Chris E.: Welcome to the Salk Talk podcast. I’m your host, Chris Emery. In today’s podcast, I interview Dr. Sam Pfaff. Dr. Pfaff is a professor at Salk Institute, as well as a Howard Hughes Medical Institute Investigator, and holder of the Benjamin H. Lewis Chair. To learn more about the Salk Institute and it’s research visit www.salk.edu.

Dr. Pfaff, thank you for joining us today. Earlier this year I was in your office and you showed me these beautiful pictures of brightly colored bundles of neurons, growing in a lab dish. And you had a video of these bundles and they were flashing like strobe lights and you told me you had dubbed them “circuitoids.” Is that how you say it?

Samuel Pfaff: That’s correct, Chris.

Chris E.: What exactly are circuitoids?

Samuel Pfaff: It’s one of the luxuries that we have in science. Sometimes we come up with something new, we have the luxury of giving it our own name. Whether the name circuitoid or not actually sticks long-term, time will tell. What we intended to convey with the name circuitoid was the notion that we had constructed what might be regarded as a very synthetic circuit. And it was constructed by starting with embryonic stem cells. In our case, mouse embryonic stem cells because we can genetically engineer those cells in order to see and track different cell types. And we were very interested in generating specific kinds of neurons that you would typically find within the spinal cord, from the embryonic stem cells. Then asking whether we could combine those purified neurons that we had derived from embryonic stem cells in ways that would begin to produce an activity that we would normally find within the spinal cord.

And the activity that we were looking for was the kind of activity that is, in the field known as a Central Pattern Generator activity. And what that activity represents is a core kind of circuit within their spinal cord that is driving the alternating step movement of our legs. And one of the hallmark features of that circuit is that it alternates. It’s on and off.
Chris E.: And that was the flashing?

Samuel Pfaff: And that was the flashing. So we had engineered cells, well first we had produced different types of neurons that you would find in the spinal cord. We would then combine them in a dish, in a way, in the right combinations. And then we exposed them to some of the drugs, some of the neurotransmitters that would normally be present within the spinal cord. And on their own they took on the activity of the Central Pattern Generator, and they began flashing. Almost as if they were trying to send the instruction that we want to produce a walking or stepping kind of rhythm. The name circuitoid is kind of goofy as it sounds, is really from a longer history of other kinds of related terms. And in the stem cell field, the term embryoid and organoid had already been coined. And you can guess that it’s because stem cells had been used to produce embryo-like and organ-like tissues. And because we had produced circuit-like tissue, we extended that to the name circuitoid.

Chris E.: So what do you do with a circuitoid? You have them, you’ve put them in a dish, they’ve grown together to form these sort of basic circuits that you might find in the spinal cord. What’s the next step?

Samuel Pfaff: I’m nodding my head yes because … No one can see that. So a variety of things. The kinds of questions that we thought were immediately accessible with the system were questions that related to understanding things like how do you regulate the speed of stepping? We don’t really have a great idea of how that’s done. And we hypothesized that having different ratios of neurons recruited within the circuit might influence how fast or how slow the rhythm that the circuit produces, might influence that rhythmicity.

So we were able to test that directly by taking purified neurons and combining them in different ratios and then measuring the speed of the flashing that you saw. And we found that a particular group of neurons, when we changed it’s concentration was capable of changing the rate of the activity of the circuit. And that would be the kind of experiment that would be very difficult to do in an animal where it’s difficult to go in and just change the ratio of neurons relative to one another. It’s the kind of thing that we could take advantage of in a dish. We can control the numbers, and that was one of the kinds of things that we did.

