Axo-glial interactions regulate the localization of axonal paranodal proteins.

Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, we have analyzed the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin was only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, was dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, were not restricted to this region but were detected throughout the internode in the galactolipid-defi- cient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, was not concentrated in the paranodal regions but was diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma.

A novel mutation in myelin-deficient mice results in unstable myelin basic protein gene transcripts.

Mice homozygous for the myelin-deficient (mld) mutation have an unusual phenotype in which the gene encoding myelin basic protein (MBP) is expressed at low levels and on an abnormal developmental schedule. In this report we describe the organization of the mld MBP locus, which results in this alteration of MBP expression. The mld MBP locus consists of two tandem MBP genes, with the upstream gene containing an inversion of its 3' region. We also demonstrate that although there are low steady-state levels of MBP RNA in mld mice, the mld MBP locus is transcribed at a rate comparable to that of the wild-type MBP gene, indicating that the MBP transcripts are abnormally unstable.

New perspectives on the function of myelin galactolipids.

A defining feature of the vertebrate nervous system is the ensheathment of axons by myelin, a multilamellar membrane containing a small group of proteins and an abundance of the galactolipid galactocerebroside (GalC) and its sulfated derivative sulfatide. Several in vitro studies have suggested that these galactolipids transduce developmental signals, facilitate protein trafficking and stabilize membranes. In addition, mice lacking the ability to synthesize GalC or sulfatide form dysfunctional and unstable myelin. These findings suggest that the galactolipids are essential components of myelin, and that functional and structural properties of myelin result from the combined contributions of galactolipids and proteins.

Cis-acting human ApoE tissue expression element is associated with human pattern of intraneuronal ApoE in transgenic mice.

Apolipoprotein E polymorphic variants (ApoE-epsilon2, epsilon3, and epsilon4) are associated with the age of onset distribution and risk of Alzheimer disease. The question of whether ApoE is expressed at a comparatively low level in human neurons compared to astrocytes, or whether ApoE enters neuronal cytoplasm via altered endosomal metabolism is important to understanding potential pathogenic roles for ApoE as a susceptibility gene in Alzheimer disease. ApoE deficient ("knock-out") mice received large human genomic DNA fragment transgenes for each of the three common apoE alleles. All transgenic mice demonstrated glial/astrocytic (normal rodent pattern), as well as cortical intraneuronal ApoE immunoreactivity with all three human isoforms and at multiple ApoE human allele doses (Xu et al. (32)). To test whether ApoE intraneuronal immunoreactivity was due to ApoE gene sequences between mouse and human, we examined another set of mice constructed using targeted replacement so that the human ApoE gene was placed under mouse gene promoters. Current analyses show that targeted replacement recombinant mice show normal rodent glial expression pattern, but no ApoE neuronal immunoreactivity through six months of age compared to large human genomic DNA fragment transgenic mice, which show neuronal content of ApoE throughout adult life. We conclude that cis-acting DNA sequences, rather than the specific sequence of the ApoE gene, may be responsible for low levels of transcription activity in cortical neurons.

The effects of interferon-gamma on the central nervous system.

Interferon-gamma (IFN-gamma) is a pleotropic cytokine released by T-lymphocytes and natural killer cells. Normally, these cells do not traverse the blood-brain barrier at appreciable levels and, as such, IFN-gamma is generally undetectable within the central nervous system (CNS). Nevertheless, in response to CNS infections, as well as during certain disorders in which the CNS is affected, T-cell traffic across the blood-brain barrier increases considerably, thereby exposing neuronal and glial cells to the potent effects of IFN-gamma. A larger portion of this article is devoted to the substantial circumstantial and experimental evidence that suggests that IFN-gamma plays an important role in the pathogenesis of the demyelinating disorder multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). Moreover, the biochemical and physiological effects of IFN-gamma are discussed in the context of the potential consequences of such activities on the developing and mature nervous systems.

Mammary tumorigenesis in C3Hf/Ki mice: examination of germinal mouse mammary tumor viruses and the int-1 and int-2 putative proto-oncogenes.

