A cellular mechanism governing the severity of Pelizaeus-Merzbacher disease.

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.

The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta.

Trophoblast cells of the placenta are established at the blastocyst stage and differentiate into specialized subtypes after implantation. 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 angiogenic and vasodilatory 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.

Myelin basic protein gene contains separate enhancers for oligodendrocyte and Schwann cell expression.

The DNA sequence between position +36 and -1907 of the murine myelin basic protein gene contains the enhancer and promoter elements necessary for abundant and cell specific expression in transgenic mice. Surprisingly, the pattern of expression promoted by this DNA fragment is a subset of that exhibited by the endogenous myelin basic protein (MBP) gene. Fusion genes prepared with this promoter/enhancer and a Lac Z reporter gene are expressed only in oligodendrocytes and not in Schwann cells, whereas the endogenous MBP gene is expressed in both cell types. The level of transgene expression measured by nuclear run-on experiments is very substantial and rivals that of the endogenous MBP gene. Furthermore, this 1.9-kb DNA fragment directs transcription on the same (or very similar) developmental schedule as the endogenous gene. These results indicate that the MBP promoter/enhancer sequences are at least tripartite: a core promoter, the oligodendrocyte enhancer elements, and a third component that either expands the specificity of the oligodendrocyte enhancer to include Schwann cells or acts independently to specifically stimulate transcription in Schwann cells.

Disrupted proteolipid protein trafficking results in oligodendrocyte apoptosis in an animal model of Pelizaeus-Merzbacher disease.

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.

Emergence of three myelin proteins in oligodendrocytes cultured without neurons.

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin-associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.

The initial events in myelin synthesis: orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes.

Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.

Age-related atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit.

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.

Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits.

Neurofilaments (NFs), the major intermediate filaments of central nervous system (CNS) and peripheral nervous system (PNS) neurons, are heteropolymers formed from the high (NFH), middle (NFM), and low (NFL) molecular weight NF subunits. To gain insights into how the expression of NF subunit proteins is regulated in vivo, two transgenes harboring coding sequences for human NFM (hNFM) with or without the hNFM multiphosphorylation repeat domain were introduced into mice. Expression of both hNFM constructs was driven by the hNFM promoter and resulted in increased levels of hNFM subunits concomitant with an elevation in the levels of mouse NFL (mNFL) proteins in the CNS of both lines of transgenic mice. The increased levels of mNFL appear specific to NFM because previous studies of transgenic mice overexpressing either NFL or NFH did not result in increased expression of either of the other two NF subunits. Further, levels of the most heavily phosphorylated isoforms of mouse NFH (mNFH) were reduced in the brains of these transgenic mice, and electron microscopic studies showed a higher packing density of NFs in large-diameter CNS axons of transgenic versus wild-type mice. Thus, reduced phosphorylation of the mNFH carboxy terminal domain may be a compensatory response of CNS neurons to the increase in NFs, and reduced negative charges on mNFH sidearms may allow axons to accommodate more NFs by increasing their packing density. Taken together, these studies imply that NFM may play a dominant role in the in vivo regulation of the levels of NFL protein, the stoichiometry of NF subunits, and the phosphorylation state of NFH. NFM and NFH proteins may assume similar functions in regulation of NF packing density in vivo.

Requirement of heavy neurofilament subunit in the development of axons with large calibers.

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H-null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H-null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.

Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content.

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.

Remarkable intron and exon sequence conservation in human and mouse homeobox Hox 1.3 genes.

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.

Antibody-induced modulation of rhabdovirus infection of neurons in vitro.

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.

Placental expression and chromosomal localization of the human Gcm 1 gene.

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 hGcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization.

Expression of myelin basic protein gene in the developing rat brain as revealed by in situ hybridization.

The developmental program controlling the expression of myelin basic protein (MBP) gene was studied in the rat using the technique of in situ hybridization. A 35S-labeled cDNA clone of mouse MBP encoding an amino acid sequence present in all four of the major forms of rodent MBP was used. The probe hybridized to the tracts of white matter with different intensities, depending on the age of the animal and the region of the brain examined. In the medulla oblongata, maximal hybridization was found in 5- and 7-day-old rats and was confined to the tectospinal tracts, fibers of the seventh cranial nerve, and the spinocerebellar tracts. By 12 days the amount of MBP mRNA had decreased in these areas. In the cerebrum, the greatest amount of MBP mRNA was observed in 17-day-old rats in the radiations of the corpus callosum. Thereafter, the levels decreased but could still be observed in the adult animals. Thus, using this technique, we have been able to demonstrate that the level of MBP-specific mRNA correlates closely with the development of myelin in different regions of the brain.

