Sarah Linnstaedt, PhD, received her doctoral degree in 2008 from Georgetown University, Washington, DC, and then completed a postdoctoral fellowship in 2012 at Duke University, Durham, North Carolina, US. She is now an assistant professor of anesthesiology at the University of North Carolina at Chapel Hill, US, where she studies the role of microRNAs in pain. Linnstaedt spoke by phone recently with Neil Andrews, PRF executive editor, to discuss how she came to the field of pain research, her microRNA studies, and what it has been like as a young investigator to set up her own lab. Below is an edited transcript of their conversation.
What was your path to pain research?
When I was a postdoctoral fellow at Duke University, I was examining mechanisms by which RNA and microRNA contribute to lymphomagenesis. I was also looking at microRNAs as predictors and biomarkers of disease. Dr. Sam McLean, who is vice chair of research in the Department of Anesthesiology at UNC, found out about my research and contacted me. He was doing research on post-trauma pain development and wanted to incorporate microRNA studies into that work, so he asked if I would consult with his group.
I started talking to Dr. McLean, and after a few conversations was fortunate to be invited to join his research group. I was extremely excited by his offer, so I joined the group and started reading about chronic pain and the burden it causes society, and the individuals who suffer from it. For me, the enormous scope of the problem of pain was then obvious, and I knew I’d have an important job in researching mechanisms of chronic pain development. I hoped that my training in RNA and microRNA biology would help to further the understanding of important pathogenic mediators that are driving post-trauma chronic pain development.
Can you say a little bit about microRNAs?
MicroRNAs are small, non-coding RNA molecules. They're called “small” because they are 21 to 23 nucleotides in length. They can bind to messenger RNAs or really to any kind of RNA, but they mostly bind to the 3’ UTR [untranslated region] of messenger RNAs. When they bind, they can either cause degradation or translational inhibition of messenger RNA. The result is altered levels of the protein that would have been made from the messenger RNA. MicroRNAs are very powerful regulatory molecules that determine how much protein is made in a cell or a biological system.
Why study microRNAs specifically in the context of chronic pain?
MicroRNAs play a role in almost all biological processes and have been shown to play pathogenic roles in a variety of diseases, from cancer to heart disease. Research to date in the pain field has shown that microRNAs are also associated with various types of pain, and appear to be important pathogenic mediators in the development of chronic pain.
What are the goals of your microRNA studies?
One major goal is to use microRNAs as predictive tools, since they are very stable in blood and easy to detect. These tools could be used in the very early aftermath of trauma to predict who will develop chronic pain before chronic pain symptoms or pathogenesis manifest. Once we can identify those individuals who are susceptible to pain, then hopefully we can treat them before the pain ensues. Because there aren’t great therapies for post-traumatic chronic pain, a second major goal is to identify specific targets for therapeutic intervention so that once we identify who is at risk, we can treat them before chronic pain develops.
What studies have you done on microRNAs and pain?
The first study I did when I started at UNC was to identify a microRNA signature predictive of chronic pain. Using RNA sequencing approaches, we can reliably detect about 400 to 500 microRNAs that circulate in the blood from individuals we have enrolled in our studies. That's a lot of microRNAs, but they're not all predictive of chronic pain development. What we found was that only a distinct subset of about 30 microRNAs could predict chronic pain outcomes.
What was interesting about that study—and what I didn't really expect to find, since we were just looking for predictors—was that there was an enrichment of microRNA originating from the X chromosome; higher levels of microRNA predicted pain in women but not necessarily in men. That suggests that there might be sex-dependent mechanisms influencing microRNA expression that then affect the development of chronic pain.
I am now looking at some of these sex-dependent microRNAs and am trying to understand the mechanisms that regulate them. It’s an important topic right now because of the need to determine why women develop increased incidence and severity of pain—microRNAs can help us understand the mechanisms a little bit better.
What have you found so far?
We have some exciting data that identify miR-19b as a pain regulatory hub—that is, it regulates more pain transcripts than expected by chance—and it predicts chronic pain and comorbid post-traumatic stress disorder (PTSD) in a sex-dependent manner following motor vehicle collision trauma, and is expressed differently in male and female animals in tissues that are important in the regulation of stress and pain.
When we look at what this microRNA regulates, there's an enrichment of genes that are associated with the circadian rhythm. Other groups have shown that alterations in circadian rhythm processing contribute to the development of neuropsychiatric diseases including PTSD, and we have recent genetic data indicating that the circadian rhythm is also important in post-trauma pain outcomes. If miR-19b regulates genes involved in circadian rhythm, then there may be differences in circadian rhythm pathways in men versus women that could influence sex differences in susceptibility to chronic pain.
Any ideas as to why there would be sex differences in microRNAs expressed in chronic pain?
