Prenatal Stressors and Epigenetics in the Development of Behavioral Disorders
"Over the last five to ten years, the plasticity has become the novel piece of epigenetics —
the fact that there are epigenetic mechanisms that are constantly responding to the environment."
Tracy Bale, PhD, is a professor of neuroscience at the University of Pennsylvania's School of Veterinary Medicine and Department of Psychiatry in the Perelman School of Medicine who studies prenatal stress and how it can translate to neurodevelopmental and neuropsychiatric diseases. Her work involves developing mouse models of stress sensitivity in order to uncover effects of prolonged stressors on future offspring's health. So far, Bale has discovered some epigenetic mechanisms that may be responsible for these effects, as well as sex-dependent differences in terms of maternal and paternal prenatal stress and identified predictive placental biomarkers of maternal stress.
After studying molecular biology and genetics at Washington State University as an undergraduate, Bale received her Ph.D. in pharmacology and neurobiology from the University of Washington, Seattle. She did her postdoctoral training in stress neuroendocrinology at the Salk Institute in California before joining the faculty at the University of Pennsylvania in 2003.
Interviewed by Meeri Kim, PhD, March 2014
Tell me about your research interests.
I'm interested in understanding how the brain either developmentally, pubertally, or in the adult responds and copes with stress. I'm a neuroendocrinologist — the study of neuroendocrinology lies within the intersection of neuroscience and hormones. So that could mean looking at hormones acting on the brain, or the brain's role in regulating hormonal production via interactions with the pituitary and other endocrine tissues. In looking at stress, we're interested in building a better mouse model to understand neuropsychiatric diseases and neurodevelopmental disorders.
An example of one of our mouse models is that of prenatal stress. We stress mice very early in pregnancy — to chronic mild stresses. So nothing painful, but more mental stressors. Ten years ago, we launched into these studies to figure out which period of gestation would be more vulnerable and produce different outcomes in offspring. We're also interested in how, during the same window that the brain might be affected, the germ cells could also be programmed. This means any effects could also be passed down to other generations.
After studying molecular biology and genetics at Washington State University as an undergraduate, Bale received her Ph.D. in pharmacology and neurobiology from the University of Washington, Seattle. She did her postdoctoral training in stress neuroendocrinology at the Salk Institute in California before joining the faculty at the University of Pennsylvania in 2003.
Interviewed by Meeri Kim, PhD, March 2014
Tell me about your research interests.
I'm interested in understanding how the brain either developmentally, pubertally, or in the adult responds and copes with stress. I'm a neuroendocrinologist — the study of neuroendocrinology lies within the intersection of neuroscience and hormones. So that could mean looking at hormones acting on the brain, or the brain's role in regulating hormonal production via interactions with the pituitary and other endocrine tissues. In looking at stress, we're interested in building a better mouse model to understand neuropsychiatric diseases and neurodevelopmental disorders.
An example of one of our mouse models is that of prenatal stress. We stress mice very early in pregnancy — to chronic mild stresses. So nothing painful, but more mental stressors. Ten years ago, we launched into these studies to figure out which period of gestation would be more vulnerable and produce different outcomes in offspring. We're also interested in how, during the same window that the brain might be affected, the germ cells could also be programmed. This means any effects could also be passed down to other generations.
Bale studies mouse models of prenatal stress.
Could you give some examples of these “chronic mild stresses”? How would these translate to humans?
We might change their cage multiple times during the day. Or we'll use restraint as a stressor by putting them into a small tube for a short amount of time. We can put novel objects in their cage that they've never seen before — for instance, marbles — or expose them to a predator odor like fox urine. Also, wet bedding can be stressful. In general, these are very psychological, and we vary them so that the animals don't habituate. This way they just continue to be stressed. So it is something stressful, but the stressor is different every day, happens at a different time of day, and for different lengths of time. The human equivalent would be anything chronically stressful: moving, job loss, death in the family, divorce, and so on.