The organization and expression of endogenous mouse mammary tumor virus (MMTV) proviruses in normal and neoplastic C3Hf/Ki tissues were examined. MMTV-containing EcoRI, HindIII, BamHI and PstI restriction fragments of C3Hf/Ki DNA were identical to those of C3H/StWi DNA. The full-length endogenous MMTV Units Ia (Mtv-7), II (Mtv-8), III (Mtv-9) and IV (Mtv-10), in addition to the subgenomic endogenous MMTV Units I (Mtv-6) and IX (Mtv-14), were germinally transmitted in C3Hf/Ki DNA. The previously uncharacterized Mtv-7 was contained in EcoRI fragments of 16.7 and 11.7 kbp. The endogenous MMTV Unit V (Mtv-1), which is responsible for virus production and mammary tumorigenesis in C3Hf/He mice, was absent from C3Hf/Ki DNA. The 9.0 kb gag-pol, the 3.8 kb env and the 1.7 kb LTR MMTV RNA transcripts were present in C3Hf/Ki mammary glands. MMTV proviruses, in addition to the endogenous C3Hf/Ki MMTV complement, were not detected in C3Hf/Ki mammary tumor DNA. The DNA organization and RNA expression of the putative mammary proto-oncogene regions int-1 and int-2 were also examined in C3Hf/Ki mammary tumors. The int-1 and int-2 regions did not appear rearranged, amplified, or expressed in C3Hf/Ki mammary tumors. These studies indicate that MMTV proviral activation of the int proto-oncogenes is not necessary for C3Hf/Ki mammary tumorigenesis.

Expression and demethylation of germinally-transmitted BALB/c mouse mammary tumor virus DNA in Abelson MuLV B-lymphoid cell lines.

Murine mammary tumor virus (MMTV) RNA expression, DNA organization and DNA demethylation were examined in BALB/c B-lymphoid cell lines produced by transformation with the Abelson murine leukemia virus (AbMuLV). The MMTV DNA sequences in AbMuLV B cell lines, based on restriction mapping with EcoRI, PstI, BglII, BamHI and SacI and molecular hybridization with cloned probes of the MMTV LTR, gag-pol or env gene regions, were identical to the germinally-transmitted MMTV DNA complement of BALB/c mice. Several AbMuLV B cell lines expressed MMTV poly(A+)-RNA at detectable levels. MMTV poly(A+)-RNA for the env gene, 3.8 kb, and the long terminally redundant (LTR) region, 1.7 kb, were detected in some AbMuLV B cell lines. MMTV DNA sequences in the AbMuLV B cell lines were at least partially sensitive to digestion by the methylation-sensitive restriction endonucleases HhaI and HpaII. HhaI-sensitive sites were present in Units I, II and III of the germinally-transmitted MMTV DNA and were localized specifically near the 5' end of the 5' and 3' LTRs of both Units II and III. HpaII-sensitive sites were localized near the 3' end of the 3' LTRs of Units II and III, and at a cellular site 2.1 kbp 5' to the 5' LTR. These observations demonstrate that the germ line MMTV DNA sequences of BALB/c mice are expressed in cells of B lymphocyte origin, and suggest a correlation between MMTV RNA expression and selective demethylation in the LTR regions of germinally-transmitted MMTV DNA sequences.

Thyroid-specific and hormone-dependent expression of rat thyroglobulin promoter fused with bacterial chloramphenicol acetyltransferase gene in transgenic mice.

The minimal promoter of rat thyroglobulin (TG) gene (168 bp) was fused with bacterial chloramphenicol acetyltransferase (CAT) gene, and transgenic mice carrying the TGCAT gene were produced. The minimal promoter is sufficient for thyroid-specific and hormone-dependent expression of TGCAT in transgenic mice. Deletion of a region between -128 and -92 bp (TGII), which is not required for the expression of TGCAT in transient expression assays but whose sequence is most extensively conserved among different species, appears to decrease frequency of the expression of TGCAT in transgenic mice. However, the same deletion apparently has no significant effect on TG promoter activity in stably transformed rat FRTL-5 cells.

Quantitative analysis of myelin protein gene expression during development in the rat sciatic nerve.

