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Aberrant accumulation of neurofilaments is a feature of human motor neuron diseases. Experimentally motor neuron disease can be induced in transgenic mice by overexpressing the mouse neurofilament light subunit (NF-L), the human heavy subunit (NF-H), or mouse peripherin. Here we describe that mice harboring a bacterial artificial chromosome (BAC) transgene containing the human midsized neurofilament subunit (NF-M) gene develop a progressive hind limb paralysis associated with neurofilamentous accumulations in ventral horn motor neurons and axonal loss in ventral motor roots. Biochemical studies revealed that all three mouse neurofilament subunits along with the human NF-M contributed to filament formation, although filaments contained less peripherin. In addition the endogenous mouse NF-M became less phosphorylated in the presence of the human protein and accumulated in the cell bodies of affected neurons even though phosphorylated human NF-M did not. Remaining motor axons contained an increased density of neurofilaments and morphometric studies showed that principally small myelinated axons were lost in the transgenic animals. Removing half of the mouse NF-M by breeding the transgene onto the mouse NF-M heterozygous null background offered no protection against the development of disease, arguing that the effect is not simply due to elevation of total NF-M. Collectively these studies argue that the human and mouse NF-M proteins exhibit distinct biochemical properties and within mouse neurons are not interchangeable and that indeed the human protein may be toxic to some mouse neurons. These studies have implications for the use of human neurofilament transgenic mice as models of amyotrophic lateral sclerosis.

The formation of the labyrinth layer is a critical step of placental development. The transcription factor glial cells missing 1 (Gcm1) plays a pivotal role in labyrinth development, but the sequence of events controlling its expression has not been identified yet. Our studies presented herein show that Gcm1 expression occurs in three distinct phases during placental development, each specific to a particular stage of chorio-allantois interaction. In the first, the pre-fusion phase, Gcm1 mRNA is expressed in isolated clusters of chorionic cells, but not efficiently translated. Upon allantois–chorion fusion, the second phase, Gcm1 expression is greatly induced in clusters of chorionic cells separated by non-expressing cells and the Gcm1 mRNA is translated to protein. In the third phase, the labyrinth formation, cells expressing Gcm1 proliferate, involute in the chorionic plate and branched villi formation begins.

Neurofilaments are central determinants of the diameter of myelinated axons. It is less clear whether neurofilaments serve other functional roles such as maintaining the structural integrity of axons over time. Here we show that an age-dependent axonal atrophy develops in the lumbar ventral roots of mice with a null mutation in the mid-sized neurofilament subunit (NF-M) but not in animals with a null mutation in the heavy neurofilament subunit (NF-H). Mice with null mutations in both genes develop atrophy in ventral and dorsal roots as well as a hind limb paralysis with aging. The atrophic process is not accompanied by significant axonal loss or anterior horn cell pathology. In the NF-M–null mutant atrophic ventral root, axons show an age-related depletion of neurofilaments and an increased ratio of microtubules/neurofilaments. By contrast, the preserved dorsal root axons of NF-M–null mutant animals do not show a similar depletion of neurofilaments. Thus, the lack of an NF-M subunit renders some axons selectively vulnerable to an age-dependent atrophic process. These studies argue that neurofilaments are necessary for the structural maintenance of some populations of axons during aging and that the NF-M subunit is especially critical.

Pelizaeus-Merzbacher disease (PMD) is a leukodystrophy linked to the proteolipid protein gene (PLP). We report a cellular basis for the distinction between two disease subtypes, classical and connatal, based on protein trafficking of the two PLP gene products (PLP and DM20). Classical PMD mutations correlate with accumulation of PLP in the ER of transfected COS-7 cells while the cognate DM20 traverses the secretory pathway to the cell surface. On the other hand, connatal PMD mutations lead to the accumulation of both mutant PLP and DM20 proteins in the ER of COS-7 cells with little of either isoform transported to the cell surface. Moreover, we show that transport-competent mutant DM20s facilitate trafficking of cognate PLPs and hence may influence disease severity.