One hypothesis we have has to do with how microRNAs are regulated. MicroRNAs are just like any other transcript in the cell in that they're regulated by transcription factors. Transcription factors are regulated by a number of different things, including estrogen levels. We're particularly interested in some of the microRNAs that are regulated by estrogen, how estrogen levels in individuals—when they experience trauma—might influence microRNA levels through estrogen-stimulated transcription factors, and then how the microRNAs are pathogenic. There are other mechanisms by which microRNAs are regulated, but there are definitely a number of sex-dependent mechanisms that can be tested.
What else are you working on now?
Another study that we're working on is looking at FKBP5, which is a gene that encodes the glucocorticoid receptor co-chaperone. Sam McLean and colleagues published a genetic association study in 2013 showing that polymorphisms in FKBP5 predict chronic pain development. This gene had been associated with a number of different neuropsychiatric disorders before, and the role of this gene in glucocorticoid resistance is a hot topic, but no studies had previously shown that FKBP5 plays a role in chronic pain development.
Building on this study, we now have data demonstrating a molecular mechanism by which FKBP5 polymorphisms influence chronic pain outcomes following trauma. This mechanism involves allele-specific regulation of FKBP5 by miR-320a, a microRNA we previously showed to influence chronic pain outcomes following motor vehicle collision. In a collaboration with Alain Laederach’s lab at UNC, we are also able to show that the allele-dependent effect on miR-320a binding is likely due to differences in RNA secondary structure in the 3’UTR of FKBP5 in individuals with the risk versus protective allele. It’s one of my favorite research studies that I've done since I've been at UNC. We're putting some finishing touches on this research and are hoping to get it out soon.
What are the most important questions that remain with regard to microRNA and pain?
There is a lot of great research showing that microRNAs are associated with pain, but there are not a lot of studies examining the regulation of pain-associated microRNA or the causal relationships behind the associations. Building up that level of evidence and showing that microRNAs are important regulatory hubs in controlling various parts of the transcriptome that then influence chronic pain levels are the data that our group and others are working on. There are already a few great examples of miRNA playing essential roles in pain development, but in general, the level of evidence needs to be bulked up so that we can better identify targets for therapeutic intervention.
Additionally, because much of the current understanding of which miRNAs are involved in pain pathogenesis stems from animal studies, it will be important to translate this knowledge to humans. Reciprocally, because many human studies use blood tissue, it will also be important to determine whether pain-miRNAs identified in human association studies also play important roles in tissues relevant to pain processing.
What’s it like to be a young investigator with your own lab?
It's very exciting. You're kind of just thrown in there: Here's your lab and have fun! And that's basically what it is—a lot of fun. I knew from working with students during my postdoc that I needed to have students in the lab to be able to produce enough data to be successful. So in the first two weeks after joining the UNC anesthesiology group, I advertised for an undergraduate student to work with. I was very fortunate to find an amazing student, Maggie Walker, who really set the stage for all my future students and who did most of the work on the first few publications from the lab (for example, the first publication I mentioned above, as well as one about ADRA2A, the gene that encodes the α2A adrenergic receptor). Having great students in the group has enabled me to focus on advancing my writing skills and submitting grants.
Since joining the UNC Anesthesiology Department, I’ve always said that it feels like a family, but it's run like a business. And I think that's a good thing—so I embrace that mentality as I’m running my own lab. I try to make the lab work fun for all of the students by giving them independent projects, taking them to conferences, and so on. But I also am very rigorous about the business aspects—I keep everyone goal oriented and focused, run "lean," scale appropriately based on funding and need, and work hard on my leadership skills. Doing all of these things while also trying to stay innovative with research ideas can be challenging, but it sure is fun.
What is the biggest challenge facing young pain researchers?
The biggest challenge is finding good mentors—and you should have a good mentor at every stage of your research. I've been very fortunate. My PhD mentor, Dr. John Casey, was amazing and taught me all of the foundations of science and research, and inspired me as a scientist and as a person. My postdoctoral mentor, Dr. Bryan Cullen, helped me learn how to focus my studies and do the right experiments to answer my questions and tell a cohesive story. My current mentor, Dr. McLean—I can’t say enough great things about him—is still teaching me how to write really effective research grants, and how to write manuscripts and deliver engaging talks. This is so very important. If you don't know how to communicate your research effectively, then you're at a loss in science.
I'll always seek the mentorship of people who are more experienced than me, even when I've hopefully transitioned to being an associate or full professor. There's always a wise soul out there who can give you advice about how to succeed in your career.
When you're not doing science, what are you doing? Any hobbies?
I love photography, and I started a photography business when I was a postdoc. Unfortunately, I haven't had time to do much with the business recently, but I still love photography, and I continue to take pictures of my kids, friends' kids, families, and some animals/landscapes.