Brian Popko - US grants

Myelination is a critical developmental process that, when altered, results in severe neurological dysfunction. The overall goal of our research is directed toward obtaining a better understanding of the role myelin proteins play in the myelination of the central nervous system (CNS). Additionally, the function of myelin proteins in the differentiation of oligodendrocytes, the myelinating cell of the CNS, will be examined. The gene encoding the myelin basic protein (MBP) will be analyzed in the dysmyelinating murine mutant myelin deficient (mld). The mld MBP gene is organized as a tandem duplication with the upstream gene containing an inversion of its 3' region. The DNA sequence surrounding the breakpoints of the inversion/duplication will be determined in an effort to identify regions associated with the recombinational events responsible for the gene rearrangement. The mld MBP gene is expressed at decreased levels and on an abnormal developmental schedule. Several in vitro and in vivo approaches will be taken to determine the basis of this altered MBP expression. The dysmyelinating murine mutation jimpy results in proteolipid (PLP) and DM-20 protein deficiencies, CNS hypomyelination, and oligodendrocyte death. Transgenic jimpy animals will be generated that express either only PLP or DM-20 in an attempt to determine the effect these proteins have on myelination and oligodendrocyte survival in these animals. Efforts will also be made to inactivate the myelin protein genes in transgenic mice. Animals will be generated that contain a vast molar excess of the cis regions involved in regulating myelin protein gene expression. By functionally depleting oligodendrocytes of the myelin protein genes trans-activator proteins, inhibition of the transcriptional activity of these genes should occur.

The long-term goal of the research in my laboratory is to gain a molecular understanding of the role glial cells (specifically Schwann cells and oligodendrocytes) play in the development and function of the nervous system. Our principal approach is to use techniques that allow for the manipulation of the mouse genome. This approach has allowed us to generate a mouse model of human oligodendrogliomas - which in turn has been useful for the establishment of an oligodendroglial cell line. Moreover, we have developed a transgenic line of animals that experience a severe, late-onset neuromuscular disorder that is likely the result of abnormal Schwann - cell axon interactions. In addition to our transgenic experiments, we have recently initiated studies using the polymerase chain reaction in an effort to isolate genes encoding members of protein families that likely play a role in glial cell function.

Among the most abundant components of myelin are the galactolipids galactocerebroside (Ga1C) and sulfatide. In spite of this abundance, the roles that these molecules play in the myelin sheath are not well understood. Until recently, our concept of Ga1C and sulfatide functions had been principally defined by immunological and chemical perturbation studies that implicate these lipids in oligodendrocyte differentiation, myelin formation, and myelin stability. Recently, however, genetic studies have allowed us to re-analyze the functions of these lipids. We have cloned the gene encoding the enzyme UDP- galactose: ceramide galactosyltransferase (CGT), which is required for myelin galactolipid synthesis, and we have generated mice that are incapable of synthesizing either Ga1C or sulfatide by inactivating the CGT gene in the mouse germline. These galactolipid-deficient animals exhibit a severe tremor, hindlimb paralysis, and electrophysiological deficits. In addition, ultrastructural studies have revealed hypomyelinated white matter tracts with unstable myelin sheaths and a variety of myelin irregularities. Longitudinal EM analysis has exposed a number of profound abnormalities at the node of Ranvier, including increased nodal length, reversed lateral loops, and compromised axo-oligodendrocytic junctions. Collectively, these observations indicate that cell-cell interactions, which are essential in the formation and maintenance of a properly functioning myelin sheath, are compromised in the galactolipid-deficient mice. The studies described in this proposal are designed to further explore the function of the myelin galactolipids, using the CGT mutant animals as a key resource. We will determine the degree to which glucocerebroside, a lipid that atypically accumulates in the CGT mutant's myelin, compensates for the loss of the galactolipids. We will further explore the role that the galactolipids play in myelinating cell development and in the early stages of the myelination process. The function of the galactolipids in node of Ranvier formation will also be examined in detail. Furthermore, the contribution that these molecules make to the stability of the myelin sheath will be studied. Together, this work will significantly increase our understanding of the function of the galactolipids in myelinating cell development, the myelination process, and in the stability of the myelin sheath.

