 The webinar today will explore how translational neurobiology research is being conducted in the Intramural Research Program of the NIH in a broad variety of disorders, including depression, age-related macular degeneration, and Gaucher disease. Our panelists all conduct translational research across the full bench-to-bedside continuum with the ultimate goal of developing novel paradigms for the treatment of a range of diseases and improving quality of life for patients. Today they will share their experiences and how they have applied their basic research in a clinical setting. It gives me great pleasure to introduce the three exceptional scientists joining me today, all from the Intramural Research Program here at the Bethesda-Marilyn campus of the NIH. They are Dr. Carlos Zirate from the National Institute of Mental Health, Dr. Ellen Sidranski from the National Human Genome Research Institute, and Dr. Anand Swaroop from the National Eye Institute. Many thanks to you all for being with us today. Thank you. Before we get started, I have some important information for our audience. Please note that you can adjust the size or hide any of the windows in your viewing console. The widgets at the bottom of the console control what you see. Click on these to see the speaker bios, additional information about the NIH Intramural Research Program, or to download a PDF of the slides. Each of our speakers will give a short presentation about their work, after which we will have a Q&A session, during which our panel will address questions submitted previously by email or from our live studio audience. We unfortunately will not be accepting questions from our online viewers today. You can also log in to your Facebook, Twitter or LinkedIn accounts during the webinar to post updates or send tweets about the event. Just click the relevant widgets at the bottom of the screen. For tweets, you can add the hashtag hash science webinar. Finally, thank you to the NIH Intramural Research Program for sponsoring today's webinar. Now I'd like to introduce our first speaker, Dr. Carlos Zarate, who is chief of the experimental therapeutics and pathophysiology branch, and of the section on neurobiology and treatment of mood and anxiety disorders at the National Institute of Mental Health. His research focuses on the pathophysiology and development of novel therapeutics for treatment-resistant mood disorders, as well as the study of biomarkers and neural correlates for treatment response. Welcome, Dr. Zarate. Thank you, Sean, for introducing me and giving me the opportunity to present our work in today's webinar. So the topic today will be depression, specifically focusing on two major points. One, how we go about developing treatments that work in hours instead of six to eight weeks, which are convention antidepressants take. And the second is, what are those cellular molecular and neural correlates of this rapid antidepressant effects in the hope of developing better treatments that work similarly within a very short period of time? But first, let's talk about the impact of depression. Depression is a major mental disorder that is associated with significant impairment and ability to function. Patients experience guilt, anedonia, lack of pleasure, drive, motivation, and have significant impairment in their ability to work, to function, and carry out their normal duties. And this goes on for weeks, if not longer, for years at a time. It is one of the leading causes of disability worldwide, much more disability than major medical disease such as cardiovascular, cerebral vascular disease. It's estimated that approximately 10% of the American population suffers from depression. And there's an increased rate of death, even after controlling for risk factors such as suicide and smoking. It's estimated that it's not only is there a significant morbidity associated with depression, but significant mortality. There are over 30,000 suicides per year in the United States alone. And individuals who experience depression and also have had severe medical illnesses such as cancer, describe the angst and the suffering and go with depression much worse than those when they experience the cancer. Now we have over 20 to 30 different antidepressants and it's fair to say that they do benefit many individuals. For those more moderate to severe depression, recent studies have found that patients do not benefit as much as previously believed to. It's estimated that if one takes a course of an antidepressant first line treatment, we're referred to as Cytallopram, for example, only one-third of individuals achieve remission within 10 to 14 weeks. Remission means absence of depressive symptoms or only a few depressive symptoms. It often takes two antidepressant trials or six months for half of the people to have a significant improvement in antidepressant treatment. So in my view, that's unacceptable. We've got to do much better. Other areas of medicine can intervene very rapidly within a matter of hours. And so we should also try to do the same in major mental disorders, particularly in depression. Now one of the limitations has to do with that most of our treatments are monominergic based have been developed based on serotonin in our epinephrine. On the left side of this panel, we see what are the number mechanistically distinct drug targets in the 1950s. In yellow and green, we see depression and schizophrenia targets, respectively. And after nearly five decades, we remain about the same number mechanistically distinct drug targets. Whereas in other areas such as cardiovascular disease, there has been a significant increase in the number of mechanistically distinct drug targets, which has resulted in a decrease in mortality from major cardiovascular diseases. That has to do with novel treatments, but also perhaps exercise and preventive measures. Whereas we have not developed a new group of medications. And that's not because the industry has not tried or government or academics, they have tried. It's just that the etiology of our major mental disorders is not really that clear. Towards the right are drugs that we use for bipolar disorder. And in essence, we have not developed a single agent for bipolar disorder based on an understanding of the molecular underpinnings of the disease. Most of the drugs have been repurposed from epilepsy, anticonvulsants, or from schizophrenia the anti-psychotic drugs. Now one of the limitations of our current medications is the lack of onset of antidepressant effects and is highlighted in this figure. Towards the bottom, we can see the natural course of the illness without receiving treatment, particularly in the beginning of the illness. It's about six to 12 months. But as we give it an antidepressant in the middle yellow line on the bottom of the graph, we see we shift the time curve a little bit sooner in terms of improvement, where one achieves remission within 10 to 14 weeks. And one third, that's what I already mentioned. The goal with our next generation of treatments would be in the beginning of major depressive episode when symptoms begin to start, specifically those with a high risk of relapse, we would intervene with a next generation antidepressant that produces a response in hours. If we're able to do that, one can see the top line of the figure. Yellow is euthymia stabilized mood. Blue refers to major depressive episodes. And each one has a toll or produces disruption in a personal, family occupational role. And also there's a considerable risk for suicide. But if we intervene with a treatment that works rapidly, we will decrease the length and severity of depressive episodes and we'll decrease the impact