“Inflamm-Aging”: Inflammatory Signals in the Brain Regulate the Lifespan of Mice

The hypothalamus is located at the base of the brain and in adult humans, it has a volume of only 4cm³, less than half a percent of the total adult human brain volume. Despite its small size, the hypothalamus is one of the most important control centers in our brain because it functions as the major interface between two regulatory systems in our body: The nervous system and the endocrine (hormonal) system. It consists of many subunits (nuclei) which continuously sense inputs and then respond to these inputs by releasing neurotransmitters or hormones that regulate a broad range of vital functions, such as our metabolism, appetite, thirst, reproduction, temperature and even our internal timing system, the circadian clock. As if this huge workload wasn’t enough, researchers have now uncovered an additional role for the hypothalamus: regulating lifespan.

The recent paper “Hypothalamic programming of systemic ageing involving IKK-β,NF-κB and GnRH” published in the journal Nature (published online May 1, 2013) by Guo Zhang and colleagues at the Albert Einstein College of Medicine in New York used elegant genetic mouse models to either continuously activate or continuously suppress the function of the NF-κB protein in the hypothalamus. This protein is a key transcription factor which is found in most organs and tissues and turns on genes in response to an inflammatory stimulus. The researchers were thus able to artificially create an internal scenario in which the hypothalamus was receiving a continuous “inflammation on” or “inflammation off” input without having to provide any external infectious or inflammatory agents. The results were quite striking. Continuous activation of the inflammatory NF-κB pathway in the hypothalamus resulted in a reduction of overall lifespan in the mice, but it also resulted in a loss of muscle mass, bone mass, and cognitive function – the mice showed signs of accelerated aging. An even more remarkable finding was that continuous suppression of the inflammatory pathway extended the lifespan of the mice when compared to their littermates that did not undergo any genetic modifications. Not only did these mice live longer (median lifespan increased by 23%), but they also exhibited significantly less physical and cognitive decline than regular mice!

To investigate the mechanism by which the suppression of inflammatory signals could result in such a profound increase in longevity and functional capacity, the researchers studied Gonadotropin Releasing Hormone (GnRH), one of the major hormones released by the hypothalamus which in turn regulates the release of reproductive hormones. They found that aging or inflammatory activation indeed suppressed GnRH release, whereas inhibition of the inflammatory signaling was able to restore GnRH levels. More importantly, simply injecting the mice with GnRH was able to prevent the physical and cognitive decline in the aging mice. How the injections of GnRH were able to restore muscle mass and even cognitive function was not evaluated in the study, but the researchers did observe that the brain showed increased evidence of neuron growth, which could explain the anti-aging effects of GnRH.

This paper is not the first to link inflammation to aging, but it is the first to show that localized inflammation signals in the hypothalamus can have such a profound effect on the lifespan of mice and it is also the first to propose that suppression of GnRH may be the reason for this inflammation-aging link. As with all important scientific papers, this study raises more questions than it answers. Is GnRH not just a regulator of sex hormones, but does it also exert effects on neurons and muscle cells that are independent of its role as a regulator of reproductive hormones? The mice with prolonged life-spans were all studied in a laboratory setting and thus not exposed to infectious agents that mice (or humans, for that matter) living in the wild commonly encounter. Would suppression of the NF-κB pathway in the hypothalamus possibly compromise their ability to fend off infections or other natural forms of inflammation? It is also not clear whether the GnRH link would apply to all mammals such humans, since aging female primates have higher, (not lower!) GnRH levels. These are all questions that lie beyond the scope of this paper and they need to be addressed in future papers.

However, there are some major limitations of this study and the proposed new hypothalamus-inflammation-GnRH-aging model. First, there is one rather obvious experiment that is missing. The researchers showed that manipulating NF-κB in the hypothalamus can have a major effect on the lifespan and the cognitive as well as physical function, but for some reason the researchers did not show the results from a rather simple experiment: Does GnRH alone extend the lifespan? If GnRH were really the main pathway by which the hypothalamus regulates aging, than giving GnRH ought to have extended the lifespan of the mice.

A second limitation of the paper is that it does not distinguish between general functional decline versus decreased regeneration. Biological aging is characterized by a gradual functional decline over time, but this is due to a combination of at least two parallel processes. Existing cells and tissues accumulate damaged and become dysfunctional and regenerative stem cells or progenitor cells become exhausted and cannot keep up with the repair. This study does not assess whether increased NF-κB activation in the hypothalamus causes more cellular dysfunction, whether it merely inhibits the regenerative repair process or whether it affects both. The researchers did not perform assessments of cellular aging, such as measuring the expression levels of the cellular aging regulator p16 or quantify oxidative stress. Therefore, it is unclear whether NF-κB activation in the hypothalamus had any impact on the cellular aging (senescence) program in the brain, muscles or elsewhere in the body.