Now, down the road, we have great hopes that, hope that we will be able to expand this to studies where we begin to think about how we might leverage it into understanding treatments for spinal cord injury. And there are projects underway in the lab right now in which we’re taking some of the cells that we’ve studied in the circuitoid context and beginning to transplant them back into the spinal cord. These are animal studies, mouse studies and looking at how the neurons integrate within their host environment. So the next steps for us, one of the next steps is to see how we might be able to apply this to something that is clinically relevant. But having said that, there’s still a lot of very fundamental issues that need to be resolved in regards to understanding how a circuit actually processes information, having a reduced system that we can look at in a dish, watch in real time, is much more accessible system than studying an animal. And we’re also trying to understand very fundamental principles about how the circuit functions.

Chris E.: So taking a step back, how did you get involved in and interested in spinal cord research? The nervous system is a large place. How did you land on the spinal cord?

Samuel Pfaff: Right. You know, it’s partly interest and it was partly good fortune, I would say. If you don’t mind, can I take a little time and tell you about my career?

Chris E.: Absolutely.

Samuel Pfaff: Okay, so this is maybe going to be a little bit of a long-winded story that gets to your eventual question here. I grew up in Minnesota, and my father worked at the Mayo Clinic. I was probably regarded as a fairly nerdy kid, although like a lot of people in Minnesota, I spent a lot of time outdoors as well.

Chris E.: In the summer?

Samuel Pfaff: In the summer. But, you know, in Minnesota people don’t talk about bad weather. You just do it. So it was summer and winter outdoors a lot. But I was very, very interested in science. You know, I had a fossil collection. I had insect collections. All kinds of things. I had my own little microscope. I was just fascinated by anything I could see and begin to understand, that especially had a biological context. And I was fortunate in that I had the opportunity to, even as a high school student, work in a research laboratory at the Mayo Clinic. And my first exposure to research was in the area of neural disease. Something called peripheral neuropathies. And it was a lab that was run by a clinician so everything was very relatable to diseases.

At that time, I didn’t necessarily formulate a specific desire to work only on the nervous system or on the spinal cord, but I really knew that I loved biological research. It just completely fascinated me. So I went to college of course. I went to college at Carleton College in Minnesota. It’s a liberal arts school. I did poorly in everything that was non-science related, which only reinforced my desire-

Chris E.: You were on the right path?

Samuel Pfaff: That I was on the right path. But it turns out, for any younger folks out there who are thinking about science that writing and communication is an incredibly important part of science, and a well-rounded education will certainly pay off. Okay, so I went to Carleton and I took, one of the classes I took was in development biology. Fetal embryology. One of the things that we did was watched a frog embryo develop from a fertilized egg.

And a frog embryo can develop in approximately 24 hours, and it can go from fertilized egg to a swimming tadpole that is reacting with it’s environment in 24 hours, and you can watch that in real time. I was so fascinated I stayed up all night in the lab watching in the microscope, the cell divisions and suddenly this thing was able to swim and sense touch and breathe. And I thought, “That is such a remarkable biological process.” There are a million questions tied up in how you go from the one cell to making all kinds of specialized cells, building a nervous system.

The kind of biology I would really love to do is biology that relates to the fetal development. So after college I went to UC Berkeley, in part because they had a phenomenal group of scientists there who studied embryology. And it was my intention to do a PhD in the area of embryology. But this was also a burgeoning time in molecular biology, when it was possible to begin to clone genes, and really study gene function. It was still early days, but it was clear that this was going to be the tool that really helped shape our understanding of biology.

And I was kind of confronted then by a difficult decision when deciding what labs to go into. Should I go into a lab that is what I would characterize as traditional embryology that really studies things at a cellular level, or should I think about getting training in more molecular systems and return back to studying developmental biology with a molecular approach. And I decided that I really needed to develop my skill sets in understand molecular biology, and return to the problems of embryology, because that was, I really wanted to study embryology at a molecular level. And so I went into a lab that studied cancer biology thinking that cancer was kind of the opposite of development. It’s really kind of an aberration of development and a misregulation of cell identity. And I learned a lot of molecular biology in that context.