on one's personal social life and increase yellow, the euthymic periods where they still well. Now, another area where we can explore besides the monomerinergic-based system is the glutamate. Complex system glutamate is an excitatory amino acid, abundant in mammalian CNS. And we can see it's a rich area to pursue targets. And already we and others have pursued targets on regulating glutamate. It's believed that in mood disorders, major depression, there is a disruption of the glutamate glutamine cyclin pathway. And we may be able to regulate this with a host of compounds. There's indirect evidence that glutamate is involved in depression. We see regional reductions in brain volumes such as prefrontal cortex, hippocampus, that translates into reductions of spine densities and the pyramidal neurons that are involved in glutamate function. This figure in the top left illustrates a summary of the presumed mechanism of current treatments. We see that serotonin norepinephrine, when I give an antidepressant serotonin reuptake inhibitor, you can change interest synaptic levels of serotonin within minutes. It takes weeks, if not longer, for antidepressant effects to take effect. Why is that? Well, there have to be these intracellular signaling cascade changes, changes in gene expression, and what perhaps ultimately matters is changes in a protein in a brain called BDNF, brain-derived neurotrophic factor, which is involved in synaptic plasticity. And most of our antidepressants do increase at BDNF, but it takes a long time to do so. Whereas with treatments that act more directly closer to BDNF, either at two targets, mammalian target or rapamycin mTOR, or eukaryotic elongation factor 2, or more proximal to what is believed to be involved in BDNF release, would result in a more rapid antidepressant effect. Now, in order to do that, one can target the yonotropic, post-synaptic glutamate receptor, NMDA, particular at the synapse. And one drug that does that is ketamine, which is a dissociative anesthetic. It's derived from PCP, and it's referred to as a non-competitive NMDA antagonist. Now, when ketamine binds to the NMDA receptor, particularly at the PCP site, while individuals receive ketamine, they experience psychomimetic or dissociative side effects. They're temporarily disconnected from their body's senses. There's a long history of safety with this agent. It's used in emergency rooms for diagnostic treatment procedures, and it's also used as an anesthetic. So, we and others came up with the hypothesis that if you target NMDA receptor directly with an NMDA antagonist, would you bring about a rapid antidepressant effect? And the answer is yes. Towards the left, we see a depression skill. Down means greater improvement. Towards the bottom left, we see the time course in minutes. So, within two hours, we see rapid antidepressant effects. Towards the right are the percentage of individuals having a 50% improvement, what we refer to as response. We can see in the right graph the bars for current antidepressants. If one gives an antidepressant, it takes about 60 to 65% response rate in six to eight weeks. We see comparable response rates to ketamine within six hours to one day. So, we see rapid, robust, and relatively sustained antidepressant effects with a single infusion of ketamine. This is to highlight towards the left. There are now several trials, but we also found the same in bipolar depression. It's rapid onset antidepressant effect within hours. Last and most of the week with one infusion and towards the bottom right, lower right, we see not only are the rapid antidepressant effects, but we see rapid anti-suicidal effects within one hour. And this could have major impact on public health because we could produce rapid antidepressant effects and rapid anti-suicidal effects within a very short period of time. Now, this figure, this cartoon illustrates that ketamine not only blocks an MDA receptor, but it enhances glutamate release. And we could see that its police and NMDA receptors are blocked, that there's enhanced throughput through amper receptors, which ultimately is involved in BDNF brain neurotrophic factor production. And towards the bottom right, work by Yale, Ron Newman, suggests that M-torch, mammalian target, a rapamycin is involved in a rapid antidepressant effect of ketamine. One can see within one hour, very rapidly within a few hours, behavioral effects in animals, increased synaptic activity, and within 24 hours increased spine density. There is synaptogenesis within 24 hours. So there are a number of compounds that are now in development because ketamine produces these dissociative side effects. Some are looking at other targets within the NMDA receptor complex I mentioned. One compound that we tested here at NIH is AZD6765. It's a lower affinity and MDA trapping blocker. We see towards the left of ketamine that has high trapping blockade believed to be involved in a psychomimetic dissociative side effects. And towards the right, we see this compound that's associated. So in theory, one would predict that there are lower dissociative side effects. So in this proof of concept with one single intravenous infusion in people with treatment-resistant depression, we see onset within hours an antidepressant effect as illustrated by the decrease in the symptoms, depressive symptoms. And more importantly, we didn't find any evidence of dissociative or psychomimetic effects. So this proof of concept study suggests that it's possible to develop rapid antidepressant effects without these dissociative side effects. The second point I mentioned in the beginning was we want to understand drugs or treatments that are radically different than existing treatments. Ketamine, as I mentioned, is one tool. And scopolamine is another agent which is a muscarinic antagonist and produces a response within a couple of days. So with a biological systems levels approach using a multitude of technologies to do very deep bio-phenotyping anywhere from the genes on the left all the way to a rapid reversal of complex behavioral phenotypes such as depression, we can look for intermediate phenotypes. And this is a summary of some of the work that's going to take place. At the genetic level, we know that BDNF, a SNP, is largely a metkaryote towards the right. We see that you have a lower degree of response to ketamine than if you have val-val BDNF. This is to show that one of the limitations of psychiatry or study in brain is we don't have a window into the brain. And so these are tasks we can not invasive such top left where you can see fearful phases that activates anterior cingulate cortex. And it's a measure of plasticity. The more activity you have the better the response to ketamine. Here we can see when individuals exposed to fearful tasks there's a greater chance of responding to ketamine and we can see pre-treatment and also ACC predicts antidepressant effects to ketamine. On the right is a cognitive task. We see the reciprocal patterns of some examples of using measures to be able to measure response. Yet another is cortical excitability. We can stimulate fingers with a pneumatic device, applies pressure. One can see in the bottom right that the sensory cortex is activated and one gives ketamine we can see to the top right which is the power spectra and in the middle is gamma. Which gamma rhythms are important to connect in different brain regions precisely at the same time. And towards the left we can see baseline and yellow non-responders and green responders. The greater the gamma activity to this simple sensory task we can predict response to ketamine and encircled is the difference. So in the last slide I'd like