11 September 2006. In the September 6 Nature online, a suite of papers reports that age-related loss of regenerative capacity is caused, at least in part, by a single protein, the cyclin-dependent kinase inhibitor p16INK4a. Most notably from a neurological standpoint, Howard Hughes Medical Institute Investigator Sean Morrison and colleagues at the University of Michigan, Ann Arbor, together with Norman Sharpless and his team at the University of North Carolina, Chapel Hill, report that increasing p16INK4a in the mouse forebrain correlates with decreased progenitor proliferation in the subventricular zone (SVZ), one of only two regions where adult mammalian neurogenesis takes place.
The discovery of active neurogenesis in the mammalian adult brain raised the possibility that neural stem cells could one day be harnessed to tackle neurodegenerative disorders, such as Alzheimer and Parkinson diseases. But there are many hurdles that need to be cleared first, not least being that neural stem cells themselves appear to eventually run out of steam. This age-related decline in adult neurogenesis does not bode well for the chances of exploiting endogenous neural stem cells in older adults. But the three papers suggest that there may be viable strategies, such as inhibiting p16INK4a, which could help boost endogenous regenerative capacity, particularly in the aged.
There are previous hints that p16INK4a, and another cyclin-dependent kinase inhibitor, p19Arf, may influence stem cells. Work from Morrison’s lab showed that expression of both is attenuated by the transcriptional repressor Bmi-1, which is required for full stem cell renewal potential (Molofsky et al., 2003). Because both kinase inhibitors increase with age in a variety of tissues, joint first authors Anna Molofsky, Shalom Slutsky, and colleagues specifically examined the SVZ to determine if expression of either protein correlated with neurogenesis. Finding that only transcription of p16INK4a significantly increased as mice age, the authors then correlated various measures of progenitor function with expression of the protein.
Molofsky and colleagues examined neural progenitors in young (60 days) and old (2 years) p16INK4a-decifient mice and controls. They found little difference between the young animals—the SVZ cells from both control and p16INK4a knockout heterozygotes formed multipotent neurospheres in culture at about the same frequency, and the ability of these cells to self-renew was the same regardless of which animal they were derived from. However, reduced p16INK4a in older mice doubled the frequency of both neurosphere formation and self-renewal potential. The authors posit that p16INK4a predominantly affects stem cells rather than downstream progenitors because a pulse-chase (8 days pulse, 4 weeks chase) experiment with bromodeoxyuridine (BrdU) labeled twice as many SVZ cells in older p16INK4a-deficient mice than controls: Studies suggest that such long pulse-chase experiments can identify stem cells because they divide infrequently and are retained within the SVZ, unlike progenitors, which divide rapidly and migrate.
Of course, having more stem cells does not necessarily translate into more neurons, but the authors found that in p16INK4a-deficient older animals, new olfactory bulb neurons formed at twice the rate as in control mice. Interestingly, the p16INK4a effect seems specific for neural stems cells—at least in the brain—because proliferation of non-neuronal cells (i.e., those negative for the neuronal marker NeuN) was unaffected.
But stem cells in the periphery also appear under the influence of the kinase inhibitor. The other two papers report that p16INK4a attenuates the regeneration of pancreatic islet cells and hematopoietic stem cells (HSCs). Janakiraman Krishnamurthy and colleagues at Sharpless’ lab and at the Joslin Diabetes Center, Boston, found that after older mice were given streptozotocin, a drug that induces diabetes, regeneration of insulin-producing β cells was significantly higher in p16INK4a-negative animals. David Scadden and colleagues at Massachusetts General Hospital, Boston, and the Harvard Stem Cell Institute in Cambridge, report that p16INK4a modulates hematopoietic stem cell function. Joint first authors Viktor Janzen, Randolf Forkert, and colleagues found that increased expression of p16INK4a with age limits the number of HSCs and their ability to repopulate—without the kinase inhibitor, proliferation of HSCs increases while apoptosis decreases.
Taken together, the three papers show that p16INK4a plays a pivotal role in regulating stem cells in aging mice and suggest that inhibiting the protein may be one strategy of increasing stem cell potential. But as Christian Beausejour, University of Montreal, Canada, and Judith Campisi, Lawrence Berkeley National Laboratory, California, write in an accompanying News & Views article, “stem/progenitor-cell proliferation is a double-edged sword. Although it ensures tissue repair and regeneration, it also puts tissues at risk of hyperproliferative diseases, the most deadly of which is cancer.” Indeed, Sharpless and colleagues report that p16INK4a-/- animals are tumor prone. Nevertheless, the findings raise the possibility of being able to compensate for the age-related loss in regenerative capability.
Curiously, Molofsky and colleagues found that p16INK4a-deficiency had no effect on stem cell proliferation or neurogenesis in the dentate gyrus of the hippocampus, the second site of mammalian adult neurogenesis. The hippocampus is more interesting from an AD perspective because it plays an important role in learning and memory and is a major site of neurodegeneration as the disease progresses. It will be interesting to see if there are other factors that might explain age-related loss in dentate gyrus neurogenesis.—Tom Fagan.
Molofsky AV, Slutsky SG, Joseph NM, He S, Pardal R, Krishnamurthy J, Sharpless NE, Morrison SJ. Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature. 6 September, 2006; Advanced online publication. Abstract
Krishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S, Sharpless NE. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature. 6 September, 2006; Advanced online publication. Abstract
Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature. 6 September, 2006; Advanced online publication. Abstract
Beausejour CM, Campisi J. Balancing regeneration and cancer. Nature. 6 September, 2006; Advanced online publication. Abstract