Wendy B. Macklin, Ph.D. - US grants

Important advances in neuroscience research are currently being made through application of molecular biological techniques. Use of molecular probes is providing new insight into brain development, brain function and a number of pathological conditions. The current studies are designed to elucidate the regulation of myelination in the central nervous system through use of molecular probes. Myelination is a crucial event for normal brain development and demyelinating diseases such as multiple sclerosis seriously affect neurological function. Because of the complexity of brain development, investigation of the regulation of oligodendrocyte differentiation and myelin formation has been difficult. Isolation and utilization of molecular probes for oligodendrocyte differentiation provides a new approach to the study of CNS myelination. In the current studies, clones of the cDNAs for the myelin proteolipid and basic proteins will be isolated, sequenced and characterized. These clones and genomic clones, isolated by screening genomic libraries with the cDNA clones, will be used to assess the control of the expression of these myelin proteins. In further studies on the regulation of oligodendrocyte differentiation and myelination, we will isolate previously undescribed cDNA clones which may function in oligodendrocyte differentiation. A cDNA library will be produced which will be highly enriched in oligodendrocyte cDNAs. Selected clones from this library will be sequenced and characterized to identify previously unknown oligodendrocyte genes. The expression of the different oligodendrocyte genes will be investigated in several experimental systems, e.g., a neurological mutant mouse strain which has altered myelination and, more extensively, a primary brain cell culture system which can be experimentally manipulated to induce differentiation of oligodendrocytes and to reduce differentiation of astrocytes. Investigation of oligodendrocyte differentiation and the regulation of expression of the proteolipid and basic protein genes as well as these other oligodendrocyte genes may have great significance for future studies on remyelination in the central nervous system, with particular reference to multiple sclerosis.

oligodendroglia; cell differentiation;

The overall aim of this project is the investigation of the molecular mechanism(s) by which myelination occurs. The expression of the myelin proteolipid protein gene will be investigated in normal mice and several mouse dysmyelination mutants. A family of proteolipid mRNAs have been identified in the mouse, which range from 1400 to 4400 nucleotides in length. cDNAs specific to each of the mRNAs will be isolated and characterized to establish the relationships among these mRNAs. Selective probes will be used to screen the proteolipid protein cDNAs to identify cDNAs specific for the DM20, a myelin protein that is very closely related to the major myelin proteolipid protein. These putative DM20 cDNAs will be sequenced to identify their exact relationship to the proteolipid cDNAs. The proteolipid protein gene has been mapped to a region of the human X-chromosome that encompasses the jimpy locus in the mouse genome, and we have demonstrated that proteolipid mRNAs in the jimpy mouse are 100-200 nucleotides shorter than normal proteolipid mRNAs. The current studies will pursue the question of whether there is a structural alteration in the proteolipid protein gene that causes this size differences in the mRNAs. The site within the jimpy proteolipid protein gene that is altered will be determined. Proteolipid protein gene expression will also be studied in the jimpy msd mouse, which is an allele of the jimpy mutation, to establish the effect of this mutation on the synthesis of the family of proteolipid mRNAs, proteolipid and DM20 protein. Proteolipid protein gene expression will be investigated in two other dysmyelination mutants, shiverer and quaking. These two mutations have distinctly different effects on myelin basic protein expression in these animals. The shiverer mouse has a deletion in the myelin basic protein gene and make no myelin basic protein mRNA, while the quaking mouse has close to normal levels of myelin basic protein mRNA. Detailed analyses of the levels of the proteolipid mRNAs and proteins during development in these animals will be conducted. In order to address certain questions on the expression of these myelin genes in the mutant animals, normal and mutant mouse oligodendrocytes in mixed primary glial cell cultures will be characterized, and the expression of the four myelin basic proteins and the two myelin proteolipids (PLP and DM20) and their respective RNAs will be studied in cultured oligodendrocytes, using a battery of antibodies and cDNA probes.