to give you a summary of the work that's been taking place. On the right we see we can produce very consistently reliably a reverse of a complex behavioral phenotype within a couple of hours, much radically different than existing treatments. Towards the left we see some very preliminary evidence that genes might be involved in a response. And in the middle at the center level increased spine density appears to be important for response to ketamine. That's evidence at the center level and towards the right of that on a circuit level because we believe that mood disorders are disorders of circuits and synapses. So we hope that by filling in the gaps of a systems level biological approach we may be able to come up with a better understanding of treatments that are radically different than existing treatments. Still gaps remain but this is very promising work. To conclude I think using ketamine scopolamine as tools is a new paradigm of research to develop across a systems biological level evidence of these effective treatments understand the cellular molecular and neuro correlates that impart this dramatic response very rapidly in anti-suicide effects and hopefully with that understand and be able to personalize treatment for patients and to come up with a better understanding of our signatures that are involved in this rapid onset of antidepressant effects. Thank you. Great. Thank you so much Dr. Zarate. We're going to move right on to our second speaker today and that is Dr. Ellen Sadransky. She is chief of the section of molecular neuro genetics and a pediatrician and clinical geneticist in the medical genetics branch of the National Human Genome Research Institute. Her work covers clinical and basic research aspect of Gauchier disease and Parkinson disease as well as studies of genotype phenotype correlation and genetic modifiers, clinical insights from mouse models and the development of new treatment strategies for lysosomal storage disorders. She also focuses on understanding the complexity encountered in simple Mendelian disorders, the association between Gauchier disease and Parkinsonism and the development of small molecule chaperones as a therapy for Gauchier disease and related disorders. Welcome Dr. Sadransky. Thank you very much, Sean. It's my pleasure to be here to tell you about our work that's being conducted here at NIH. I've chosen to focus on our projects on Gauchier disease and Parkinsonism, which you'll see is an evolving story. What I hope to show you today is how studies of rare recessive disorders can provide a window into more complex disorders, where in our case, by focusing a detailed examination of a single gene disorder, Gauchier disease, we've come up with insights that are applicable first to monogenic disorders, but ultimately might help unravel complex disorders like Parkinson disease. So just to introduce the two disorders that I'm focusing on are really quite different. Gauchier disease is a rare recessive single gene disorder. It's the deficiency of an enzyme leading to the accumulation of a lipid. There's variable age of onset, multi-organ involvement, and symptoms include enlarged livers and spleens, low platelet and blood counts, and at times, bone and brain involvement. In contrast, Parkinson disease is a common disorder affecting 1.5% of the population over age 65, and it's a complex multi-gene disorder with a late onset. It results from loss of dopaminergic neurons in the brain, and you see the accumulation of aggregates of proteins, including one that you'll hear about, alpha-synuclein, within bodies inside the brain that are known as Lewy bodies. The symptoms of Parkinsonism and Parkinson disease include bradykinesia, which is slow movements, rigidity, tremor, and sometimes dementia, and it's a disorder that primarily affects the substantia nigra and brainstem regions. So how are these two disorders associated? I'm going to show you that by using an integrated translational approach with pathologic studies, clinical studies, genetic studies, imaging, cell biology, etc., we're beginning to gain some insight into this. So to begin with Gauchier disease, it's the inherited deficiency of the enzyme Glucocerebrosides, which cleaves the glucose moiety off of the lipid Glucocerebroside. It's the most common lysosomal storage disorder and the most common inherited disorder among Ashkenazi Jews. It's a disorder primarily of the reticulandothelial system, where lysosomes within macrophages become engorged with the stored lipid, giving rise to what you see on the left, the characteristic appearing Gauchier cell. And on the right, you're looking at an electron micrograph of a Gauchier cell, and the distorted organelle that you see there is actually a lysosome, which is engorged with this tubular storage material. There's fast clinical heterogeneity encountered in this single gene disorder. It's classically divided into three types, type 1 being non-neurologic, type 2 being acute neuronopathic, and type 3 being chronic neuronopathic. But having studied patients with this disorder for more than two decades now, I've really come to see it much more as a spectrum ranging from asymptomatic octogenarians to fetuses that succumb in utero with a wide range of associated manifestations. And one of the groups that we started to appreciate was patients that developed Parkinsonian manifestations. So this association between what I'll call GBA, or the gene for glucosaribrositis and Parkinsonism, was a story that really began here at the NIH Clinical Center with the observation of actually one particular patient who we were seeing for Gauchier disease who had pretty progressive Parkinsonism. We then noted that these two phenotypes were encountered sometimes in other rare patients. And then we also started to appreciate that Parkinson disease was seen in relatives of our patients with Gauchier disease more often than we might expect. Then we and other groups around the world started to appreciate that there was an increased incidence of GBA mutations in patients with Parkinson disease and with associated Lewy body disorders. However, actually many of these initial studies were greeted with skepticism because of limitations of power and controls, and also because large genome-wide association studies had not identified this gene. But the associations persist and now glucosaribrositis is considered the most common genetic risk factor for Parkinson disease. In fact, if you just look in the last decade doing PubMed scans, the number of papers and studies on this gene related to Parkinson disease is growing exponentially. Though I do want to emphasize that the vast majority of the patients that we see with Gauchier disease and the majority of Gauchier carriers or people with GBA mutations never develop Parkinson disease. So it's a risk factor but not a predictive gene. Well, one of the reasons why this began to become more accepted was several years ago we spearheaded a multi-center study of glucosaribrositis mutations in large groups of patients with Parkinson disease. We collected genotypes from 16 centers spanning four continents and ultimately had over 5,000 genotypes from patients with Parkinson disease and about the same number from controls. The bottom line was we determined that subjects with Parkinson disease are over five times more likely to have a mutation in glucosaribrositis, giving an odds ratio of over 5.4. We also noted that patients with Parkinson disease that carried mutations tended to have a little bit earlier Parkinson onset about four or five years and we had the impression that there were more cognitive deficits. Just recently, we've actually gone back and done a very