And how does this kind of stress manifest itself as a physical change in the germ cell? Is there a window of susceptibility for the germline?
Stress can have an effect likely via epigenetic programming. A change in the environment that causes a readout to some epigenetic programming process such as a methylation point or a histone mark. Or it could involve processing of microRNAs — short, non-protein coding RNAs — that then end up in mature sperm. They bind to messenger RNAs that have already been processed within a protein complex in the cytoplasm of the cell, and this then degrades the mRNA. So basically the more microRNA, there will probably be less mRNA of that target because it has been chewed up. It is just another way that an epigenetic process can limit the amount of protein eventually made.
As for windows of susceptibility — that's kind of an unknown right now. There's evidence that there are windows of time when the male germline might be more or less vulnerable, but it seems from more recent studies that the germline can be vulnerable at almost any point. The bigger factor might instead be the type or level of stress, or whatever manipulation it is, that will determine if it affects the germline or not. It's still a big question, and no one really knows.
What have you found through your experiments of prenatal stress?
We have a model of prenatal stress that we implement very early in gestation, within what would be equivalent to the first trimester in humans. In this model, it's the male offspring that end up showing enhanced sensitivity to stress. As adults themselves, they are physiologically and behaviorally more sensitive to stress. They can then pass that onto their male offspring, so we know that the effect is somatically programmed and epigenetically germ cell programmed.
With our subsequent studies, we've been focusing on mechanistically how that happens. We have looked at the placenta as a target tissue because when we're stressing the mom, the brain is not even developing yet, so the stress has to be affecting an earlier tissue. The placenta is developing during that window, so that may be a point at which the function of the placenta could be reprogrammed. We first identified gene candidates that were affected specifically in males, and then used transgenic mice to target those genes. We're able to confirm that many of the key features we see in these early stress males are a result of specific genes in the placenta.
Also, we were able to identify this same gene and its regulation in human placental tissue. Obviously we can't do any manipulations in human subjects, but the way this gene is regulated and the level of expression is the same in human placenta which highlights the translational potential of the animal model.
What about exposing fathers to stress? Does that have an effect on offspring health as well?
The paternal model, where we exposed fathers to stress, passed on stress effects to both their male and female offspring. But they were much more limited effects as opposed to the maternal model, which had a much more profound, broad effect on the offspring. The males were exposed to stress during puberty, since that is when some of the epidemological studies suggest that there exists a window of germ cell vulnerability. But we also used another group of males that were exposed only as adults, and surprisingly this group was also able to pass on effects to their offspring.
Germline development is a highly evolutionarily conserved process. Is the biology and vulnerability of the human germline different from that of other mammals?
Probably it's not any more vulnerable, but because as humans we live longer than most other mammals and are exposed to greater variety of perturbations in our environment on a more frequent basis, we may be at a greater risk.
Are there any research topics you would like to delve into further that you have not explored yet?
One newer concept in the field is thinking about in utero exposures, but distinguishing between the somatic programming — meaning programming of that first generation — and germ cell programming. You can miss the first generation. As in, the exposed fetus may not have shown much of an effect, but yet the exposure still affects the germ cell such that that first generation can pass on an effect to the second generation. Of course, the problem is that you now have disconnected that exposure with the outcome, and this can lead to incredible difficulty in figuring out mechanisms.
For instance, take the recent rise in autism rates. This sudden increase in the number of children with autism could be related to a germ cell exposure that occurred in utero. You didn't see it in the first generation, but that first generation is able to pass it on to the next generation, so now you're seeing the effect. Mechanistically, that's a really interesting thing to examine and really nobody has explored this at all in animal models. Even epidemiological human studies become very difficult because then you have to retrospectively look back two generations and ask mothers what drugs they took. In most cases, those women can't remember. So it's a really important question that needs to be better looked at in animal models for sure. I don't have current funding to start doing toxicology, but we do think about this concept.
