Akiko Nishiyama - US grants

The goal of this proposal is to characterize a unique population of glial cells in the central nervous system (CNS) that can be identified by antibodies to NG2 proteoglycan and the alpha receptor for platelet- derived growth factor (PDGF). in vitro the two molecules are expressed o the surface of oligodendrocyte progenitor cells but are downregulated as they differentiate into mature oligodendrocytes. The presence of a physical interaction between NG2 and PDGF a receptor has been demonstrated by bioclleluical rllelhoils. Furthermore, incubation of oligodendrocyte progenitor cells with antibody to NG2 diminishes their ability to proliferate in response to PDGF. In the developing rat brain in vivo NG2 and PDGF a receptor are co- expressed on process-bearing cells that begin to appear during late embryonic stages and continue to increase in density through the first postnatal week. Our preliminary results indicate that the antibodies to the two molecules label a large population of process-bearing glial cells in the gray and white matter of the mature rat brain which appears to be distinct from microglia, mature oligodendrocyte, or mature astrocytes that express glial fibrillary acidic protein (GFAP). The specific aims of this proposal are designed to address the following questions: 1. Are the NG2=/PDGF ct receptor (N+P+) cells oligodendrocyte progenitor cells? (specific aim 1) 2. How do these cells respond to pathological conditions of white matter? (specific aim 2) and 3. what are the functions of N+P+ cells in gray matter? (specific aim 3). In specific aim la, the relationship between N+P+ cells and oligodendrocytes will be investigated by examining the changes in N+P+ cells in the dysmyelinating mutant jimpv, the phenotype of which is characterized by oligodendrocyte, death and increased proliferation of immature oligodendrocytes. In specific aim 1 b, immunopurified NG2+ cells carrying a genetic marker will be transplanted into the brain to determine whether they differentiate into oligodendrocytes and/or atrocytes in white and gray matter. In specific aim 2a, changes in N+P+ cells will be examined in the chronic relapsing mouse model of experimental autoimmune encephalomyelitis in order to determine whether N+P+ cells proliferate and differentiate into oligodendrocytes in response to myelin damage. The possibility that N+P+ cells interact with microglia will be investigated in vitro in specific aim 2b. In specific aim 3, a lcinic acid-induced gray matter lesion will be used to determine whether N+P+ cells in the gray matter can be induced to become GFAP-positive astrocytes. The possibility that N+P+ cells promote neuronal survival will be tested in vitro in specific aim 3b. These studies will enhance our current knowledge of glial cells and may have important clinical implications in devising strategies to promote remyelination in demyelinating diseases.

Funds are requested for the purchase of a Bio-Rad Radiance 2000 Confocal Imaging System and Nikon Eclipse TE300 Inverted Microscope to be used in the Department of Physiology and Neurobiology. Currently, there is no confocal imaging system in the department. The only existing confocal imaging system on the Storrs campus is located in the Laboratory of Dr. Knecht in the department of Molecular and Cell Biology in the Life Science Annex Building. However, the building is located one mile away from the Department of Physiology and Neurobiology, and parking by the building is not available. Thus investigators in the Department of Physiology and Neurobiology must either take the campus shuttle or walk twenty minutes to access the confocal unit, which severely limits their use of the Department of Physiology and Neurobiology. There will be four NIH-funded major users, whose projects will require 85% of the total usage. The remaining availability time will be used by three minor users with grants from the National Science Foundation and the American Heart Association. The proposed Confocal Facility in the Department will be operated by Dr. Akiko Nishiyama who has extensive experience with confocal systems. An advisory Committee consisting of two members of the Department and Dr. David will be formed to ensure fair use of the instrument. There is a rapidly growing demand for a confocal imaging system with the Department as the members have begun to carry out detailed localization studies for multiple molecules in tissue sections and organotypic cultures and have initiated studies on living cells using green fluorescent protein to examine cell. migration and subcellular transport of molecules. Radiance 2000 is the least expensive through three fluorescence channels and for rapidly and independently regulating the power for each laser line. These two features are needed for the triple labeling and live cell imaging proposed in the application. The Dean of Liberal Arts and Sciences and the Office of the Vice Provost for Research and Graduate Education have agreed to share the cost of the purchase of the instrument. Support for the maintenance of the equipment will be provided by the user group, the Department, the Dean's Office, and the Research Foundation.

