, said lead author Tyson Ruetz, PhD, a formal post-doctoral scholar in Brunet’s lab. “In these more dynamic parts of the brain, at least in young and healthy brains,” he said, “new neurons are constantly being born and the more transient neurons are replaced by new ones.”
As brains age, however, NSCs become increasingly less adept at making these new neurons, a trend that can have devastating neurological consequences, not just for memory, but also for degenerative brain diseases such as Alzheimer’s and Parkinson’s, and for recovery from stroke or other brain injury. “Aging impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain,” the team continued. “Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during aging.”
Ruetz developed a way to test genetic pathways that might be involved in new neuron production in vivo, “where the results really count,” Brunet said. To do this, Ruetz took advantage of the distance between the part of the brain where the neural stem cells are activated, the subventricular zone (SVZ), and the place the new cells proliferate and migrate to, the olfactory bulb, which is many millimeters away in a mouse brain. “It’s allowing us to observe three key functions of the neural stem cells,” Ruetz said. “First, we can tell they are proliferating. Second, we can see that they’re migrating to the olfactory bulb, where they’re supposed to be. And third, we can see they are forming new neurons in that site.”
To systematically identify genes that boost NSC activation as a function of age the team developed screening platforms that allowed them to investigate gene knockouts that boost NSC activation specifically in old mice. “Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs,” they wrote. “One of the most consistent gene knockouts that boost old NSC function both in vitro and in vivo is Slc2a4 knockout.”
By knocking out the glucose transporter genes in the SVZ, waiting several weeks, then counting the number of new neurons in the olfactory bulb, the team demonstrated that knocking out GLUT4 had an activating and proliferative effect on neural stem cells, leading to a significant increase in new neuron production in live mice. “The knockout of the insulin-sensitive glucose transporter GLUT4 was consistently a top hit for both in vitro and in vivo screens, which led to a twofold increase in neurogenesis in old mice in vivo,” the investigators pointed out.
The glucose transporter connection “is a hopeful finding,” Brunet said. For one, it suggests not only the possibility of designing pharmaceutical or genetic therapies to turn on new neuron growth in old or injured brains, but also the possibility of developing simpler behavioral interventions, such as a low carbohydrate diet that might adjust the amount of glucose taken up by old neural stem cells.”
Commenting on their collective results the team noted, “Thus, expression of the glucose transporter GLUT4 increases during aging in NSCs in vivo, and knockout of this transporter boosts NSC number and neurogenesis in old mice. Together, these data raise the possibility that the age-dependent increase in GLUT4 could be detrimental for NSC function and neurogenesis in old brains.”
The researchers found other provocative pathways worthy of follow-up studies. Genes relating to primary cilia, parts of some brain cells that play a critical role in sensing and processing signals such as growth factors and neurotransmitters, also are associated with neural stem cell activation. This finding reassured the team that their methodology was effective, partly because unrelated previous work had already discovered associations between cilia organization and neural stem cell function. It is also exciting because the association with the new leads about glucose transmission could point toward alternative avenues of treatment that might engage both pathways, Brunet said.
“There might be interesting crosstalk between the primary cilia—and their ability to influence stem cell quiescence, metabolism and function—and what we found in terms of glucose metabolism,” she noted. “The next step,” Brunet continued, “is to look more closely at what glucose restriction, as opposed to knocking out genes for glucose transport, does in living animals.”
The same technique could also be applied to studies of brain damage, Ruetz pointed out. “Neural stem cells in the subventricular zone are also in the business of repairing brain tissue damage from stroke or traumatic brain injury.”