The studies presented in this proposal are designed to provide molecular reagents to assist in the classification of human glial tumors. Astrocytes and oligodendrocytes are the predominant glial cells of the central nervous system and are believed to represent the transformed cells of origin in most gliomas. The classification of these tumors is currently based solely on histological criteria. Our preliminary studies indicate that the RNA transcripts that encode the proteins of myelin should serve as sensitive markers of tumors of oligodendrocyte origin. Moreover, these studies also suggest that a subset of human glial tumors currently believed to be of astrocytic origin may actually be of oligodendroglial origin. We speculate, based on our studies with a transformed oligodendrocyte cell line, that the phenotypic properties of CNS glial tumors are influenced by the environment in which these tumors reside. The environment of these tumors is altered due to the disruption of the blood-brain barrier and the infiltration of immune cells, which secrete a multitude of cytokines. We propose to correlate the molecular and histological phenotype of human CNS gliomas with the degree of lymphocyte infiltration into these tumors and the local concentration of various cytokines. In turn, these results will be correlated with the clinical outcome of the patients. Together, these studies should provide us with molecular reagents that should help in the classification of human gliomas such that the clinical course of afflicted individuals may be more accurately predicted and treatment protocols designed accordingly.

DESCRIPTION (From the Applicant's Abstract): In the human demyelinating disorder multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE), there is a breakdown of the blood-brain barrier and an infiltration of immune cells into the CNS. Infiltrating T lymphocytes and macrophages are believed to be key mediators of the disease process. Considerable circumstantial and experimental evidence has suggested that the pleiotropic cytokine interferon gamma (IFN-gamma), which is exclusively expressed by T cells and natural killer cells, is a deleterious component of the immune response in these disorders. We have shown that when ectopically expressed in the CNS of transgenic animals IFN-y promotes many of the pathological changes that occur in immune-mediated demyelinating disorders. The harmful actions of IFN-gamma on CNS myelin are likely mediated, at least in part, through direct effects on the myelinating cells. In support of this hypothesis we have shown that this cytokine elicits a number of effects on oligodendrocytes in vitro, including their apoptotic cell death. We have shown also generated in vivo data that suggests that one detrimental effect of IFN-gamma involves overloading the endoplasmic reticulum (ER) of oligodendrocytes through the induction of major histocompatibility complex (MHC) class I heavy chain gene expression. In the current application we plan to build on these studies to further explore the role that this cytokine plays in immune-mediated demyelinating disorders. We will establish second generation transgenic animals, using the tetracycline-induction system that will allow us to control IFN-gamma delivery into the CNS. Using these animals we will examine the effect of the presence of IFN-gamma on the remyelination process exploiting the cuprizone model of demyelination. We will also examine whether members of a newly discovered family of proteins, termed suppressors of cytokine signaling (SOCS), are able to protect oligodendrocytes from the deleterious actions of IFN-gamma. Moreover, we will examine biochemically and genetically the possibility that the primary effect of IFN-gamma on oligodendrocytes is mediated through the induction of ER stress. Together, these studies will significantly further our understanding of the cellular and molecular effects of immune cell infiltration into the CNS. Such information is critical to the rationale design of therapeutic strategies.

