Interferon-gamma protects against cuprizone-induced demyelination.

Evidence suggests that interferon-gamma (IFN-gamma), a proinflammatory cytokine secreted by activated T lymphocytes, contributes a deleterious effect to immune-mediated demyelinating disorders such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Nevertheless, mouse strains that are normally resistant to EAE induction become susceptible when the gene encoding either IFN-gamma or its receptor is mutated, demonstrating that the role that this cytokine plays in demyelinating disorders is complex. We have examined the effect of IFN-gamma in a chemically induced model of CNS demyelination. Mice that receive through their diet the copper chelator cuprizone display extensive demyelination of the corpus callosum. Remarkably, transgenic mice that ectopically express low levels of IFN-gamma in the CNS did not display evidence of demyelination when treated with cuprizone, nor did they shows signs of oligodendroglial death, astrogliosis, or microgliosis, which are typically seen in treated animals. Myelin protein gene expression was, however, dramatically reduced in both the treated control and the transgenic animals, indicating that demyelination is not an obligatory consequence of a large diminution of myelin protein synthesis. Interestingly, the CNS of the IFN-gamma-expressing mice contained elevated levels of insulin-like growth factor I, which has been demonstrated to have a protective effect against the demyelinating action of cuprizone.

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.

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.

Biochemistry and neuropathology of mice doubly deficient in synthesis and degradation of galactosylceramide.

We have generated mice doubly deficient in both synthesis and degradation of galactosylceramide by cross-breeding twitcher mice and galactosylceramide synthase (UDP-galactose:ceramide galactosyltransferase, CGT) knockout mice. The prediction that the phenotype of the doubly deficient mice should be the same as the cgt -/- mice, since the degrading enzyme should not be necessary if the substrate is not synthesized, proved to be only partially correct. In early stages of the disease, the doubly deficient mice (galc -/-, cgt -/-) were essentially indistinguishable from the cgt -/- mice. However, the doubly deficient mice had a much shorter life span than cgt -/- mice. Both galactosylceramide and galactosylsphingosine (psychosine), were undetectable in the brain of the cgt -/- and the doubly deficient mice. The characteristic twitcher pathology was never seen in the galc -/-, cgt -/- mice. However, after 43 days, neuronal pathology was observed in the brainstem and spinal cord. This late neuronal pathology has not been seen in the CGT knockout mice but has been described in some long surviving bone marrow-transplanted twitcher mice. Furthermore, the motor segment of the trigeminal nerve of the galc -/-, cgt -/- mice showed severe degeneration not seen in either twitcher or CGT knockout mice. Thus, the galc -/-, cgt -/- mice, while primarily showing the cgt -/- phenotype as predicted, develop late pathology that is seen only in twitcher mouse and also a unique pathology in the trigeminal nerve. These observations indicate that the functional relationship between galactosylceramidase and galactosylceramide synthase is complex.

Twitcher mice with only a single active galactosylceramide synthase gene exhibit clearly detectable but therapeutically minor phenotypic improvements.

Cross-breeding of mouse mutants, each defective in either synthesis (CGT knockout) or degradation (twitcher) of galactosylceramide, generates hybrids with a genotype of galc -/-, cgt +/-, in addition to doubly deficient mice. They are ideally suited to test the potential usefulness of limiting synthesis of the substrate as a treatment of genetic disorders due to degradative enzyme defects. The rate of accretion of galactosylceramide in the brain of CGT knockout carrier mice (cgt +/-) is approximately two-thirds of the normal, suggesting a gene-level compensation for the reduced gene dosage. Phenotype of twitcher mice with a single dose of normal cgt gene was indeed milder with statistical significance, albeit only slightly. Compared among 10 paired littermates, the difference in the life span was 7+/-3.9 days (S.D.) and the difference in the maximum attained body weight was 1.9+/-1.2 g (S.D.). Neuropathologists were able to distinguish blindly galc -/-, cgt +/- mice from galc -/-, cgt +/+ mice. The brain psychosine level in galc -/-, cgt +/- mice was also approximately two-thirds of the galc -/-, cgt +/+ mice. These observations indicate that reduction of galactosylceramide synthesis to two-thirds of the normal level results in minor but clearly detectable phenotypic improvements. Because of the detrimental consequences of drastic reduction in galactosylceramide synthesis that may be required for pragmatically meaningful improvements, this approach by itself is unlikely to be useful as the sole treatment but may be helpful as a supplement to other therapies.

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.

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.

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.

Myelination in the absence of galactolipids and proteolipid proteins.

The galactolipids galactocerebroside and sulfatide and the proteolipid protein (PLP) and its splice variant DM20 are the most abundant lipid and protein components of central nervous system myelin. Recent studies have found that mice lacking either the galactolipids or PLP are able to form myelin sheaths with apparently normal periodicity and near normal compaction. Here, we have generated galactolipid/proteolipid double mutants to examine the possibility that these molecules have overlapping functions. We show that the absence of the galactolipids and PLP has pleotropic effects on myelin formation. While oligodendrocytes in the postnatal day 20 galactolipid/proteolipid-deficient mouse are able to elaborate myelin with close to normal intraperiod lines, there is an increased frequency of uncompacted myelin sheaths as well as unmyelinated axons. Moreover, the double mutants display extensive white matter vacuolization of the cerebellum that initiates around postnatal day 16, which correlates with the onset of a severe ataxic phenotype and an increased percentage of apoptotic nuclei in the cerebellar internal granule cell layer. These data indicate that the galactolipids and PLP/DM20 are not required for intraperiod line formation, but they suggest a role for these molecules in mediating myelin compaction and in maintaining the integrity of the cerebellum.

