 All right, I'm going to get us started right now. So it is my pleasure to welcome Holly for her second Steenbach lecture, which is going to be on Hormone Responsive Brain Module Power Movement and Skeletal Strength. And so I'm going to steal this. These events are made possible from the generous donations to the Harry Steenbach Lectureship Fund. And these are some photos of Harry Steenbach in his younger years when he grew up on a 120-acre farm here in Wisconsin. He later went on to be a prolific scientific researcher. He published over 250 papers through his academic career. And he made some fundamental discoveries. And some of those included discoveries in vitamin D and vitamin A. And from the patents of those, he was able to start the Wisconsin Alumni Research Foundation. And but I think what really makes Harry Steenbach stand out is his vision for the future of science. Worf was one of the first patent institutes at any university. And then also he trained over 135 different graduate students throughout his time. And so to celebrate his prolific career, we have started this lecture ship. And I have invited Holly as part of this. Holly is a phenomenal researcher who has a really diverse array of scientific stories. Yesterday, we heard a lot about lipids in the intestine and also the liver and also the brain. I did not anticipate we'd be talking about rectal balloons, but there we were at the end of the talk. But today, she's going to share a slightly different story. And I hope you guys have realized she has made a phenomenal contribution to science, not just through the trainees in her scientific research, but also the programs that she started and headed, which have been recognized both at UCSF and also nationally. So without further ado, thank you so much, Holly. Right. Well, OK. This is less crowded than it was yesterday, but I actually think that this story is really quite, this area of research, I'm very excited about doing. So I'm going to talk to you today about hormones and nerves in female physiology. And it sort of extends what I've been talking about or where we're going in terms of the gut. But today, what I'm going to talk about is the brain. And so why did I start doing this? So I started this project, in part because the nuclear receptors that I talked about, one of them is highly enriched in the ventral medial hypothalamus. But I also, as I got more and more into this, I began understanding that this was a region that expresses, is very important for estrogen signaling. And then as I thought more about it, especially as an aging woman, I decided that it was quite, that what we don't know in female physiology is what estrogen is really doing in the brain to give us a metabolic benefit. And a benefit in many, many ways in terms of our bone, cognition, et cetera. And I think that this hasn't been important, but it will become more and more important. And because I put together this chart showing that really, back in 1700, this really wasn't an issue because very few women were going to be in estrogen depletion because they would be dead. But as things antibiotics and other things have come about, we now, a significant population of the women in this country and other industrialized countries are going to be alive for at least four decades, three to four decades in an estrogen depleted state. And the issue is, do you take hormone replacement therapy and there's controversy surrounding that because of breast cancer? So you have women that have naturally gone through menopause that will be estrogen depleted, but you also have three billion breast cancer survivors that are going to be on aromatase inhibitors to eliminate estrogen for five to 10 years of their life and essentially send them into premature menopause. So I felt that we needed to understand, because I'm a very mechanistic person, I thought, OK, I'm going to start understanding this at a mechanistic level to really understand how estrogen in the brain is important for counteracting diseases of aging. And the goal really is to understand and define these estrogen-regulated neural pathways so that we could perhaps exploit them. And I just show this for those, because I was in Rome. I saw this bust of only, there's only three women, real women, that are busts, wait, I'm saying that wrong. And there's three busts of real women in Rome. And she is one of them. And so I always ask the trainees or somebody to guess who she is, because the amazing thing is she lived till she was 86 years old. And so she was not only physiologically fit, but she was also smart to not get killed off by her brothers and parents. I mean, they always killed each other off. So we can talk about that afterwards after wine. OK, so what I'm going to talk about today are three, an area of the brain called the medial basal hypothalamus, and it's just shown here in pink. And of course, for people that think about estrogen signaling, there's three major estrogen receptors, ER alpha, ER beta. Both of those are nuclear receptors, like I talked about yesterday. And then there's a seven-trans membrane G per estrogen receptor. So in this part of the brain, the only receptor that really does anything is ER alpha, which makes it simpler. And so I'm just going to show you here amino fluorescence of this part of the brain, a coronal section shown here, shown with the ventral medial hypothalamus, this ventral lateral region, and then the arcuate nucleus. If you've thought about satiety, this is where leptin is thought to act in terms of feeding behavior. So what you see are these two regions that are highly enriched in estrogen signaling, but you also see this in