similar analysis with 11 different centers participating where we looked for the frequency of glucosaribrositis mutations in patients with an associated disorder with Lewy bodies. Here there's a much more rapid progression of cognitive impairment and it's a rarer disorder, so in this series we collected about 700 cases and actually the odds ratio was greater than eight, suggesting that mutations in this gene play an even larger role in the dimensions with Lewy bodies. At the Clinical Center, we've been following these patients for about a decade now and our studies focus both on clinical features and pet imaging. We collaborate with Karen Berman's group in the National Institute of Mental Health. The goals of the study are to look at fluoridopa uptake and to evaluate pet as a surrogate marker in patients and subjects that have glucosaribrositis mutations and to see if we can find the earliest signs of Parkinson disease in this at-risk cohort. So in our studies, we recruit patients that have both Gauchier disease and Parkinson disease. We're also looking at patients in Gauchier carriers who have a positive family history of Parkinsonism. Patients come to the NIH and undergo fairly routine physical, neurologic and neurocognitive evaluation each time. We do olfactory testing and screens for non-motor symptoms of Parkinsonism to see if we can find signs of early involvement. The imaging studies include MRI, fluoridopa pet studies for fluoridopa metabolism. We do radioactive water studies to evaluate cerebral blood flow and we're evaluating transcranial sonography. We just recently published the results in our first 40 patients that we've studied and basically we found that patients with Parkinsonism that also had Gauchier disease had fluoridopa uptake that was very similar to patients that just had sporadic Parkinson disease. Where we did see differences, we're in the cerebral blood flow studies where we see some changes that are more characteristic of disorders with cognitive impairment and this study is ongoing. So how can mutations in a metabolic enzyme lead to Parkinson disease? Well, the verdict is not out but there's certain hypotheses to consider. One is that we know that the formation of insoluble alpha-synuclein aggregates contribute to the neuronal cell death that occurs in Parkinsonism. So the gain of functional hypothesis is that having this mutant enzyme around could somehow lead to an increase of an aggregate formation as you see on the right or it could lead to organelle dysfunction, particularly the lysosome, leading to decreased aggregate clearance, both cases contributing to these aggregates that contribute to neuronal cell death. But another hypothesis would be that this is a loss of function, that having the mutant Lucas ribosidase around leads to an unstable or deficient protein that's degraded and then you don't have enough enzyme. So the lipid accumulates and the accumulation of this lipid could lead to neuronal cell death. And then another theory that was recently published by our group in collaboration with the group at Mass General is that there could be, it could be even more involved, there could be something like what we call the bidirectional feedback loop where there's indications that having increases in this lipid level, the glycosyl ceramide lead to increased in soluble alpha-synuclein oligomers and fibrils and having these around would contribute to alpha-synuclein aggregates and neuronal cell death. The same time having these insoluble aggregates around seems to block the ER Golgi trafficking of the enzyme, which again would lead to increased lipid accumulation and compounding the problem in a vicious cycle. In collaboration with Jennifer Lee's group and NHLBI, we've also done some biophysics studies and we feel that there is likely a molecular link between alpha-synuclein and our enzyme glucose ribosidase. This was shown by several different techniques including fluorescence spectroscopy, NMR, and co-immunoprecipitation studies. The association only appears to occur at pH 5.5 and not pH 7. The interaction between the two proteins appears to occur at the c-terminus of alpha-synuclein. So this binding at lysosomal pH could facilitate alpha-synuclein degradation or prevent aggregation. Also, this GBA story implicates the lysosome NPD pathogenesis. Now I'm going to move on a little bit towards some of the work that we've been doing in therapeutics and one approach that we've been looking at as a therapy for Gauchier disease is chemical chaperone therapy. The protein glycosuribrosidase is synthesized in the ER and it's glycosylated and folded but it doesn't reach its tertiary functional structure until it's actually in the lysosome as you see the top panel on the right. If you have a mutation in the enzyme, it's likely that it will not fold correctly and it will be degraded and none of it will get to the lysosome. So our strategy is to come up with small chemicals that are known as chemical chaperones that can bind to the mutant protein, stabilize it so that it's at least partially corrected and it can get to the lysosome where it can still function. So in collaboration with the NCGC, the National Chemical Genomic Screening Center, we've been conducting high throughput screening of large libraries of small molecules to see what might impact the enzyme activity in glycosuribrosidase. In fact recently we took a new approach and we actually used a patient's spleen sample as our source of mutant enzyme. In this high throughput screen we evaluated 250,000 compounds at seven different concentrations. Fortunately there's robots to do this work and identified 30 new non-inhibitor chaperones that we're very excited about. Our lead chaperones look like they can improve the translocation of the enzyme to the lysosome and patient fibroblasts and in macrophages I'm going to show you the compound seems to reverse storage. So the small molecule therapies like this may stabilize mutant glycosuribrosidase and also be used to treat Gauchier disease as well as possibly Parkinson disease. Well one problem that we had with this drug development is that we didn't have a really good model for showing reversal of storage which is what you targeted in Gauchier disease. So in the last few years we've been working to develop induced pluripotent stem cells as a model for Gauchier disease beginning with patient fibroblasts. We generated the appropriate embryoid bodies and then showed that our cells make the appropriate markers, have the right karyotype, they can go on to form teratomas. We differentiated them first into monocytes and then macrophages. And to our excitement we were able to determine that Gauchier macrophages can show the storage which we were never able to demonstrate before. We think this model will be very useful for drug development and for understanding pathophysiology. And to demonstrate this, so what we do if you see the two fluorescent images below one is a control macrophages generated from induced pluripotent stem cells and on the right is a Gauchier. When you feed these cells with labeled erythrocyte ghosts you can appreciate that only the fluorescent storage is much, much greater in the Gauchier macrophages. Then we take these macrophages and we treat them with our best chaperones. So the top panel is the control and the second two panels, the two lower panels are macrophages from patients. In the very last column to the right we've added our lead chaperone and you can see that we're seeing a reversal of the storage indicating that this does seem to have promise. So I hope that I've shown you that understanding the links between these two disorders can prove to be quite