Neurofilamentous changes in select groups of neurons are associated with the degenerative changes of many human age-related neurodegenerative diseases. To examine the possible effects of aging on the neuronal cytoskeleton containing human proteins, the retinas of transgenic mice expressing the gene for the human middle-sized neurofilament triplet were investigated at 3 or 12 months of age. Transgenic mice developed tangle-like neurofilamentous accumulations in a subset of retinal ganglion cells at 12 months of age. These neurofilamentous accumulations, which also involved endogenous neurofilament proteins, were present in the perikarya and proximal processes of large ganglion cells and were predominantly located in peripheral retina. The presence of the human protein may thus confer vulnerability of the cytoskeleton to age-related alterations in this specific retinal cell type and may serve as a model for similar cellular changes associated with Alzheimer's disease and glaucoma.

In ten transgenic lines, expression of a human mid-sized (M) neurofilament (NF) transgene was restricted to neurons in the central and peripheral nervous systems. However, no two lines gave identical expression patterns and none exactly matched the expression of mouse NF(M). These varied expression patterns within the neural compartment likely result from interactions of the transgene with enhancer elements located in the regions flanking the insertion site. Unexpected patterns of enhancer activity included an enhancer active in subsets of cerebellar basket cells as well as others preferentially active in subsets of motor or sensory neurons.

The primary structure of the proteolipid protein (PLP) from the central nervous system (CNS) myelin of mammals is highly conserved with only three amino acid differences between the mouse, rat, dog, bovine, and human proteins. Furthermore, within a particular species no polymorphisms in the protein have been identified. Recent interest has focused on the targeting of PLP in oligodendrocytes and the role that mutant forms of this protein play in generating dysmyelinating or hypomyelinating diseases. We previously expressed the human cDNA encoding PLP in transiently transfected Cos-7 cells and characterized the subcellular distribution of the protein in this simple heterologous system. In the current study we have used the same paradigm to examine the effect of five missense mutations in the PLP gene on processing of the encoded protein. The mutations chosen span the carboxy-terminal half of PLP and encompass that part of the protein in which most mutations have been identified. Our results show that transport of all mutations examined was arrested in the secretory pathway at an early stage, causing the mutant proteins to accumulate in the endoplasmic reticulum. Thus, a common mechanism of protein misfolding and failure of PLP to reach the cell surface of oligodendrocytes rather than the inability of the mutant protein to perform some crucial function at the cell surface may be responsible for the diseases caused by many PLP mutations. Our results, together with those of others, prompt us to speculate that the pathobiology observed in PLP mutants may result from oligodendrocyte cell death caused by the accumulation of misfolded protein in the endoplasmic reticulum. This speculation is consistent with the observations that oligodendrocytes bearing misfolded PLP, as in the jimpy mutant, proliferate but die rapidly while oligodendrocytes from PLP deletion survive and produce a myelin-like membrane which lacks PLP. © 1994 Wiley-Liss, Inc.

Transgenic mice of line MβP3 express bacterial β-galactosidase in oligodendrocytes but not other cells of the CNS. The marker enzyme, demonstrated histochemically or by immunostaining in oligodendrocyte cell bodies and along myelin internodes, appears at the time of myelination and persists thereafter; in transplantation experiments, the marker may serve to indicate both the source of particular cells and their state of differentiation. The subventricular zone of the lateral ventricle, grafted from transgenic to wild-type perinatal recipient mice, yields histochemically labeled oligodendrocytes in surrounding host tissue. When grafts are placed in cerebral cortex near callosal radiations, graft-derived oligodendrocytes are found in cerebral cortex and subcortical white matter as far as 1.5 mm from the site of implant but not in nearby caudoputamen. This study is the first to document differentiation of transplant-derived oligodendrocytes in normal developing CNS. Our results are consistent with the well- established notion that oligodendrocyte progenitors migrate during normal development and suggest that such migration might be guided or restricted by mechanisms yet to be identified.

By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5′ upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a β-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.

Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibited either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.5 to E13.5), there was no evidence of embryonic lethality. The dysraphisms develop during the period and within the zone of transgene expression. We present evidence that these dysraphisms result from an inhibition of neuropore closure and a stimulation of secondary neurulation. After transgene expression ceases, the spinal dysraphisms are progressively resolved and the neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column. The multiple spinal cords remain but are enclosed in a single spinal column as in the human diastematomyelia. The animals live a normal life time, are fertile and do not exhibit any obvious weakness or motor disabilities.