males. So part of what I'm going to tell you about are endpoints that are definitely sexually dimorphic, but it's really not just because of the presence or absence of estrogen receptor alpha. OK, so what we have known, and this is work that our lab contributed, as well as Joel Elmquist and Zhu has made a really important contribution with Deborah Clegg and others in rats, showing that, in fact, if you just sort of think about these two regions, the arcuate nucleus was suggested to regulate food intake, so estrogen is going to suppress food intake. And the VmHVL is going to, but we actually are going to show that it's not involved in food intake. And then the VmHVL region is involved in energy expenditure, such as locomotion and bat thermogenesis. So basically, brown adipose tissue thermogenesis. OK, so the team that did this work were Stephanie Correa, who's now an assistant professor at UCLA, Bill Krause, who really was the pioneer on this second story that I'm going to tell you about. Candice, who just received a K01. And then Ruben Rodriguez, who just recently joined who's an Aractus scholar. And this was the team that did these two stories that I am really excited to present here. So what I'm going to tell you is that estrogen engages two nodes, two independent nodes, to essentially allocate the way energy is, the way you allocate energy in females. And one is this sort of surprising finding that estrogen in the brain is important for this neural skeletal node, and it basically decreases bone remodeling. And the other story is that estrogen is very important for this spontaneous activity mode, sort of like, instead of a trainer coming to you and telling you have to do exercise, it's like, I want to get up and move. So that's really different. This is a spontaneous activity node. OK, so there's these two stories I'm going to tell you about. But what I want to first tell you is that within this brain region, the effects of estrogen on the neuronal activity is quite different in these two regions. And I think this is where, when we think about hormone signaling, we really have to think about what they're doing in each cell type, because it's not always the same. So if we go in and we stage an animal, a female in estrus, which is low estrogen, you can see this is ER alpha. And what we've done here is we've stained with phospho S6, which is really a surrogate or a marker linked to the mTOR pathway that is basically a surrogate for neuronal activity. And so everything is quiescent. Nothing is really going on. And now if we then take an animal in proestrus where it's high estrogen, all of a sudden you see this phospho S6 light up in the VmHVL. So you can do this. You can play the trick of taking the ovaries out and then giving super physiological doses of estrogen. But here we're just taking an animal in its normal estrus cycle and seeing this effect. And what I want to note is that in the arcuate nucleus where we have plenty of ER alpha, you don't see this neuronal activation. So right away we have this large difference in these two regions of the medial basal hypothalamus. So what we've done is we've taken two approaches to look at this problem. We've used genetic tools like a genetic Cree. Here we used NKX 2.1 Cree, which comes on early in development. And essentially we've eliminated all ER alpha in this area of the brain. And I would say that other models that have used these different Crees really don't, they have not achieved a full knockout. And to be perfectly honest, though, anybody who uses a Cree knows that they're not as specific as they say they are, and they're hitting other tissues. So we're always faced with this, especially if you work in the brain. And therefore, we have other methods, stereotactic viral Cree injections to go in specifically to a geographical area and knock out the gene that you want to knock out. And so the beauty of this is that you know if you do this, it's from the brain. It's not from a peripheral tissue. And the other thing is that you can do this at different stages and different times. So you can do this in adult mice. So you're not dealing with some of the developmental aspects of knockouts. So in fact, this is what we did. We went in and knocked out the estrogen receptor alpha in the VmHVL in the arcuate that's shown here. And what we see if you knock out estrogen receptor alpha in the VmHVL, you see this loss of ambulatory activity during the dark phase and not when you do the arcuate. In contrast, when you knock out ER alpha in the arcuate, what you see is an increase in lean mass as shown here. But we actually saw nothing else. We saw no change in food intake, no change in activity, only a change in lean mass. So we thought about that for a minute. And we did more experiments. And we looked at our genetic knockout, our NKX 2.1. And we see the same increase in lean mass. And this is, I mean, so we had to sort of deal with this. Why is there an increase in lean mass? And so Stephanie, who was about ready to leave the lab, was doing these analyses. And she tells me that she knew right away that she wanted to use DEXA to look at these mice. The people in the lab tell me, no, the ECHO MRI machine was broken, and she had to use the DEXA. So the ECHO MRI, for those of you who don't know the difference, is that it's much simpler. It's much faster. You don't have to put the animals to sleep. But the one thing that you're missing in an ECHO MRI that you're not getting with a DEXA is bone mass. And I was just giving a talk in Las Vegas. So I put this in because, in fact, it was having to use the DEXA that really revealed this phenotype. So Stephanie came to