fruitful teaching us about the pathogenesis of both disorders providing some clues into the role of lysosomes in the development of Parkinsonism and then ultimately it may yield improved genetic counseling and new therapeutic strategies. And I just want to briefly acknowledge all the people in my group who've done this work my close collaborators here at NIH and around the world and of course to give special thanks to patients, family members and the referring physicians who have contributed to these studies. Thank you. Wonderful. Thank you so much Dr. Sudranski. We're going to move on to our final speaker for this webinar and that is Dr. Anand Swaroop he's Chief of the Neurobiology, Neurodegeneration and Repair Laboratory at the National Eye Institute. His laboratory primarily focuses on photoreceptor development and retinal macular degeneration diseases including elucidation of transcriptional regulatory pathways involved in cell fate and homeostasis the genetic basis of retinal defects and the development of treatments using cell gene or small molecule based approaches. Welcome Dr. Swaroop. Thank you Sean and I'm delighted to be here. Blindness generally ranks second or third among all the fear or scary diseases in this world in many many surveys after cancer or cardiovascular diseases people are very scared of going blind. What I'm going to tell you today is some of the research that we are doing on transcriptional regulatory networks to produce a photoreceptor cell that captures light and also going to tell you how some of this research is leading to new paradigms for finding treatment for retinal and macular degeneration. Retina in fact is our window to this visual world and also to brain. Dr. Zalate earlier said we have no window to the brain. Actually retina is its most approachable part of the central nervous system. At any point in time and space you can look at thousands in fact over hundred thousand individuals of different size shapes color you can look at their location in a visual field you can see them moving around and despite all these objects you are able to focus on a single individual if you chose to. All of this visual information is processed through cells in the back of our eye called retina. In fact light is focused through various optical elements called cornea and lens that many of you are aware of. And that focus light goes to retina which is is relatively simple but architecturally a beautiful stratified part of central nervous system. There are six major types of neurons as shown on the right side of this slide. These neurons are organized in three layers of cells. The layer which is at the bottom actually is an epithelial layer called retinal pigment epithelium or RPE which is extremely important for supporting the cells next to those that and this is retina. These are photoreceptors and I'm going to talk to you more about the photoreceptors a little later. The information that is captured by photoreceptors goes through bunch of different kinds of neurons in different types of interneurons and then these neurons convey information to ganglion cells and axons of these ganglion cells from optic nerve and that takes information to different parts of brain. All of this information actually is captured integrated processed to certain extent at least in the retina. In fact 30% of our brain is devoted to processing of visual information. As one can imagine degeneration of these cells which are post-myototic will lead to blindness even though we have treatment for certain kind of blinding disorders like cataract and to certain extent glaucoma retinal and macular degeneration are still a major cause of untreatable blindness. They're highly heterogeneous both clinically and genetically. If you look at the left rather right the top part shows you the picture of a fundus. If you look in the eye of an individual this is what you will see. Beautifully nicely colored uniformly colored part you can see optic disc which is where optic nerve goes through and then optic vessels come in the retina. The center of this retina is fovea that's where highest visual acuity is and that's where the light actually gets focused. The area around fovea is called macula and degeneration of photoreceptors and underlying pigment epithelium cells will lead to macular degeneration and as you can see in the picture below a degeneration of photoreceptors in the macular region will lead to loss of central vision and you will not be able to see or drive or watch TV. Whereas on the right side of that you have another picture of the fundus where there is degeneration of photoreceptors in the peripheral retina and that leads to loss of peripheral vision even though your central vision is OK you will not be able to see on the periphery. The many many different genes that can lead to retinal and macular diseases as written here over 200 genes have been mapped and more more than 150 have already been identified. Many genes can lead to same phenotype and sometimes the same gene and even the same mutation in a family can lead to distinct phenotypes for a variety of regions. Retinal degeneration is also observed as part of numerous syndromic diseases and in fact we have been working on many other many disease like nephronostasis and others where you have kidney disorders or other neurological disorders along with retinal degeneration and then you have multifactorial these are like age-related macular degeneration. So in majority of these diseases this function of death of photoreceptors leads to loss of vision. There are two kinds of photoreceptors which allow you to see in the night and cones which are actually much less in number in humans is only five percent of all photoreceptors but they allow you to see in bright light they're responsible for high resolution and also color vision and color vision is mediated by different kinds of cone receptors because they include different visual pigments short wavelength for like S cones, M cones have medium wavelength visual pigment and long wavelength are L cones in human. In mice we have only two kinds of cones S cones and M cones. Photoreceptors are highly active metabolically active cells and the reason is that these highly polarized cells have got these membrane disc as you can see on top of these photoreceptors ten percent of these discs are shed every day that means the whole outer segment is regenerated every year rather every ten days and even though these cells are post-myototic and they do not regenerate the outer segment part which is where the light is captured has to be replaced every ten days and these outer segment disc are like membrane disc which contain phototransduction material. Now my lab over the last 20 plus years has focused on all aspects of photoreceptor biology we look at photoreceptor differentiation, look at aging of photoreceptors many many different diseases that are caused by defects in photoreceptor function and then eventually trying to look at the treatment for these photoreceptor diseases. Today however I will briefly focus in this short duration on networks that are involved in differentiation of photoreceptors and how we are trying to identify treatments. Photoreceptors and in fact all retinal neurons and glia are generated from common pool suspended cells and as on the left you can see rod photoreceptors we dominate the retina there are over 70 percent of all cells in the retina their birth overlaps with the birth of all other cells. There is a conserved order of birth and as you can see on the right this photoreceptor differentiation like other differentiation of different cell types proceeds in a very sort of simple manner you have a dividing