DESCRIPTION (Provided by the Applicant): The long-term goal of these studies is to establish the mechanism by which mutations affect oligodendrocyte differentiation and overall brain development. The focus is on the myelin proteolipid protein (PLP) gene, which constitutes 50 percent of myelin protein. Even conservative amino acid mutations in this protein are devastating to norrnal brain development. We will test the hypothesis that these PLP mutations induce oligodendrocyte cell death by affecting intracellular translocation of the protein. In particular, we have evidence that defective PLP increases its association with the chaperone, calreticulin, which is also involved in calcium homeostasis. Up- or down regulation of calreticulin induces calcium dysregulation and increased sensitivity to apoptosis. Calcium homeostasis is dysregulated in jimpy oligodendrocytes, and we will study whether the increase in calreticulin is involved in that dysregulation and in the increased apoptosis found in mutant oligodendrocytes. In addition, we have identified a new DM20-related protein expressed in the peripheral nervous system. We will investigate what this protein is and whether it has unique functions with respect to glial cell survival and/or differentiation. These studies develop from basic investigations on the translation and translocation of the PLP protein to studying how the oligodendrocyte expressing defective PLP/DM20 protein changes its developmental pattern and eventually dies. We have generated transgenic mice that overexpress enhanced green fluorescent protein (EGFP), which can be visualized in live tissue. In the mutants, oligodendrocytes begin to myelinate axons, but there is a high level of oligodendrocyte cell death, and eventual hypomyelination. We will use these mice to visualize this process, i.e., the differentiation and interaction of mutant oligodendrocytes with axons. We will be able to assess when and where these cells start to die. We have been investigating one element in the interaction of oligodendrocytes and axons that normally enhances their survival: neuregulins. We will study whether the mutant cells can respond to neuregulins and whether the downstream survival signaling system for neuregulins, activated Akt, can enhance survival of mutant oligodendrocytes.