me with these data. She was leaving the next week for UCLA. And she showed me this four star difference. And I loved it because you see it in females, but you don't in males. And I thought, wow, this is a four star difference. You just can't ignore that. But I don't know much about bone. And I'm not sure that this is a significant statistically, but is it really significant? So I thought about this and thought about, oh, gee, do I really want to do anything in bone? And so I reached out to some of the experts at UCSF. And we did what you're supposed to do, which is to do a three dimensional micro CT to look at the distal femur, or the L5 as shown here. And the reason that you do this is because this is trabecular bone. Both of these sites are where you have trabecular bone. And this is bone that's going to degrade with osteoporosis. So all of the bone people really look at the distal femur, or L5, to look at trabecular bone. And this is what bone looks like in a normal, wild-type mouse. And surprisingly, at about 12 weeks, you're almost seeing peak bone mass. So in a mouse, it really starts disintegrating after that. This is what our mutant look like. So we have this amazing bone density as shown here, both in the L5 and the distal femur. And all I know is that this was significant, because I got a call from the bone people, Bob Nissenson, saying, what did you do? We want to know. We rarely, rarely see bone like this. The only bone phenotype that looks close to this is a sclerostin knockout, which is now being used in the clinics. Anti-sclerostin antibodies are being used for severe osteoporosis in older women and men. So this was highly significant. And more importantly, during aging, you can see that this bone phenotype persists quite nicely. So we let's see. OK, so this was great. But of course, we needed to know more about it. And this is just to say that, again, because that other model was using the NKX 2.1 Cree. We don't know if it's coming from the periphery or not. We went back in with stereotaxic techniques to knock out ER alpha in these two regions to find out where it was being mediated from. And it's from the arcuate nucleus. So not the VmHVL, but from the arcuate nucleus. OK, so we then spent about another year because within the arcuate nucleus, there are five major neuronal subtypes, all of them which express ER alpha. And we had to figure out which neuronal subtype was mediating this phenotype. And we thought at first it had to be dopaminergic neurons. We did profiling. And we saw changes in kiss neurons, in dopaminergic neurons, in tyrosine hydroxylase neurons, as well as two other subtypes. So we went through and got all the Crees to redo the experiment to figure out what neuronal subtype might be mediating this phenotype. And I was in Thailand when I got these data from Candace showing that, in fact, when we use a kiss Cree that was recreated by Paul Meiter and Steiner, a very specific Cree that was really quite good, we can really see this bone phenotype. And this even exceeds the bone phenotype we saw before. In fact, you can see it with the naked eye. So you don't even have to genotype these mice. Or you don't have to put them through a micro CT. You can see this by a naked eye. And so much so that I forgot to mention that in our other model, as well as this model, we do not see this phenotype in males. So this is a very female specific phenotype. And in looking at this phenotype a little bit, the kiss Cree model, we can see that we start getting bone marrow failure. The spleen starts enlarging. So we see extra medullary hematopoiesis, which is what you might expect when you have this very dense bone and very little bone marrow. OK, so we've published this. And there's a lot that if you're interested in the aspects of the bone and the bone phenotype, the different aspects of the bone people like to think about, which is bone formation. What it looks like is this is causing new bone formation rather than causing a decrease of bone loss. So as a person that thinks about hormones and endocrinology, one of the first things I wanted to know is is this mediated by a neuronal circuit or is this mediated by a hormone? So a circulatory factor. So we did two experiments. And one is, I'm sorry, this might look gross to some people. But essentially what you do is you take a two week old wild type female femur and you plant it in the backs of your mutant animal or your wild type and you look to see what happens to this bone. And this is really just taking a page from people that do this on a kidney capsule. They take these small bones and they put them on the kidney capsule and look at bone. So if you do this, the scheme is just shown here. And you take a wild type bone, put it on to a wild type mouse, you see a normal looking bone. But as soon as you take the wild type bone, put it into a mutant mouse, all of a sudden you get this very nice increase in bone density and bone volume. So that was one experiment. And then we did a second experiment that actually is a person that thinks about endocrinology. I've always wanted to do, which is a parabiosis experiment. So this is where you're fusing mice together. And it's not that complicated. It sounds really complicated. But you really sew their legs, muscles together, and then pretty soon their circulation fuses. And this, of course, is the way Coleman and his founder helped Jeff Friedman identify leptin. So it was this classic parabiosis experiment. So we did this and we sewed them together and then did a baseline scan at week zero and then at week three up to week nine. And parabiosis itself is hard on these animals and hard on their