multiple potent product cells at some point in their differentiation they become linear-districted then when they exit cell cycle they have their fate specified and then through a variety of regularity pathways these photoreceptors acquire function multiple transcription factors are involved in generating in this pathway however let us focus towards the right only on transcription factors that are involved in photoreceptor cell fate determination the primary factor there is NRL along with that you have a bunch of other factors CRX which is a homeodomain transcription factor very critical for both rod and cone photoreceptors TR beta 2 is primarily for cone differentiation and NR2 E3. Several years ago an excellent postdoc in my lab Alan Mears made a knockout for this NRL gene and showed that if you knock out this gene a loss of function of this specific gene leads to a cone only retina you have no longer any type of rods there is a complete fate switch at the bottom of the slide you could see the ERG or electro retinogram that shows the functional characteristics of these photoreceptors in wild type you can see the dark adapted ERG is very high but in knockout its flat dark adapted ERG it shows the response of rods like light adapted is for cone cells and you could see there is a huge increase in cone response in this NRL knockout retina and what is even more exciting for us was when this graduate student at oh what he did was he took NRL and expresses under the control of CRX promoter which is both in rods and cones and showed that now all cones become rods so NRL alone is sufficient to convert cone photoreceptors to rod photoreceptors and in fact if you drive another expression under the control of SOPS promoter which is when the cone cells are actually even at a different stage in differentiation even then some of these differentiating cone photoreceptors can get converted to rods as much as 40% of these cells something which is even more exciting for us working with Douglas forest here at an IDDK we showed that TR beta 2 and NRL these two transcription factors are present in certain photoreceptor precursors at the same time as I mentioned earlier TR beta 2 is responsible for M cone differentiation whereas NRL is for rod differentiation what are they doing in the same cell what we believe is happening is that there is some sort of tug of war going on between different transcription factors and they can then sort of determine what they're going to be and this particular slide shows us the transcriptional regulatory network I'm not going to go into detail of that but what it shows is that the default pathway is the S cone pathway and if you have NRL you're going to make a rod photoreceptor if you have no NRL you're going to go towards the S cone or if TR beta 2 is there you're going to make an M cone now another post talk in the lab showed that if you can take the NRL promoter and drive GFP you could label the rod photoreceptors as soon as they are born this is extremely important because now we can flow sort these cells and these flow sorted photoreceptor cells can be utilized to develop gene regulatory networks during differentiation and under N disease processes you could use these purified photoreceptor for cell replacement and for drug discovery now what we have been doing in this brief slide I'm going to give you a whole lot of background a whole lot of information what we have been doing is we have been trying to look at the networks that guide the differentiation of newborn photoreceptors to a functional photoreceptors by doing RNA seek and other profiling global profiling we are doing chip seek with using different transcription factors histone modification studies and DNA methylation and I've listed the names of post talks and fellows who have been involved in this particular work now several years ago we collaborated with the group in London and showed that we could take these photoreceptor precursors the newborn NRL positive photoreceptors and we can transplant in degenerating retina and these cells will then not only differentiate but also integrate within the retina and could give you some function but you need to have these immature developing rods once the cells differentiate that means fully differentiate they have outer segments they can no longer function or integrate within this degenerating retina this has been very exciting development a large number of labs have now been trying to use this technology to approach stem cell based therapy for retinal repair here is another slide working with David Zaks we showed that you could put that in a degenerating retina and these cells can integrate as shown here and they are viable for several months Kohai Homa in the lab has recently I've got a paper now in press we showed that this NRL GFP positive developing rod photoreceptors when you integrate them in a degenerated retina they are functional and their function is very similar their membrane properties by using patch clamp and other studies are very similar to native rods now how do you make these rod photoreceptors if you want to do transplantation therapy you could do that from human embryonic stem cells, IPS or induced blood stem cells and you could develop these immature photoreceptors for a variety of different uses at a later stage now this is just one of the slides that I wanted to put in here pioneering work in the lab of Dr. Sasai in Japan showed last year or in 2011 actually that you could make eye in a dish from both mouse and later on he showed it from human ear cells and this we have been using their protocols to develop human retina in a dish and Rasukan and Kohai and Jessica in the lab have been working on the strategies to look at rather to generate neural retina now what we have also discovered very recently is that the retinal photoreceptors if you make them in a dish do not have outer segments and they will not respond to light for that you require retinal pigment epithelium integrity and this was another work that we have published very recently in development where we show RP is critical for only outer segment morphogenesis cell fate is still conserved but outer segments are not there if RP is not properly polarized so what do we need to have cell based replacement therapies for retinal and macular degeneration we need to generate photoreceptors but we can make these cells but we still need to do a little bit more work on epigenetics and other characteristics of these cells we should also be able to purify these cells without using GFP otherwise it will be hard for us to transplant these in humans transplanting methods have become pretty good these days but we need to find ways so that the cells do not clump and we might require some sort of biomaterial or scaffold cell integration is still relatively poor only small number of cells get integrated in the retina we need to figure out ways to improve that we also need to find fundamental methods to generate their connections and we are also working on many different methods for better assessment for efficacy in animal models eventually we believe that we need to have some sort of 3D reconstruction of outer retina if we want to have treatment for retinal and macular degenerative diseases I'm going to stop there this is our group a large number of people have been involved in this work along with that we have several collaborators I'm going to stop with this quote from Helen Keller the only thing worse than being blind is having sight but no vision thank you thank you so much Dr Swaroop and many thanks to all of our speakers for their excellent presentations we're going to move right on to the Q&A portion of the webinar