[unreadable] DESCRIPTION (provided by applicant): Support is requested for the next annual meeting of the American Society for Neurochemistry, the 2006 meeting to be held in Portland, Oregon from March 11-15, 2006. To accommodate the breadth of neurochemistry as well as cellular and molecular neurobiology, the ASN programs traditionally build its scientific program around four interwoven, but distinct, themes. 1) Building the Nervous System: Sessions in this theme address the neurobiology of stem cells, the generation of cell diversity, elaboration of neuronal cytoarchitecture, specificity of synapse formation and the role of growth factors during development. 2) Metabolism and Cell and Molecular Neuroscience: Sessions under this theme deal with basic mechanisms applicable in a wide range of health issues ranging form metabolism to neurotransmitter function and from cell motility to molecular cell structure. 3) Glial Mechanisms and Injury: The crucial role of non-neuronal cells in neural development and pathogenesis are explored in sessions that address basic glial biology and the role of the glia in diseases such as Multiple Sclerosis, Alzheimer's Disease, and CNS injury. 4) Neuronal Degeneration and Disease: This theme addresses studies on the molecular mechanisms of neuron development, neurodegeneration, and the contributions of neurotransmitters to disease. These themes have been selected to increase our understanding of the cellular and molecular bases of neural development and disease. They provide a framework for in depth presentation of important new studies in molecular and cellular neurobiology. Additionally, we have a day-long Pre-Meeting Workshop, which this year focuses on "Identifying Glial: Neuronal Interactions: New Approaches and New Insights". ASN has traditionally focused on involving junior investigators, and NIH support for previous ASN meetings has been invaluable for supporting the high quality ASN scientific programs and for making these programs accessible to graduate students, postdoctoral researchers, junior faculty and minorities. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The current studies focus on the signaling pathways controlling myelination. Transgenic mice that overexpress constitutively active Akt (protein kinase B) in oligodendrocytes produce increased amounts of myelin. Furthermore, there are effects on the neurons in these mice. The current studies focus on identifying the signaling pathways by which Akt regulates myelination, and the impact of Akt expression in oligodendrocytes on neurons. The hypothesis to be tested is that Akt signaling directly regulates oligodendrocyte differentiation and myelination. This will be investigated initially by studying the signaling pathways and gene expression changes that occur in these mice. Thus, the first aim will focus on the signaling pathways downstream of Akt that regulate CMS myelination. Initial studies will investigate the activation/inactivation state of a series of Akt phospho-substrates, including transcription factors, mTOR and other known Akt substrates. Our studies demonstrate that Akt overexpression in these mice induces the MAP kinase pathway. We will therefore investigate this pathway as well, focusing on Erk1/2 signaling. The main focus of this aim is on mRNAs and proteins that regulate the hypermyelination phenotype. We will investigate whether this is exclusively controlled by an inability to stop myelinating, or if there is also an impact on differentiation. Thus, we will study whether and how Akt overexpression impacts oligodendrocyte differentiation, in addition to impacting the long-term myelination process. We will investigate how Akt signaling drives CMS myelination as assessed by expression of known oligodendrocyte lineage and differentiation markers, with particular interest in the transcription factors regulating myelination. Putative major Akt substrates controlling myelination or oligodendrocyte differentiation will be confirmed by over- expressing or knocking down expression of these proteins in cultured oligodendrocytes. The second aim of the proposal focuses on the effect of elevated Akt expression and increased myelination on developing neurons. Studies focus on axonal development in optic nerve, investigating morphological maturation and organization of nodes of Ranvier. Our preliminary data demonstrate strong interactions between oligodendrocytes overexpressing Akt and developing axons. Interactions of oligodendrocytes and neurons will be studied in culture, using cells obtained from PLP-Akt-DD mice. The importance of these studies is that identifying the crucial Akt substrate(s) that regulates myelination may lead to therapeutic approaches to enhance remyelination in multiple sclerosis.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is the most devastating neurological disease in young adults, affecting more than 2.5 million people worldwide. Current therapies to treat MS and other demyelinating diseases are primarily focused on control of the immune component and inflammation in the disease. These treatments are not directed at repairing the damage in the central nervous system through remyelination. Studies are currently underway in many laboratories to identify small molecules that will enhance remyelination. Some of these studies are focused on specific identifiable pathways, while others are essentially black box screens, for which the molecular target may not be known, but the phenotypic target, myelination, can be identified. Once such lead molecules are identified, their optimization is needed. Optimization includes medicinal chemistry, but the screening of derivatives of lead compounds can be a lengthy and possibly unsuccessful process, if the in vivo impact of these compounds is not established at an early stage. The current project focuses on developing a new in vivo screen for drugs that will enhance myelination. The zebrafish model is becoming an important in vivo screen for drugs. Myelination occurs in the zebrafish over a rapid time frame, with significant myelination of the spinal cord by seven days post fertilization. Given the transparency of the embryo, fluorescent protein tags are an easy screen for increases in myelination. In order to accomplish our main goal, we will develop a new transgenic zebrafish line that will be an excellent readout of myelination per se. We will drive fluorescent protein reporter expression with the Protein zero (P0) promoter, which in the zebrafish is expressed early and strongly in oligodendrocytes and only much later in the peripheral nervous system. This will specifically mark mature oligodendrocytes as well as their myelin sheaths, allowing fluorescent protein imaging as a rapid readout of drug efficacy. Using an in vivo approach will allow us to screen out numerous drugs for toxicity or other negative impacts. We will optimize imaging techniques as complementary readouts of myelination per se, studying in vivo time lapse imaging to establish optimal time frames and regional assessment of drugs enhancing myelination.

