Epigenetic mechanisms regulating pluripotency from embryonic to adult neurogeneisis

Neurological diseases are amongst the most devastating since the brain does a very poor job in healing itself. In the large majority of cases, new nerve cells cannot be produced to replace injured tissue. With the increase in the population's average age, diseases such as Alzheimer's, Parkinson's and stroke will take an increasingly burdensome toll on individuals, families and society. Hope for a therapeutic solution has arisen with the discovery of brain stem cells and increased understanding of how they are controlled. Stem cells are found in two specific regions of the brain and continue to generate nerve cells in those restricted areas. Our goal is to understand how to enhance this process and entice it to be of more general relevance throughout the brain. In the past decade, laboratories around the world including ours (Shen in Beijing, Szele in Oxford) have discovered many molecules that stimulate stem cells to grow during normal development and molecules that stimulate them to repair a damaged brain. Use of this knowledge will allow us to develop medicines that mimic these molecules and thereby stimulate the growth of stem cells for brain repair. Since the repair of the adult brain is conceptually similar to the development of the brain, we also seek to understand fundamental molecular mechanisms regulating the generation of nerve cells from human embryonic stem cells (hESC). Amazingly, hESC can be grown in cell culture dishes, allowing us to discover molecules that increase nerve cell production. Similarly, adult brain stem cells can be grown in cell culture to study their generation of nerve cells. In this grant, we will use and compare both approaches in our goal to increase stem cell-derived nerve cell generation.

Most research in the 20th century has uncovered how individual molecules regulate stem cells one at a time. In this century, a shift in emphasis has occurred, and the field is now seeking to understand how molecules work together to coordinate stem cell nerve generation. This is an intelligent shift since dozens to hundreds of molecules usually are required to coordinate stem cell nerve generation. But how are so many molecules regulated in the same space and at the same time in such a coordinated way? The answer is in a fashion analogous to how a conductor organizes an orchestra: she may increase the loudness of the string section whilst repressing the horns. So-called "epigenetic" mechanisms coordinate dozens to hundreds of molecules by causing some to be emphasized and some to be repressed. Epigenetic mechanisms can turn molecules completely on or off, or they can more subtly turn them up or down. Importantly, they do this in stem cells and thereby cause the cells to either proliferate or to turn into nerve cells.

In our laboratories, we study how epigenetic mechanisms increase nerve cell production from embryonic stem cells (Shen, Beijing) and from adult brain stem cells (Szele, Oxford). In this collaborative project, we will seek to understand the extent to which the same molecules govern nerve cell production in the embryo as we as in the adult. We will also use this novel molecular knowledge to increase stem cell-mediated nerve cell production in a model of stroke. Brains of infants have a far higher plasticity than those in the adult and aged population. However, even infant brains do not completely repair themselves in diseases such as cerebral palsy. Thus, we will also seek to understand how epigenetic molecules enhance stem cell-mediated repair of a model of infant cerebral palsy.

Our overarching goal is to eventually use this information to increase nerve cell production from stem cells. This exciting branch of regenerative medicine is one of the best hopes for the future of treating neurological disorders.

Recently, it was confrmed that the human SVZ continues to be neurogenic throughout life, but that instead of migrating to the olfactory bulbs as in other species, progenitors migrate into the adjacent caudate putamen and become calretinin+ neurons. The human SVZ can be cultured as neurospheres, proving that latent stem cells, which could be stimulated via epigenetic mechanisms, reside in the tissue. These findings make targeting human SVZ stem cells for neural repair more relevant than ever, since it proves that they are stem cells and migrate naturally to clinically relevant areas.
This project is designed to uncover epigenetic mechanisms that can be used to control and enhance stem cell-mediated neurogenesis and repair. We have shown that loss of Enhancer of Zeste Homolog 2 (Ezh2) function, the Polycomb Repressive Complex 2 (PRC2) histone methyltransferase, significantly reduces neurogenesis in the adult subventricular zone (SVZ)/olfactory bulb (OB) system. We now plan to extend these data by studying how PRC2 is functionally related to lnRNAs. Genomic analysis indicated that an unanticipated large percentage of genomic DNA encodes for lnRNAs. It has been estimated that about 20% of human lncRNAs bind to PRC2 and help direct them to specific nuclear sites. lncRNA expression in turn can be modulated by PRC2 and PRC1 suggesting recursive functional interactions between these molecules. Because of the large concentration of lncRNAs in the SVZ, we hypothesize that in this system they also functionally interact with PRC2 function. It is becoming increasingly evident that lncRNAs have clinical relevance via lncRNA interactions with PRC2. We will focus on PRC2 and two lncRNAs, RIAN and YSD14/Haunt. The Shen lab has generated both these knockouts. We will use complementary in vivo and in vitro approaches to study their function in ES cells as well as in normal postnatal and adult neurogenesis.

The hope in this - and all of our work - is that those suffering from diseases of the brain will benefit. The diseases we study are incurable, difficult to treat and rob people of the most important core aspects of life. Anyone who has experienced serious brain injury or disease would readily divert resources from many human endeavours to developing treatments. Most pharmacological therapeutics only modify symptoms, providing relief while the body does the real job in healing itself. Endogenous tissue-specific stem cells are one of the ways through which the body heals itself, and learning how to stimulate them to do so even more offers great hope. Pharmaceutical companies are closing neuroscience research programmes, decreasing the prospect of new small molecule therapies for brain disease. New paradigms for treatment must be developed, and stem cell therapies offer a potential alternative. We must approach them with caution, however; they may do harm and are unlikely to be as widely beneficial as expected. The brain is a particularly difficult target for stem cell therapy because of its great cellular, anatomical and functional complexity. In this project, we seek to combine forces in order to learn how novel and powerful lncRNA and epigenetic mechanisms may be harnessed to stimulate stem cell-mediated repair.

Policy makers interested in the relative benefit of endogenous adult stem cells versus embryonic stem cells will be interested in this work and benefit from it.

The proposed research is unlikely to immediately lead to commercially exploitable results. Any techniques we use will be available to the University of Oxford, Tsinghua University as well as the international scientific community.

The postdoctoral fellows recruited to carry out the work will be trained in state-of-the-art techniques. Though this grant will fund postdocs, which in and of itself will attract students. Many PhD students are interested in our work on stem cells. Thus, young national and international scientists will benefit from it.
Because the work is translational in nature, it will foster links between basic scientists and clinicians, and the latter will benefit from it as well.

Shareholders and employees of companies that manufacture the reagents and equipment used in the project will also be beneficiaries. We have developed a very effective 2-photon time-lapse microscopy system. A significant part of work will use the advanced software, Volocity, developed by the company Improvision, who are based in the UK (Coventry). I regularly speak at international meetings and transparently discuss how central the Volocity software is to our studies.