now we have probably about 10 minutes so I'm going to get through as many questions as we can so the first question I'm going to put to all of you and maybe we'll start with Dr Swaroop and we'll work our way back down the table is how might some of the processes and techniques that you're developing right now in your research be applicable to some other fields of biomedicine? so just three very quick points as I mentioned earlier retina is part of brain in fact retinal disease research has been at the forefront as a poster child for human genome project first disease that was mapped by using GVAS was isolated macular degeneration gene therapy has been highly successful in case of you know this retinal degeneration caused by defects in RP 65 gene and so what we are hoping is that some of the work that we are trying to do in discovering transcription regulating network some of these will also be sort of will be directly applicable to research on other neurodegenerative diseases specifically Alzheimer's and Parkinson's yes I think that as we're trying to understand the genetic basis of many different disorders we're beginning with work on Mendelian disorders can give us sort of an anchor whereas when if we can try to understand how we get this great spectrum of variability in a single gene disorder looking at modifiers or other contributing factors it'll be helpful when we go to tackle complex disorders that have multi genes involved and I also think that some of the strategies and techniques that we're looking for also will have wide applicability first of all things like the IPS models and also what high throughput screens for small molecule targets with regards to understanding more about the circuits or synapses we find that there's a lot of comorbidity with certain of our disorders within other psychiatric disorders one might see more substance abuse one might see more comorbid medical conditions neurological conditions and it's that this function at the synapse may apply not only to mood disorders but might apply to PTSD might apply to other disorders and the more we understand how we stabilize a synaptic dysfunction at the synapse level and also at the circuit level might be applicable to other disorders some of our medications for example lithium is neuroprotective and is being studied in Alzheimer's disease and other neurodegenerative disease and it's also been studied in eye conditions where as a model for infarction so we can see that some of our drugs are being applied to other disorders and particularly it's neuroprotective properties So a question I'm going to stay with you Dr. Zarate that I think is very interesting and hopefully not too controversial is have you encountered any barriers or biases in the research amongst colleagues or other people in the same area who might not appreciate the molecular underpinnings of mood disorders? Well I think that one of the limitations we have in psychiatry is that we don't have clear ideology and there's certain assumptions on what might be the causes the issue is that the lack of targets has been a hindrance in terms of drug development for psychiatric disorders and when we go from target to hit target to lead we're assuming this based on some secure notion of how our disorders work mental diseases and the other aspect of that is we were most of our research was based on animal models which were largely developed to identify compounds that modulate serotonin or benevolent monominergic systems and that's led to development of me too drugs over the years so now that there is a move to more social effective models, animal models other ways of assessing, you know, develop a more sophisticated model than we previously had the other is to work backwards, I mean if we find treatments are radically different then we can become to understand, you know, what might be the circuit synapses or genes involved and that might not, that perhaps will lead to an understanding of the pathophysiology of the illness but if not then at least we might be able to develop better treatment so I think there's considerable progress in recent years it's quite exciting and where industry has been moving out of psychiatry now there seems to be a greater interest of going back particularly in the area of mood disorders Dr. Zdranski I got a question for you what kind of therapy for Parkinson's do you predict from your research on Gauchy? Well I think that our research is helping us focus appreciate the role of lysosomal pathways in the etiology of Parkinsonism I also think that there's now more and more evidence in the last few years that ways of, that because of this association between glucosaribrosidase and alpha-synuclein that if we can find ways to enhance glucosaribrosidase it may ameliorate the aggregation that you see with alpha-synuclein so strategies like the chaperone to therapy or other ways to increase enzymatic activity in the brain may have a role in Parkinson's of course we have a long way to go and unfortunately we still don't totally understand the mechanisms and I think that a lot of basic science work is needed before we can extrapolate totally Dr. Swaroop what do you think is currently the biggest what do you think is the biggest breakthrough that will be needed to occur before we are capable of growing complex organs for replacement such as eyes or possibly brains? Yeah, I think we still do not understand the basic physiology of each of these cells and how they behave in vivo we look at cell, we look at biochemistry or we do the biology in isolated cell cultures and many times it does not reflect the biology in vivo so what we have been trying to do is to develop sort of systems in vitro like we could do, explain cultures of the retina we have been thinking to sort of combine biomaterials and nanotechnology based methods with the cell culture protocols in order to sort of generate these tissues in vitro 3D in 3D so that they can then be used to study biology first and what we are lacking is really a collaboration among different scientists unfortunately and I came from extramural site until very recently I was at the University of Michigan and in extramural science we are always concerned about an R01 grant our own funds how to do research but what we are not able to do many times is come together as a group and write large projects and many times those large projects are thought to be oh you know it's impossible to do this fortunately NIH has taken note of that and now we are having larger projects or program projects that we are thinking about NEI has recently started this audacious goals project and trying to sort of bring in people from many many different areas together in order to really solve a problem and I think that's what we need we need for people to come together from different areas in physics and engineering to biology and sort of do work together to solve a problem and not focus on a very tiny part of the big picture we should look at the big picture Any other comments Dr. Szeranski? I think one of the strengths that we have here at NIH is the ability to collaborate with so many people in so many different fields and I think it's greatly helped all of our individual work and it's also given us an opportunity now that there's initiatives to collaborate with extramural and have these joint partnerships with the clinical center so I think that's going to go a long way and facilitate and discover Just one comment and that was the primary reason for me to come here NIH is a wonderful place if I don't know anything I can go around to different institutes even in different people I collaborate with folks in seven different institutes already and