Drastically abnormal gluco- and galactosylceramide composition does not affect ganglioside metabolism in the brain of mice deficient in galactosylceramide synthase.

Mice that are genetically deficient in UDP-galactose: ceramide galactosyltransferase are unable to synthesize galactosylceramide. Consequently, sulfatide, which can be synthesized only by sulfation of galactosylceramide, is also totally absent in affected mouse brain. Alpha-hydroxy fatty acid-containing glucosylceramide partially replaces the missing galactosylceramide. A substantial proportion of sphingomyelin, which normally contains only non-hydroxy fatty acids, also contains alpha-hydroxy fatty acids. These findings indicate that alpha-hydroxy fatty acid-containing ceramide normally present only in galactosylceramide and sulfatide is diverted to other compounds because they cannot be synthesized into galactosylceramide due to the lack of the galactosyltransferase. We have examined brain gangliosides in order to determine if alpha-hydroxy fatty acid-containing glucosylceramide present in an abnormally high concentration is also incorporated into gangliosides. The brain ganglioside composition, however, is entirely normal in both the total amount and molecular distribution in these mice. One feasible explanation is that UDP-galactose: glucosylceramide galactosyltransferase does not recognize alpha-hydroxy fatty acid-containing glucosylceramide as acceptor. This analytical finding is consistent with the relative sparing of gray matter in the affected mice and provides an insight into sphingolipid metabolism in the mouse brain.

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 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.

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.

Demyelination and altered expression of myelin-associated glycoprotein isoforms in the central nervous system of galactolipid-deficient mice.

Vertebrate myelin is enriched in the lipid galactocerebroside (GalC) and its sulfated derivated sulfatide. To understand the in vivo function of these lipids, we analyzed myelination in mice that contain a null mutation in the gene encoding UDP-galactose:ceramide galactosyltransferase, the enzyme responsible for catalyzing the final step in GalC synthesis. Galactolipid-deficient myelin is regionally unstable and progressively degenerates. At postnatal day 30, demyelination is restricted to the midbrain and hindbrain, but by postnatal day 90, it spreads throughout the central nervous system. Activated microglial cells and reactive astrocytes appear with the loss of myelin in older animals. Nonetheless, major myelin protein gene mRNA levels are normal throughout the life of these animals, suggesting that widespread oligodendrocyte death is not the primary cause of demyelination. The developmental switch in myelin-associated glycoprotein isoform expression, however, does not occur normally in these mice, suggesting an alteration in oligodendrocyte maturation. Taken together, these findings indicate that GalC and sulfatide are required for the long-term maintenance of myelin and that their absence may have subtle effects on the development of oligodendrocytes.

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.

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.

The cytoplasmic domain of the large myelin-associated glycoprotein isoform is needed for proper CNS but not peripheral nervous system myelination.

The myelin-associated glycoprotein (MAG) is a member of the immunoglobulin gene superfamily and is thought to play a critical role in the interaction of myelinating glial cells with the axon. Myelin from mutant mice incapable of expressing MAG displays various subtle abnormalities in the CNS and degenerates with age in the peripheral nervous system (PNS). Two distinct isoforms, large MAG (L-MAG) and small MAG (S-MAG), are produced through the alternative splicing of the primary MAG transcript. The cytoplasmic domain of L-MAG contains a unique phosphorylation site and has been shown to associate with the fyn tyrosine kinase. Moreover, L-MAG is expressed abundantly early in the myelination process, possibly indicating an important role in the initial stages of myelination. We have adapted the gene-targeting approach in embryonic stem cells to generate mutant mice that express a truncated form of the L-MAG isoform, eliminating the unique portion of its cytoplasmic domain, but that continue to express S-MAG. Similar to the total MAG knockouts, these animals do not express an overt clinical phenotype. CNS myelin of the L-MAG mutant mice displays most of the pathological abnormalities reported for the total MAG knockouts. In contrast to the null MAG mutants, however, PNS axons and myelin of older L-MAG mutant animals do not degenerate, indicating that S-MAG is sufficient to maintain PNS integrity. These observations demonstrate a differential role of the L-MAG isoform in CNS and PNS myelin.

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.

Myelin galactolipids are essential for proper node of Ranvier formation in the CNS.

The vertebrate myelin sheath is greatly enriched in the galactolipids galactocerebroside (GalC) and sulfatide. Mice with a disruption in the gene that encodes the biosynthetic enzyme UDP-galactose:ceramide galactosyl transferase (CGT) are incapable of synthesizing these lipids yet form myelin sheaths that exhibit major and minor dense lines with spacing comparable to controls. These CGT mutant mice exhibit a severe tremor that is accompanied by hindlimb paralysis. Furthermore, electrophysiological studies reveal nerve conduction deficits in the spinal cord of these mutants. Here, using electron microscopic techniques, we demonstrate ultrastructural myelin abnormalities in the CNS that are consistent with the electrophysiological deficits. These abnormalities include altered nodal lengths, an abundance of heminodes, an absence of transverse bands, and the presence of reversed lateral loops. In contrast to the CNS, no ultrastructural abnormalities and only modest electrophysiological deficits were observed in the peripheral nervous system. Taken together, the data presented here indicate that GalC and sulfatide are essential in proper CNS node and paranode formation and that these lipids are important in ensuring proper axo-oligodendrocyte interactions.