bones. And in fact, what happens over time is that you start losing bone in the wild type fuse to a wild type, as shown here. But when you do this with a wild type fuse to a mutant, you start seeing this buildup of bone mass. So we know that we have a circulatory factor. And we are now, I think, this is pretty, we don't know where it's coming from. We know that manipulation of these neurons causes this circulatory factor, whether it's coming directly from the brain or the brain is sending a signal to another organ remains to be determined. And so we are developing an assay to actually look at that now and doing some biochemistry. So we also wanted to know what's going on in these bones a little bit more closely. And we fiddled around with looking at some of the niches in bone. It turns out that there's a lot of different ways to do that. And so we started working with Chuck Chan and Tom Ambrose, in part because Chuck, with long acre, had defined a new skeletal stem cell population that essentially takes the skeletal stem cell and can foam cartilage or bone. So after fiddling around with asking if the other cell types might change and getting nowhere, we turned to them and said, maybe these skeletal stem cells are different in this massive bone. And I should say this bone is not only dense, it's also very strong. So if you take a machine and you try to crush the L5, which is what they do to measure strength, the engineer, the bioengineer that did it for us said that basically the bone almost broke the parameters of the machine that they would have to change them because it was so dense and so strong. So we have dense, strong bones, and we need to know why. So with Tom and Chuck, we looked at these different niches, including the mesenchymal stem cell, adipocyte progenitor cells, as well as these skeletal stem cells. And you can see quite nicely that we don't have changes. We have actually a drop in the bone adipocytes, which are bone fat, which is from a metabolism point of view, bone fat is quite fascinating. You can come talk to me later. It's this really underexplored area. But you have this very prominent increase in these skeletal stem cells and not in males. So we then did two more things with Tom and Chuck, which are really cool experiments, which is you take these skeletal stem cells and you put them into culture, different media, that's going to allow you to go towards bone mineralization and cartilage as detected by these two stains. And you can see that if you put the same amount of skeletal stem cells from the mutant and the wild type, you actually see more bone and more cartilage. And if you do another experiment that I think is just a beautiful experiment that they did for us, which is you take a package of these skeletal stem cells and you take wild type or mutant, you put them on a kidney capsule and transplant them. And then six weeks later, you look to see what it's differentiated into. And so you can look at, you can stain with this pentachrome stain for cartilage, the mesenchymal area, the stromal area, and bone. And you can see in our mutant that we have much more bone and cartilage than the wild type. So not only do we have more cells, but these cells are really programmed to go into bone and cartilage. So we've done single cell seek. And I will just say that of these skeletal stem cells, and we see with our mutant skeletal stem cells, we see an enrichment in this cluster. And now, of course, we're taking that genomic information. And we're trying to figure out if we've got a surface plasma, a surface receptor that might be the target for this osteogenic program. So we still don't know how we get so much bone formation. It's not through some of the classic programs. And obviously, we hope that this information, molecular information, is going to give us a clue along with our biochemistry. So what I've told you about in terms of the arcuate nucleus is there's a subset of neurons in the arcuate nucleus where ER alpha signaling, estrogen signaling, is essentially restraining bone growth in females. And it's really opposing the action of peripheral estrogen, which is going to be preventing the loss of bone. And in mice, it's an anabolic factor for bone. And then when you knock this whole system out, you get this remarkable increase in bone and increase in these skeletal stem cells. And our question really now is what is we're calling this brain-derived osteogenic factor. And we, of course, want to find out what it is. And I get an email every week from a woman, usually a woman who is suffering from premature, from osteoporosis, asking if we have found, can we reproduce in humans what we have done in mice? So hopefully, we are working very hard on this. And then you might ask yourself, why would you have this signaling system? And we think that it comes on at different periods, life stages. You might imagine that you want bone changing, going into the fetus, for instance, the energy going into the fetus in late-stage pregnancy when you do start losing bone in the pre- and post-lactation period where you see really large bone changes in bone density in the pre- and post-puberty growth period. So it's a counter, it's a really for a very counter-intuitive finding, but one that we think is actually going to be important physiologically at different life stages. OK, so that is sort of this neural skeletal node. And now I'm going to tell you about this new activity node that we just uploaded to BioArchives. It's in review, and I'm hoping that the reviewers like it. I think this story is pretty cool. So back in 1924, Sloan Ochre did lots of experiments on rats where he just looked