Mature oligodendrocytes make myelin sheaths that insulate axons and allow for fast conduction of electrical signal. Recent in vivo studies by the PI and other investigators have shown that oligodendrocyte progenitor (immature) cells do not all differentiate into mature oligodendrocytes, but a significant number of them persist in the adult brain and spinal cord. There is extensive cellular contact between the progenitor cells and differentiated oligodendrocytes, indicative of an active signaling between the immature and mature cells. It remains unclear how some progenitor cells are stimulated to differentiate into mature myelin-forming oligodendrocytes while others in the same region remain undifferentiated. The objective of the proposed study is to investigate whether a local contact-dependent signal induces differentiation of oligodendrocytes and simultaneously triggers a signal to inhibit differentiation of neighboring progenitor cells. The following hypotheses will be tested: 1) increased progenitor cell density and contact inhibits the intercellular Notch signal; 2) Notch activity represses transcriptional activation of Nkx2.2, thereby inhibiting OL differentiation; 3) Nkx2.2 promotes OL differentiation and activates transcription of the Jagged1 gene, which in turn, activates Notch1 on neighboring progenitor cells and inhibits their differentiation, thus counteracting the density and contact-dependent stimulus for differentiation. The proposed studies using the oligodendrocyte system as a model will address the fundamental question in developmental biology of how differentiation and maintenance of the immature cell type are regulated. At the same time, there will be training opportunities for a graduate student and two undergraduate students. In addition, this project will impact a diverse group of over 400 students each year, including minority students, through the PI's large lecture class. The PI also participates in outreach activities to girls in middle school, through an annual university-wide program to promote women in science.

DESCRIPTION (provided by applicant): Recent studies suggest that glial cells in the central nervous system (CNS) actively participate in the neural network. Most of the previous studies on the functions of glia have focused on astrocytes identified by the presence of glial filaments. However, in the mature CNS, there exists another major macroglial cells type, the NG2-expressing glia (NG2 cells) which comprise at least 10% of the cell population. Morphological, immunological, and electrophysiological studies indicate that NG2 cells are distinct from astrocytes, mature oligodendrocytes, and microglia. NG2 cells have the potential to differentiate into mature oligodendrocytes in vitro and in vivo and are hence referred to as oligodendrocyte progenitor cells. However, the persistence of a large number of NG2 cells throughout the gray and white matter of adult CNS raises the possibility that they carry out functions besides generating myelinating oligodendrocytes. Based on the ability of purified NG2 proteoglycan to inhibit axonal growth and cause growth cones to collapse, it has been speculated that NG2 cells also play a negative role in axonal growth. On the contrary, preliminary results generated in the Pi's laboratory indicate that growing axons extensively contact NG2 cells and do not immediately collapse upon encountering NG2 cells. The goal of this proposal is to determine whether NG2 cells (and not the molecule itself) promote, inhibit, or cause branching of developing, regenerating, and sprouting axons, and whether these effects are altered by the level of NG2 expression. The hypothesis is that NG2 cells promote or inhibit axonal growth depending on the level of NG2 expressed at the surface, which might be regulated by metalloproteinases. The ability of NG2 cells to promote or inhibit axonal growth as a function of NG2 level will be investigated in dissociated culture (aim 1), in developing corpus callosum in vivo and in living slices (aim 2), and in white matter and gray matter lesion models (aim 3). These studies should elucidate the role of NG2 glia in axonal development and regeneration and may lead to novel concepts and design in therapeutic strategies to promote axonal regeneration following injury.

This is a request for funds to support a satellite meeting "Glial Meeting" during the Annual Meeting for the Society for Neuroscience. The PI currently has funds from NSF (grant #0316893 titled 'Molecular mechanisms of glial progenitor cell differentiation') awarded to Akiko Nishiyama through the Integrative Biology and Neuroscience Division.

The funds will be used to support a Glial Meeting, which is listed as a satellite/ancillary meeting to the 2004 Society for Neuroscience meeting in San Diego. The following description of the meeting will be printed in the Program: 'There is increasing recognition that glia is not glue. The Glial Meeting is an informal gathering for investigators from diverse backgrounds to share new and exciting findings in glial biology and identify unsolved problems. There will be one or two short presentations followed by informal discussion and interactions among all attendees. Light refreshments will be served.'

It will be held from 6:30 to 9:00 pm on October 25, 2004, in one of the meeting buildings in San Diego, CA, in the middle of the week during which the Annual Meeting of the Society for Neuroscience will be held. This meeting attracts more than 25,000 participants and is an excellent occasion to promote informal interaction among a diverse group of investigators interested in glial biology. The first Glial Meeting was held in 2003 and was organized by Dr. Harold Kimelberg in New Orleans, also during the Annual Neuroscience Meeting.