Another key limitation is that the hypothalamus has so many functions other than GnRH release, which could all contribute to aging and changes in the lifespan of the mice. The authors themselves have previously published that NF-κB in the hypothalamus regulates the link between obesity and high blood pressure and multiple other groups have already shown that the hypothalamus may affect aging via its role in metabolic regulation. Unfortunately, the current study glosses over the potential role of metabolism and high blood pressure, which could explain the observed longevity effects and instead just focuses on the more provocative but less substantiated idea of GnRH as the aging regulator.

Due to these limitations, we still have to await additional studies that confirm the role of GnRH as the target for NF-κB activation in the hypothalamus and this link between inflammation, aging and the hypothalamus.

We should also remember that biological aging is just one aspect of aging. As André Maurois once wrote, “Old age is far more than white hair, wrinkles, the feeling that it is too late and the game finished, that the stage belongs to the rising generations. The true evil is not the weakening of the body, but the indifference of the soul.”

 

Image credit: A GIF depicting the Hypothalamus “BodyParts3D”, © The Database Center for Life Science – Creative Commons license via Wikimedia Commons, Painting by John Haberle (1856-1933) – Time and Eternity, via Wikimedia Commons

 

Zhang G, Li J, Purkayastha S, Tang Y, Zhang H, Yin Y, Li B, Liu G, & Cai D (2013). Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH. Nature, 497 (7448), 211-216 PMID: 23636330

Advertisement

Cellular Alchemy: Converting Fibroblasts Into Heart Cells

Medieval alchemists devoted their lives to the pursuit of the infamous Philosopher’s Stone, an elusive substance that was thought to convert base metals into valuable gold. Needless to say, nobody ever discovered the Philosopher’s Stone. Well, perhaps some alchemist did get lucky but was wise enough to keep the discovery secret. Instead of publishing the discovery and receiving the Nobel Prize for Alchemy, the lucky alchemist probably just walked around in junkyards, surreptitiously collected scraps of metal and brought them to home to create a Scrooge-McDuck-style money bin.  Today, we view the Philosopher’s Stone as just a myth that occasionally resurfaces in the titles of popular fantasy novels, but cell biologists have discovered their own version of the Philosopher’s Stone: The conversion of fibroblast cells into precious heart cells (cardiomyocytes) or brain cells (neurons).

 

Fibroblasts are an abundant cell type, found in many organs such as the heart, liver and the skin. One of their main functions is to repair wounds and form scars in this process. They are fairly easy to grow or to expand, both in the body as well as in a culture dish. The easy access to large quantities of fibroblasts makes them analogous to the “base metals” of the alchemist. Adult cardiomyocytes, on the other hand, are not able to grow, which is why a heart attack which causes death of cardiomyocytes can be so devastating. There is a tiny fraction of regenerative stem-cell like cells in the heart that are activated after a heart attack and regenerate some cardiomyocytes, but most of the damaged and dying heart cells are replaced by a scar – formed by the fibroblasts in the heart. This scar keeps the heart intact so that the wall of the heart does not rupture, but it is unable to contract or beat, thus weakening the overall pump function of the heart. In a large heart attack, a substantial portion of cardiomycoytes are replaced with scar tissue, which can result in heart failure and heart failure.

A few years back, a research group at the Gladstone Institute of Cardiovascular Disease (University of California, San Francisco) headed by Deepak Srivastava pioneered a very interesting new approach to rescuing heart function after a heart attack.  In a 2010 paper published in the journal Cell, the researchers were able to show that plain-old fibroblasts from the heart or from the tail of a mouse could be converted into beating cardiomyocytes! The key to this cellular alchemy was the introduction of three genes – Gata4, Mef2C and Tbx5 also known as the GMT cocktail– into the fibroblasts. These genes encode for developmental cardiac transcription factors, i.e. proteins that regulate the expression of genes which direct the formation of heart cells. The basic idea was that by introducing these regulatory factors, they would act as switches that turn on the whole heart gene program machinery. Unlike the approach of the Nobel Prize laureate Shinya Yamanaka, who had developed a method to generate stem cells (induced pluripotent stem cells or iPSCs) from fibroblasts, Srivastava’s group bypassed the whole stem cell generation process and directly created heart cells from fibroblasts. In a follow-up paper published in the journal Nature in 2012, the Srivastava group took this research to the next level by introducing the GMT cocktail directly into the heart of mice and showing that this substantially improved heart function after a heart attack. Instead of merely forming scars, the fibroblasts in the heart were being converted into functional, beating heart cells – cellular alchemy with great promise for new cardiovascular therapies.