We determined the temporal profile of expression of the genes encoding the P0 glycoprotein, the myelin-associated glycoprotein (MAG), the myelin basic protein (MBP), the proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in the sciatic nerve of rats. The level of expression of the MAG gene occurred maximally in animals 13 days of age, approximately one week earlier than the peak expression of the MBP and P0 genes. The genes encoding PLP and CNP were not expressed developmentally in a manner that correlated with the myelination of the sciatic nerve. Furthermore, using RNA synthesized in vitro, specific for each of the myelin protein genes, we have determined the absolute amounts of messenger RNA for the various myelin proteins in total RNA from sciatic nerves. P0 and MBP RNA were present at very high levels, whereas the amount of MAG, PLP and CNP RNA were much less.

Galactolipids in the formation and function of the myelin sheath.

Among the most abundant components of myelin are the galactolipids galactocerebroside (GalC) 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 GalC 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. Two laboratories have independently generated mice that are incapable of synthesizing either GalC or sulfatide by inactivating the gene encoding the enzyme UDP-galactose:ceramide galactosyltransferase (CGT), which is required for myelin galactolipid synthesis. These galactolipid-deficient animals exhibit a severe tremor, hindlimb paralysis, and display electrophysiological deficits in both the central and peripheral nervous systems. In addition, ultrastructural studies have revealed hypomyelinated white matter tracts with unstable myelin sheaths and a variety of myelin abnormalities including altered node 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 these galactolipid-deficient mice.

Genetic analysis of myelin galactolipid function.

The CGT enzyme is responsible for catalyzing the final step in GalC synthesis. The isolation of the CGT cDNA has allowed for the genetic analysis of galactolipid function by providing the opportunity to generate null mutants deficient in CGT enzymatic activity. The detailed analyses of CGT mutant mice demonstrate that the galactolipids are essential for the formation and maintenance of normal CNS myelin, but neither GalC or sulfatide appear to be required for the development of structurally normal PNS myelin. These studies also show that the differentiation of myelinating cells is not dependent on galactolipid function, in contrast to the conclusions drawn from prior antibody perturbation studies. The abnormal node of Ranvier formations present in the CNS likely explain the disrupted electrophysiological properties displayed by mutant spinal cord axons and the tremoring phenotype of these mice. The abnormal myelin structures present in the mutant animals are consistent with the possibility that the galactolipids play a role in regulating or mediating proper axo-glial interactions. The further detailed analysis of these animals should help refine our understanding of galactolipid function in the myelination process.

Myelin galactolipids: mediators of axon-glial interactions?

The precise alignment of myelin segments along the length of the axon is essential for the saltatory propagation of an electrical impulse. Furthermore, node of Ranvier formation and function are dependent on the proper interactions between myelinating glial cells and the axon. Nevertheless, the molecules that regulate the placement and association of myelinating cells with axons remain largely unidentified. Recently, however, the analysis of mutant mice incapable of synthesizing the galactolipids of myelin has revealed defects in these processes. The galactolipid-deficient mice display alterations in the spacing of internodal segments along the axon: large unmyelinated gaps are common and overlapping myelin segments are observed. Moreover, the normal tight association between the lateral loops of the myelinating cell and the axonal membrane at the paranode region is also disrupted in these animals. Strikingly, there is a complete absence of transverse bands at the axon-glial junction, with the lateral loops frequently turning away from the axon. These data indicate that the galactolipids play an essential role in axon-glial interactions and node of Ranvier formation.

In situ analysis of myelin basic protein gene expression in myelin-deficient oligodendrocytes: antisense hnRNA and readthrough transcription.

Mice homozygous for the recessive mutation myelin deficient (mld) exhibit a severe deficit in the synthesis of myelin basic protein (MBP). The primary defect in mld mice is an unusual rearrangement of the MBP gene. The mld MBP locus consists of two tandem MBP genes that span approximately 90 kb of DNA; the upstream gene contains an extensive inversion of its 3' region, while the downstream gene appears identical to the wild-type gene. In this report, the aberrant expression of the mld MBP locus was examined by in situ hybridization with MBP genomic and cDNA probes. In situ hybridization with a single-stranded genomic probe from the inverted region of the mld MBP gene revealed that primary transcripts initiating from the upstream MBP promoter elongate through the inverted region generating abundant antisense MBP heterogeneous nucleus RNA (hnRNA) transcripts in mld oligodendrocytes. Little or no antisense MBP RNA was detected in cytoplasmic regions in mld brain sections indicating that the antisense MBP hnRNA transcripts are not processed and transported out of the nucleus. Furthermore, we provide evidence that these abnormal transcripts elongate through the downstream MBP gene's promoter region, suggesting that transcription of the downstream mld MBP gene is inhibited by readthrough transcripts that originate at the upstream promoter.