Among the most abundant components of myelin are the galactolipids galactocerebroside (Ga1C) and sulfatide. In spite of this abundance, the roles that these molecules play in the myelin sheath are not well understood. Until recently, our concept of Ga1C and sulfatide functions had been principally defined by immunological and chemical perturbation studies that implicate these lipids in oligodendrocyte differentiation, myelin formation, and myelin stability. Recently, however, genetic studies have allowed us to re-analyze the functions of these lipids. We have cloned the gene encoding the enzyme UDP- galactose: ceramide galactosyltransferase (CGT), which is required for myelin galactolipid synthesis, and we have generated mice that are incapable of synthesizing either Ga1C or sulfatide by inactivating the CGT gene in the mouse germline. These galactolipid-deficient animals exhibit a severe tremor, hindlimb paralysis, and electrophysiological deficits. In addition, ultrastructural studies have revealed hypomyelinated white matter tracts with unstable myelin sheaths and a variety of myelin irregularities. Longitudinal EM analysis has exposed a number of profound abnormalities at the node of Ranvier, including increased nodal length, reversed lateral loops, and compromised axo-oligodendrocytic junctions. Collectively, these observations indicate that cell-cell interactions, which are essential in the formation and maintenance of a properly functioning myelin sheath, are compromised in the galactolipid-deficient mice. The studies described in this proposal are designed to further explore the function of the myelin galactolipids, using the CGT mutant animals as a key resource. We will determine the degree to which glucocerebroside, a lipid that atypically accumulates in the CGT mutant's myelin, compensates for the loss of the galactolipids. We will further explore the role that the galactolipids play in myelinating cell development and in the early stages of the myelination process. The function of the galactolipids in node of Ranvier formation will also be examined in detail. Furthermore, the contribution that these molecules make to the stability of the myelin sheath will be studied. Together, this work will significantly increase our understanding of the function of the galactolipids in myelinating cell development, the myelination process, and in the stability of the myelin sheath.

DESCRIPTION (provided by applicant): Oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) are responsible for producing the multilayered myelin sheath that surrounds the majority of nerve axons. The myelin sheath promotes rapid, saltatory nerve conduction and is essential for proper nervous system function in higher vertebrates. The process by which axons are ensheathed with the multilayered myelin membrane is exceptionally complex. A number of molecules have been shown to be essential for the formation of the myelin structure. For example, there are myelin-specific proteins and lipids that appear to play critical roles in myelin formation and maintenance. Additionally, the interactions of myelinating cells with the extracellular environment and with their target axons are critical for proper myelin sheath function. The underlying hypothesis of this proposal is that glycosphingolipids represent an important class of molecules in myelinating cell function. We believe that glycosylated lipids facilitate many key steps in the formation and maintenance of the myelin sheath, including mediating the interactions of myelinating cells with axons as well as with their extracellular environment. Until recently, the glycosphingolids have largely been characterized using biochemical techniques. The focus of the proposed studies is to use genetic approaches to better understand the role that these molecules specifically play in the myelination process. We will examine the myelinating cell function of enzymes that transfer either glucose (UDP-glucose:ceramide glucosyltransferase) or galactose (UDP-galactose: ceremide galactosyltransferase) to ceramide. Glucosylceramide serves as the backbone for the majority of the gangliosides (sulfated glycolipids), and galactocerebroside, and its sulfated derivative sulfatide, represent the most abundant glycolipid components of the myelin sheath. In myelinating cells gangliosides and the galactolipids have been shown to be residents of lipid rafts, along with a class of cell adhesion proteins that are attached to the plasma membrane through a glycan linkage, (glycophosphatidylinositol [GPI]-linked proteins). We will examine whether the function of the glycolipids, at least in part, is mediated through the transport of the GPI-linked proteins by these raft domains. Together the studies described in this proposal should provide us with a better appreciation of the importance of glycosphingolipids in the complex biological processes of myelinating cells

6. Project Summary/Abstract The myelination of CNS axons during development and the remyelination of demyelinated axons in adults require oligodendrocyte progenitor cells (OPCs) to migrate to their target axons where they mature into myelinating cells. Although a number of critical factors have been identified for these processes, our understanding of the molecular control of CNS myelination and remyelination remains incomplete. We have identified a zinc finger protein (Zfp191) that when mutated in mice results in the absence of CNS myelin despite the presence of normal numbers of mature, process-extending oligodendrocytes. Zfp191 mouse mutants express an array of myelin-related genes at significantly reduced levels, suggesting that this protein participates in the control of the CNS myelination program. Zfp191 belongs to a family of nuclear proteins whose members contain both DNA binding zinc finger domains and SCAN domains, which are responsible for protein-protein interactions. The goal of this proposal is to gain a better understanding of the role that Zfp191 plays in the myelination process. Zfp191 is expressed in all tissues and cell-types examined, including astrocytes and neurons, and the level of Zfp191 mRNA does not change as OPCs differentiate into mature, myelinating oligodendrocytes. Thus, a critical question that we will address in the studies outlined in this proposal is whether Zfp191 has a cell autonomous function in oligodendrocytes or whether other cell types contribute to the myelin abnormalities displayed by the Zfp191 mutants. Moreover, we will determine if the continued expression of this protein is required for the maintenance of the myelin sheath, and we will also assess if this protein has a similar essential function in the remyelination process. We will also explore the molecular mechanism by which ZFP191 controls the myelination program by determining its DNA and protein binding potential. Relevance: The studies described in this proposal will focus on the molecular control of the final stages of oligodendrocyte maturation, which result in the initiation of the myelination program. A better understanding of the factors that enhance oligodendrocyte maturation is essential in our effort to develop strategies to promote axonal remyelination in demyelinating neurological disorders (e.g. multiple scelrosis).

