Dr. Ricardo Dolmetsch, Stanford University

Ricardo Dolmetsch, Ph.D., is an assistant professor of neurobiology at Stanford University. In 2007 he received the Society for Neuroscience Young Investigator Award, and in 2008 he received the NIH Pioneer Award for his work with induced pluripotent stem cells to study autism. Dr. Dolmetsch was a keynote speaker at IMFAR 2011. ASF intern Max Rolison interviewed Dr. Dolmetsch about his research and his unique role as both a scientist and the parent of a child with autism.

Max Rolison: How did you first become interested in autism research?

Ricardo Dolmetsch: I became interested in autism research because my son has autism. We always knew there was something different about him, but when he was four he was formally diagnosed. I think we went through the kind of stages that every parent goes through. The first stage was denial. That was followed by, “what can we do,” so we tried all of the available treatments. I happen to be a neurodevelopmental biologist, so I started to look at the literature and in those days there was very little in the way of any biological understanding of the disease, and there were no drug targets. So I decided to change the direction of my lab to do that.

MR: You previously researched calcium channels. How does your previous work relate to your autism research?

RD: Serendipitously, it turns out there are a couple of mutations in one calcium channel, and possibly mutations in another calcium channel that are associated with syndromic forms of autism. For me, it was actually sort of helpful because it allowed me to connect stuff I knew about to stuff I knew nothing about. Specifically, there is a mutation in the CaV1.2 channel, also called the L-type calcium channel, which leads to something called Timothy Syndrome, which is a very penetrant, but very rare form of autism. There are also much more common mutations, in the same channel, that are associated with bipolar disorder. And there are mutations in a different member of the same family that are also associated with autism. So that’s how the two parts of my career are related.

MR: What is your lab currently researching?

RD: We basically have two different projects. The overall goal is to identify the molecular and cellular basis of autism spectrum disorders, and to identify molecular targets that we can use to develop new drugs. We have started by studying genetic mutations that have been identified in populations of children with autism. So the question is, “how do you go from a mutation to trying to understand what it is that that mutation does.” There are basically two approaches. One approach is to make an animal model, and we’ve done that. We’ve taken, for example, some of the common copy number variations, where there are microdeletions or microduplications in the genes, and we’ve introduced them into mice, or into flies, and we look to see in the mice how that affects behavior and the development of the mouse brain. But we know that mice aren’t really great models for humans, and so the other approach has been to study people. A big part of my lab has been devoted to developing a new approach for studying the molecular architecture of the human brain. Basically we don’t have access to brain cells from children with autism, and so this has impeded the field for a long time. But relatively recently it has become possible to take skin cells or blood cells and reprogram them so they are no longer skin cells or blood cells, instead they are stem cells. And they are pluripotent stem cells, meaning that they have the capacity to form essentially any tissue in your body. So we have generated many pluripotent stem cells from many different children with autism. The next step is to coax these cells into making little brains, or sometimes little hearts, because some of these kids also have cardiac defects. Once we have those miniature brains in a dish, then we try to characterize them, using some of the molecular and cellular tools that have been developed to study brain development. We also collaborate with a lot of people who have been helping us with assays that we can’t do ourselves. So that’s our human work, and then in our mouse work, we have been collaborating with people to test the behavior of some of these mouse models with some kind of autism, and also trying some potentially new treatments.

MR: What is the process involved in turning a skin cell into an induced pluripotent stem cell, and then differentiating it into a brain cell?

