Mapping the Human Protein Atlas: Inside the Brain Profiling Lab at Karolinska

Part I: Introduction to the Human Protein Atlas and Mulder Lab

For thousands of years, maps and atlases have been drawn to navigate uncharted waters, to guide travelers through unknown territory and provide insight into better understanding the landscape and the world we live in. At the Biomedicum at the Karolinska Institute, the third oldest university in Sweden and one of the leading research institutions in the world, the Mulder Lab seeks to chart one of the biggest unknowns, impossible to see with the naked eye–the molecular landscape of the mammalian brain.

The Human Protein Atlas (HPA), which is a Swedish-based project that began development in 2003, was set forth with an ambitious goal: to map every protein in the human body and visualize their distribution in cells, tissues, and organs relative to time and space. Over the course of two decades, the HPA has expanded to include international collaborations and landmark functionalities, such as the addition of the Pathology Atlas in 2017, which provides information on the role of certain genes in development of cancer. The HPA has since become the largest open-source database that maps the spatial distribution of proteins, and the accessible nature of the online platform has enabled scientists from across the world to utilize its insights for biomedical research with ease.

An example of insight into which tissues a protein is expressed in from the Human Protein Atlas. The HPA has over 10 million high-resolution images that are invaluable to understanding the frontier of proteomics and gene expression.

The brain profiling lab at the Biomedicum has been one of the fundamental contributors to the Human Protein Atlas, with the generated transcriptomics data serving as a connective to other functionalities on the HPA such as the tissue and single-cell tissue atlases. This section of the international HPA collaboration is overseen by Jan Mulder, who earned his PhD at Rijksuniveristeit Groningen in the Netherlands and was a postdoctoral associate at Karolinska under Swedish physician and neuroscientist Tomas Hökfelt. In 2010, Jan returned to the Karolinska Institute to guide researchers in the newly-built SciLifeLab, a national institute and facility with state-of-the-art technologies–and has led the brain profiling group at Biomedicum ever since.

Moving Forward: Translational Approaches and Spatial Transcriptomics

Millions of people around the world every year suffer from neurodegenerative disease, which causes progressive damage to nerve cells in the brain, which can lead to debilitating loss of normal functions such as walking and speaking. Unfortunately, these conditions are incurable–and it is the hope that the HPA may one day aid in unearthing information about the underlying cellular mechanisms of these disorders, which may potentially be used in formulating preventative measures.

The HPA provides diagrams of gene expression in the brain that aid in ease of visual comprehension. Tau, a microtubule-associated protein implicated in Alzheimer’s disease, has enriched RNA expression throughout the brain, with the lowest pictured distribution in the choroid plexus.

During his postdoctoral position at the University of Aberdeen in Scotland, Jan’s focus shifted to better understand the complexity of neurodegenerative disease in human tissue, rather than the animal tissue he had previously worked with. “Neurodegenerative diseases are about changes in people’s brains and their functions, which became an increasing motivation to work on these diseases and try to find ways to cure them,” Jan said. “Once a person has Alzheimer’s disease, it’s too late because the symptoms are caused by the death of most of their cells. So if there is something we can understand about how those diseases start–and it’s something we don’t understand yet–and what kind of mechanisms are involved in the start of those diseases, we could find ways to detect early changes. And I think that’s where research into curing Alzheimer’s disease needs to go.”

In addition to being the group leader for the HPA brain profiling project at Karolinska, Jan has been an instructor at DIS since 2017–and he currently co-instructs the DIS summer course Molecular and Cellular Neuroscience. Since 2020, the Molecular and Cellular Neuroscience class has been co-taught with postdoctoral researcher Nikolaos (Nick) Mitsios, who has been with the Mulder Lab since 2011. During his PhD at Manchester, Nick studied ischemic stroke across different species and found significant differences, which has implications for designing treatments in animal vs. human models.

Recently, the Mulder Lab has been focusing more on understanding and comparing protein expression levels between different species. Often, animal models are used to understand human diseases; thus, there is a need to understand how effective the “translation” of findings are between species. The current functionality of the HPA contains transcriptomic information on humans, mice, and pigs, which may reveal the translational potential of medicine and treatments across species.

