Vance Handley

Expression of Myelin Protein Genes and Other Myelin Components in an Oligodendrocytic Cell Line Conditionally Immortalized with a Temperature-Sensitive Retrovirus

Abstract: We have conditionally immortalized oligodendrocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (G418) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase mRNA. These cell lines grew continuously at the permissive temperature (34°C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39°C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin‐specific protein mRNA levels were observed in N20.1 cells grown at 39°C for >9 days compared with cells maintained at 34°C. Immunocytochemical staining revealed N20.1 cells to be positive for the oligodendrocyte surface markers—galactocerebroside, A007, and A2B5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell‐free protein synthesis experiments indicated that the MBP mRNAs isolated from N20.1 cells were translatable and directed the synthesis of the 17‐, 18.5‐, and 21.5‐kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a “mature” oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of “maturation’. This cell line has now been passaged >40 times with fidelity of phenotype and genotype.

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.

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.

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.

Embryonic expression of vasoactive intestinal peptide (VIP) and VIP receptor genes.

Abstract: Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a 33P‐labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct‐size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.

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.

Multiple Kinase Pathways Regulate Voltage-Dependent Ca2+ Influx and Migration in Oligodendrocyte Precursor Cells

It is becoming increasingly clear that voltage-operated Ca2+ channels (VOCCs) play a fundamental role in the development of oligodendrocyte progenitor cells (OPCs). Because direct phosphorylation by different kinases is one of the most important mechanisms involved in VOCC modulation, the aim of this study was to evaluate the participation of serine–threonine kinases and tyrosine kinases (TKs) on Ca2+ influx mediated by VOCCs in OPCs. Calcium imaging revealed that OPCs exhibited Ca2+ influx after plasma membrane depolarization via L-type VOCCs. Furthermore, VOCC-mediated Ca2+ influx declined with OPC differentiation, indicating that VOCCs are developmentally regulated in OPCs. PKC activation significantly increased VOCC activity in OPCs, whereas PKA activation produced the opposite effect. The results also indicated that OPC morphological changes induced by PKC activation were partially mediated by VOCCs. Our data clearly suggest that TKs exert an activating influence on VOCC function in OPCs. Furthermore, using the PDGF response as a model to probe the role of TK receptors (TKr) on OPC Ca2+ uptake, we found that TKr activation potentiated Ca2+ influx after membrane depolarization. Interestingly, this TKr modulation of VOCCs appeared to be essential for the PDGF enhancement of OPC migration rate, because cell motility was completely blocked by TKr antagonists, as well as VOCC inhibitors, in migration assays. The present study strongly demonstrates that PKC and TKrs enhance Ca2+ influx induced by depolarization in OPCs, whereas PKA has an inhibitory effect. These kinases modulate voltage-operated Ca2+ uptake in OPCs and participate in the modulation of process extension and migration.

Expression of a Novel Transcript of the Myelin Basic Protein Gene

Abstract: A cDNA (M41) corresponding to a mouse myelin basic protein (MBP) mRNA with a longer 5′‐untranslated region than predicted from earlier studies of MBP gene structure has been isolated and characterized. The additional 5′‐untranslated region is encoded by two previously unidentified exons upstream of the major transcription start site of the gene. Using a DNA probe specific for M41‐MBP mRNAs, Northern blot analysis indicated that expression of this transcript follows a developmental course in mouse brain similar to that of the majority of MBP mRNAs, but that the level of expression varies between brain and spinal cord. Expression of MBP mRNAs similar to the mouse M41‐MBP also was identified in rat brain. The results suggest that the structure of the MBP gene is more complex than originally thought, containing at least two more exons. There appears to be at least one more MBP gene promoter that directs the synthesis of a subset of MBP mRNAs with a unique 5′‐untranslated region.

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.