Jul 17, 2023 / Neuroscience

Demystifying the complex interactions between stress, genetics, and PTSD

Josh Azevedo

Stress. The word conjures images of looming deadlines and dangerous situations, followed by a sigh of relief when it’s over. In an ideal stress response, after each of these events (termed “insults”), the person then returns to their pre-insult baseline. However, some individuals don’t fully return to baseline, and these insults can precipitate psychiatric disorders such as post-traumatic stress disorder (PTSD) and depression.

This raises important questions: why does this only happen in some people? How does an individual’s genetic makeup predispose (or protect) them from these environmental impacts? And what causes some people to develop PTSD, while others develop depression?

That’s where researchers like Harvard University’s Nikolaos Daskalakis, MD, PhD, come in. He recently gave a webinar that speaks to these concerns: “Brain single nucleus RNA sequencing of PTSD dissects the role of cortical neurons in the stress response.” Here, his group examined both bulk and cell type–specific gene expression profiles in PTSD and major depressive disorder, compared and contrasted cell-type responses in these disorders to stress hormones, and demonstrated the convergence of cell type–specific gene expression with neuroimaging genetics.

We were fortunate enough to sit down with him for a few minutes and learn about his background, why he chose to research these debilitating disorders, and how new technologies are pushing his field further than ever before.

So how did you get started in science, and what led you to where you are now?

I grew up in Athens, the capital of Greece. There was this trajectory of all good students to either go into medicine, law, or electrical engineering. I didn't know what a doctor was, to be accurate, but I chose medicine at University of Athens. The biggest deficit (in Greece) is research money, so I had to volunteer. There are individuals who are so curious—or they don’t care about being paid (like me!)—that they’ll find any location in the building they can do some experiments in.

In my case, I ended up in the Institute of Molecular Biology and Biotechnology in Crete. Later on, the European Union created exchange programs to do education and research abroad. One requirement for a medical student was to have research experience. So when I showed up and said, “Oh, I can participate in a research program because I have this research experience from volunteering,” I got this little fellowship to go to the Netherlands for four months. The mediating professor was a neuroscientist, and I started research—doing stress and behavior experiments in her lab. She collaborated with a famous professor at Leiden University, Ron de Kloet—a giant in the stress research field. I did good work and they said, “Oh, do you want to stay after your studies and do a PhD?” So, you know, that was easy.

What do you want to tell us about your current research approach?

That there is a new age of doing gene environment interaction studies. So basically, the idea that started from my PhD times is that you have some genetic background that operates at multiple time points in your life. The environment impacts the organism, but how does it program functions in the future? And can we use genome-wide association studies (GWAS), big genetic studies, or postmortem brain molecular studies, or model systems to answer these questions? That is what fascinates me the most.

So I think, now, looking at the data we have seen, we recognize that the genetics that make you susceptible to a stress-related disorder are those that are most likely to be impacted by stress at the first stop. Those genes that actually make you respond to stress are the ones that will accumulate epigenetic alterations when stress comes your way. But then the fascinating thing is that, when stress comes at different times, it might affect things differently.

Another concept is, if we envision gene networks with genes that deal with environmental interactions, what are the other nodes? Is it that you have some genetic nodes, but then you have some more plastic nodes, and, if so, how is this all connected at the edges?

Obviously, this doesn't take place in only one cell type. So then you have the cell communication network. You need to think about natural stress and about linking the various networks with hormones (endocrinology). About the stress hormones that travel the entire body and pass through the blood brain barrier—through many cellular membranes—and deliver the stress messages of homeostasis. Putting all this together is where I want to go.

What can you tell us about your current research projects? What are you most excited about right now?

We can start with what we’re discussing today. A prime example is that we established, in some postmortem brains, that they have the labels of PTSD with aspects of depression or major depressive disorder (MDD) with no aspects of PTSD. But in both groups, there are various degrees of stress exposure. So you construct various group comparisons, and you find some molecular alterations and pathways that are unique or directionally different for adults with each disorder, particularly in cell types.

So you say, “Oh, but what could be the primary factor driving these alterations?” Are there medication differences, is there less exposure, is the postmortem interval different, is the type of death different? There’s all these complicated factors, and the sample sizes are against you. But I realized I can go in vitro into the same cell type(s) and, for instance, use stress agents to study the effect of a stressor more accurately. And then we saw this fascinating opposing direction in the correlation of the molecular effects of stress hormones in stem cell–derived neurons in PTSD in brain neurons compared to MDD.

So how did you find out about 10x Genomics, and what made you decide to use our technologies specifically for your work?