“It’s quite interesting to see how many of the proteins that we know are expressed in humans, which types of cells are in which regions of humans–to see the relationships between those animals, and to see how useful the research that is now happening in those animal models can be for humans,” Nick said. In the future, the Mulder lab hopes to add information about protein distribution in the brains of dogs, rats, and macaques.

According to the HPA, my protein, early-growth response 4 (EGR4), is differentially expressed in humans, mice, and pigs. For example, there is high expression of EGR4 in the amygdala and basal ganglia in mice and pigs but not humans, implying that this protein may play a different role between species.

The HPA has seen stunning progress since its initial development, with the ability to target 87% of human gene-coding proteins as of the last update. What’s next for the Mulder lab is to work forward towards meeting the ambitious goal laid forth twenty years ago–and to also use these findings to access and understand human diseases. Recently, the lab has started using an innovative technique called spatial transcriptomics, which measures the expression levels of gene activity and also the tissues in which activity is occurring. In the future, the lab also hopes to have more tools and good-quality material (e.g. human brain tissue) to work with to continue expanding the map.

Ultimately, Jan’s hope is to share the lab’s findings with the community and with industries to find solutions. “If I do this spatial transcriptomics data, I will share it again, like I’ve shared all my data. Why should I do it with only my brain–why not use the collective brain of the entire world?” Jan said. “We have to use our collective brain and share data to really find the answers.”

Part II: A Week in the Mulder Lab

As part of the Molecular and Cellular Neuroscience course, we had the opportunity this past week to conduct an experiment at the Karolinska Institute under the guidance of Jan, Nick, and Saga (a postgraduate associate working in the lab). Initially, it may seem a bit intimidating for American students to be introduced to a new laboratory in a foreign country, but Karolinska is a hub for international communication and exchange–a place where many different cultures, nationalities, and backgrounds interact. Neither Jan nor Nick are originally from Sweden – Jan hails from the Netherlands, while Nick is from Greece. With members from all around the world, from France to China to the United States, the lab group prides itself on its diverse background, which is no hindrance to the flourishing of science that occurs.

Learning from Jan how exactly the experiment we carried out works to “mark” where our protein is expressed within the tissue with fluorescence, which can then be viewed with a scanning microscope.

At the beginning of the week, we were given two human tissue samples: one from a healthy “control” brain and tissue from either an aging brain or one with fronto-temporal dementia (FTD), a neurodegenerative disease. The idea behind this was to see whether our protein is differentially expressed–which might be an indicator of molecular changes or perhaps implications of disease. Armed with the knowledge from our previous research into our protein of interest from multiple atlases (including the HPA), we began the process of fluorescent immunohistochemistry, an antibody-based technique that allows for visualization of localization, distribution, and cell types of a protein of interest.

First, we incubated our assigned protein in a primary antibody that recognizes and binds to our protein. In order to ensure that only the primary antibody binds to our protein, we had to perform multiple steps of “blocking” (to ensure that nothing else binds) and washing with reagents. “When you work with antibodies, you can have a nonspecific binding, so the signal that you expect may not be the one that you see,” Nick said.

The next step involves the addition of the secondary antibody, which is raised to specifically recognize and bind to the primary antibody. The secondary antibody is then conjugated with a molecule called horseradish peroxidase, which produces a colored fluorescence when it reacts with a dye-labeled tyramide, that allows for detection and quantification analyses. Since the primary antibodies should be bound to our protein of interest, the secondary antibody is indirectly attached to the protein, and if things went right, we would be able to see clearly where our protein is expressed in the brain tissues.

The process of immunohistochemistry requires utmost patience due to waiting periods designed to maximize the effectiveness of binding – but we were able to explore the essence of Karolinska and the research in the Mulder lab more during this time. During this time, we were given the opportunity to learn other techniques that the researchers use frequently, such as using a microtome to precisely section brain tissue into thin slices. For the first time, I saw robots and machines that can aid in the long process of RNA sequencing. We also participated in a science fika, where we discussed topics ranging from the role of AI in neuroscience research to the ethics of animal models in research. We ended the fika by sharing our favorite memories of the class and our choice to pursue science–something that was inspiring to hear and a reminder of why we do what we do.