And then I went on to do a post-doctoral fellowship in which I started to work in embryology on frog embryos, and applying my molecular skills. But felt I wasn’t quite at the point where I could get the sort of faculty positions that I wanted. And a dream position for me was to come to a place like the Salk Institute, which is pure, unadulterated science with no other distractions. And since I wasn’t going to be able to make that step directly, I decided to do another post-doctoral fellowship. And at that time, there was a very well-known scientist, still early in his career, named Tom Jessell, who is a, also a nonresident fellow of the Salk Institute, who was one of the leading people applying molecular techniques to understanding development.

And the development that he was very interested in was the development of the nervous system. And he had made the very conscious decision to study the spinal cord thinking that the spinal cord, knowing that the spinal cord was functionally important, of course, for many of the activities that we, and behaviors that we perform as humans. But beyond that, knowing that the spinal cord is part of the central nervous system but perhaps a relatively simple part of the nervous system. And if you’re going to get started some place, let’s work out basic principles in the simpler system.

So I went into the Jessell lab and that really cemented my, which is at Columbia University … That really cemented my interest in molecular developmental studies of the nervous system, and really focused my attention on the spinal cord, and in particular, the part of the spinal cord that controls movement. And since that training with Tom Jessell, which is now a little bit over twenty years ago, my lab has taken a variety of approaches to studying the spinal cord and in particular studying movement. But it’s been very heavily shaped by that career path I just described and especially the mentor Tom Jessell. So that was the long answer, I apologize.

Chris E.: No, don’t. That’s exactly what I wanted to know, how you got to this place. You know, it’s funny, I think a lot of people, myself included, when we think of the spine, or the spinal cord and the motor system, there’s a tendency to think of it as kind of incoming wires and outgoing wires. The brain’s doing everything, but from talking to you and others here at Salk, you quickly learn that there’s a lot more going on outside of the brain. A lot more computation and more complex circuits that things are happening that we’re unaware of at the conscious level, you know, below our neck.

Samuel Pfaff: Right, so and now you’re getting into areas that keep me going late into the night, and what I think about when I take a shower, so sorry to get so personal. Yes, when it comes to our ability to control our movements, which is one of the fundamental things that we do. This, most people, I think, intuitively realize that as we interact with our world, as we behave, it’s really as a consequence of our ability to move.

And there are many different parts of the nervous system that contribute to our ability to move. And you can break it down in a variety of ways, in terms of different parts of the nervous system, so the Motor Cortex is a very critical part of the area of the nervous system that controls movement, but you’ve also touched on something that is increasingly apparent, and that is that there are multiple layers of circuitry within the brain, the brain stem and the spinal cord that each weigh in in the processing of commands for regulating movement.

And they do it in a variety of ways, so rather than getting into a very long-winded didactic lecture on the subject, maybe there are a few intuitive things that we can touch on. So one of the intuitive things is that when you touch a hot pan, you, we all recognize the fact that we’ll move our hand away from that very rapidly, and often before we even perceive the pain itself. And that kind of response, known by everyone as a reflex, is so rapid that it cuts out a lot of the circuitry that is controlled at the level of the brain. It’s simply a signal of the brain that is sent from the skin back into the spinal cord, and then processing has to occur, then the spinal cord relays out a signal “Move your hand.”

Now as simple as it sounds to move, just move your hand, the reality is that the way that we move our hand, is even in the context of this subconscious kind of response, done in a very, very precise way. We don’t push our hand further into the pan. We pull our hand back. And depending on where your hand starts out, the response that you activate is very precisely controlled. And the reason I’m pointing that out is to try and bring up the issue that even though reflexes are very simple kind of response, that leads to movement of a limb, it’s highly regulated in that many different muscles within your hand, within your wrist, within your forearm, within your bicep and tricep and shoulder, are all working together and that is a lot of neuro processing that has to happen. And it happened without having to use any portion of the brain. It happened all at the level of the spinal cord.