DESCRIPTION (provided by applicant): Currently, the diagnosis and monitoring of multiple sclerosis (MS) are based on clinical evaluation aided by MRI3. Although MRI offers great spatial resolution, the signal on an MRI is non-specific and it can be difficult to interpret. In compariso, PET imaging is much more sensitive and specific4. It would therefore be ideal to have a PET tracer for MS to complement MRI. At present, there are very few PET markers under investigation for MS5-7 and none in the clinic. 4-aminopyridine (Ampyra(r), 4-AP) is a recently approved drug for MS that is believed to bind to newly exposed K+ channels in demyelinated lesions8. We have evidence that there is a higher uptake of 4-AP in demyelinated white matter areas than in normally myelinated areas suggesting that a PET-active derivative of 4-AP could serve as a PET tracer for demyelination. We also have evidence that two fluorinated analogs of 4-AP that we designed have very similar biological properties as 4-AP suggesting that, once labeled with fluorine-18, these molecules could be excellent PET tracers for demyelination. In this project we propose to generate these molecules and test if they can be used to trace demyelination in animal models of MS non-invasively. If, as we predict, these tracers effectively localize to demyelinated axons, it would provide clinicians with an unprecedented method to image the key pathologic event responsible for MS symptoms. In addition, our data shows that these fluorinated derivatives have similar affinity to Kv1 channels and possess greater brain permeability and metabolic stability suggesting that these molecules may be superior therapeutics to 4-AP (safer or more effective), which currently only benefits about one third of MS patients. Therefore in the first part of this project we propose to compare the beneficial effects of these drugs on the neurological function of mouse models of demyelination. If successful, these drugs could help restore neurological function in a greater number of people with MS.

? DESCRIPTION (provided by applicant): Advanced medical interventions have resulted in greatly increased survival of severe preterm infants. Unfortunately, diffuse white matter injury (DWMI) is a frequent complication in these children resulting in chronic neurological disability. There are no effective therapies for the prevention or treatment of DWMI. Selective damage to oligodendrocyte progenitor cells (OPCs) and the resultant impaired myelinogenesis are central to DWMI. Strategies that would increase the survival and function of oligodendrocyte lineage cells would likely be of clinical benefit. DWMI is initiated by hypoxia, ischemia, and infection tht lead to oxidative stress, glutamate toxicity, and inflammation, which appear to be selectively toxic to OPCs. The integrated stress response (ISR) is a conserved stress-induced signaling pathway that is activated by, and provides protection to, a variety of cytotoxic insults. ISR signaling leads to the phosphorylation of the a subunit of eukaryotic translation initiation factor2 (eIF2a), resulting in inhibition of global protein synthesis and the selective expression of cytoprotective genes. The focus of this application is to investigate the ISR's ability to protect OPCs from stresses associated with premature birth using in vitro and in vivo model systems. Our hypothesis is that the enhancement of ISR activity in oligodendroglial lineage cells could provide therapeutic benefit to severe preterm infants, resulting in the amelioration of DWMI. In the proposed studies we will determine whether DWMI-associated cytotoxic insults activate the ISR in oligodendroglial lineage cells and whether the manipulation of the ISR modifies the response of these cells to the cytotoxic insults. In aim 1 we will examine the response of OPCs in vitro to either oxygen-glucose deprivation (OGD) that models hypoxia- ischemia or in vitro intermittent hypoxia, both of which are associated with DWMI induction. These studies will include OPCs isolated from mouse mutants with genetic perturbations that either diminish or enhance the cell's ISR response. Survival and maturation of the OPCs will be quantitated. In aim 2 we will use two mouse models of DWMI: an intermittent hypoxia model and an inflammation model, both of which result in delayed CNS myelination of the CNS. The role that the ISR plays in the oligodendroglial cells response in these models will be examined using the ISR mutants: OPC and oligodendrocyte numbers will be examined, as well as myelination and behavioral parameters. In aim 3 we will determine if the drug guanabenz, which has been show to enhance/prolong the ISR response, will provide protection to OPCs in the in vitro and in vivo models described in the first two aims. These proof of principle drug studies will test the hypothesis that the pharmacological enhancement of the ISR will have therapeutic benefit for DWMI. In total, these studies will provide us with considerable insight into the role that the ISR plays in the response of oligodendroglial cells to the cytotoxic insults that are believed to be critical to the induction of DWMI. Importantly, the work has the potential to provide the foundation for a novel therapeutic approach for this devastating neurological disorder.

