Scientist Stories: Fred Gage, Disease Modeling

So I think we should pass on to the— move on to the next speaker, which is, I believe it's Fred Gage. So as I said, Fred needs no— it's hard to call you Fred. Rusty needs no introduction to this audience. Thanks for the great introduction. I can go on for another half hour if you would like. It's all right. I'll use the time. Look at my talk. Thanks for staying. I'm going to talk about something different today than I planned on talking about earlier when I said I was going to talk about something. And I'd first like to, a little advertisement for another meeting that's going to be held in the Mesa Friday. CARTA, which is a local organization that's the Center for Academic Research on Training in Anthropology, and the search in this group is to look for How did humans evolve? What was the features that make us different from our closest relatives, living relatives being chimps and bonobos, and how do we differ from those that passed since, being Neanderthals, etc.? It's a great seminar. This is an open seminar on Friday afternoon, and there are such topics. This one's on mind reading or human origins of a concept called theory of mind, and it has a really wonderful cast of characters. If you have any interest in this at all, you should come. These are really great speakers. So, I'm going to talk about this, actually, as it relates to iPS cells. What? Is this the one? Or is this the one? Yeah, okay. So, the oldest questions about humans really are, who are we? What are we doing here? Where do we come from? How did we get here? And where are we going? Many of these questions are more philosophical than they are experimental. We're sort of thinking about where do we come from and how do we get here, maybe more tractable. We know, for example, that the major split for primates occurred 33 million years ago with Old World monkeys separating out. Then the gibbons and orangutans at 16 million, our gorillas at 9, and And chimps and bonobos, our closest relatives, occurred 7 or 8 million years ago. Now, the way people usually look at this and try to figure out what's unique about humans in this evolutionary process, in part because we are— we don't have any really close relatives anymore, we got rid of most of them, is to look at DNA. Or look at tissue, brain, postmortem tissue in chimps or humans. We can go back in fossils and look at bones with Neanderthals. Or even closer, we can look at the emergence of art and other things like this. What's really missing is to study the cell and molecular basis of this, is some kind of live cell issue approach to this. So chimps and bonobos are our closest relatives. They split as I said, about 7 million years ago, and we share 96% of our genomes. We have difference in brain size and language, susceptibility to viral infection, probably inheritance of certain diseases that Larry's interested in. Cancer susceptibility are different in this concept of neoteny, which is so important to our early development. So if we can identify cellular and molecular differences between humans and non-humans, it may be essential for getting a better understanding of our own evolution. So with that in mind, many years ago, Carol Marchetto and Alison Motri in the lab, we envisioned this idea of making a zoo, a primate zoo basically, of reprogrammed cells. And over the years, we've been able to do this. It actually got us Took us quite a bit of time to get these fibroblasts, believe it or not, approved to be used in the lab. But we've now been able to reprogram most of the cells using early Yamanaka factors, same, all humanized, same conditions. We reprogrammed chimps and bonobos and humans. And what I'll be talking to you about is this subgroup of 2 bonobos, 2 chimps, 4 reprogrammed humans and comparing those to HUE6 and HUE9. Things reprogrammed well without any major differences in timing and other features. As anticipated, karyotyping the chimps and bonobos gave a greater number of chromosomes and than humans, which have a fewer number. It's an interesting feature that happens. They easily differentiate in a variety of different assays, including the standard transplantation assays. They make rosettes. We can make rosettes, neural progenitor cells, and they look very much like normal cells in their ability to give rise to neurons. And they have action potentials and really at a, you know, first-pass difference, we don't really see any difference between our close relatives doing this. Obviously, there are limitations. Anybody that uses iPS cells comparing disease certainly has these caveats in mind, but— and you can imagine we have even greater caveats when comparing across species. And we're aware of that. But really the question is, are there differences that are detectable at the cellular level that are relevant to understanding human origins. So first of all, let's just go through and see whether or not some of the key things we already know that are genetically different reveal themselves in these biological assays. Ajit Varki showed some time ago that humans are unable to make N-glyconuraminic acid, which is required for placing these little acids on the— or the enzyme is required for placing these sialic acids on the surface of of membranes. And that mutation occurred uniquely in humans, and it is present— the enzyme is present in all other primates, all other apes. So we took our cells, differentiated them into neurons, and our primates, all our chimps and bonobos, easily labeled for these, but the