As exciting as these discoveries were, many researchers remained skeptical because the cardiac stem cell field has so often seen paradigm-shifting discoveries appear on the horizon, only to later on find out that they cannot be replicated by other laboratories. Fortunately, Eric Olson’s group at the University of Texas, Southwestern Medical Center also published a paper in Nature in 2012, independently confirming that cardiac fibroblasts could indeed be converted into cardiomyocytes. They added on a fourth factor to the GMT cocktail because it appeared to increase the success of conversion. Olson’s group was also able to confirm Srivastava’s finding that directly treating the mouse hearts with these genes helped convert cardiac fibroblasts into heart cells. They also noticed an interesting oddity. Their success of creating heart cells from fibroblasts in the living mouse was far better than what they would have expected from their experiments in a dish. They attributed this to the special cardiac environment and the presence of other cells in the heart that may have helped the fibroblasts convert to beating heart cells. However, another group of scientists attempted to replicate the findings of the 2010 Cell paper and found that their success rate was far lower than that of the Srivastava group. In the paper entitled “Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5” published in the journal Circulation Research in 2012, Chen and colleagues found that very few fibroblasts could be converted into cardiomyocytes and that the electrical properties of the newly generated heart cells did not match up to those of adult heart cells. One of the key differences between this Circulation Research paper and the 2010 paper of the Srivastava group was that Chen and colleagues used fibroblasts from older mice, whereas the Srivastava group had used fibroblasts from newly born mice. Arguably, the use of older cells by Chen and colleagues might be a closer approximation to the cells one would use in patients. Most patients with heart attacks are older than 40 years and not newborns.

These studies were all performed on mouse fibroblasts being converted into heart cells, but they did not address the question whether human fibroblasts would behave the same way. A recent paper in the Proceedings of the National Academy of Sciences by Eric Olson’s laboratory (published online before print on March 4, 2013 by Nam and colleagues) has now attempted to answer this question. Their findings confirm that human fibroblasts can also be converted into beating heart cells, however the group of genes required to coax the fibroblasts into converting is slightly different and also requires the introduction of microRNAs – tiny RNA molecules that can also regulate the expression of a whole group of genes. Their paper also points out an important caveat.  The generated heart-like cells were not uniform and showed a broad range of function, with only some of the spontaneously contracting and with an electrical activity pattern that was not the same as in adult heart cells.

Where does this whole body of work leave us? One major finding seems to be fairly solid. Fibroblasts can be converted into beating heart cells. The efficiency of conversion and the quality of the generated heart cells – from mouse or human fibroblasts – still needs to be optimized. Even though the idea of cellular alchemy sounds fascinating, there are many additional obstacles that need to be overcome before such therapies could ever be tested in humans. The method to introduce these genes into the fibroblasts used viruses which permanently integrate into the DNA of the fibroblast and could cause genetic anomalies in the fibroblasts. It is unlikely that such viruses could be used in patients. The fact that the generated heart cells show heterogeneity in their electrical activity could become a major problem for patients because patches of newly generated heart cells in one portion of the heart might be beating at a different rate of rhythm than other patches. Such electrical dyssynchony can cause life threatening heart rhythm problems, which means that the electrical properties of the generated cells need to be carefully understood and standardized. We also know little about the long-term survival of these converted cells in the heart and whether the converted cells maintain their heart-cell-like activity for months or years. The idea of directly converting fibroblasts by introducing the genes into the heart instead of first obtaining the fibroblasts, then converting them in a dish and lastly implanting the converted cells back into the heart sounds very convenient. But this convenience comes at a price. It requires human gene therapy which has its own risks and it is very difficult to control the cell conversion process in an intact heart of a patient. On the other hand, if cells are converted in a dish, one can easily test and discard the suboptimal cells and only implant the most mature or functional heart cells.

This process of cellular alchemy is still in its infancy. It is one of the most exciting new areas in the field of regenerative medicine, because it shows how plastic cells are. Hopefully, as more and more labs begin to investigate the direct reprogramming of cells, we will be able to address the obstacles and challenges posed by this emerging field.

