Celia W. Campagnoni

The major myelin protein genes are expressed in the human thymus

Demyelinating diseases, such as multiple sclerosis (MS) in man or experimental allergic encephalomyelitis (EAE) in rodents, may include an associated immune response directed against myelin protein antigens such as the proteolipid protein (PLP) and the myelin basic protein (MBP). Development of an immune response has been attributed, in part, to the sequestration of central nervous system antigens behind the blood‐brain barrier. Recently, we identified a nova gene, the golli gene, which overlaps the mbp gene. The Golli transcription unit produces a family of mRNAs, and their corresponding proteins possess MBP epitopes known to be encephalitogenic in EAE. Transcription of the golli gene was detected in immune system tissue. Therefore, we wished to determine whether genes that encode the two major myelin protein components, PLP and MBP, were expressed in the human thymus. Our data demonstrate that both the plp and golli genes are transcribed in the fetal human thymus. Moreover, both the PLP and DM‐20 transcripts are produced from the plp gene, and the HOG 7 and HOG 5 transcripts are produced from the golli gene. Confocal fluorescent immunohistochemistry using antibodies for the PLP/DM‐20 and Golli proteins, co‐localized expression of these antigens to thymic macrophages. Thus, the plp and golli genes are expressed, and their corresponding protein produced, in an antigen presenting cell in the human immune system.

Visualization of corticofugal projections during early cortical development in a τ-GFP-transgenic mouse

The first postmitotic neurons in the developing neocortex establish the preplate layer. These early‐born neurons have a significant influence on the circuitry of the developing cortex. However, the exact timing and trajectory of their projections, between cortical hemispheres and intra‐ and extra‐cortical regions, remain unresolved. Here, we describe the creation of a transgenic mouse using a 1.3 kb golli promoter element of the myelin basic protein gene to target expression of a τ–green fluorescent protein (GFP) fusion protein in the cell bodies and processes of pioneer cortical neurons. During embryonic and early neonatal development, the timing and patterning of process extension from these neurons was examined. Analysis of τ‐GFP fluorescent fibers revealed that progression of early labeled projections was interrupted unexpectedly by transient pauses at the corticostriatal and telencephalic–diencephalic boundaries before invading the thalamus just prior to birth. After birth the pioneering projections differentially invaded the thalamus, excluding some nuclei, e.g. medial and lateral geniculate, until postnatal days 10–14. Early labeled projections were also found to cross to the contralateral hemisphere as well as to the superior colliculus. These results indicate that early corticothalamic projections appear to pause before invading specific subcortical regions during development, that there is developmental regulation of innervation of individual thalamic nuclei, and that these early‐generated neurons also establish early projections to commissural and subcortical targets.

Embryonic Expression of the Myelin Basic Protein Gene: Identification of a Promoter Region That Targets Transgene Expression to Pioneer Neurons

The myelin basic protein (MBP) gene produces two families of structurally related proteins from three different promoters—the golli products, generated from the most upstream promoter, and the MBPs, produced from the two downstream promoters. In this report we describe the expression of golli proteins within some of the earliest neuronal populations of the brain, including Cajal–Retzius cells and preplate neurons of the forebrain, representing a new marker for these cells. To identify elements responsible for neuronal expression of the golli products, we generated transgenic animals from constructs containing different portions of the upstream promoter. A construct containing 1.1 kb immediately upstream of the golli transcription start site targeted expression of β-galactosidase to preplate neurons and a subset of Cajal–Retzius cells in transgenic mice—the first reported genetic element to target expression to these pioneer cortical populations. Although expression in Cajal–Retzius cells declined with embryonic development, preplate cells continued to express the transgene after arriving at their final destination in the subplate. Interestingly, expression persisted in subplate neurons found within a distinct layer between the white matter and cortical layer VI well into postnatal life. Birth dating studies with bromodeoxyuridine indicated that these neurons were born between E10.5 and E12.5. Thus, the transgene marked subplate neurons from their birth, providing a fate marker for these cells. This work suggests a role for the MBP gene in the early developing brain long before myelination and especially in the pioneer cortical neurons important in the formation of the cortical layers.