humans didn't. So that's good evidence that if there's a a known mutation. We can pick it up biologically. Another one is a very interesting gene that was found by this Chinese group called Neuropsin that has a splice variant that's unique to humans, so that they have two splice variants and all other species only have one. This is from their original data. They did chimps, orangutans, white-bow gibbons, all with a single variant, but but humans have this second variant. So we took our reprogrammed CNS tissue, derived tissue, and chimps and bonobos only have the single isoform, and we have both isoforms being formed in our humans-derived neurons from IPS cells. So at least at first glance, we are able to karyotype and demonstrate that the cells are differentiated and they can recapitulate some of these differences. Now, we're doing a variety of experiments to try to delve into this a little bit, to try to understand more and reveal things, but one of the features of the differences between the species that we find most interesting is that there is greater genomic variability in the chimp species than there is in the human species. In fact, If you look at mitochondrial DNA, the genetic differences between chimps in the same group substantially exceeds differences in all living humans between humans and Neanderthals. Here's these cladograms showing the mitochondrial differences between humans and Neanderthals. This is just one group of Eastern chimps. Here's the Central chimps. This is the whole four-chimp clade. So they're genomically, at the mitochondrial level, very different. And just a few months ago, this paper was published in Nature where they deep sequenced 76 great apes, including humans, and mapped CNVs and SNVs and other types of differences that that are different across the pan against bonobos, gorillas, and humans. And just to give you— just look at something like number of CNVs. Here's human, and here are the chimps, and bonobos, and gorillas. And across— almost across all features, genetic features, we as a human species have a tighter relationship genomically to each other than the other species do across all their domains. Now, how could we look at this? How could we start looking at genomic variability and— or even mechanisms for generating genomic variability with regard to the cells that we're dealing with? So, iPS cells are, we'd like to think, equivalent to ES cells. Well, at least they're in that domain of things, right? They're pre-germline, we'll call them. ES cells originated in the inner cell mass of the blastocyst, and the epiblasts give rise to the ganglion germ cells. Temporally, our iPS/ES cells fall in this domain in human embryologic development, whereas the germ cells are much later out. So if we can find things that are going on at this early stage, pre-germ cell, these would be events that would be carried on in the germ line and then be passed on to generations. So we're going to do RNA-seq on the IPS cells and see if we can see any expression differences in this that might give us a clue to what might underlie variability in the genome. Again, these are our populations— Homo sapiens, bonobos, and chimps— and we have our ES cells that we're going to put in here as well to keep ourselves honest. And this is the number of mapped read totals. And you can see well over 1 million for each one. This is total poly-A. Now, first-pass analysis on the clustergram turns out real interesting. Here is humans, and these are humans plus the ES cells. And then here, this is where the clustergram splits, and here's the chimps and bonobos. So they cluster together. And there— whoops. There's the human's neural progenitor cells, which are different nicely from the other ones. So far, so good. Now, let's just look at— for the purposes of this short presentation, let's talk about protein coding sequences. First thing to notice is that chimps and bonobos overlap really, really well, as one might expect, and together they differ from humans in significant ways. And this is just another way of looking at it. Here are the transcripts, these transcripts here, that differ between human and chimps. Now, we're all learning bioinformatics, but we're relying heavily on our colleagues to do it. But we're biologists at heart, so what do you do? You look at the top 50 genes and see if there's anything that looks interesting, right? So look closely. See if you can see the two that I think are the most important. Yes, you're right. There it is. PIWI2 and APOBAC3B. And these are highly expressed in humans relative to both chimps and bonobos. And why do I think they're interesting? Well, these are documented major repressors of something called LINE-1 retrotransposition. And then the next question is, "What are retrotranspositions?" Retrotranspositions are part of a larger family of elements called mobile elements or jumping genes that were discovered by Margaret McClintock in the '40s and '50s. And the transposon— this is a cartoon of the transposon, which has enzymes that cleaves itself out and allows it to transport itself to another part of the genome, and that is how you get this variegation in corn, so it's a somatic— and often both germline and somatic events can occur. These transpositions occur in the germline, and people have hypothesized that they may be involved in increasing genomic complexity. It's actually proposed in that way, but most investigators, I think, up until recent times have thought of this as junk DNA or part of the selfish gene. Remarkably, however, when