at giving them different chow, giving them, I mean, he did everything you could think about. And in 1924, you can imagine that the tools for looking at these rats was fairly crude. But what he developed was a system to look at their activity over time. So no clams, no TSE chambers, no any, none of the techniques that we use today. And it was actually pretty ingenious. And if you want to read this, it's really fun to read. And what he noted is that there has to be this intrinsic rhythmic change in animals, in females, because what he would see is that every four days you would have this spike in activity and a reduction of food intake. So every four days in a young adult, this is what the activity looks like. And then as the rat ages, you can see that these activity spikes go down. So we now know that in part because of Wisconsin farmers, there the dairy farmers now really use this, what's known in a lot of agricultural and in agricultural setting, is that there is this activity spike that correlates with ovulation. And so they not only use these Fitbits for cows to look at their health, but they use them to figure out when they're going into heat. And that's because as I read someplace, the mounting activity is a 5.8 hours. So if you have a big herd, I don't know if that's what you call these cows, dairy cows, you have limited time to make it happen, either with artificial insemination. So in fact, it was on 60 minutes, I urge you all to look at Wisconsin. They did a whole thing on Wisconsin dairy farms on 60 minutes about Fitbits and cows. And you can just see here, this is sort of what that activity spike looks like in terms of every 21 days is when they ovulate. So this has been known for a long time, and it's being exploited now in terms of these Fitbits. And yet, we don't really know why you get this surge of activity right before ovulation. And so we know that if you get rid of ER alpha and the VmHVL, as I showed you earlier, you get this lowered activity during the dark cycle. So Bill Krauss started working on this problem. And one of the first things we did was to profile that micro-dyssected VmHVL in an OVX female where we're giving them back estrogen. And we just are saying, what are the estrogen responsive genes in this area? And the one gene that we focused in on was melanocortin-4 receptor. And we verified that, yes, during estrus, it's low. During proestrus, it goes up. And in males, it's quite low. And then we did in situ hybridization in this region showing that this is true. You can see this beautiful staining that occurs in the VmHVL during proestrus. And it's often males. And I should say that melanocortin-4 is not a new gene for anybody who thinks about obesity. It is one of the most common form of monogenic obesity out there in the human population. But people really have not looked at it in this region, in part because if you go on to Alan's brain atlas, this is what you'll look at because most people are looking at males. So this is what I've just said, that melanocortin-4 is, leptin, there's 12 individuals with a leptin mutation. This is not true for melanocortin-4. There's a lot of individuals with a partial loss of function or loss of function. And now they've actually found gain of function so that the melanocortin-4 doesn't recycle into the cell but stays out on the membrane. And those people are protected and are lean. So it's really clear that it's linked to obesity because of hyperphasia or overeating. And this is just a young child with one of these mutations. But what they know from both human genetics and from the mouse genetics where they went and knocked out melanocortin-4 is that there's a sex difference. So humans, females, women with melanocortin-4 defects really seem to suffer from more diabetes as well as all the other symptoms that go along with this mutation. And what Paul Meiter in another group showed very early on is that if you take a male mouse, a mutant male mouse, and you parapheed them, so let's... Okay, so if you parapheed a male mouse, a mutant male mouse, you can get it back to normal. But you can't really do that with a female mutant. So you can see here it's improved but not totally improved. So there's this female male difference that was noted very early on. I mean, this paper was from 2000, but nobody had really pursued that. Okay, so Bill and Andreas Rodriguez looked a little bit more closely at the hotspots of melanocortin-4 receptor expression. And these are just the different regions where we have expression. This is the VmHVL region where we have ER alpha. The PVH is the region that really regulates satiety and you don't see any overlap. And this is the medial amygdala where there is some overlap. And if you look at it a bit more closely, the only important aspect of this slide is that in the VmHVL, every neuron that expresses ER alpha expresses melanocortin-4. So there's a complete concordance of expression of these two signaling molecules. And that's not true in other regions of the brain. And this just shows that the VmHVL neurons are highly sensitive to estrogen with respect to melanocortin-4 but not in other regions of the brain. Okay, we have this nice thing where we show that melanocortin-4 is an estrogen responsive gene. And that's always nice if you're thinking about correlations. But as mechanistic as I am, I wanted to know whether estrogen receptor alpha can be recruited to the melanocortin-4 promoter. And that's not so simple to do, especially in cell lines. I was not really happy with doing that. So we reached out and Jessica Tolkien, who has spent about a year and a half working out cut and run with the subcortical