Chris E.: There’s no conscious thought that “I’m going to do this”.

Samuel Pfaff: That’s right. And so it’s things like that that have revealed to us that the spinal cord itself is not just a relay or cabling center, but really is an important layer of circuitry that is controlling our movements. And maybe to just take this one step further … The challenge that we deal with in trying to understand how we control our movements, you can kind of break into a, the computational problem of really trying to understand how approximately 650 muscles in our body are being regulated. And whenever we perform even a simple task like I have a cup of coffee and I’ve just lifted it up … That simple sort of task is using probably on the order of a hundred different muscles.

And it’s not simply just “Let’s activate one hundred muscles.” It’s “Let’s activate a hundred muscles with the right relative timing and relative forces compared to one another.” And we can perform those sorts of movements with very little thought. You can be driving, walking down the street, be thinking about all kinds of other things and look like a pretty coordinated person as you’re moving through the world. If you reduce that to a problem that a supercomputer had to solve, that would be enormously challenging for a supercomputer because of all of the computations about okay, which muscle am I gonna use? Which order am I going to use it in? How am I going to get some feedback so I know whether I’m not crushing the cup or holding it so lightly I’m going to drop it. And it’s one of the reasons why if you look at even pretty sophisticated robots, they look fairly robotic and stiff. It’s because the computations are enormous.

The nervous system and in part the spinal cord have presumably found ways to solve these computations in a very efficient way. Where, you know, supercomputers use enormous amounts of power and they have air conditioners to cool it, you know, we’re not built that way. So presumably biology has found some fairly clever solutions to running some very complex computations. And it’s that kind of complexity that really intrigues us. We want to get in there and understand how is the neural circuitry within the spinal cord processing information, and how is it doing well and so efficiently.

Now I touched on the fact that we’re interested in neural development, and I mentioned things like stem cells, and now when I talk about circuitry you might wonder is there any kind of relationship between the two. And there is, partly because in order to understand how the system operates, one of the approaches that we have taken is to try and see how nature put it together in the first place. And the logic there is if we can stand at the assembly line, which is fetal development, we can see all the parts being built and put together that we might have a better understanding of how the machine actually works. And so a lot of our studies combine developmental studies with circuit studies to better understand how the spinal cord and nervous system is processing information.

Chris E.: So understanding how it works also lays the groundwork for understanding what goes wrong when we become ill. Your lab recently discovered the, when a molecule called MicroRNA is missing it causes motor neurons in the spinal cord to just degenerate. So-

Samuel Pfaff: Yes.

Chris E.: First of all, what is a MicroRNA? For our listeners.

Samuel Pfaff: A MicroRNA is a cute RNA because it is small. They are RNAs that are only about 22 nucleotides in length. And it’s a whole world of gene regulation that biologists and molecular biologists didn’t even know existed until around twenty years ago. Through very simple genetic experiments that were being done in sea elegans. And it revealed a type of gene regulation that we never could have predicted had we not just stumbled into it.

It was first found in simple worms, and we now know that this type of regulation also exists in most species, including humans. And that it’s an important additional way that we control the level that genes are produced within each of our cells. We became interested in this particular MicroRNA which they don’t have very catchy names, they’re just numbered. And so the one that we worked on, have worked on and still work on is MicroRNA 218. And we found that because we were very interested in spinal motor neurons. These are the neurons that reside within the spinal cord and have long, thin axons that extend from the spinal cord out to muscles. And they’re the neurons that you need to activate if you want a muscle to be able to contract.

They’re also the neurons that are affected by diseases. Diseases like ALS, so we’re recording this podcast right now, and it’s about a week after Sam Shepard passed away … Actor and playwright. He passed away from ALS. It’s a neurodegenerative disease and it selectively leads to the loss of motor neurons. And the consequence of that disease is that many aspects of the brain are preserved, so individuals with the disease are often very aware of what is happening, maintain all of their cognitive functions, but they can’t move. And if you can’t move, that’s incredibly inconvenient, but it’s also life-threatening, because eventually you won’t be able to breathe, you won’t be able to eat, you won’t be able to swallow. And therefore it becomes a lethal disease. We have absolutely no treatments that are effective for preventing ALS.