DESCRIPTION (provided by applicant): The current studies are a multiple-P.I. proposal that focuses on the role of mTOR in oligodendrocyte development and CNS myelination. This is an important research area because of the devastating consequences of demyelination in humans and the need to understand the molecular details of how myelination and remyelination are regulated, in order to repair such damage. The Macklin laboratory has investigated the role of Akt and mTOR in CNS myelination, while the Wood laboratory has investigated the role of mTOR in oligodendrocyte differentiation and has identified an mTOR-regulated proteome in oligodendrocytes. There is inconsistency in the literature and in the preliminary data from the two laboratories as to when in the developmental program of oligodendrocytes mTOR becomes a major regulator. Thus, the current proposal is designed to answer unequivocally when and how the mTOR signaling complexes regulate oligodendrocyte development including potential actions on both differentiation and myelination. Rather than compete to address these questions, we propose a collaborative project using complementary mouse lines, and standardized reagents and techniques. The two laboratories have complementary sets of conditional mutant mice that will be used collaboratively to investigate these questions. In the first specific aim, we will investigate the mechanisms by which mTOR regulates oligodendrocyte differentiation, testing the hypothesis that mTOR directly regulates oligodendrocyte differentiation via specific actions of both mTORC1 and mTORC2. This will be investigated by studying the signaling pathways and the differentiation events that are modulated in mTOR, raptor or rictor conditionally-deleted mice. In the second specific aim, we will investigate the mechanisms by which mTOR regulates CNS myelination and myelin maintenance. We will test the hypothesis that mTOR directly regulates myelination via both mTORC1 and mTORC2, with differential control by each complex. Studies will additionally investigate how active myelination shifts to myelin maintenance in the CNS. In the third specific aim, we will investigate the upstream regulation of the two mTOR complexes by TSC1/2 in developing oligodendrocytes. These studies will test the hypothesis that TSC signaling regulates oligodendrocyte differentiation and CNS myelination through upstream inhibition of mTORC1 and activation of mTORC2. TSC1/2 are considered to be negative regulators of mTOR signaling, yet in some contexts loss of TSC activity induces hypomyelination rather than the expected hypermyelination. Establishing how they impact mTOR signaling in the oligodendrocyte is therefore important. In the final aim, we will determine the mechanisms by which the mTOR pathway regulates remyelination. The crucial questions in this aim will be whether the role of this pathway in the regulation of oligodendrocyte differentiation and myelination recapitulates its function during development, or whether there are unique elements of mTOR regulation of remyelination in adult tissue. This aim clearly has significant impact on our understanding of remyelination in multiple sclerosis. PUBLIC HEALTH RELEVANCE: Myelination is essential for normal nervous system development, and demyelination in the adult causes serious diseases such as multiple sclerosis. The current proposed studies address the intracellular signaling pathways that regulate central nervous system myelination during oligodendrocyte development, and following a demyelinating event in the adult.

Central nervous system (CNS) myelin is produced by oligodendrocytes, which are glial cells that arise from oligodendrocyte progenitor cells (OPCs). By published transcriptome analysis, the orphan nuclear receptor Nur77 (nr4a1 gene) is highly expressed by OPCs, and downregulated as newly formed oligodendrocytes become myelinating oligodendrocytes. Nur77 is a nuclear receptor, and it also has nontranscriptional roles in cell growth and survival, which are largely determined by its subcellular localization. Phosphorylation or protein interactions such as with other nuclear receptors may control the localization and thus the function of the receptor. Nur77 is thought to bind directly to DNA as monomers or dimers, but can also bind with other nuclear receptors, including retinoid X receptors (RXRs), which are known modulators of OPC differentiation and may be critical transcriptional regulators of myelination. It has also been shown that Akt and ERK2, which are master regulators of myelination, phosphorylate the receptor. The current proposal will establish 1) the role of Nur77 in oligodendrocytes, and 2) which pathways/binding partners regulate Nur77 function in oligodendrocytes. The first specific aim will test the hypothesis that Nur77 downregulation is necessary for proper differentiation of OPCs to myelinating oligodendrocytes. First, we will characterize the expression profile of Nur77 in oligodendrocyte lineage cells during differentiation in primary rat OPC cultures and during myelination in embryonic zebrafish. Next, using pharmacology, over/under expression studies and CrispR/Cas-9 mediated genomic editing, we will test the role of Nur77 as a regulator of OPC maturation/differentiation, using oligodendrocyte-specific transgenic reporter fish and live imaging. In the second specific aim, we will test the hypothesis that Akt and/or ERK1/2 signaling increases Nur77 phosphorylation, to promote Nur77 interaction with RXRs and nuclear localization of Nur77/RXRs heterodimers during OL lineage progression. Here, we will use zebrafish and rodent cell culture models to test 1) if AKT and/or ERK1/2 signaling control Nur77 phosphorylation and localization during OPC differentiation; and 2) whether formation of heterodimers with RXRs mediates the impact of Nur77 on OPCs and myelination.