the whole genome was sequenced, human genome was sequenced, it became clear that these mobile elements played a large portion, made up a large portion of our genome. So, more than 50% of the whole human genome is made up of repeat elements. Now, they're not all active, but only 2% of your genome is coding sequence for proteins. Our interest is this, or just these line elements, they're called non-LTR lines, long interspersed nucleotides. There are about 150 of these that are active in humans and over 3,000 of them active in mouse. So what are the LINE elements? They are about a 6-kb sequence. For those of you that are virologists, they have a lot of features of viruses. They have a promoter and two open reading frames. They make an RNA copy of themselves and they They then translate the two open reading frames into proteins that bind back onto the RNA, and they are then taken back in, chaperoned in some way back into the genome, where one of the open reading frames is an endonuclease, nicks the DNA, and the other open reading frame is a reverse transcriptase, which then reverses the RNA back into DNA and inserts it into the genome. We have hypothesized over the years that— and demonstrated this occurs not just in germline, but also in early development and even in the adult.. And we have speculated, and others are joining with us or opposing us, whatever, in suggesting their role in generating diversity. But in addition to this dangerous thing of inserting genes all over the place in the brain, because we're such adaptive organisms, we've developed over the evolutionary period multiple mechanisms for suppressing this activity. And suppression, another way to look at it is just regulating it. And many of these mechanisms have been evolved to regulate this rather dangerous event, also have been adapted to do other things as well. But the two genes that we found to be highly upregulated are PIWI and APOBAC3B. APOBAC3B is a cytosine deaminase, but more importantly, and whether or not this is the mechanism by which it does it or not, It is one of the strongest suppressors of LINE retrotransposition available. And it's— for your interesting point of view, this is a polymorphic gene, so it's still changing. We have variability with our own populations. PIWI2 is an important enzyme that cooperates with these small piRNAs, or 26 to 33 nucleotides. That are essential for suppressing transposons early in development. That's how they were discovered and described as germline suppression of transposon activity. And they also work to suppress LINE activity in an effective way. This is just sort of the schematic ping-pong event that leads to the elevated levels of the piRNAs so they can most effectively, in conjunction with PIWI2 binding to them, can effectively suppress transposition. So the first thing Indigo did was to clone all 3 of these proteins from the human, chimps, and bonobos, finding a remarkable amount of similarity, conserved similarity between them. And then we went back to begin the biology. And the first thing is that the human protein and message for APOBAC is is, as shown from the expression, highly expressed in humans relative to chimps and bonobos. And remarkably, it's just 3B, and that's the one that has some unique features on line elements. Likewise, PIWI2 is confirmed in the biological experiments showing upregulation multifold over the chimps and bonobos. And again, It's unique. It's just PIWI2 that's upregulated. There are other members in the human family of PIWIs. So the generation— we've generated them. There are genetic differences, and they seem to be revolving around— our focus has been on these two, which are suppressing LINE activity. So is this biologically active and meaningful? We have an assay system where we can monitor this, and what we do is we use a reporter system where we have an RFP, a GFP in the 3' end which has an intron so that when this RNA is expressed, it splices out the intron. And if it's inserted back by this mechanism I described to you previously, it's now an active non-spliced-out, this intron, so now it actively can make GFP. So you can use this as a reporter of a cell that had an insertional event. And sure enough, when we do this, we find a dramatic increase in insertional events within the chimp and the bonobo relative to human. And it's not some mutation that's occurred in the chimp over time that enabled or disabled the function, because remarkably, Inigo actually tracked down cloned tested many chimp presumed LINE elements but found an active one that could actively be transposed in both human— in chimps and bonobo cells but not in human cells. So it is a feature of the cells, the human cells themselves, not the LINE. And when we say that, we would say, then what about the cells? It's the overexpression of APOBEC3. 