RD: The first part of the process is converting a skin cell into an induced pluripotent stem cell. There are several basic approaches, that all depend on introducing into a somatic cell, (which is a cell that is already differentiated) a set of genes that changes the packaging of the DNA. You can do this in several different ways; you can use viruses that have these reprogramming genes, or you can introduce RNA, or you can use a kind of RNA called a microRNA. We then we put the cells in growth conditions that favor the growth of pluripotent stem cells, and then we wait, and look for cells that form colonies that look as if they were human stem cells. We then take those put them through a series of comprehensive tests to determine whether they really have been reprogrammed or not. Once we are convinced that they really have been reprogrammed, we can use those cells to try to generate neurons, or cardiomyocytes. Here we are standing on the shoulders of giants, in the sense that we know quite a lot about the conditions required to make brain cells in a dish. You do is you start off with these little clusters of induced pluripotent stem cells, and you convert them into embryoid bodies, which are like the very early stages of the development of an embryo. You take those embryoid bodies and put them in special culture conditions that favor the development of the brain, and when you do that they form the very earliest stage of the development of the brain, something called the neural tube. You can take these little neural tubes that are forming in a dish, and isolate them mechanically, and grow them in a suspension culture and eventually you get these little floating balls that are called neurospheres. We develop our little balls of neurons in such a way that we promote the formation of cells from the cortex, and not the spinal cord or other parts of the brain. And that’s because we are very interested in the cells in the frontal lobe, which people think are important for autism. So that’s basically the way it works. There are hundreds and hundreds of man-years of work in developing these methods, not just by our lab but other labs, but that’s more or less the way it works.

MR: Are all of the cells you study formed from induced pluripotent stem cells? Do you compare the synthetic brain cells with autism mutations to harvested brain cells, or are both autism and neurotypical brain cells synthetic?

RD: In the ideal world, we would like to be able to compare the induced pluripotent stem cells derived neurons to neurons that are actually in the brain of somebody with autism. Typically we don’t have access to neurons in the brain of a child with autism, except in some very rare and tragic cases. So we do a couple of things, because, of course, one of the key questions is, “is this really a good model?” You have to understand that this isn’t just a scientific project for me, I want to make sure that I’m working on the right disease and I’m moving forward, so I want to know if induced pluripotent stem cells are a useful tool or not. So how do you validate them? You can validate them in three ways. One way is to compare them to a mouse model of the same disease. What we found in that case is that there are some defects in the human cells that you can also see in the mouse neurons, and the mouse neurons are of course developed in a mouse, so they are actually real brain cells. The other approach that we are taking is to try to compare these cells in a dish to cells in a human brain. We are facing the fact that we just don’t have very many brains from people with autism, especially brains from people with defined mutations. Finally we would like to see if a drug that fixes the defects in neurons from an individual in the lab also changes the behavior of that individual. This is the ultimate validation.

MR: What are you comparing between the cells? What are you looking for?

RD: The question is, “how would you find something wrong” and “what class of neurons do you look for”. There are basically two approaches. One approach is to compare which genes are activated in neurons from kids with autism to those from neurotypical controls. That is helpful because we know a lot about a lot of genes, so sometimes we can use that as a way of identifying which signaling pathways and which kinds of cellular processes might be altered in a particular type of autism. In addition to looking at the activation of genes, we can do other things, like, for example, looking at the production of metabolites. There are certain metabolites that are produced by different metabolic pathways and those might be different. We can also look at differences in the secretion of neurotransmitters. We can measure all these things in a relatively unbiased way, where we don’t start off with a preconceived notion of what is wrong. The other approach is to make a hypothesis based on whatever set of mutations we are working on. So, for example, the most common mutations in autism are on chromosome 16 and chromosome 15, and that encompasses a whole bunch of genes. Based on what is known about the function of those genes we can look at specific processes in the neurons. An example of that is that we have cells from children with mutations in a protein called Shank3. Shank3 is known to be important for the function and formation of synapses, so it makes sense for us to look at synapse function and compare the synapses of the patients to those of neurotypical controls.

MR: It is uncommon to use induced pluripotent stem cells to study autism. Can you discuss why you think this is, and what you think the future of this technology in the field is?

RD: I think it’s uncommon just because it’s new. We have pioneered this area. I don’t think it will be uncommon for long. In fact, I know there are all manner of other people that are doing this as well. I think that it’s like other developments in science. You pioneer them, and if they look like they work and produce interesting, valuable information, other people will do them, and I’m pretty sure this will happen here as well.

MR: What can we learn from studying induced pluripotent stem cells that we can’t learn from other research techniques?