Organization and expression of mouse mammary tumor virus sequences in normal and neoplastic C3Hf/HeSed mouse tissues.

The organization and expression of germinally transmitted mouse mammary tumor virus (MMTV) proviruses in C3Hf/HeSed mouse tissues were examined. Digestion with the restriction enzymes EcoRI, BamHI, and HindIII and hybridization with cloned probes specific for the long terminal repeat and the 5' and 3' regions of the MMTV genome revealed three full-length (units Ib, II, and V) and two subgenomic (units I and IX) MMTV proviruses in C3Hf/HeSed mouse germ line DNA. The EcoRI fragments (15.0 and 5.7 kilobase pairs [kbp]) that contained unit Ib were previously described as separate, subgenomic MMTV proviruses. The methylated state of each full-length MMTV provirus was examined in DNA from C3Hf/HeSed mouse livers, spleens, mammary glands, and mammary tumors by digestion with EcoRI or BamHI in combination with the methyl-sensitive restriction enzymes HhaI or HpaII. Unit Ib contained HhaI- and HpaII-sensitive sites in spleen, mammary gland, and mammary tumor DNA but was completely methylated in liver DNA. Units II and V contained HhaI- and HpaII-sensitive sites in mammary gland and mammary tumor DNA, but the sites were extensively methylated in spleen and liver DNA. The HhaI-sensitive sites were mapped to the 5' end of the 5' and 3' long terminal repeats of each full-length MMTV provirus. C3Hf/HeSed mouse tissue RNA was examined for MMTV transcripts. Mammary glands contained MMTV RNA species of 9.0, 3.8, and 1.7 kb. Mammary tumors contained high levels of the 9.0- and 3.8-kb transcripts but lacked the 1.7-kb species. A very low level of the 3.8-kb MMTV transcript was present in spleens. Livers lacked detectable MMTV RNA. These results implicate mammary tissue as the site of unit V activation in the formation of MMTV virions.

Developing and mature oligodendrocytes respond differently to the immune cytokine interferon-gamma.

Increasing evidence suggests that the immune cytokine interferon-gamma (IFN-gamma) plays a deleterious role in immune-mediated demyelinating disorders. To further understand the effects of IFN-gamma on oligodendrocytes, we have compared and quantitated the response of developing and mature oligodendrocytes in vitro to IFN-gamma and have observed several differences. Morphological changes and cell death occurred in developing cultures after 2 days in IFN-gamma, and in mature oligodendrocytes after 4-7 days. Developing oligodendrocytes underwent significantly increased apoptotic cell death in the presence of IFN-gamma, but mature oligodendrocytes exposed to IFN-gamma died of necrosis. Prior to morphological changes or cell death in mature oligodendrocytes exposed to IFN-gamma, steady-state levels of myelin-specific mRNAs and proteins were reduced. Thus, these results indicate that the sensitivity of oligodendrocytes to IFN-gamma is related to the developmental state of the cell. Such information is crucial for understanding the response of oligodendrocytes in immune-mediated demyelinating disorders and during remyelination in these diseases.

Advanced transgenic and gene-targeting approaches.

Over the past two decades the techniques associated with the manipulation of the mouse genome have provided a powerful approach toward the better understanding of gene function. Conventional transgenic and gene targeting approaches have been used extensively, and these techniques have been particularly rewarding for neuroscientists. Nevertheless, the traditional approaches toward genome manipulation have certain limitations that diminish their usefulness for studying more sophisticated biological processes. Therefore, variations to these techniques have recently been developed. The improvements are focused on two areas: one provides regulated control of transgene expression using an inducible expression system; and the other provides the opportunity to inactivate genes in specific cells and at predetermined developmental stages with a conditional gene targeting system. This review summarizes the advantages as well as some of the technical difficulties of these new approaches. The application of these advanced approaches in biomedical research, particularly neuroscience, are also discussed.

Oligodendroglial response to the immune cytokine interferon gamma.