We used the yeast two-hybrid assay to identify novel proteins that interact with the D1dopamine receptor. The third cytoplasmic loop (residues 217–273) of the rat D1receptor was used as bait to identify clones encoding interacting proteins from a rat brain cDNA library. This identified two clones encoding the C terminus of rat neurofilament-M (NF-M) (residues 782–846). The NF-M clone did not interact with the third cytoplasmic loops of the rat D2, D3, or D4receptors, but showed weak interaction with that of the D5receptor. Coexpression of full-length NF-M with the D1receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D1receptor-mediated cAMP accumulation. NF-M had no effect on the expression of other dopamine receptor subtypes. Using a D1receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D1receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol. Interestingly, the D1receptors that were expressed at the cell surface in the presence of NF-M were resistant to agonist-induced desensitization. Cellular colocalization of NF-M and the D1receptor in the rat brain was examined by epifluorescence microscopy. These experiments showed that ∼50% of medium-sized striatal neurons expressed both proteins. Colocalization was also observed in pyramidal cells and interneurons within the frontal cortex. Similar immunohistochemical analyses using NF-M-deficient mice showed decrements in D1receptor expression compared with control mice. These results suggest that NF-M interacts with the D1receptorin vivoand may modify its expression and regulation.

Schwann cells and oligodendrocytes produce myelin sheaths of widely varying sizes. How these cells determine the size of myelin sheath for a particular axon is incompletely understood. Axonal diameter has long been suspected to be a signal in this process. We have analyzed myelin sheath thickness in L5 lumbar root and spinal cord white matter of a series of mouse mutants with diminished axonal calibers resulting from a deficiency of neurofilaments (NFs). In the PNS, average axonal diameters were reduced by 20 ‐37% in the NF mutants. Remarkably, the average myelin sheath thickness remained unchanged from control values, and regression analysis showed sheaths abnormally thick for a given size of axon. These data show that a genetically induced reduction in axonal caliber does not cause a reduction in myelin sheath thickness in PNS and indicate that Schwann cells read some intrinsic signal on axons that can be uncoupled from axonal diameter. Interestingly, myelin sheaths in the spinal cord of these animals were not abnormally thick, arguing that axonal diameter may contribute directly to the regulation of myelination in the CNS and that oligodendrocytes and Schwann cells use different cues to set myelin sheath thickness. J. Neurosci. Res. 65:493‐ 499, 2001. © 2001 Wiley-Liss, Inc.

Trophoblast cells of the placenta are established at the blastocyst stage and differentiate into specialized subtypes after implantation1,2. In mice, the outer layer of the placenta consists of trophoblast giant cells that invade the uterus and promote maternal blood flow to the implantation site by producing cytokines with angiogenic3 and vasodilatory4 actions. The innermost layer, called the labyrinth, consists of branched villi that provide a large surface area for nutrient transport and are composed of trophoblast cells and underlying mesodermal cells derived from the allantois. The chorioallantoic villi develop after embryonic day (E) 8.5 through extensive folding and branching of an initially flat sheet of trophoblast cells, the chorionic plate, in response to contact with the allantois. We show here that Gcm1, encoding the transcription factor glial cells missing-1 (Gcm1), is expressed in small clusters of chorionic trophoblast cells at the flat chorionic plate stage and at sites of chorioallantoic folding and extension when morphogenesis begins. Mutation of Gcm1 in mice causes a complete block to branching of the chorioallantoic interface, resulting in embryonic mortality by E10 due to the absence of the placental labyrinth. In addition, chorionic trophoblast cells in Gcm1-deficient placentas do not fuse to form syncytiotrophoblast. Abnormal development of placental villi is frequently associated with fetal death and intrauterine growth restriction in humans, and our studies provide the earliest molecular insight into this aspect of placental development.