 

Image credit: Painting in 1771 by Joseph Wright of Derby – The Alchymist, In Search of the Philosopher’s Stone via Wikimedia Commons

 

Nam, Y., Song, K., Luo, X., Daniel, E., Lambeth, K., West, K., Hill, J., DiMaio, J., Baker, L., Bassel-Duby, R., & Olson, E. (2013). Reprogramming of human fibroblasts toward a cardiac fate Proceedings of the National Academy of Sciences, 110 (14), 5588-5593 DOI: 10.1073/pnas.1301019110

Radical Tails: Antioxidants Can Prevent Regeneration

Amphibians such as frogs or salamanders have a remarkable ability to regenerate amputated limbs and tails. The regenerative process involves the formation of endogenous pluripotent stem cells, which then expand and differentiate into the tissue types that give rise to the regenerated body part. The complex interplay of the cell types and signals involved in this regenerative response to the injury are not fully known and there is considerable interest in identifying all the necessary steps. The ultimate hope is that by identifying the specific mechanisms of injury response and regeneration, one might be able to activate similar repair processes in humans, who lack the extraordinary regenerative capacity of amphibians.

The recent paper “Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration” by Nick Love and colleagues published online in the journal Nature Cell Biology on January 13, 2013 elegantly demonstrates that reactive oxygen species (ROS), also known as oxygen radicals or oxidants, play a critical role in the regeneration of amphibian tails. Using a rather elegant approach, the researchers generated Xenopus tadpoles with a genetically integrated sensor of the oxidant-sensitive protein HyPerYFP that emits fluorescence upon contact with ROS, and is thought to be rather specific for the oxidant H2O2, more commonly known as hydrogen peroxide. This allowed them to study the hydrogen peroxide levels in all cells of the live tadpole, while it was responding to an injury. They found that within 6 hours after the tail amputation, the residual tail tissue was flooded with high levels of the hydrogen peroxide and that as the tail started growing back, the regenerative edge of the growing tail continued to show high levels of this oxidant.

After excluding the possible confounding phenomenon that the increase in ROS was merely a bystander effect of increases in inflammatory cells, the researchers then performed a pivotal set of experiments in which they used anti-oxidants to see if these would impact the tail regeneration. The researchers first utilized pharmacological inhibitors that reduce the production of oxidants as well as the therapeutic antioxidant MCI-186 (its trade-name is Edaravone and is marketed for use in patients in Japan). These pharmacological agents were all very effective in terms of lowering the hydrogen peroxide levels in the regenerating tail, but they also significantly impaired the regeneration itself. In another intriguing set of experiments, the researchers treated the tadpoles with these agents immediately after the injury and then withdrew them after three days, to see if the regeneration would set in after their removal. Interestingly, when the tails were exposed to agents that prevented the generation of the oxidants, the regenerative program remained blocked even when they were removed. On the other hand, the antioxidant scavenger that soaks up oxidants being produced did not permit regeneration while it was present, but regeneration resumed after the antioxidant was removed.

The researchers also performed complementary genetic experiments in which they reduced oxidant revels by suppressing the enzymes that produce oxidants. The results all point to an important conclusion: There is a burst of oxidants that are released after injury and that are necessary to initiate the regenerative program. The exact molecular targets of the oxidant hydrogen peroxide that enable regeneration remain unknown, but some of the data in the paper points to the Wnt protein pathway as a potential oxidant-sensitive regenerative signal in the tadpole tail.

One has to bear in mind that this work was performed in tadpoles and may not be necessarily fully applicable to the human setting, but Wnt is a key regulator of stem cell renewal, differentiation and regeneration in human tissues. This does suggest that there may be some key similarities between the tadpole regeneration pathways and those found in humans. Despite the shared Wnt signals in tadpoles and humans, building a bridge from this work in Xenopus tadpoles to research and therapeutic applications in humans will be quite challenging. After all, the elegance of this study lies in the genetically integrated oxidant sensor that allows live tracking of oxidants as well as the fact that tadpoles can regenerate whole limbs and tails. Current tools do not permit real-time tracking of human oxidant levels in tissues and humans can usually only regenerate very small amounts of tissue, such as superficial skin injury.

Nevertheless, this work is an important milestone in understanding the role of oxidants as promoters of regeneration and it is very likely that at least some similar pro-regenerative role of oxidants may also be present in human tissues. One of the most important take home messages of this work is that we need get rid of the common “oxidants are bad guys and antioxidants are good guys” myth. Oxidants can be harmful in some context, but they can also serve as important regenerative signals. Indiscriminate use of antioxidants can actually impair these important endogenous signals. Instead of consuming large quantities of non-specific antioxidants, we need to use antioxidants in a very targeted, context-specific and perhaps time-limited manner so that they only prevent oxidative damage without affecting beneficial oxidant signaling.