neurons to actually figure out what are all the estrogen responsive genes and figuring out where estrogen receptor alpha is recruited in a ligand-dependent way. And she called me up right as we were ready to get this paper uploaded. And she said, I found it. We have the PIC, the estradiol benzoate PIC with ER alpha being recruited right to the proximal promoter of the melanocortin-4 promoter and with some of our other targets. So this is wonderful. We no longer need to hand wave. ER alpha is regulating melanocortin-4. And this is just what it looks like. There's a half ERE along with an SP-1 motif, which is also found in the progesterone receptor. So that's sort of interesting for those of you who think about this. So we then use three methods to sort of get at the connection of melanocortin-4 signaling and estrogen with our physiological endpoint or activity. So we've done three things and I'm gonna go through this because I know that many of you are not neuroscientists. We wanted to stimulate these neurons with dreads. So dreads for those of you who don't know it, Brian Roth developed these. These are these designer receptors that are activated by a synthetic ligand. So you can basically put these in stereotactically, turn them on in a cre dependent way, give your synthetic ligand and activate these neurons or you can inhibit neurons. Okay, so we did that in these neurons and we also then restored melanocortin-4 to a null mouse that has no melanocortin-4 receptor in it. And then finally we increased the dosage of melanocortin-4 via a CRISPR-A technology. So I'm gonna go through each one of these experiments. Okay, so this is what I thought a movie's worth a lot. So here we're activating these neurons with these dreads, giving CNO and you can see an hour later after we've given a single injection of CNO, the mice on the right look like all of us wanna be active all the time. Although I guess you wouldn't wanna be active all the time. But even five hours later, you can see this is when we begin the experiment, an hour later, this movie of course is sped up, and then five hours later these mice are still moving around. Okay, so this is really powerful to activate these neurons. We see activity in males, okay, so we're sort of bypassing estrogen and we can see activity in males as well and the data are just shown here. If we chronically stimulate these neurons, so there we're just, we're giving one bolus of CNO and then we're looking five hour, and that lasts for about five hours. Here what we're doing is we're chronically stimulating those neurons by adding CNO to the water and you can see we have this drop in weight that persists, it's about a 15% or 12% drop in the weight and then as soon as we get rid of CNO, we normalize. Now we've also looked at bat thermogenesis and we don't see any changes. So we think that these sets of neurons are clearly distinguished from those that are gonna contribute to bat thermogenesis. Okay, so we then restored central melanocorn for a receptor only in the VMH and looked to see if we could get an effect. So remember, these mice are overeating by a lot. Let me just show you, their food intake is enormous and you can see here's a null mouse, I mean that body weight difference is quite large but we can drop it by about 10 to 12% just by restoring melanocorn for only to the VMH so these neurons, they're not gonna offset this hyperphasia but they are gonna have an effect on the overall end point, physiological end point and we do not see any effect in males when we do this. We think this of course is activity because we can see this spike up in activity but we did a lot of this in clams and as I was telling some people earlier today I'm really, it's not really happy with clams data, it's somewhat all over the place and we were thinking what else do we have to do to really think about this story and link melanocorn for signaling to activity because I think that's what you wanna be able to do, take a gene and link it to activity and you can't do that by just activating the neurons. That doesn't work even with optogenetics or dread chemogenetics like we've used. You need something else. So we turned our attention to using a CRISPR-A technology and the idea would be that if we could increase the dosage in a wild-type mice in this VMH-VL region would we see a change in activity and so we were extremely fortunate that Navanit and Nadav had really gone through all the promoter of the melanocortin-4 to figure out what the best guide RNA is in terms of activation. So I heard him talk at a retreat about this and said, okay, I wanna use this in our setting and okay, so if we essentially if you, what we do, it's a dual virus system. So one virus carries the guide RNA with an M-Cherry reporter. The other carries the Cas9 VP64. VP64 is simply VP16 times four. It's an activation domain and what we didn't know is that before we even started this experiment but just turned out to be the case is that the guide RNA sits right on top of that ERE. So we really are bypassing any estrogen receptor, alpha recruitment and the need for estrogen. So okay, so we target, we know how to hit the VMH-VL. Great, what happens? So I should say that this is one of those times when you do an experiment and you're waiting, we're expecting to see weight loss and so we two months go on, two and a half months go on and we are seeing no weight loss. And so I just thought this had to work. So I said to Bill, let's take these mice and put them in an inemaze system and look at their activity. And when you do that, you can see, this is four months after we've injected the CRISPR-A, you can see this, and we