Golli Myelin Basic Proteins Regulate Oligodendroglial Progenitor Cell Migration through Voltage-Gated Ca2+ Influx

Migration of oligodendrocyte progenitor cells (OPCs) from proliferative zones to their final location in the brain is an essential step in nervous system development. Golli proteins, products of the myelin basic protein gene, can modulate voltage-gated Ca2+ uptake in OPCs during process extension and retraction. Given the importance of process extension/retraction on movement, the consequences of golli expression on OPC migration were examined in vivo and in vitro using time-lapse imaging of isolated OPCs and acute brain slice preparations from golli KO and golli J37 overexpressing mice (JOE). The results indicated that golli stimulated migration, and this enhanced motility was associated with increases in the activity of voltage operated Ca2+ channels (VOCCs). Activation of VOCCs by high K+ resulted in a significant increase in the migration speed of JOE OPCs versus control cells and golli-mediated modulation of OPC migration disappeared in the presence of VOCC antagonists. During migration, OPCs generated Ca2+ oscillations that were dependent on voltage-calcium influx and both the amplitude and frequency of these Ca2+ transients correlated positively with the rate of cell movement under a variety of pharmacological treatments. The Ca2+ transient amplitude and the rate of cell movement were significantly lower in KO cells and significantly higher in JOE cells suggesting that the presence of golli promotes OPC migration by increasing the size of voltage-mediated Ca2+ oscillations. These data define a new molecule that regulates Ca2+ homeostasis in OPCs, and are the first to demonstrate that voltage-gated Ca2+ channels can regulate an OPC function, such as migration.

EXPRESSION OF THE MYELIN BASIC PROTEIN GENE LOCUS IN NEURONS AND OLIGODENDROCYTES IN THE HUMAN FETAL CENTRAL NERVOUS SYSTEM

The myelin basic protein (MBP) gene locus is composed of two overlapping transcription units that share all of the MBP exons. One of these transcription units expresses the MBPs and the other expresses a family of proteins structurally related to the MBPs. This second transcription unit is called the Golli gene, and the entire complex is called the Golli‐mbp gene. In this study, the expression of the Golli gene was examined in the human fetal central nervous system (CNS). By using reverse transcriptase‐polymerase chain reaction cloning we have identified eight new members of the Golli gene family of transcripts expressed in the human CNS. Golli gene expression was examined by in situ hybridization and immunohistochemistry, and surprisingly, Golli products were found to be expressed in neurons as well as oligodendrocytes. Furthermore, the subcellular distribution of Golli immunoreactivity in fetal spinal cord interneurons shifted between the various laminae. Golli protein was localized within the nuclei of interneurons in the posterior horn, but was found in the cell bodies and processes of interneurons in the anterior horn. Within oligodendrocytes, Golli protein was detected in the cell bodies and processes, including processes which were wrapping axonal segments. Golli mRNA expression was also observed in neurons within the cerebral cortex between 18 and 20 weeks postconception, prior to myelination of this brain region. During this period, there was a striking developmental increase in the numbers and in the locations of neurons expressing Golli mRNAs within the cortical plate. The diverse distribution of Golli proteins within neurons and oligodendrocytes indicates that their function is quite different from that of the MBPs to which they are closely related.

Increased Expression of Golli Myelin Basic Proteins Enhances Calcium Influx into Oligodendroglial Cells

The myelin basic protein (MBP) gene encodes two families of proteins: the classic MBP constituents of myelin and the golli-MBPs, the function of which is less well understood. Previous work suggests that golli proteins may play a role in Ca2+ homeostasis in oligodendrocytes (OLs) and in T-cells. Overexpression of golli in OL cell lines induces elaboration of sheets and processes. Live imaging of these cells revealed a rapid retraction of the processes and sheets after depolarization with high K+. This phenomenon was associated with a significant increase in [Ca2+]int without changes in cell viability. The results indicated that golli produced its effect through Ca2+ influx, rather than Ca2+ release from intracellular stores. Furthermore, a specific [Ca2+]int chelator (BAPTA) or Cd2+, a specific blocker of voltage-operated Ca2+ channels, abolished the ability of golli to promote process extension in a dose-dependent manner. Analysis of the golli protein identified a myristoylation site at the C terminus of the golli domain, which was essential for the action of golli on Ca2+ influx, suggesting that binding of golli to the plasma membrane is important for modulating Ca2+ homeostasis. High-resolution spatiotemporal analysis along N19 processes revealed higher-amplitude local Ca2+ influx in regions with elevated levels of golli. These findings suggest a key role for golli proteins in regulating voltage-gated Ca2+ channels in OLs during process remodeling. Our observations are consistent with the hypothesis that golli proteins, as a part of a protein complex, modulate Ca2+ influx at the plasma membrane and along OL processes.