 

Image credit: Image of a Xenopus hybrid from Figure S1 in Narbonne P, Simpson D, Gurdon J (2011). “Deficient Induction Response in a Xenopus Nucleocytoplasmic Hybrid“. PLOS Biology. 

Love, N., Chen, Y., Ishibashi, S., Kritsiligkou, P., Lea, R., Koh, Y., Gallop, J., Dorey, K., & Amaya, E. (2013). Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration Nature Cell Biology DOI: 10.1038/ncb2659

Can The Heart Regenerate Itself After A Heart Attack?

Some cardiovascular researchers believe that the heart contains cardiac stem cells or progenitor cells which can become mature cardiomyocytes (beating heart cells) following an injury and regenerate the damaged heart. The paper “Mammalian heart renewal by pre-existing cardiomyocytes” published in the journal Nature by Senyo and colleagues (online publication on December 5, 2012), on the other hand, suggests that the endogenous regenerative potential of the adult heart is very limited. The researchers studied the regeneration of cardiomyocytes in mice using a genetic label that marks cardiomyocytes with a green fluorescent protein and they also used the nonradioactive stable isotope ¹⁵N (Nitrogen-15) to track the growth of cardiomyocytes. They found that the adult mouse heart has a very low rate of cardiomyocyte regeneration and projected the annual proliferation rate to be only 0.76%. This means that less than one out of a hundred cardiomyocytes in the adult heart undergoes cell division during a one year period. Even though this number is derived from studying the turnover of cardiomyocytes in mice, it correlates very well with the proposed rate of annual cardiomyocyte self-renewal (0.5% to 1%) that Bergmann and colleagues estimated for the human heart in a 2009 paper published in Science. The key novelty of the paper by Senyo and colleagues is that they identified the source of these new cardiomyocytes. They do not arise from cardiac stem cells or cardiac progenitor cells, but are primarily derived from pre-existing adult cardiomyocytes. Does this low rate of cardiomyocyte turnover increase after an injury? Senyo and colleagues found that eight weeks after a heart attack, only 3.2% of the mouse cardiomyocytes located near the injured areas had undergone cell division.

 

This low rate of self-renewal in the adult heart sounds like bad news for researchers who thought that the adult heart had the ability to heal itself after a heart attack. However, the journal Nature also published the paper “Functional screening identifies miRNAs inducing cardiac regeneration” by Eulalio and colleagues on the same day (online publication on December 5, 2012), which indicates that the low levels of cardiomyocyte growth can be increased using certain microRNAs. A microRNA is a small RNA molecule that can regulate the expression of hundreds of genes and can play an important role in controlling many cellular processes such as cell growth, cell metabolism and cell survival. Eulalio and colleagues performed a broad screen using 875 microRNA mimics in new-born rat cardiomyocytes and identified 204 microRNAs that increase the growth of the cells. They narrowed down the number of microRNAs and were able to show that two distinct microRNAs increased the growth of cardiomyocytes after heart attacks in mice. The effect was quite significant and mice treated with these microRNAs had near-normal heart function 60 days after a heart attack.

Based on these two Nature papers, it appears that the cardiomyocytes in the adult heart have a kind of “brake” that prevents them from proliferating. Addition of specific microRNAs seems to relieve the “brake” and allow the adult heart cells to regenerate the heart after a heart attack. This could lead to potential new therapies for patients who suffer from heart attacks, but some important caveats need to be considered. MicroRNAs (and many other cardiovascular therapies) that work in mice or rats do not necessarily have the same beneficial effects in humans. The mice in the study by Eulalio and colleagues also did not receive any medications that patients routinely receive after a heart attack. Patients usually show some improvement in their heart function after a heart attack, if they are treated with the appropriate medications. Since the mice were not treated with the medications, it is difficult to assess whether the microRNAs would have a benefit beyond that what is achieved by conventional post-heart attack medications. Finally, the delivery and dosing of microRNAs is comparatively easy in mice but much more challenging in a heterogeneous group of patients.

The studies represent an important step forward towards identifying the self-renewal mechanisms in the adult heart and suggest that microRNAs are major regulators of these processes, but many additional studies are necessary before their therapeutic value for patients can be assessed.

 

Image credit: Wikimedia Commons