One of the first difficulties when you start a lab is that the initial investment of your funds has a great impact. One other limitation I faced was the scarcity of postmortem human brain tissue. So I narrowed it down to using the single nuclei RNA-seq protocol.

Compared to other single cell technologies, I am a little bit greedy in the sense that I'm primarily doing this to confirm biology, but I also picked this technology because it can examine many things at the same time. So I wanted to be somewhere in the RNA-seq category of techniques. I’m an RNA guy primarily. Putting all this together, I ended up doing your single nucleus RNA-seq protocol in collaboration with researchers at the Boston Veterans Affairs (VA) Medical Center.

We got the 10x Chromium Controller, then did our pilot there in the VA. Then, the initial pilot experiments, with my suggestion, kind of convinced McLean Hospital to have another 10x Controller here. Since then, we have been doing these studies systematically.

I know working with brain tissue can be really challenging. When you started up with some sample prep and QC for nuclei from brain tissue, what challenges did you run into, and how did you circumvent them?

In the beginning, I was very afraid. But I could appreciate a very well written protocol, and I think it improved even more over the years—with the troubleshooting notes in the protocol. That was amazing. But when I see a very long protocol, I still feel scared.

So what we did was actually set up the training with the specialists that came from 10x Genomics in the brain bank to meet us. That was a key experience. I followed it—I'm a PI, but I was there and I tried to do my own samples. And two postdocs and I did this protocol. The initial samples were quite okay. Then I thought about how to build it up; we did one sample, three samples, nine, and then we were comfortable!. Then we did an experiment together with another lab, and they were doing their own libraries. We compared our libraries to their libraries bioinformatically. The protocol is straightforward enough that, if you pay attention to the details, you get good data. The cell type–specific signals are very robust, at least for the cortical region we have done many times. We know what to expect.

How do you feel your field—and the public perception of it—has changed in the last decade, especially with the stigma around psychiatric illness?

I feel that all these contributions we’re collectively making, knowing the biological aspects of psychiatric disease, help remove that stigma. Now we also know that we should not group all patients in one bucket. From a research perspective, it’s very hard right now to remove all the known diseases. Theoretically speaking, you can remove them all and have a more individualized approach [based] on the unique combination of symptoms that each patient has. But that includes factoring in these groups’ social health, as well as research and treatment plans, and we don't know how soon we can remove all those things.

I think publicly recognizing those facts, having also given tools to advocates by defining how stress works, how genetics affect your brain, makes psychiatric disorders like any other illness. Advocacy within the health sciences will help in this domain.

What do you wish more people knew about PTSD, depression, and these types of illnesses?

I want them to understand that stress is not a bad thing. Stress is an umbrella term we use for how our systems respond to challenges. That isn’t, itself, a negative process. Positive experiences can also be a stress response. It's not that the people that have stress-related disorders get exposed to stress and have an immediate problem, it’s that they have some underlying biology that [means] their systems don’t return to balance. So that understanding is the biggest miscommunication about stress: we would basically die without a cortisol response.

So, one final question: we love hearing scientists talk about their work. Is there anything you haven’t had the chance to talk about today, either with this work or otherwise, that you really want people to know?

Something that was not part of our webinar, but I think is important, is that there is the whole translational path. We get better at GWAS, but then you have the silo of GWAS people that are focusing on improving association results, and you have the functional genomics people that are willing to kind of improve their respective molecular resolution and sample size. Then, when you have people that do the biological experiments, and the people that do regular clinical studies, they need biomarkers to deploy because they want to understand the clinical topic and stratify patients to improve clinical trials. It's hard because the way that the funding goes, it normally funds the experts in the silos. Sometimes translational efforts are not appreciated, but we think it’s important.

In my lab, we’ve put a lot of effort towards creating an international team with diverse backgrounds and expertise. We have people that are focusing on various aspects of the entire trajectory from genetic discovery all the way to deploying biomarkers in a clinical study. Only by being active in the entire trajectory can we say to a collaborator, “I think we offer something you could add in your study that might help you understand a little bit more about environmental versus genetic influences in the disease.”

Also, it's kind of impossible to measure all these exciting variables, but we can use genomic data to get (impute) some of those variables in a clinical study. In recent years, we’ve put a lot of work into that space. To conclude, I think developing this lateral thinking to address a question from every way you can is something that I want to add to the field.

Thank you very much for taking the time to talk with us today!

Thank you.

This interview has been edited for length and clarity.

To learn more about Dr. Daskalakis’ work, visit the Neurogenomics & Translational Bioinformatics Laboratory (NG-TBL), rewatch his recent webinar, or explore some of the methods his group used in their research here.