But we continue to look at mechanisms as well as biomarkers in the placenta that could help identify women who are at an increased risk. On the male side, we're performing more studies to pinpoint when in spermatogenesis and what the marks are that are being reprogrammed.
We might change their cage multiple times during the day. Or we'll use restraint as a stressor by putting them into a small tube for a short amount of time. We can put novel objects in their cage that they've never seen before — for instance, marbles — or expose them to a predator odor like fox urine. Also, wet bedding can be stressful. In general, these are very psychological, and we vary them so that the animals don't habituate. This way they just continue to be stressed. So it is something stressful, but the stressor is different every day, happens at a different time of day, and for different lengths of time. The human equivalent would be anything chronically stressful: moving, job loss, death in the family, divorce, and so on.
And how does this kind of stress manifest itself as a physical change in the germ cell? Is there a window of susceptibility for the germline?
Stress can have an effect likely via epigenetic programming. A change in the environment that causes a readout to some epigenetic programming process such as a methylation point or a histone mark. Or it could involve processing of microRNAs — short, non-protein coding RNAs — that then end up in mature sperm. They bind to messenger RNAs that have already been processed within a protein complex in the cytoplasm of the cell, and this then degrades the mRNA. So basically the more microRNA, there will probably be less mRNA of that target because it has been chewed up. It is just another way that an epigenetic process can limit the amount of protein eventually made.
As for windows of susceptibility — that's kind of an unknown right now. There's evidence that there are windows of time when the male germline might be more or less vulnerable, but it seems from more recent studies that the germline can be vulnerable at almost any point. The bigger factor might instead be the type or level of stress, or whatever manipulation it is, that will determine if it affects the germline or not. It's still a big question, and no one really knows.
What have you found through your experiments of prenatal stress?
We have a model of prenatal stress that we implement very early in gestation, within what would be equivalent to the first trimester in humans. In this model, it's the male offspring that end up showing enhanced sensitivity to stress. As adults themselves, they are physiologically and behaviorally more sensitive to stress. They can then pass that onto their male offspring, so we know that the effect is somatically programmed and epigenetically germ cell programmed.
With our subsequent studies, we've been focusing on mechanistically how that happens. We have looked at the placenta as a target tissue because when we're stressing the mom, the brain is not even developing yet, so the stress has to be affecting an earlier tissue. The placenta is developing during that window, so that may be a point at which the function of the placenta could be reprogrammed. We first identified gene candidates that were affected specifically in males, and then used transgenic mice to target those genes. We're able to confirm that many of the key features we see in these early stress males are a result of specific genes in the placenta.
Also, we were able to identify this same gene and its regulation in human placental tissue. Obviously we can't do any manipulations in human subjects, but the way this gene is regulated and the level of expression is the same in human placenta which highlights the translational potential of the animal model.
What about exposing fathers to stress? Does that have an effect on offspring health as well?
The paternal model, where we exposed fathers to stress, passed on stress effects to both their male and female offspring. But they were much more limited effects as opposed to the maternal model, which had a much more profound, broad effect on the offspring. The males were exposed to stress during puberty, since that is when some of the epidemological studies suggest that there exists a window of germ cell vulnerability. But we also used another group of males that were exposed only as adults, and surprisingly this group was also able to pass on effects to their offspring.
Germline development is a highly evolutionarily conserved process. Is the biology and vulnerability of the human germline different from that of other mammals?
Probably it's not any more vulnerable, but because as humans we live longer than most other mammals and are exposed to greater variety of perturbations in our environment on a more frequent basis, we may be at a greater risk.
Are there any research topics you would like to delve into further that you have not explored yet?
One newer concept in the field is thinking about in utero exposures, but distinguishing between the somatic programming — meaning programming of that first generation — and germ cell programming. You can miss the first generation. As in, the exposed fetus may not have shown much of an effect, but yet the exposure still affects the germ cell such that that first generation can pass on an effect to the second generation. Of course, the problem is that you now have disconnected that exposure with the outcome, and this can lead to incredible difficulty in figuring out mechanisms.