tried to do this by clans and the data were all over the place so we turned the inemaze, which is just a wonderful system for looking at locomotion and activity. You can see that you have this activity in the dark time so we haven't disrupted their diurnal rhythms. That essentially if you walk 10,000 steps, you'd be walking 20,000 steps a day. So this is just the total distance traveled and some of these mice really travel a lot during the dark period. And we see this in males as well. It's not quite as robust, but we can see this in males, which was what we would expect because we bypassed estrogen in terms of where we targeted. So we have these mice moving twice as much and because of that, their bone actually gets denser and we measure that because remember, mechanical loading, everybody says if you wanna preserve your bone, go out and run and walk. Okay, go out and run and walk because these bones are denser. But in fact, when you look at this, one of the perplexing things that we still don't quite understand is that the body weight ad lib is not changed and it's only when we parapheed them that we start dropping their body weight. They're moving twice as much and this is just continuous. And their food intake, this is a bit perplexed. I mean, their food intake is not that different. It's not statistically different. So why is it that you parapheed? Then you see this drop in body weight. And it does remind me that in fact, if you just exercise without changing diet, it's really hard to lose weight. I mean, all the human studies support that. And this Ohio teen, he walked to school every day, rain or shine or snow, but he also had to change his eating patterns and he had to restrict his eating. So we tell people what we'd like to think of is, if you think about this system now, we have these neurons and there's only about 200 neurons in the brain of a mouse that have this melanocortin-4 and estrogen alpha in the VMHBL. And essentially estrogen comes on board, gets increases at transcription of melanocortin-4 and that you have this integration of this system of the melanocortin signal and estrogen signal to then cause your mouse to run, female mice to run around more. And what of course we'd like to do now with this system is ask, is this operational in a very old female or male mouse? Can we see this? And can we, we're not losing weight without parapheeding them, but exercise does much more than that. It's not just about your weight, it's about your whole health. And one of the things that we think is gonna be quite informative is to look at this model in Alzheimer's disease model as well as a stroke model because those are two things that have exercises been reported to offset these diseases. So, and then we really wanna know how long this effect will last. We sacrificed some mice at four months and they were still moving around, but how long will it last? And I mean, I guess NADOV has a patent to actually fix haploinsufficient diseases with this CRISPR-A technology. And so, you could think 10 years in the future that maybe you could go in and manipulate neurons for, you know, I mean, this is all sci-fi, but where you, rather than doing gastric bypass, which is really major surgery, maybe manipulation of neurons is gonna get you where you wanna go. It's something to think about and I know the technology is gonna get better and what's great about this is it's long lasting. You're not putting in anything, you're just asking the gene to do its normal thing. So, and then we wanna know very much it's there in males, what's triggering this in males? Is it triggered in that postnatal surge of estrogen that they see in males before you get the male masculinization? So we don't know that yet. And then this is just to show you that in fact, if you give estrogen to males, you can see melanocortin-4 coming on. So the system's there, it's just when is this engaged? Okay, and then if you wanna look at this, we've uploaded this to bioarchives. So we think that this activity node that was seen almost 100 years ago, that these neurons really are the main focal point for this activation in females. And of course, there's many other aspects that require this activity node. There's a lot of other things that need to go on for mating. And so all of these behaviors need to be coordinated in order to mate. And if you think about this part of the brain, you know, there's only two things you wanna do really as an animal. You want to preserve fuel, or intake fuel, and you wanna reproduce. And so this is a node that we think is absolutely essential for reproduction. And it is, we found just this in June, 2019, melanocortin-agonists are one of the first, maybe the second drug approved for premenopausal women for libido, or hyposexual dysfunction. And so it's only in premenopausal women, and perhaps it is working through this circuit to then increase melanocortin-4 and increase overall activity. So we don't know, and I'm not gonna speculate in any questions about this. So what I've told you about are these two nodes. One is to increase energy expenditure involved in mate seeking, maternal behavior, exercise. And then the other node, of course, is to regulate the allocation into bone because putting energy in bone is energetically costly. So we think just continuing to do this and understand what estrogen is doing in the different regions of the brain is really gonna be very fruitful in terms of normal female physiology, but also in disease states, such as Alzheimer's disease, which shows the strong sex bias, as well as other psychiatric diseases. So with that, the people that did this work, our Candace and Bill helped