There is another fetal form, I’ll say, of ALS, which is a disease called Spinal Muscular Atrophy, abbreviated SMA. And it’s also due to the loss of motor neurons. So from many past studies on SMA and ALS, we were starting to gain some clues about the genetic risk factors that cause those diseases. And one of the things that was becoming more and more apparent in this accumulating information was that the way that RNA was being processed might be affected in these diseases. Now I’m speaking in generalities, so my apologies to some of the real experts out there, but this was the kind of clue that prompted us to wonder if there wasn’t a MicroRNA that was present in motor neurons that might be critical for regulating gene activity in motor neurons and might be affected in these diseases where motor neurons degenerate.

And so one of the things that has absolutely exploded over the last decade or so was the ability to do what is called next generation sequencing, is now very feasible to go in and sequence all of the RNA transcripts in a cell type, and understand what genes are being activated in a cell. And we use that technology to identify or find that there was a MicroRNA within motor neurons that was very, very selective for those motor neurons. The ones I’ve referred to as MicroRNA 218. And from there, we went on to show that if you prevent MicroRNA 218 from being active within motor neurons, that motor neurons begin to degenerate. And this is now a smoking gun that indicates to us that maybe one of the underlying issues with motor neuron degeneration is the inability to properly activate or regulate how MicroRNA 218 is being produced and function within motor neurons.

So we have a number of projects underway in the laboratory to really begin to try and determine whether there is a critical link between motor degeneration and MicroRNA 218. Beyond that, MicroRNAs are generally quite important for regulating cell function and we’re fortunate to work in an institution that is really well-suited for these kinds of molecular studies, and some of our experiments are really turning up new, fundamental information about how MicroRNAs are processed and how the MicroRNAs actually regulate gene activity.

Hopefully we’ll learn some important things about motor diseases and I’m also hopeful that some of that will extend to understanding how gene regulation in other cell types is also controlled. Even if it’s not directly controlled by miRNA 218, it may be controlled by other analogous MicroRNAs.

Chris E.: So you are a cyclist?

Samuel Pfaff: Yes.

Chris E.: Obviously a movement-oriented activity. Do you ever-

Samuel Pfaff: A bicyclist, yes.

Chris E.: A bicyclist. There’s a distinction there?

Samuel Pfaff: I have to confess I also love riding motorcycles, so.

Chris E.: Two wheels.

Samuel Pfaff: Yeah, I spend most of my time on two-wheeled bicycles.

Chris E.: Do you ever find yourself, as you’re pedaling, contemplating using yourself as an experimental model?

Samuel Pfaff: Yes and no. There are times when I am suffering badly going up hills and I’m not sure what’s going through my brain other than “I hope I get to the top.” My daughter actually challenged me recently with a question of why do you ride your bicycle? Why do you like to ride it so much? And while I was thinking she said, “Dad I bet it’s because you’re just trying to keep your telomeres long.” Which made me laugh because I thought she was going to say something like it’s just a healthy way for general fitness. But instead she came back with the telomere response which is kind of an inside joke because the Salk Institute, at Salk Institute, our president is Elizabeth Blackburn who won the Nobel Prize for her seminal work on telomeres. So I think perhaps it was appropriate that she made the telomere reference.

Chris E.: She was very plugged in.

Samuel Pfaff: Yes.

Chris E.: Well Dr. Pfaff, thank you so much for joining us today and sharing your career path and some of the work you’re doing. Thank you.

Samuel Pfaff: Chris, my pleasure. Thank you.

Chris E.: To learn more about the Salk Institute and it’s research, visit www.Salk.edu.