In the human demyelinating disorder multiple sclerosis, and its animal model experimental allergic encephalomyelitis, 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. When experimentally introduced into the CNS IFN-gamma promotes many of the pathological changes that occur in immune-mediated demyelinating disorders. In vitro, this cytokine elicits a number of effects on oligodendrocytes, including cell death. The harmful actions of IFN-gamma on CNS myelin are likely mediated through direct effects on the myelinating cells, as well as through the activation of macrophages and microglia. In this review we summarize relevant studies concerning the action of IFN-gamma in demyelinating disorders and discuss possible mechanisms for the observed effects.

Genetic dissection of myelin galactolipid function.

The roles that the myelin galactolipids galactocerebroside (GalC) and sulfatide play in cellular differentiation, myelin formation and maintenance have been investigated for nearly 3 decades. During that time the primary approach has been to perturb lipid activity using antibodies and chemical agents in artificial systems. Recently, the isolation of the gene that encodes UDP-galactose:ceramide galactosyltransferase (CGT), the enzyme that catalyzes an essential step in the synthetic pathway of GalC and sulfatide, has enabled the generation of mice that lack myelin galactolipids. These mice display a severe tremor, hindlimb paralysis and electrophysiological defects. In addition, the CGT null mutants exhibit: 1) impaired oligodendrocyte differentiation, 2) myelin sheaths that are thin, incompletely compacted and unstable, and 3) structural abnormalities in the nodal and paranodal regions including disrupted axo-glial junctions. Collectively, these findings suggest that GalC and sulfatide are essential in myelin formation and maintenance, possibly by mediating intra- and intercellular interactions.

Major histocompatibility complex heavy chain accumulation in the endoplasmic reticulum of oligodendrocytes results in myelin abnormalities.

The immune cytokine interferon-gamma (IFN-gamma) is believed to be a key agent in the pathogenesis of immune-mediated demyelinating disorders. We have examined the possibility that one effect of this cytokine involves overloading the endoplasmic reticulum (ER) of oligodendrocytes through the induction of major histocompatibility complex (MHC) class I heavy chain (HC) gene expression. For these studies, we have utilized several genetic mouse models that yield different subcellular localizations of HC in oligodendrocytes. We show that transgenic mice that ectopically express HC in oligodendrocytes (MBP/MHC class I mice) fail to transport HC past the ER. These mice are hypomyelinated and have a tremoring phenotype. When oligodendrocytes deficient in beta-2 microglobulin (beta2m), which is required for MHC class I assembly and transport, were treated with IFN-gamma in vitro, HC was transported past the ER to the trans-Golgi network but not onto the cell surface. When an asymptomatic line of mice that expresses MHC class I in the CNS due to transgene-derived IFN-gamma (MBP/IFN-gamma mice) was crossed onto the beta2m-/- background, the resulting mice were asymptomatic. In contrast, increasing the amount of MHC class I protein transported through the ER in MBP/MHC class I transgenic mice, by crossing them to the asymptomatic MBP/IFN-gamma mice, exacerbated their phenotype. Taken together, these data indicate that the ER is a sensitive site in oligodendrocytes for accumulation of MHC class I HC and suggest a molecular mechanism for IFN-gamma's deleterious effects on these cells.

Effects of galactolipid elimination on oligodendrocyte development and myelination.

The galactolipids galactocerebroside and sulfatide, which require the enzyme UDP-galactose:ceramide galactosyltransferase (CGT) for their synthesis, are among the most prevalent molecules in the myelin sheath. Numerous studies, mainly using antibody perturbation methods in vitro, have suggested that these molecules are crucial mediators of oligodendrocyte differentiation and myelin formation. Although we have previously demonstrated that myelin formation occurs in CGT null mutant mice, which are incapable of synthesizing the myelin galactolipids, here we show that there are developmental alterations in the CNS of these animals. There is a significant decrease in the number of myelinated axon segments in the mutant spinal cord despite normal levels of myelin gene-specific mRNAs and proteins. Also, there is an increased cellularity in the mature mutant spinal cord and the distinctive morphology of the additional cells suggests that they are actively myelinating oligodendrocytes. Using in situ hybridization techniques, we show that there is a 50% increase in the number of oligodendrocytes in the mutant spinal cord. The data suggest that galactolipids play an important developmental role in regulating the maturation program and final number of oligodendrocytes.