This year, we are fortunate to have Dr. James Goldman from Columbia University who will present a talk on his recent studies on glial cell migration in living slices. Dr. Goldman has been studying the development and pathology of various types of glial cells for a number of years and has been one of the key contributors to uncovering the origin and migratory behavior of astrocytes and oligodendrocyte lineage cells.

The meeting will include a number of minorities and women as participants. Since the organizer is a women and from a non-white background, the speaker was chosen from a general the population and was selected on the basis of one with the best scientific qualifications.

ABSTRACT

The NSF Research Experience for Undergraduates site in Physiology and Neurobiology at the University of Connecticut (Storrs, CT) offers paid summer research opportunities in the areas of renal, cardiac, and muscle physiology as well as developmental neurobiology, synaptic plasticity and neural circuits. Interested students, especially underrepresented minorities and students with disabilities and from institutions with limited research opportunities are invited to apply. Ten undergraduates will be recruited each year to participate in graduate level research projects on physiological systems in animals ranging from invertebrates and marine fish to birds and mammals. All students receive a stipend, room and board, research supply money, and assistance with funds to travel to and from the University. REU students will be part of a campus multidisciplinary REU community consisting of several programs in the physical sciences and engineering as well as physiology and neurobiology. The 10-week program begins in early June with orientation and training programs in laboratory safety and animal care protocols as well as ethics programs and Diversity in Science workshops. Students receive hands-on practical research experience guided by an individual faculty mentor. Objectives for student participants include being able to: (a) apply the scientific method in hypothesis-driven research; (b) engage in self-directed research; (c) communicate research results in verbal and written form; and (d) make informed decisions about a career in science. The REU program also includes seminars, an end-of-summer research symposium, and a variety of recreational activities. Each student prepares a scientific paper as well as a poster of their research for presentation at an on campus REU symposium. Students can expect to acquire skills and knowledge to help them enter graduate research programs. UConn is located midway between New York City (2.5 hours) and Boston (2 hours) in scenic eastern Connecticut. More information is available at http://www.pnb.uconn.edu/reu or by contacting Dr. J. Larry Renfro at 860-486-4119 or larry.renfro@uconn.edu.

DESCRIPTION (provided by applicant): Glial cells in the mammalian central nervous system (CNS) that express the NG2 proteoglycan (NG2 cells) appear during late embryonic stages and rapidly expand to uniformly occupy the entire CNS. These cells are distinct from mature oligodendrocytes, astrocytes, or resting ramified microglia and represent a fourth major glial population. Cultured NG2 cells give rise to oligodendrocytes and are thus called oligodendrocyte precursor cells (OPCs). In vivo fate mapping studies using our newly generated NG2creBAC transgenic mice revealed that NG2 cells in the white matter and dorsal forebrain generate exclusively oligodendrocytes, while those in the gray matter of the ventral forebrain generate both oligodendrocytes and protoplasmic astrocytes. When the basic helix-loop-helix transcription factor Olig2 is deleted in NG2 cells, almost all the NG2 cells in the dorsal forebrain differentiate into astrocytes at the expense of oligodendrocytes, resulting in myelin loss. Thus, NG2 cells retain lineage plasticity, and a single transcription factor Olig2 plays a critical role in maintaining the oligodendroglial fate of NG2 cells. Deletion of Olig2 in more differentiated oligodendrocytes does not alter the level of neocortical GFAP expression, in contrast to Olig2 deletion at earlier stages, suggesting that there is a developmental window in which oligodendrocyte lineage cells can be converted into astrocytes in the absence of Olig2. Aim 1 will test the hypothesis that NG2 cells lose their lineage plasticity and become incapable of generating astrocytes as they mature into oligodendrocytes, and that restriction of lineage plasticity in NG2 cells is regulated by epigenetic mechanisms. Aim 2 will test the hypothesis that there is a signaling pathway that maintains the expression of Olig2 in NG2 cells in the normal brain and prevents them from differentiating into astrocytes. This will be explored by screening small-molecule libraries for a compound that alters Olig2 transcriptional activity. Identified compounds will be used in future studies to delineate the endogenous signaling pathways that regulate Olig2 expression. The pathway could then be manipulated in future injury repair paradigms to promote differentiation of NG2 cells into the desired cell type. PUBLIC HEALTH RELEVANCE: NG2 cells represent a glial progenitor population that is ubiquitously distributed throughout the gray and white matter of the central nervous system. The proposed studies are aimed to identify the mechanisms that regulate their lineage plasticity through the transcription factor Olig2. The results from these studies can be used in future experiments in which the fate of endogenous NG2 cells can be manipulated to maximize their contribution to lesion repair in various types injury.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a demyelinating disease that affects 350,000 people in the U.S. and is a major cause of chronic neurological deficit, affecting adults during their most active period of their lives. Remyelination failure is a characteristic of long-standing and primary progressive lesions of MS and is associated with impulse conduction failure and axonal pathology. Despite the debilitating clinical effects of remyelination failure, the reason why some lesions are effectively remyelinated while others are not remains unclear. Glial cells that express the NG2 proteoglycan (NG2 cells) exist widely throughout the mature central nervous system. Recent genetic fate mapping studies have provided direct demonstration that they generate oligodendrocytes not only during development but also in the mature central nervous system. Using new transgenic mouse lines that we have generated, we have observed that deletion of the basic helix-loop-helix transcription factor Olig2 specifically in mature NG2 cells reduces the number of oligodendrocytes that are produced from NG2 cells in the adult brain. We have also performed a high throughput screen to identify compounds that upregulate Olig2 transcription. We will use these newly acquired tools to test the hypothesis that a critical level of Olig2 is required for successful remyelination in experimental autoimmune encephalomyelitis (EAE), which is a clinically relevant rodent model of MS. This will be tested in the following three specific aims. In Aim 1, we will determine whether loss of Olig2 will compromise the ability of NG2 cells to produce new oligodendrocytes in EAE lesions. In Aim 2, we will determine whether the newly identified compounds that increase Olig2 transcription activate the Sonic hedgehog-Gli pathway or the mitogen-activated protein kinase pathway and test compounds that affect different pathways for their ability to promote remyelination in EAE. In Aim 3, we will determine whether loss of Ezh2, which is a member of the Polycomb Repressor Complex responsible for methylating lysine 27 on histone H3, will promote remyelination by derepressing genes required for myelination.