Although gcm was first recognized for its role in specifying glial cell fate in Drosophila melanogaster, its mammalian counterparts are expressed predominantly in non-neural tissues. Here we demonstrate expression of the mouse and human GCM 1 proteins in placenta. We have prepared a highly specific antibody that recognizes the GCM 1 protein and have used it to assess the temporal and spatial expression profile of the protein. In both mouse and human placenta, the protein is associated with cells that are involved with exchange between maternal and fetal blood supplies: the labyrinthine cells of the mouse placenta and the syncytio- and cytotrophoblasts of the human placenta. Using the full-length h Gcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization.

The 129 mouse strain has become of increasing interest to neurobiologists due to its importance in gene targeting studies. However it has been pointed out that 129 mice suffer from a number of neuroanatomical idiosyncrasies that may make them less attractive as animal models in neurobiology. Here we show that 129 mice also differ from other commonly used strains in possessing large numbers of unmyelinated axons in their lumbar motor roots. By contrast in all other strains of mice (C57BL/6, C3H, Swiss-Webster) that we studied the axons in the L5 roots are all myelinated. Additionally we show that 129 mice have smaller myelinated axons than other mouse strains and perform poorly in the rotorod test. These characteristics must be kept in mind in studies of mutant mice that are frequently performed on a mixed genetic background containing a129 contribution.

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease resulting from mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Distinguishing features of PMD include pleiotropy and a range of disease severities among patients. Previously, we demonstrated that, when expressed in transfected fibroblasts, many naturally occurring mutant PLP alleles encode proteins that accumulate in the endoplasmic reticulum and are not transported to the cell surface. In the present communication, we show that oligodendrocytes in an animal model of PMD, the msd mouse, accumulate Plp gene products in the perinuclear region and are unable to transport them to the cell surface. Another important aspect of disease in msd mice is oligodendrocyte cell death, which is increased by two- to threefold. We demonstrate in msd mice that this death occurs by apoptosis and show that at the time oligodendrocytes die, they have differentiated, extended processes that frequently contact axons and are expressing myelin structural proteins. Finally, we define a hypothesis that accounts for pathogenesis in most PMD patients and animal models of this disease and, moreover, can be used to develop potential therapeutic strategies for ameliorating the disease phenotype.

To understand the function of the myelin oligodendrocyte glycoprotein (MOG), a myelin specific protein of the central nervous system, transgenic mice were produced. The transgene is a fusion gene containing 1.9 kb of murine myelin basic protein promoter, 430 bp of rat MOG cDNA in the reverse orientation and 4.5 kb of human proteolipid protein gene. In spite of high expression of antisense MOG mRNA in the oligodendrocytes, MOG synthesis was not inhibited in transgenic mice. This lack of inhibition of MOG underlines the difficulties encountered with antisense transgenic strategies.

qkI, a newly cloned gene lying immediately proximal to the deletion in thequakingviablemutation, is transcribed into three messages of 5, 6, and 7 kb. Antibodies raised to the unique carboxy peptides of the resulting QKI proteins reveal that, in the nervous system, all three QKI proteins are expressed strongly in myelin-forming cells and also in astrocytes. Interestingly, individual isoforms show distinct intracellular distributions: QKI-6 and QKI-7 are localized to perikaryal cytoplasm, whereas QKI-5 invariably is restricted to the nucleus, consistent with the predicted role of QKI as an RNA-binding protein. Inquakingviablemutants, which display severe dysmyelination, QKI-6 and QKI-7 are absent exclusively from myelin-forming cells. By contrast, QKI-5 is absent only in oligodendrocytes of severely affected tracts. These observations implicate QKI proteins as regulators of myelination and reveal key insights into the mechanisms of dysmyelination in thequakingviablemutant.