RD: A lot of things. Some classes of autism are going to require new therapeutics, new drugs. And in those cases, we have to be able to identify enzymes or biochemical signaling cascades or cellular defects that we can use to screen for compounds. The way we discover drugs is we identify a defect in a protein or defect in a cell, and then we design an assay based on that that to screen half a million compounds. You can’t really do that kind of screen in a mouse or in a fly or in a human being, so it is important to identify cellular and molecular defects associated with autism so that we can look for new drugs. The other thing that has become apparent is that sometimes we can make recommendations about possible therapeutic options from what we find in the cells of the patient. For example in one class of patients, we made some neurons and we discovered that this mutation causes them to generate too many cells that produce norepinephrine. Norepinephrine is a neurotransmitter that is really important, it activates the fight or flight response and causes anxiety. In fact, these children are very anxious so it is probably a good idea to treat them with blockers of the receptors of norepinephrine. While these blockers may not work for the majority of children they might work for these kids. It is reasonable to try some of these ideas specially if there are no good therapeutic options and the drugs are relatively safe. I think it will be possible to do this for other kinds of autism where we may be able to identify misregulated signaling cascades and formulate hypotheses quickly. If there are drugs that already available, the turnaround from the study back to the patients could be really fast. Even in cases where that isn’t true, this approach will help us look for new drugs, and this is something I think you can’t really do in any other way. This is the way we discovered the cancer drugs that we have. We’ve just never been able to do that in the brain because we never had access to brain cells.

MR: What value does this research have for families?

RD: There are three areas where I think we provide value to families. In some cases, but they may be rare, there may be some immediate value if we can determine that there is a particular signaling cascade or particular class of neuron is misregulated in a specific child we might be able to direct therapeutic interventions. It might allow us to suggest treatments that would never have been suggested because basically those drugs are not normally used to treat autism. The other way in which we provide value is by identifying new therapeutic targets for the development of drugs. This will take a while but if you never start, then you will most certainly never get there. Finally I think that patient-derived stem cells might be useful as a diagnostic by helping us answer question like “how many classes of autism are there,” and which kinds of treatments work for which kind of autism.

MR: What are your personal goals for the field?

RD: I want to cure the disease. I want to cure a kind of autism. In fact, I’d like to cure my son’s autism, to be perfectly honest with you. But in terms of my kid it might be really hard. I would like to identify a therapeutic target, and I would like to be able to drug it, or make some suggestions about therapy that are really valuable to families. That’s a primary goal. A secondary goal is a mechanistic goal. I would like to know what is different; what changes in the brain of a child with autism. But I have to say, biology is by necessity opportunistic. We work with this general goal in mind, but there are so many things about how the brain that we don’t know that you can’t really say, “I am going to do this”. You work on something, and if something else appears along the way, you take advantage of that.

MR: You have a unique position as both an autism researcher and the parent of a child with autism. How has that affected your research and your parenting?

RD: Well it has affected my research A LOT. For one thing, I wouldn’t have entered the field except for the fact that I am a parent. I also get to interact with a lot of families, so I spend a lot of time thinking about what matters the most to them. So problems with social interactions might be at the core, but maybe they aren’t as big of a problem as difficulty sleeping, or irritability, or intractable epilepsy, maybe those are worse, maybe I should focus on that. So I think about that a lot. Being a researcher has changed my parenting… of course I have tried to base my parenting on what I know about the science, and there are some things where I have thought about it more deeply than I would have thought about it otherwise. On the other hand there is so much that we don’t know that I navigate blind like other parents.

MR: As a parent, do you think that some of the scientific research that is being conducted is too inefficient or too esoteric?

RD: I think it is a difficult question to answer, because a lot of research that may seem esoteric turns out to be really important in the future. I can think of all sorts of examples in fields where we have made more progress, like cancer or cardiovascular research, where basically everything we know, all the drug targets we have, came not from studying people with cardiovascular people or cancer, but came from studying for example fly signaling pathways. People were interested in understanding the fly eye, and it turns out some of those same pathways are important in cancer. A lot of the clues we have about what genes do comes from what would be considered really esoteric research looking at fundamental development of the brain. So I think you always have to balance applied research with basic research. You are making bets. Yes, there probably is some research that will not be useful at all and is very esoteric, but I think that that research is just as likely to be very basic research as it is to be very applied research. There is a lot of applied research that is also not very good and not going to move us forward very much. Sometimes we are trying the same therapeutic strategies over and over, or we’re making minor modifications and that is kind of like beating the dead horse.