3B and PeeWee. So is that true? If we overexpress PeeWee2 or ApoBac, we can in fact suppress LINE activity. So we know we can do that. And then if we go in regular cells like 3T3 cells and human cells, but if we go back specifically into our chimp lines, two different lines, bonobos and chimps and humans, and overexpress ApoBac3V in all of them, we can actively suppress. And overexpressing PWE2 does the same thing, and obviously there's no impact on the human. If we knock down APOBAC3B, then in humans, do we see an elevation of the level of jumping that occurs in those cells? Carol and Inigo and Ahmet made up some shRNAs that work effectively. You know, one works better than the other. And it turns out that the relative increase in jumping is proportional to the effectiveness in blocking the ApoBec3b in these cells. So it works both directions. Now, with regard to the PeeWee, if it really is the piRNAs that are involved, we should be able to detect those piRNAs elevated and in association with the PeeWee protein. Indigo made a really amazing cell where he overexpressed a tagged DOX-inducible PWE2 that he could then use to pull down and do an analysis of the piRNAs. And he found in this messy band here an induction or an increase in the amount of piRNAs just in the DOX-induced PWE2 and not in the others. So it does look like they're there, but that wasn't quite enough. So, we did RNA-seq on just small RNAs from these cells to see whether or not the annotated versions of piRNA that are annotated off what Laird told you, the ENCODE analysis, was there. So, here's the sequence of the LINE element, and here are the ENCODE— this little black dots correspond to the annotated versions of the existing piRNAs. We see them in high abundance with these reads consistently, so they are there. Those annotated ones are there, but we actually believe there's a lot more. These are the ones we actually see in addition to the ones that are up there, so there's— we believe there's actually many more of these piRNAs that are involved in this process than before. So we have high levels, higher levels of L1 in nonhuman primates. Knocking down the overexpressed proteins increases activity in humans, and overexpressing these in the nonhuman primate suppresses LINE activity. And we get this complementary effect with piRNAs. So what about the endogenous level? All of this is cells, so we have to do a little bit more work here to see whether or not this is an endogenous effect. Remarkably, Ahmet shows that there was a consistent upregulation of the expression of the endogenous LINE activity in the chimps and bonobos, across all the chimps and bonobos relative to humans. And he made primers to the 5' UTR to the two coding sequences, and this was true for all of them. So we really believe that there's a serious upregulation of the expression of these elements in these primates. Now, in this last part, So, if you're still paying attention, you're still following this, we're looking now into the chimp genome, the sequenced chimp genome to see whether or not the predicted— there would be a predicted increase in the number and the amount of LINE sequences in the genome of the chimp relative to the human. If they're more active in the chimp and they've been jumping in there more often, There should be more of them, and if we go back to the human versus the chimp genome, we should see more genomes. Okay. So remember, these are the time elements about when these changes, when the splits occurred. And interestingly, and I'm not going to go too philosophically into this, these splits correspond to mutations that occurred in line elements. So the oldest line element, the most The oldest one that exists at the time of the split is called the L1PA primate A5. There was a mutation after the split causing— with a new nomenclature of PA4. Then another split that occurred at this. So these are all line ones that can be demarcated as different because of primarily mutations in the 3' end. And some of them become functionally dead. And others then take over and become active in the next species as it moves on. So you can use these actually as evolutionary markers for the change in the species. Are these lineups— they can be used. I'm not making a causal statement there, I'm just saying that they can be used correlationally. Importantly, the PA1, L1PA1 is the active one in chimps at at the point of the conversion. But then the humans adapted and developed and evolved their own special line element, which is L1HS. That's the one that we have in us. That's the one that's active in us. And the non-human primates, the chimps and bonobos, continued on with L1PT1, and we don't have that. That's not active in us very much. It's primarily the L1H1 which is active in ours. So if we look at the total line elements in the genome now, mapping the genome sequences, there's no difference between humans and chimps in the total line sequence in the documented genomes at PA4. So that's here. So those are dead for the most part. They're not moving around. They already happened to all of us, both chimps and us. And that's true for PA3, PA21, PA2, but at PA1, there are thousands more PT in the chimps than there are 1Hs in humans. Our 1H is very, very low active. And if you look at the— this is a divergence map, so these are the most recent mutations that occur. You can plot the recency of mutations that occur in these line elements by sequence. And this just shows that there's no change in P4, P3, P2, and it's just in the most recent mutations where this variance occurs between the two. So the summary is that L1 is the significant and more abundant more active in nonhuman primates than humans. L1 mobility is more active in the nonhuman primates. There's a species-specific— L1 elements are more abundant in the genome endogenously. This is not looking in the IPS cells. This is looking back in the genome. It's the newer ones, the most recent events, the most recent changes that are that are changing the genome of the chimpanzee genome more than ours. And the conclusion is that there's a greater genomic diversity in these non-human primates than others. So those are the summary of the data that we have. And this last slide is just