Previous studies have revealed that the transgenic mouse line expressing the human neurofilament-mid-sized (NF-M) gene evidences age-dependent and cell-specific pathological neurofibrillary accumulation in the central nerve system. In the current study, we investigated the learning and memory processes of NF-M transgenic mice at 3 and 8 months of age in a modified Morris water maze using a series of tasks including those primarily related to reference memory (i.e., spatial learning, reversal learning and probe trials) and to working memory (i.e., matching to sample tasks with or without delays). At 3 months of age, NF-M transgenic mice were indistinguishable from age- and litter-matched non-transgenic wild-type controls on any of the tests of reference and working memory. At 8 months of age, however, the NF-M transgenic mice exhibited significantly poorer performance than the age- and litter-matched wild-type control mice on both reference and working memory tasks. Immunohistohistological study of the brains of the 8-month-old NF-M transgenic mice revealed spherical and tangle-like neurofilamentous accumulation in their cerebral cortices. These results suggest that NF-M transgenic mice express both age-related histopathological changes and age-dependent learning and memory deficits. Whether NF-M transgenic mice exhibit even more severe behavioral impairments when they become aged is currently under study.

Alterations in neurofilaments are a common occurrence in neurons of the human nervous system during aging and diseases associated with aging. Such pathologic changes may be attributed to species-specific properties of human neurofilaments as well as cell-type-specific regulation of this element of the cytoskeleton. The development of transgenic animals containing human neurofilament subunits offers an opportunity to study the effects of aging and other experimental conditions on the human-specific form of these proteins in a rodent model. The present study shows that mice from the transgenic line NF(M)27, which express the human midsized neurofilament subunit at low levels (2–25% of the endogenous NF-M), develop neurofilamentous accumulations in specific subgroups of neurons that are age dependent, affecting 78% of transgenic mice over 12 months of age. Similar accumulations do not occur in age-matched, wild-type littermates or in 3-month-old transgenic mice. In 12-month-old transgenic mice, somatic neurofilament accumulations resembling neurofibrillary tangles were present predominantly in layers III and V of the neocortex, as well as in select subpopulations of subcortical neurons. Intraperikaryal, spherical neurofilamentous accumulations were particularly abundant in cell bodies in layer II of the neocortex, and neurofilament-containing distentions of Purkinje cell proximal axons occurred in the cerebellum. These pathological accumulations contained mouse as well as human NF subunits, but could be distinguished by their content of phosphorylation-dependent NF epitopes. These cytoskeletal alterations closely resemble the cell-type-specific alterations in neurofilaments that occur during normal human aging and in diseases associated with aging, indicating that these transgenic animals may serve as models of some aspects of the pathologic features of human neurodegenerative diseases.

Bacterial beta-galactosidase is widely used as a marker for gene expression and in cell tracing experiments. In a survey of three transgenic mouse lines expressing beta-galactosidase in the central nervous system (CNS) under the control of different promoters, we find substantial variation in the intracellular distribution of the lacZ protein. In line M beta P5, transgene beta-galactosidase expression is driven by a promoter/enhancer fragment from the oligodendrocyte-specific myelin basic protein gene; however, electron microscopy of histochemically stained preparations reveals transgene expression not only in oligodendrocytes but also in some neurons. Immunofluorescence and immunoperoxidase staining show the beta-galactosidase protein distributed throughout the perikaryal cytoplasm of oligodendrocytes and in processes reaching to myelin sheaths. By contrast, immunoreactive protein appears restricted in neurons to one or a few small perikaryal immunoreactive granules. The granules are visible in the electron microscope as amorphous inclusion bodies of moderate electron density and lack a limiting membrane. Histochemical staining patterns with X-gal and Bluo-gal echoed the protein distribution: diffuse distribution of enzyme protein yielded cells filled with substrate, while punctate enzyme distribution yielded restricted or punctate histochemical staining. Examination of two other lines using different promoter/enhancers to drive expression in the CNS showed both diffuse and punctate beta-galactosidase immunolocalization and histochemical staining. The amount of protein synthesized or other properties, yet unidentified, intrinsic to the target cells may determine the intracellular distribution of beta-galactosidase. In retroviral marking studies, clone members have been identified as those cells filled with X-gal reaction product. This approach may underestimate both clone size and the minimum number of divisions separating the members of each clone.