there are top-notch scientists here sometimes that's not easy to do in extramural that's why NIH is a great place to come So talking of interacting with other people in different fields we have a question on the potential role that epigenetics might play in neurodegenerative diseases and how these therapies... how therapies can maybe overcome such epigenetic alterations Dr. Zaraitse? We'd like to go ahead there Dr. Swaroop? Epigenetic right now is still in very early stage and what we are trying to do is understand what it really means in many cases but right now what we are looking at is that we can manipulate the histone methylation or DNA methylation and we can change patterns of gene profile but we don't still have much control over that phenomenon of epigenetics so I think it'll be few more years from phenomenology that we move to really real benefits of understanding epigenetics so I think we'll take another few years to understand how epigenetic changes are really giving you a specific phenotype and how you can alter or manipulate that to give you a very distinct treatment or different phenotypes Dr. Zdranski, any thoughts? I agree, I think we're now well poised to look at the contribution of specific genes to phenotypes and as we start to appreciate the limitations of this I think that'll give us openings for other epigenetic components of these disorders At least in the mental illness it's going to be much more challenging and there are interesting findings already that how your reared might affect or aspects of abuse and trauma might reflect later in greater suicide rates for example in bipolar disorder but understanding epigenetics is really early on and you need to do epigenetics in the specific cell type you cannot use the whole tissue for that because there'll be multiple different... there are a lot of differences among different cell types and for many diseases particularly in psychiatric and neurological diseases it's hard to get those cells and if you have them cells in culture the epigenetic landscape is going to be very different than in vivo and again I'm going to say that now hopefully will provide some of the early sort of conclusions for that kind of studies So we're pretty much out of time so I'm going to just fire one more question at you to start with Dr. Zarate What do you see as the biggest challenge in moving proof of concept basic research into the clinic and how can this process possibly be improved I know the NIH is very focused on this right now Yes and I think that there's initiatives at least by our institute in IMH and also by NCATS wonderful initiatives where most of the trials that were taken place have been were either industry run for many years and of course a lot of times there are refinements over existing treatments which I mentioned it's not that industry, academia and government has not tried in coming up with new targets and testing them through but we can see that the process of drug discovery and development is quite costly it takes a long time and at least in mental health or CNS disorders the targets are unclear and so in the models as we talked are imperfect but I think there's a renewed excitement and enthusiasm now where one can in places like the clinical center do very specific hypothesis driven questions where with drug target X or Y if we go after this would that lead into an improvement in symptoms and one example several examples I talked about today and I think that's possible here a lot of times these studies are done in patients taking many medications with comorbidity and it's unclear if you find something not only that the clinical center does permit a heavy biophenotype and integrated translation using multimodalities to really not only to test certain hypothesis but hypothesis is generated and this is a wonderful place you can find a lot of questions that you can pursue and that can be rapidly we can do collaborations with our colleagues here you know go in the eye or at a metabolic level and so this is a very wonderful place to do that and I think part of our mission intramural is to come up with a signal some kind of spark through our work which is very difficult to do outside but once you have that spark you can ignite discovering development out there and I think this place does very wonderfully Dr. Zyranski? I concur I think there's certain things that are very special about the clinical center that help us with translational research one it's the opportunity to become really expert at one thing and to also do natural history protocols where we longitudinally follow patients and really get to understand the disease and through that we can find targets or biomarkers that can be used when we eventually have therapeutics I also am very excited about the new NCAT Institute and the concept of programs like the trend program which will enable us to develop some of the new drugs and targets that we're working on directly through people here and I also would emphasize the clinical center gives us an opportunity to do therapeutic trials so that you can really actually go from the bench to the bed to the bedside and then learn something go back to the bench and it really facilitates this evolution Dr. Zyranski? Yeah so I mean we have come a long way I think basic research still has to be forming the trigger or forming the basis of all the translational studies that we do and translational studies are very expensive and therefore as it was pointed out earlier by Dr. Zarate we need to have a good collaboration with industry whether it's a small biotech or large pharma there are certain things we cannot do in academic institutions whether it's a university or even at NIH however NIH offers a very unique environment here that you have people from various areas who can come together I do hope that those sort of approaches are available at other institutions as well sometimes the money comes into play and we can't do that but I do hope that other universities and institutions take note of that and try to create the right environment where basic scientists can work very closely with clinicians and also with folks in industry I mean I was in a clinical department and I can tell you in Michigan both in ophthalmology and then in genetics many times everyone is so busy in whatever they do it's very hard for them to find time to collaborate with each other even within a department and I think it's up to the institutions to create that in NIH we don't have that sort of problem as much we can go to our colleagues and it's much much easier to do and I do hope that NIH becomes a trigger or helps in creating that kind of environment for people to come together Fantastic, well a lot to discuss but unfortunately we are out of time for this webinar so on behalf of myself and our viewing audience I wanted to thank our speakers for being with us today Dr. Carla Serrate from the National Institute of Mental Health Dr. Ellen Cedranski from the National Human Genome Research Institute and Dr. Anand Swaroop from the National Eye Institute Please go to the URL now at the bottom of your slide viewer to learn more about exciting research being carried out within the NIH Intramural Research Program and look out for more webinars from science available at webinar.sciencemag.org This webinar will be made available to view again as an on-demand presentation within approximately 24 to 48 hours from now We'd love to hear what you thought of the webinar Send us an email at the address now up in your slide viewer webinar at tripleas.org Again thank you to our panel and to the Intramural Research Program at the NIH for their kind sponsorship of today's educational seminar Goodbye