Abstract Myelin is essential for normal nervous system function in higher vertebrates, and recent data suggest that myelin remodeling is critical for motor learning. Moreover, CNS myelin sheath and the oligodendrocytes responsible for its synthesis are the targets of a number of neurological conditions, including genetic (e.g. leukodystrophies) and acquired (e.g. multiple sclerosis) disorders. Therefore, it is critically important that we gain a complete understanding of the pathways and mechanisms that regulate oligodendrocyte development and myelin formation. Here, we propose to explore the epigenetic regulation of oligodendrocyte development, function and response to environmental changes. Chromatin remodeling by histone deacetylases, DNA methylation and gene silencing by non-coding RNAs are epigenetic mechanisms that have already been shown to play a critical role in CNS myelination. In the studies described here the role that the reversible methylation of RNA plays in oligodendrocyte lineage cells will be examined. Recently, N6-methyladenosine (m6A) was shown to be the first example of reversible RNA methylation. Protein ?writers?, ?erasers? and ?readers? of this RNA mark have been discovered, strongly suggesting that these dynamic RNA modifications play a regulatory role. Readers have been shown to influence the stability, translation, splicing and intracellular localization of m6A-containing mRNA, such that this modification is ideally positioned to rapidly fine-tune gene expression. We propose to take a genetic approach to determine if RNA methylation influences oligodendrocyte lineage cell development and function. A multiprotein complex catalyzes the m6A methylation of eukaryotic mRNA. Methyltransferase like (METTL) 3 and 14, which form a heterodimer in the m6A writer, have been shown to be the enzymatic components of this complex, with the genetic inhibition of either resulting in a substantial reduction of m6A-containing mRNA. Although Mettl14 null mice display embryonic lethality, we have mice that carry a floxed allele of the Mettl14 gene that we will use in these studies. The Mettl14 conditional mutant mice will be used in combination with a number of distinct Cre driver lines to test the hypothesis that reversible RNA methylation plays a crucial regulatory role in oligodendrocyte development and function. In addition, the methylated RNA transcripts expressed by oligodendrocyte lineage cells will be profiled using an m6A-containing RNA pull-down approach in combination with RNA-sequencing. The degree to which the m6A marks alters the stability, splicing, translation and intracellular transport of specific mRNAs in oligodendrocytes will also be determined. Moreover, the Mettl14 gene will be inactivated in oligodendrocyte lineage cells in adult mice to examine the requirement of methylated RNA in the maintenance of oligodendrocyte function, as well as the response of these cells to demyelination and inflammation. These animals will also allow us to begin to explore the potential role that reversible RNA methylation plays in motor learning. Together, the studies described in this proposal will provide considerable insight into the function of m6A RNA methylation in oligodendrocyte lineage cells.