The induction of autoantibodies and their possible role in the pathogenesis of autoimmune disease are poorly understood. Involvement of infectious agents has been suspected, but direct evidence is sparse. Whether immunological unresponsiveness to self by antibody-forming B cells is maintained by clonal abortion, clonal anergy or suppression, or how the scenario of interactions between helper T cells, B cells and antigen-presenting cells is distorted in autoantibody responses, is being analysed and widely debated. To evaluate tolerance of neutralizing B-cell responses we used transgenic mice expressing the cell membrane associated glycoprotein (G) of vesicular stomatitis virus (VSV) as self-antigen. We show that autoantibodies to VSV-G cannot be induced by VSV-G in adjuvant or by recombinant vaccinia virus expressing VSV-G, but are triggered by infection with wild-type VSV. The data show that helper T-cell tolerance is crucial in maintenance of B-cell non-reactivity and that cognate T-B recognition is necessary to break tolerance of self-reactive B cells. These results may help to understand mechanisms of virus-induced autoimmunity.

We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

In multiple sclerosis, a demyelinating disease of young adults, there is a paucity of myelin repair in the central nervous system (CNS) which is necessary for the restoration of fast saltatory conduction in axons1,2. Consequently, this relapsing disease often causes marked disability. In similar diseases of small rodents, however, remyelination can be quite extensive, as in the demyelinating disease caused by the A59 strain of mouse hepatitis virus (MHV-A59)3,4, a coronavirus of mice. To investigate when and where oligodendrocytes are first triggered to repair CNS myelin in such disease, we have used a complementary DNA probe specific for one major myelin protein gene, myelin basic protein (MBP), which hybridizes with the four forms of MBP messenger RNA in rodents5. Using Northern blot and in situ hybridization techniques, we previously found that MBP mRNA is first detected at about 5 days after birth, peaks at 18 days and progressively decreases to 25% of the peak levels in the adult5–7. We now report that in spinal cord sections of adult animals with active demyelination and inflammatory cells, in situ hybridization reveals a dramatic increase in probe binding to MBP-specific mRNA at 2–3 weeks after virus inoculation and before remyelination can be detected by morphological methods. This increase of MBP-specific mRNA is found at the edge of the demyelinating area and extends into surrounding areas of normal-appearing white matter. Thus, in situ hybridization with myelin-specific probes appears to be a useful method for detecting the timing, intensity and location of myelin protein gene reactivation preceding remyelination. This method could be used to elucidate whether such a reactivation occurs in multiple sclerosis brain tissue. Our results suggest that in mice, glial cells react to a demyelinating process with widespread MBP mRNA synthesis which may be triggered by a diffusible factor released in the demyelinated areas.

cDNA clones encoding proteolipid protein (PLP) were isolated from a mouse brain library and sequenced. We describe two transcripts arising from the PLP locus by alternative splicing: the major one encodes the 277-amino acid PLP protein and the minor one corresponds to the DM-20 protein, a PLP-like protein of 20,000 Mr that shares both amino and carboxyl regions with PLP. These two transcripts lack approximately 70 bases in PLP mRNA from the dysmyelinating jimpy mutant. The deletion spans amino acids 208-232; however, this region is present in the jimpy PLP-encoding gene. We propose that the jimpy mutant suffers a point mutation or the deletion of a few bases in the PLP gene that alters the normal splicing pattern and generates partially deleted PLP transcripts.

The complete primary structure of the largest mammalian neurofilament component, NF-H, is predicted from mouse cDNA and genomic clones, revealing a protein of molecular weight ca. 115,000. A central filament-forming domain structurally typical of all intermediate filament proteins is present, but anomalies are noted which may place constraints on the mechanism of NF-H assembly into filaments. The COOH-terminal portion of the protein is extremely long (661 amino acids) by comparison to non-neuronal intermediate filament components and has a remarkably monotonous, highly charged composition (Glu and Lys at 20% each). Its most remarkable feature is a tandem repeat of a 6 amino acid sequence containing the motif Lys-Ser-Pro that extends for more than half the length of the COOH-terminus. The Lys-Ser-Pro motif appears 48 times and since it is now known that the serine therein is a target for in vivo kinases, the masssive axonal phosphorylation of NF-H is explained. Comparison of mouse and human NF-H reveals that otherwise conserved proteins have been subjected to evolutionary mutation within their multiphosphorylation repeat domains, although the Lys-Ser-Pro motif has been conserved.