Project Summary Myelination is essential for normal nervous system development, and the juvenile period is a major period of myelination. Differentiation of the oligodendrocyte, the myelinating cell of the CNS, is tightly regulated, and disruption of oligodendrocyte development leads to neurologic problems, such as periventricular leukomalacia and retardation. How injury during this period impacts myelination is poorly understood, although ischemic damage in the perinatal period or adults damages oligodendrocytes along with other cells, driving major tissue damage. We initiated studies to investigate the impact of juvenile ischemia on CNS myelination, as this period has distinctly different metabolic demands. We found that despite extensive death of striatal neurons, oligodendrocytes and myelinated axons were remarkably preserved in the juvenile brain, in stark contrast to the adult which exhibits extensive oligodendrocyte and myelin injury (Ahrendsen et al., Glia 64:1972, 2016). Quite intriguingly, in addition to oligodendrocyte resistance to ischemic injury in the actively myelinating juvenile brain, a transgenic mouse line that continues active myelination throughout life also has relatively protected oligodendrocytes. In the proposed studies, we will test the hypotheses that actively myelinating oligodendrocytes have high levels of anti-oxidant pathway molecules that protect them from ischemic damage, and that such damage increases their expression of insulin-like growth factor (IGF-1), which acts in an autocrine m manner to additionally protect myelinating oligodendrocytes. We have three related specific aims that test these hypotheses by investigating 1) the role of the Nrf2 signaling pathway in actively myelinating oligodendrocytes in response to ischemia; 2) the role of IGF-1 in protection of actively myelinating oligodendrocytes; and 3) the role of IGF-1 signaling in conjunction with reactive oxygen species in driving oligodendrocyte progenitor cell differentiation.

PROJECT SUMMARY Enhancing remyelination is a critical strategy for restoring brain function after demyelination in multiple sclerosis (MS) patients; however, despite concerted efforts, the ability to stimulate remyelination in MS brain has remained elusive. While signaling pathways that promote oligodendrocyte precursor differentiation have been identified, the experimental milieux under investigation do not replicate the mechanisms limiting remyelination following MS-specific inflammatory CNS injuries. The current proposal builds on our new models of demyelination/remyelination using pathogenic recombinant antibodies (rAbs) generated from MS patients. Myelin-specific MS rAbs direct complement-mediated demyelination in vivo and ex vivo, all of which spontaneously repair in association with microglial activation. However, demyelinated explants that are continuously exposed to myelin-specific MS rAb fail to activate microglia, and oligodendrocyte maturation is inhibited. Similarly, targeted depletion of microglia following rAb-mediated demyelination blocks oligodendrocyte maturation preventing active remyelination. Using single cell RNASeq (scRNASeq) on microglia isolated from remyelinating explants, we identified transcriptionally distinct microglial subsets that are associated with successful or failed remyelination. Hence, we hypothesize that microglial signals are critical for oligodendrocyte responses during the transition from early myelinating to actively myelinating oligodendrocyte, and myelin-specific MS autoantibody modulates these signals to arrest remyelination. To test our hypothesis, we propose three complementary specific aims. In Aim 1, we will evaluate microglial and oligodendrocyte responses in in vivo models of MS rAb-mediated demyelination and compare those responses to those seen in toxin-mediated demyelination. Intrathalamic or corpus callosum injection of myelin-specific MS rAb plus HC will be performed in conjunction with pharmacologic microglial depletion and chronic administration of MS rAb to validate the impact of microglial responses on remyelination in the intact nervous system. Comparable studies will be done following lysolecithin-induced demyelination, which has a very different time course of microglial activation and remyelination. In Aim 2, we will study the dynamics of demyelination, microglial responses and oligodendrocyte regeneration in situ using intravital imaging following cortical demyelination. This real-time analysis of myelin loss, microglial activation and remyelination will be compared to that seen following cuprizone-mediated demyelination. Finally, in Aim 3, we will investigate the mechanisms by which microglia impact remyelination using ex vivo cerebellar slices demyelinated with myelin- specific rAb plus human complement (HC). We will focus on investigating the role of several microglial genes identified by scRNASeq that are expected to promote or impair remyelination. Normal appearing white matter and MS lesion tissue with varying degrees of demyelination and remyelination will be evaluated to determine the abundance and localization of functionally-important microglial subsets. The results of these studies will provide insights into novel mechanisms controlling remyelination after inflammatory injury. In addition, the knowledge gained may identify novel therapeutic approaches that will result in clinically-meaningful myelin repair.