by Stephanie and others. We were helped again by a whole slew of collaborators. And this is the bridge we go across. And then what I wanna say for anybody it's not as crowded as it was yesterday, but we just received two, what are the odds, this year we got two grants that received 2%ile scores. And so I am looking for postdocs, because I need to. Okay, thank you. Thank you for an absolutely fascinating talk. I will open it to the floor for questions and James will start us off. Thank you very much for that exciting talk. So I did notice that in the Merino-Cortico four knockdown, right, there was increased food intake. In what? In the receptor knockout, the MCR are four. The MCR are four. Okay, so yeah, the MCR, no, I mean, you mean if we just in the, if you knock out Melana-Cortico four globally, you see this profound change in food intake, right? So you see increased food intake in really profound hyperphasia when you knock out the receptor. And I think the puzzle has been for males, all you need to do is restrict their food and you'll normalize their weight. But that has not been true for females. Even though you're normalizing, you're increasing their hyperphasia, you, when you parapheed them, you can't get them to where the wild types are. I see. So there's these other parameters, these other metabolic parameters that have been out there and it's probably true, it's probably not just food intake that Melana-Cortico four affects. There's sort of emotional eating. For the human data, it says there's more than just food intake. There's a whole list of other things that comes with that mutation. I see. So I was just wondering what happens today, leptin signaling. Ah, to the leptin signaling. Well, we haven't looked at the leptin signaling per se because there's not, I mean that would be an interesting thing to look at. We're not seeing this really huge change in body weight so there's sort of no reason to think there would be a change in leptin signaling because leptin is primarily delivered to the brain from fat. Yeah, so good question though. What about affect mania or depression? Yeah, so that's a great question and the only thing I'll say is that we did some, we did marble bearing assays. We started to look at anxiety in part and we really need to pursue this a little bit more because when we activate those neurons, the mice run around like crazy but they also go to the food hopper and they start gnawing at it but they don't eat anymore food. So they just get, they run around but they almost get hyper-like and anxious. So as well as you can measure that in a mouse so behavior, anxiety in a mouse is not always so easy. I mean we can do open field but we want to pursue that a little bit more. That's a great question. And you know I'm intrigued as you said earlier because estrogen seems to be doing the opposite in the brain. Yes. So I mean what if you do the really crude experiment of delivering estrogen to the brain? So that's a wonderful idea and that's what I actually got funded to do is to not only estrogen but also what we want to do is give tamoxifen because tamoxifen is bone sparing for those who have breast cancer and it's always been thought that it works as an agonist in bone but it's an antagonist in the breast and that's what makes it good. It's not as effective as an aromatase inhibitor. So we're wondering if the bone effect is actually mediated by the brain. So we are doing those experiments and we're also giving a pure ER alpha antagonist to see what we can see. So tamoxifen does the pure ER alpha antagonist doesn't. So we're gonna go in right to the arcuate and the other thing, cool thing we're doing is we're taking those skeletal stem cells and we're gonna deliver right into the arcuate nucleus and look to see if we build bone right in the brain. We're gonna do a kind of biobiosis. Yeah. I'm not exactly familiar with how a high fat diet is controlled for carbs but I had known there were some carbohydrate genotype by environment effects with MC4 on humans. Did you try to vary the carbohydrates? No, we did not. We haven't done, we just, we haven't done anything with a high fat diet. We just wanted to look on chow and, one of the things that we do wanna do with the CRISPR-A females is to challenge them with a high fat diet and I take your suggestion we should use put in high carbohydrate. It was amazingly dense bones. How do the animals react to that? Do they have, are they less active? No, and we did grip strength and they're fine. The one thing that we'd like to do is look at the calcification of their vasculature because that's one of the things in osteoporosis you get calcification in your vascular system so we probably should look at that. They seem to be fine. I mean, they're doing fine. All right, I will ask the last question. So in terms of, I guess, so one of the major things you saw was a decrease in the marrow adipose tissue depot and I am curious if, is it known that there are differences between males and females and marrow adipose tissue and? Yeah, I don't, not that I know of. The Tom Ambrose in Chuck's lab is really starting to look at that. The weird thing about bone fat is that it's really high in anorexia, it's really high in starvation, it's really high in obesity and it's really high in type two diabetes. So essentially, as soon as you have fatty bone, you're unhealthy. So it's really an indication of your overall metabolic health and what is, rather than saying it's being lean or obese, it's about your metabolic health. So I think in that sense, it's a very cool fat depot that is poorly understood. All right, please join me in thanking Holly one last time.