A high degree of conservation exists between the Hox 1.3 homeobox genes of mice and humans. The two genes occupy the same relative positions in their respective Hox 1 gene clusters, they show extensive sequence similarities in their coding and noncoding portions, and both are transcribed into multiple transcripts of similar sizes. The predicted human Hox 1.3 protein differs from its murine counterpart in only 7 of 270 amino acids. The sequence similarity in the 250 base pairs upstream of the initiation codon is 98%, the similarity between the two introns, both 960 base pairs long, is 72%, and the similarity in the 3' noncoding region from termination codon to polyadenylation signal is 90%. Both mouse and human Hox 1.3 introns contain a sequence with homology to a mating-type-controlled cis element of the yeast Ty1 transposon. DNA-binding studies with a recombinant mouse Hox 1.3 protein identified two binding sites in the intron, both of which were within the region of shared homology with this Ty1 cis element.

We have isolated cDNA clones encoding three separate forms of human myelin basic protein (MBP), 21.5, 18.5, and 17.2 kDa, and have determined the nucleotide sequence of each. The three forms share a common sequence but differ by the inclusion of a 26-residue amino acid sequence near the N terminus of the 21.5-kDa protein or by the absence of an 11-residue amino acid sequence near the C terminus of the 17.2-kDa protein. The sequences either added to or deleted from the major 18.5-kDa MBP correspond exactly to exons 2 and 5 of the mouse MBP gene, suggesting that the human and mouse genes have similar exon structures. We have also identified the 21.5-kDa human MBP on immunoblots using antisera raised to a peptide encoded by the mouse exon 2 sequence. Southern blotting studies of human genomic DNA reveal a simple pattern consistent with a single human MBP gene. Thus, the three MBP mRNAs are likely to arise from alternative splicing of a primary human MBP transcript. Conservation of the 26 amino acid mouse exon 2 sequence in human MBP suggests an important role for this sequence in myelination.

Using a family of synthetic tetradecamer oligonucleotides as a primer for cDNA synthesis and a second family of tetradecamers as a hybridization probe, we have prepared and isolated a cDNA clone of mouse myelin basic protein (MBP). The clone, pNZ111, corresponds to the region of the mRNA that codes for an amino acid sequence present in all four major forms of MBP. The relative abundance of MBP mRNA, estimated by dot blot hybridization, increased with the age of the mouse to a maximum at 18 days, then decreased to about one-fourth of that amount at later ages. Mouse MBP mRNAs, selected by their ability to hybridize to the clone, translate into the four forms of myelin basic protein. In RNA blot analyses, pNZ111 hybridized to multiple species of mouse mRNA. The predominant hybridization is to a broad band of RNAs ranging in length from 2,350 to 2,100 bases. These mRNA species are extremely long, considering that the largest MBP could be encoded by approximately 600 bases. In addition to these, there are also minor bands that hybridize with pNZ111, including a band of 4,100 bases and smaller ones of 1,900, 1,500, and 1,200 bases.

Shiverer (shi) is an autosomal recessive mutation in the mouse characterized by an almost total lack of central nervous system myelin. While small amounts of other myelin components are present in the brain of the shi mouse, the four forms of myelin basic protein (MBP) are not detectable. Previous investigations by us and others indicate that the MBP gene has undergone a major rearrangement in the shi mutant. Herein, we report in detail the nature and extent of the rearrangement: a 20-kilobase region within the MBP gene is missing in the mutant. We map the 5' breakpoint of the deletion to the second intron and the 3' breakpoint to a site 2 kilobases beyond the last MBP exon. The junction of the upstream and downstream portions of the gene contains only one nucleotide not accounted for by the wild-type MBP gene sequence. The 3' side of the deletion occurs in the 3rd of 11 tandem repeats of a 31-base-pair sequence. This region is rich in alternating purine and pyrimidine stretches, sequences that have been associated with both Z-DNA structures and gene rearrangements. The recombination junction shares several features with the junctions characterized by Anderson et al. [Anderson, R., Kato, S. & Camerini-Otero, D. (1984) Proc. Natl. Acad. Sci. USA 81, 206-210] in mouse L cells and is consistent with their model for a partially homologous recombination event. The structure of the shi recombination junction suggests that the donor DNA molecules were aligned in a partially homologous region before staggered cutting and joining occurred.