DESCRIPTION (provided by applicant): Glial cells in the mammalian central nervous system (CNS) that express the NG2 proteoglycan (NG2 cells) represent a fourth major glial cell population that is distinct from mature oligodendrocytes, astrocytes, or resting ramified microglia. They differentiate into oligodendrocytes in gray and white matter and provide an endogenous source of myelinating cells during development and repair of demyelinated lesions. While all NG2 cells express platelet-derived growth factor receptor alpha and the basic helix-loop-helix transcription factor Olig2, there are some regional differences in their fate and proliferative behavior. One example is the astrogliogenic fate of NG2 cells in the dorsal and ventral forebrain. A subpopulation of NG2 cells in the embryonic ventral forebrain generates 40% of the protoplasmic astrocytes in the region, whereas the fate of NG2 cells in the dorsal forebrain is restricted to the oligodendrocyte lineage. It has been shown that NG2 cells in the ventral and dorsal forebrain arise from distinct germinal zones that are specified by distinct transcription factors. Specific Aim 1 will investigate whether the dorsoventral differences in the astrogliogenic fate of NG2 cells are established by the transcription factor code of the germinal zone of origin. Another example of NG2 cell heterogeneity is the difference in the rate of proliferation and oligodendrocyte differentiation between NG2 cells in the gray and white matter. NG2 cells in the white matter are known to undergo greater expansion and oligodendrocyte differentiation than those in the gray matter. Our recent slice culture experiments indicate that NG2 cells in the white matter proliferate to a greater extent in response to platelet-derived growth factor compared with their gray matter counterparts. Furthermore, proliferation of NG2 cells in the gray but not white matter is increased by activation of a family of potassium channels. Specific Aim 2 will investigate the mechanisms that lead to differences in proliferation and oligodendrocyte differentiation of NG2 cells in gray and white matter during development and remyelination.