Region-Specific Myelin Pathology in Mice Lacking the Golli Products of the Myelin Basic Protein Gene

The myelin basic protein (MBP) gene encodes two families of proteins, the classic MBP constituents of myelin and the golli-MBPs, the function of which is less well understood. In this study, targeted ablation of the golli-MBPs, but not the classic MBPs, resulted in a distinct phenotype unlike that of knock-outs (KOs) of the classic MBPs or other myelin proteins. Although the golli KO animals did not display an overt dysmyelinating phenotype, they did exhibit delayed and/or hypomyelination in selected areas of the brain, such as the visual cortex and the optic nerve, as determined by Northern and Western blots and immunohistochemical analysis with myelin protein markers. Hypomyelination in some areas, such as the visual cortex, persisted into adulthood. Ultrastructural analysis of the KOs confirmed both the delay and hypomyelination and revealed abnormalities in myelin structure and in some oligodendrocytes. Abnormal visual-evoked potentials indicated that the hypomyelination in the visual cortex had functional consequences in the golli KO brain. Evidence that the abnormal myelination in these animals was a consequence of intrinsic problems with the oligodendrocyte was indicated by an impaired ability of oligodendrocytes to form myelin sheets in culture and by the presence of abnormal Ca2+ transients in purified cortical oligodendrocytes studied in vitro. The Ca2+ results reported in this study complement previous results implicating golli proteins in modulating intracellular signaling in T-cells. Together, all these findings suggest a role for golli proteins in oligodendrocyte differentiation, migration, and/or myelin elaboration in the brain.

The golli-myelin basic protein negatively regulates signal transduction in T lymphocytes

Protein kinase C (PKC) plays a critical role in signal transduction controlling T lymphocyte activation. Both positive and negative regulation of signal transduction is needed for proper control of T lymphocyte activation. We have found that a golli product of the myelin basic protein (MBP) gene can serve as a negative regulator of signaling pathways in the T lymphocyte, particularly the PKC pathway. Increased expression of golli BG21 in Jurkat T cells strongly inhibits anti-CD3-induced IL-2-luciferase activity, an indicator of T lymphocyte activation. Golli BG21 can be phosphorylated by PKC in vitro and its phosphorylation increases in PMA-activated Jurkat cells. BG21 inhibits the PMA-induced increase in AP-1 or NF-kappaB activation, consistent with golli acting in a PKC-mediated cellular event. Golli BG21 inhibition of the PKC pathway is not due to a direct action on PKC activation but in the cascade following PKC activation, since BG21 neither reduces PKC enzyme activity nor blocks the membrane association of PKCtheta brought on by T lymphocyte activation. The inhibitory function of BG21 is independent of its phosphorylation by PKC because a mutant BG21, in which the PKC sites have been mutated, is as effective as the wild type BG21 in inhibiting the PMA-induced AP-1 activation. Structure-function assays indicate that BG21 inhibitory activity resides in the golli domain rather than in MBP domain of the molecule. These results reveal a novel role for MBP gene products in T lymphocytes within the immune system.

Autoreactive T cells can be protected from tolerance induction through competition by flanking determinants for access to class II MHC

It is not clear why the N-terminal autoantigenic determinant of myelin basic protein (MBP), Ac1–9, is dominant in the B1O.PL (H-2u) mouse, given its weak I-Au-MHC binding affinity. Similarly, how do high-affinity T cells specific for this determinant avoid negative selection? Because the MBP:1–9 sequence is embryonically expressed uniquely in the context of Golli-MBP, determinants were sought within the contiguous N-terminal “Golli” region that could out-compete MBP:1–9 for MHC binding, and thereby prevent negative selection of the public response to Ac1–9, shown here to be comprised of a Vβ8.2Jβ2.7 and a Vβ8.2Jβ2.4 expansion. Specifically, we demonstrate that Ac1–9 itself can be an effective inducer of central tolerance induction; however, in the context of Golli-MBP, Ac1–9 is flanked by determinants which prevent its display to autoreactive T cells. Our data support competitive capture as a means of protecting high-affinity, autoreactive T cells from central tolerance induction.

Thymocytes express the golli products of the myelin basic protein gene and levels of expression are stage dependent

The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5+ cortical/MTS10+ medullary epithelial cells and NLDC145+ dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. Golli mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4−CD8− (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4−CD8− (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8− or CD4+ (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.