Expression of the murine homeobox containing gene Hox-1.3 was analyzed in mouse embryos using polyclonal antisera to peptides predicted from cDNA and genomic sequences. At the earliest stage examined, 7.5 days gestation, cell nuclei throughout the three embryonic germ layers and in extraembryonic structures derived from the fertilized ovum were strongly immunoreactive. Rostro-caudal gradients or other patterns of regional differentiation in levels of expression could not be seen. Surrounding maternal tissue showed only weak immunoreactivity. At 8.5 days gestation, immunoreactivity was present in all embryonic structures including neural tube, somites and lateral plate mesoderm, ectoderm and endoderm. Immunoreactivity was progressively restricted thereafter. At 17 days gestation, strong immunoreactivity was largely restricted to the nervous system, both central and peripheral. Spinal cord was well stained, with a dramatic reduction in intensity near the junction of spinal cord and brain. In addition to this overall pattern, enhanced immunoreactivity appeared in limited populations of newly-formed neuroblasts of spinal cord and brain, suggesting that Hox-1.3 might serve to regulate the development of specific types of neurons following cessation of precursor cell mitosis.

Dissociated neuron cultures of mice were inoculated with vesicular stomatitis virus (VSV) and subsequently fed with medium containing sufficient antiviral antibody (AB) to neutralize all free virus. In contrast to control acute neuronal infection, which lasts one to two days, AB-treated neuron cultures were maintained as long as two weeks. This protection was not obtained in infected monkey kidney cells treated with AB. Protected neuron cultures were examined with immunolabeling and EM techniques. During the first days of AB treatment, viral buds and surface antigens were often grouped in clusters instead of being diffuse on the neuronal surface. In addition, phagocytic cells often in contact with viruses released by neurons progressively engulfed aggregates of these viruses in their lysosomal systems. Dendritic processes in AB-treated cultures contained more viral antigen and ribonucleoproteins, but less assembly sites than in acutely-infected neurons. However, budding sites were frequent on the side of the post-synaptic density and some viruses seemed to enter directly into the lateral side of the presynaptic terminal to which the productive dendrite was connected. Removal of AB at this stage resulted in reactivation of viral infection in more neurons than those originally infected, but complete viral maturation and release occurred only 2 to 3 days after removal of AB. Chance of reactivation decreased with increasing length of AB treatment, and no viral antigens or budding sites were detected during the second week of AB treatment. Continuous treatment of VSV-infected neuron cultures with antiviral AB first induces (1) activation of non-neuronal cells to phagocytize clusters of viruses forming at the neuron surface, and (2) restriction of virus maturation sites mostly to the postsynaptic area where virus spreading to presynaptic endings seems favored. Prolonged treatment with AB later results in the apparent curing of the infection.

Infection of dissociated neuron cultures of mice with VSV and its defective particle DI-T was studied using fluorescent light microscopy as well as transmission and scanning electron microscopy. When cultures are infected with wild virus, VSV replicates selectively in neurons, producing cell death within 24-48 hr. Sensory and immature neurons express viral antigen most rapidly. Viral antigen and viral budding sites are detected along the neuron soma and dendrites. When large amounts of DI-T particles are added to the wild virus inoculum, viral growth is completely suppressed in mature neurons, the cell killing effects of VSV are considerably delayed and co-infected cultures survive for 5-16 days. Viral antigen accumulates in cytoplasmic inclusions and on the membrane of neuron cell somas and dendrites in the virtual absence of viral assembly. Identical modulation of VSV infection in mature neuron cultures is obtained when DI-T particles are added before or after the wild virus, but ultraviolet inactivation of DIs completely abolishes their protective effect. Immature neurons or Vero cells cannot be protected from acute cytopathic changes by an equivalent amount of DI particles. Thus DIs interfere with replication and assembly of the wild virus and attenuate cell killing effects in mature neurons in vitro.