DESCRIPTION (provided by applicant): Funds are requested for the purchase of a Leica TCS SP8 AOBS 405 UV Spectral Confocal Microscope to be used in the PNB Confocal Facility in the Department of Physiology and Neurobiology at the University of Connecticut, Storrs. The Department currently has a Leica TCS SP2 Spectral Confocal Microscope, which was purchased in 2001 with funds from NIH (1S10RR015684-01). The instrument is maintained as a departmental facility that is open to the entire Storrs campus community. During the past eleven years, it has been heavily used, averaging 7 hours a day, resulting in 73 peer-reviewed publications by four to eight NIH-funded major users. In 2006 Leica stopped manufacturing this model, and some parts are no longer available. There is not enough capacity on the existing confocal microscopes on the Storrs campus to accommodate the needs of the current users of the SP2 system in the event that a critical part cannot be replaced. Even if the existing instrument were to remain functional, many of the users critically need a 405 nm laser line to image the nuclear dye DAPI, along with the other fluorophores, but a 405 nm laser cannot be added to our existing SP2 unit. The other existing microscope on this campus with this capability is housed in a separate building and is too heavily used to accommodate our users' needs. The user group has grown from four NIH-funded investigators at the time of the original application in 2000 to eight major users and two minor users, and we have two new faculty members who will join our department this fall, whose research heavily depends on confocal microscopy. Confocal microscopy has now become an indispensable tool for the research program of the users in our department and a standard tool in cellular, molecular, and developmental neuroscience. Thus, there is a pressing need to acquire a new confocal system to ensure that the current users of the existing SP2 system have uninterrupted access to a functional confocal microscope and avoid delays in data collection and publication. The new confocal microscope will be housed in the center of the first floor of the Pharmacy Biology Building where the department is housed. It will be maintained by user fees and additional support from the Department, the Dean of the College of Liberal Arts and Sciences, and the Office of Vice President for Research.

Project summary ? Homeostatic regulation of NG2 cell dynamics NG2 cells represent a fourth major glial cell population in the mammalian central nervous system that is distinct from neural stem cells, mature oligodendrocytes, astrocytes, or microglia. They generate oligodendrocytes and hence are an important source of remyelinating cells. They respond robustly to changes in the density of oligodendrocyte lineage cells or myelin and restore the correct number of oligodendrocytes needed to make the correct amount of myelin in the neural network. They are highly integrated in the neural network and interact not only among the cells in the oligodendrocyte lineage but also with neurons and other glia. The proposed project will investigate the mechanism of feedback signaling that maintains homeostasis of oligodendrocytes and myelin. In Aim 1, we will test the hypothesis that microglia play a critical role in regulating NG2 cell density under normal physiological conditions, as well as during remyelination. In Aim 2, we will test the hypotheses that the Hippo signaling mediates NG2 cell density-dependent proliferation and that cell adhesion molecules and exosome signaling between myelin and NG2 cells provide feedback signaling to NG2 cells that regulates oligodendrocyte differentiation. These hypotheses are based on findings from both systems analyses and specific cellular studies. The outcome of the study will advance our knowledge by establishing fundamental new principles related to oligodendrocyte lineage cell intrinsic and extrinsic mechanisms of homeostatic regulation of oligodendrocytes and myelin in the context of other cellular constituents.

Multiple sclerosis is a chronic demyelinating disease affecting primarily people during their active years of life. Long-lasting demyelination leads to axonal degeneration with severe neurological deficits, and in most cases remyelination is limited. Remyelination failure could occur due to the inability of existing oligodendrocytes (OLs) to myelinate demyelinated axons or failure of oligodendrocyte precursor cells (OPCs) to generate myelinating OLs in the lesion. The dynamics of OLs lineage cells is intricately modulated by the local neural activity. OPCs receive synaptic and non-synaptic signals from neurons and undergo depolarization or increase intracellular Ca2+. However, exactly how OPCs sense the level of neuronal activity and initiate a signaling cascade that triggers a terminal differentiation and survival response has remained unclear. In neurons, depolarization-induced Ca2+ entry into axons triggers release of neurotransmitters from synaptic vesicles clustered at the presynaptic terminal by a series of molecular events that involve SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. OL lineage cells also express various SNARE proteins and transcripts. Our preliminary results of inactivating vesicular SNARE proteins in OPCs and their progeny revealed that SNARE- dependent mechanisms are critical for the proper generation of viable OLs. These observations led us to hypothesize that vesicular SNARE-dependent exocytosis in late OPCs is triggered by neuronal signals and has a critical autocrine function to promote OL differentiation and survival of new OLs. This will be tested by 1) fate analysis of divided OPCs that have defective vesicular SNAREs to determine whether loss of Vesicle-associated membrane protein 2 and/or 3 (VAMP2/3) function compromises OL differentiation and survival (Aim 1); 2) imaging SNARE- containing vesicles and exocytosis events in cultured OPCs and in vivo to determine whether neuronally derived signals promote SNARE-mediated exocytosis and clustering of vesicles (Aim 2); and 3) a proteomics approach to identify the autocrine signal(s) that is released from OPCs in a SNARE-dependent manner (Aim 3). The project will establish experimental evidence for a novel principle regarding the cellular mechanism by which late OPCs trigger their terminal differentiation and survival programs in response to neuronal signals.