Myelin protein expression in the myelin-deficient rat brain and cultured oligodendrocytes.

The myelin-deficient (MD) rat does not express the major protein of CNS myelin, proteolipid protein (PLP). Here we further analyze whether this defect is reflected at the level of mRNA and whether the expression of other myelin proteins is affected in oligodendrocytes in vivo and in vitro. Both myelin basic protein (MBP) and PLP message levels were reduced in MD rats to 10-20% of the normal littermate controls, while the level of expression of an astrocyte-specific gene, glial fibrillary acidic protein (GFAP), was normal. Although MBP and PLP mRNAs were equally depressed, only MBP was detected with immunolabeling of corpus callosum, while PLP was absent in oligodendrocytes both in vivo and in vitro. A reduced number of MD rat oligodendrocytes express MBP in vitro compared to controls. The MD rat optic nerve contains normal numbers of 0-2A progenitors, but they tend to differentiate into GC-positive oligodendrocytes faster than oligodendrocytes from control littermates. In conclusion, the absence of PLP and reduced levels of MBP in the MD rats point to similarities with the jimpy mouse lesion. Moreover, the defect influences the expression of other myelin proteins and the oligodendrocyte developmental pathway.

Multiple nuclear factors interact with the promoter of the human neurofilament M gene.

In order to identify potential regulatory elements of the human mid-sized (M) neurofilament (NF) gene we preformed DNase I footprinting, gel mobility shift assays and methylation interference studies with probes from the NF(M) immediate 5' flanking region. These studies identified multiple sites for DNA-binding proteins including four Sp1 sites, and single sites each for members of the NF-1 and AP-1 families of DNA binding proteins. In addition a binding site within a pyrimidine tract likely binds a novel DNA-binding protein which also interacts with the human NF(H) gene promoter. Factors that bind to these sites are found in both neural and non-neural cells suggesting that the NF(M) promoter may not contain tissue specific regulatory signals. In transient assays, addition of these binding sites to an NF(M) minimal promoter containing only a TATA box lead to a greater than 40-fold activation of transcription over background. Progressive 5' deletions reduced expression in a step wise manner suggesting that all the factors likely act synergistically as positive regulators of transcription.

Age-dependent spatial memory deficits in transgenic mice expressing the human mid-sized neurofilament gene: I.

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. Immunohistological 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.

Enhancer trapping by a human mid-sized neurofilament transgene reveals unexpected patterns of neuronal enhancer activity.

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.

Novel DNA binding proteins participate in the regulation of human neurofilament H gene expression.

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 beta-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.

Expression of human mid-sized neurofilament subunit in transgenic mice.

We have created transgenic mice which carry and express the gene encoding the human NF(M) subunit. RNAase protection assays reveal that the transgene is abundantly expressed in CNS and PNS but also, at very low levels in some non-neural tissues as well. Although the neurospecificity of transgene transcription was not absolute, we are able to detect the protein only in neurons with immunocytochemical techniques. Glial and endothelial cells do not contain immunoreactive materials. Interesting subtle differences in the relative level of the human transgene encoded and endogenous murine encoded NF(M) proteins were noted in different regions of the brain. Similar differences were found in the levels of transgene and endogenous gene mRNA suggesting that these differences may be traceable to differences in RNA transcription or stability. Our data demonstrate, within the sensitivity of the immunocytochemical techniques we used, that the human NF(M) protein is present only in the neurons of the transgenic mice and that it is present in the same neurons as the endogenous NF(M). Furthermore, immunoelectron-microscopic examination of isolated neurofilaments shows that the human NF(M) coassembles with the endogenous NF(M) during filament formation. Thus, although the human NF(M) possesses a much larger multiphosphorylation site in its carboxy terminus, it seems to be the functionally equivalent to the mouse protein, even in the murine neuron.