Finally, on Friday, the slides were scanned and ready to view. Against the darkness of the room, our screens began to glow with dazzling colors. Reading about a protein is so much different from exploring its expression in the brain and seeing it light up in the brain scan right before your eyes. For example, we were encouraged to look for co-expression with certain cell types such as neurons and astrocytes, or if the protein is expressed only in certain regions of the brain. All of this information is something that can aid our understanding of this protein and place it on the map, which we’ll use for our final presentations next week. It sure feels like we have gone a long way since then, and I’m excited to see what insights I can present with the help of the images I took.

Part 3: Q&A (DIS Version) with Jan and Nick:

To end this blog, I wanted to share some of Jan and Nick’s insights on Molecular and Cellular Neuroscience, science careers, and studying abroad. Next week, I’ll be wrapping up the blog and saying farewell to Stockholm (which I honestly don’t feel ready for yet). I feel very lucky to have been able to sit down and have these kinds of conversations with my teachers, whose expertise, mentorship, and compassion in teaching were truly a core component of my time here at DIS Stockholm.

What is your favorite part about teaching this class at DIS?

JM: What I like about this class is that everyone comes in not knowing each other and each other’s dynamics. I also like all the questions that come from the students–they really make me think. Sharing my enthusiasm about neuroscience and thinking about the questions that come from all of you is hopefully making me a better neuroscientist as well.

NM: The quality and potential of the students is really good, and I like that you can provide knowledge to students who already have a basic foundation. And that’s something that you can enjoy; it’s different when you feel that the students can really learn something from you. I like this interaction a lot. The students come from lots of different backgrounds. And I come from a place where professors don’t interact much with the students. But here, it’s the other way around, because you have a lot of team-building activities. Professors are closer to the students, and so the students feel more confident. The mentality here is nice, so I definitely enjoy teaching like this and interacting with the students. 

What advice would you give to DIS students studying STEM?

JM: Science can be really frustrating. Many of the same things don’t work. There’s a lot of disappointment, there’s a lot of struggle, there’s a lot of hard work. You have to be lucky. If you’re not lucky, then you have to work harder. You have some kind of battery, I would say, sort of like emotional energy. Every time you get this disappointment, it kind of goes down. But then you should really be able to charge the battery to full capacity once you switch on the microscope and you see what you’re expecting to see, which is something that nobody’s ever seen before. And then your battery goes to four bars. And that means that you have some time to slowly get all the frustrations to lower your battery. So if these are things that you are really enthusiastic about doing, then I think you’re okay. If not, then it’s really tough stuff. So really do what you like, and enjoy it, so that you can recharge your battery. 

NM: Don’t be afraid to be ambitious. We live in a time where technology is doing wonders, and it’s really impressive specifically in the field of biology and biomedicine. So my advice is just to have an open mind and be really ambitious.

What is your advice on studying abroad?

JM: I think it’s good for the student. But I think it’s also good for the world. I think one of the problems in the world is that we don’t know what happens on other sides of the world. Everybody’s living in their own little bubbles. I think you should go to as many places and experience as many things as you can while studying, during the independent stage of life, because then you’ve started understanding things much better. You really notice the difference between people who have had experience abroad versus people who have always stayed in a small little community.

NM: Definitely don’t be afraid to travel. Just by going to a different country, you can socialize with other foreigners who live there. You can really learn a lot. I studied in England, which was far from my home, and I’ve had the chance to meet new people from other countries. Obviously, it’s different what you know about education and about other things about other countries from a documentary than from when you discuss it with these people all the time. It’s nice to be in an international environment. I know it’s difficult to leave home, to leave family and friends, but in the end, it’s a great experience.

Song Recommendations:

The Scientist by Coldplay, while waiting in between washes

Never Forget You by Zara Larsson, on the train to Solna

The Winner Takes it All by ABBA, lunch at Mae Thai

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