the speculation and the interpretation, I would say, of what I think this means. Early humans— I'm speculating, okay? Bear with me here. Early humans, we hypothesize, and others have too, that we went through a bottleneck. There was some event that occurred that narrowed our species and made us unique from our other closest relatives. And we're not sure when that occurred, but it was maybe— it was some time prior to the migration out of Africa. And one speculation— and this hasn't been ours only, this is well embedded in the evolutionary theory— is that there may have been some sort of viral infection or something for which a mutation in the human genome had a survival impact that would protect us against these kinds of viral infections. So a subgroup of us survived with elevated mechanisms for suppressing viruses, and exaptically, suppressing these mobile elements and other things associated with them. And at that point, we feel that through some mechanism, upregulation of APOBAC3B and PIWI2. This led to less genomic diversity between the existing humans. So we are all significantly more like each other than other species are to any extent at all. This less genomic diversity, I hypothesize, put in red and everything else, leads to a more common inheritable phenotype which facilitates cultural evolution. So not only— so this, this more similarity between us allows us then to have children, offspring, that are not varying so much that they can't carry on any cultural adaptations that we then pass along to them. I'll leave you with that thought to ponder, and these are the fabulous group of lead authors on it: Indigo, Carol Marchetto, and Ahmet Danli. I'd like to thank Chris Benner. He should have been in the picture but wasn't well that day. He's our trustee director of bioinformatics. We're very helpful members of the lab, and this was done in collaboration with Gene Yao's lab at UCSD, and of course Allison Motri as well as a continued collaborator on it. That's the end. So, questions? Here's one. So very interesting. So you think that the selection— that the lesser genetic diversity in humans has not really been selected, comes out of bottleneck. But is there any scenario in which it actually could have been selected? Still in the speculation, of course, field of speculation. You mean it was selected? I mean, there are events that happen that then are selected on by virtue of the environment. I mean, something that may— these to be positively selected because of some advantage that it gives and— Well, you know, no, I think the point I'm making, the highly speculative comment I'm making is that that is the selected— that is what you're selecting on. You're selecting on low diversity because that is that which allows us to develop language, develop common traits that can be passed on, cultural events that can be passed on. By virtue of our similarity, we can share common things across large differences without having this continuing variability in population that miss some of these events that occur. That was not the cause of the bottleneck, however, so— No, the bottleneck— the cause of bottleneck, you're allowing me to speculate, was some challenging event to the genome, to the species, and then like most things, we genetically adapt to some bottleneck and then adapt acceptation occurs, that is that which is selected upon. Are there any other— so interesting that the surname of that gentleman was Paternostro, our father, which fits in nicely with— So in the case of the elements that are very actively transposing, what keeps them under control so they don't just pulverize the genome? Well, I gave you one slide. That showed you that there's probably, there's at least 7 known highly effective mechanisms that suppress LINE activity, and there are probably more. The other part about LINE activity is that they have no nuclear import machinery, so there's only a very small window in time during which a cell divides that has the opportunity to transport the whole complex in. As a nuclear complex, the RNA protein complex containing the RNA and the two proteins, they can't get in under normal conditions in the cell. An interesting speculation that is emerging now because people are seeing this is as the nuclear membrane becomes leaky with age, these pores are sitting oftentimes retained on the outside of the nucleus unable to get in, but they're retained in neurons, long-lived neurons for long periods of time. Times, another thing we can talk about. But there's a lot of control, and like in the germline that Ruth Lehrman has shown, for example, that the LINE elements are highly regulated, but they're allowed to come on at certain moments in time. And she's speculating why would you let them come on at all. And that's in Drosophila. Yeah? So if you look in these different subspecies of— okay, if you look in these different subspecies of chimpanzees and now you try to look at the specific line element that you're interested in, the L1 subtype that's in the chimps and not in humans, does it make sense? Well, that's actually— Apua and Jenny are looking— we're looking at that now. We're looking to see by doing A targeted paired-end sequencing for the elements to identify where they— the new ones have landed in recent times, try to see whether or not the pattern of insertion is different. I mean, we'll see. We'll see. I think it's one of the great questions. The other question is, what is it that changed in the regulation of APOBEC3B and and peewees that elevated their activity. Yeah.