For instance, take the recent rise in autism rates. This sudden increase in the number of children with autism could be related to a germ cell exposure that occurred in utero. You didn't see it in the first generation, but that first generation is able to pass it on to the next generation, so now you're seeing the effect. Mechanistically, that's a really interesting thing to examine and really nobody has explored this at all in animal models. Even epidemiological human studies become very difficult because then you have to retrospectively look back two generations and ask mothers what drugs they took. In most cases, those women can't remember. So it's a really important question that needs to be better looked at in animal models for sure. I don't have current funding to start doing toxicology, but we do think about this concept.
But we continue to look at mechanisms as well as biomarkers in the placenta that could help identify women who are at an increased risk. On the male side, we're performing more studies to pinpoint when in spermatogenesis and what the marks are that are being reprogrammed.
The dominant neo-Darwinian paradigm of random variation and natural selection omits environmental responsiveness and dynamism of germline. Is this paradigm shifting?
I think the original controversy was more around how dynamic epigenetic mechanisms were, and I think that has sort of become more well-accepted now. Epigenetic mechanisms such as DNA methylation are what drives things like imprinted genes. Imprinted genes are largely not plastic, meaning that they're established very early in development and they tend to stay that way. So the controversy originally arose regarding that any epigenetic mechanism could change over the course of a lifetime. Over the last five to ten years, the plasticity has become the novel piece of epigenetics — the fact that there are epigenetic mechanisms that are constantly responding to the environment.
Should the germline epigenetics paradigm change our approach to testing and regulating chemicals and pharmaceuticals?
I do not think that effects on the germline are well-focused on when testing and regulating chemicals and pharmaceuticals. A lot of the original toxicology studies done in animal models have a lot of controversy around them based on the doses and how those drugs are administered. Studies are definitely improving — but there's still a lot we don't know, and a lot of research yet to be done.
And lastly, do you have any recommendations for parents-to-be based on your work with stress?
I get asked this question a lot, but as I'm a Ph.D. and not an M.D., I am not in a position to provide medical recommendations. However, I think broadly speaking, managing your stress is a healthy suggestion no matter what. Also, that means managing it in a healthy way — so via a healthy diet and exercise rather than, say, alcohol. I think everybody on this planet could do well to find healthy stress coping strategies. You can't avoid stress — that's probably not very realistic, so finding healthy ways to manage it is probably your best bet.
I think the original controversy was more around how dynamic epigenetic mechanisms were, and I think that has sort of become more well-accepted now. Epigenetic mechanisms such as DNA methylation are what drives things like imprinted genes. Imprinted genes are largely not plastic, meaning that they're established very early in development and they tend to stay that way. So the controversy originally arose regarding that any epigenetic mechanism could change over the course of a lifetime. Over the last five to ten years, the plasticity has become the novel piece of epigenetics — the fact that there are epigenetic mechanisms that are constantly responding to the environment.
Should the germline epigenetics paradigm change our approach to testing and regulating chemicals and pharmaceuticals?
I do not think that effects on the germline are well-focused on when testing and regulating chemicals and pharmaceuticals. A lot of the original toxicology studies done in animal models have a lot of controversy around them based on the doses and how those drugs are administered. Studies are definitely improving — but there's still a lot we don't know, and a lot of research yet to be done.
And lastly, do you have any recommendations for parents-to-be based on your work with stress?
I get asked this question a lot, but as I'm a Ph.D. and not an M.D., I am not in a position to provide medical recommendations. However, I think broadly speaking, managing your stress is a healthy suggestion no matter what. Also, that means managing it in a healthy way — so via a healthy diet and exercise rather than, say, alcohol. I think everybody on this planet could do well to find healthy stress coping strategies. You can't avoid stress — that's probably not very realistic, so finding healthy ways to manage it is probably your best bet.