Pregnancy modulates precursor cell proliferation in a murine model of focal demyelination.

In mice, pregnancy has been shown to have a beneficial effect on the endogenous repair of focal lysolecithin-induced CNS demyelinative lesions, enhancing the genesis of new oligodendrocytes and the degree of remyelination. To identify local cells undergoing mitosis in response to such lesions, we examined the time course of phospho-histone H3 (PH3) and myelin basic protein (MBP) expression by immunohistochemistry. After lysolecithin injection into the corpus callosum of virgin female mice, the number of dividing cells peaked about 48 h after injection and declined gradually to baseline by day 7; in pregnant mice, this initial peak was unchanged, but a new delayed peak on day 4 was induced. Colocalization data using PH3 and NG2 proteoglycan, or bromodeoxyuridine (BrdU) and oligodendrocyte transcription factor 1 (Olig1), suggested that about 75% of the proliferating cells on day 2, and about 40% of the cells on day 4, were likely of oligodendrocyte lineage; these differential percentages were of the same magnitude in both virgin and pregnant animals. Notably, the heightened proliferative response to focal lysolecithin injection during pregnancy was specific to gestational stage (early, but not late) and to lesion location (in the corpus callosum of the periventricular forebrain, but not in the caudal cerebellar peduncle of the hindbrain).

Educating the next generation of physicians about stroke: incorporating stroke prevention into the medical school curriculum.

BACKGROUND AND PURPOSE: In response to the need to educate physicians about stroke, we have implemented an educational program on stroke prevention for undergraduate medical students within the first-year neuroscience course. This study investigated whether first-year students learned and retained key information about stroke, and used students' feedback both to identify effective curricular components and to explore their attitudes regarding stroke prevention. METHODS: Stroke knowledge and self-assessed confidence in that knowledge before, immediately after, and 8 months after participation in the stroke curriculum were analyzed and compared for 3 classes, using paired t tests and repeated-measures ANOVA. Student feedback about the effectiveness of specific parts of the curriculum and about the importance of stroke prevention was solicited and evaluated. RESULTS: First-year medical students in 3 classes more than doubled their overall stroke knowledge scores (pretest total mean of 8.2; posttest mean 18.0), and retained significant improvement 8 months later (mean 15.7). Subscores in all 4 areas of stroke knowledge tested significantly increased (P<0.001). Students' confidence in their knowledge of stroke risk factors and warning signs, as well as in their knowledge itself, increased (P<0.001). Each of the 3 cohorts demonstrated similar improvements. Feedback indicated heightened awareness and interest in stroke prevention, which was maintained after completion of the curriculum. CONCLUSIONS: These results demonstrate that when instruction on stroke prevention is incorporated into the first-year curriculum, students learn and retain key information. Because entire classes of medical students are involved, this type of approach has the potential to reach all future physicians and therefore to meaningfully impact future stroke care.

Towards the reconstruction of central nervous system white matter using neural precursor cells.

Epidermal growth factor-responsive neural precursor cells were used as donor cells for transplantation into wild-type and myelin-deficient shiverer (shi) mice. The cells engrafted robustly within the CNS following intracerebroventricular and cisternal transplantation in neonatal mice. The cells adopted glial phenotypes, and some functioned as oligodendrocytes, producing myelin basic protein and morphologically normal internodal myelin sheaths. When individual shi mice received two transplants (on post-natal days 1 and 3), donor-derived cells disseminated widely and expressed myelin basic protein in central white matter tracts throughout the brain.

Rumpshaker behaves like juvenile-lethal Plp mutations when combined with shiverer in double mutant mice.

The phenotypes of double mutant mice whose genomes are homozygous for an Mbp (myelin basic protein) mutation and hemizygous for a juvenile-lethal Plp (proteolipid protein) mutation were compared in earlier studies. The results suggested that the shiverer Mpb mutation might have some unexplained ability to partially rescue oligodendrocytes (OLs) from the 'death sentence' that is imposed by the Plp mutations. Conversely, they also indicated that the juvenile-lethal Plp mutations may normalize shiverer OL morphology by reducing the numbers of microprocesses. The Plp mutation rumpshaker produces a mild hypomyelination without reduction in OL numbers and a normal lifespan. This report describes double mutant mice combining two Mbp mutations with rumpshaker, utilizing a common B6C3F1 hybrid-based genetic background. Initial studies on B6C3F1 rumpshaker optic nerve and spinal cord white matter showed unanticipated signs of OL death, with morphologic criteria suggestive of an apoptotic mechanism. In shiverer*rumpshaker double mutant mice, this small class of dying cells could not be identified. White matter morphology was similar to that of mice expressing only the shiverer mutation, except that OL microprocesses were far less abundant. This evidence suggests that, despite their distinctive phenotypic differences, rumpshaker may share more characteristics with the juvenile-lethal Plp mutations than has previously been recognized.

Evidence that CNS hypomyelination does not cause death of jimpy-msd mutant mice.

Mice expressing three of the proteolipid protein (Plp) mutations in the mouse (jimpy, jimpy-msd, and jimpy-4J) all have a severe deficiency of CNS myelin and oligodendrocytes (OLs), and die sometime in their 4th postnatal week. The prevailing view has been that the animals' shortened life span and lack of myelin are causally related. Here we describe the survival of jimpy-msd males for as long as postnatal day (P) 210. Although these spontaneously occurring longer-lived jimpy-msd males show a 2- to 8-fold increase in numbers of myelinated axons in many CNS regions, this does not protect them from a later but still premature death. Investigating the cause of premature death may reveal previously undiscovered properties of the myelin genes or the cells that express them, or perhaps additional unsuspected cellular responses that contribute to the disease. This study identifies small accumulations of inflammatory cells in the brain parenchyma of jimpy-msd mice as young as P14 and as old as P60, suggesting that the pathology of the disease produced by at least this Plp mutation may be far more complex than has been previously recognized.

Quaking*shiverer double-mutant mice survive for at least 100 days with no CNS myelin.

Mice expressing mutations that produce CNS hypomyelination often die prematurely: the more severe the hypomyelination, the shorter the life span. However, we have previously described jimpy-msd mice that survive twice as long as usual; although they acquire significantly increased amounts of myelin, they still succumb long before their unaffected littermates. This result contradicts any postulated causal relationship between extent of CNS hypomyelination and premature death of the animal. Here we have addressed this question in another way, by using an animal model that does not involve a proteolipid protein (Plp) gene mutation. We demonstrate that quaking*shiverer double-mutant mice can survive for at least 100 days without any CNS myelin whatsoever. Therefore, at least for a mouse, absence of CNS myelin is not lethal per se.

Jimpy-4J mouse has a missense mutation in exon 2 of the Plp gene.

We previously showed that the jimpy-4J mouse mutation is located on the X chromosome, in or closely linked to the proteolipid protein (Plp) gene. The phenotype is characterized by the most severe hypomyelination of any of the naturally occurring myelin mutant mice, sharp reduction in oligodendrocyte number, and virtual absence of PLP protein. Affected animals show tremor, seizures, and die at about 24 postnatal days. We now report that sequencing of Plp genomic and cDNAs identifies a single nucleotide substitution in exon 2 that predicts an Ala38Ser substitutions in a hydrophilic region of PLP/DM20 protein close to a transmembrane domain. This mutation occurs in a very different region of the mouse Plp gene than that jimpy-msd mutations, yet all three produce qualitatively similar phenotypes.

Regulation of cytoskeleton by myelin components: studies on shiverer oligodendrocytes carrying an Mbp transgene.

Oligodendrocytes from the shiverer mutant mouse are missing most of the myelin basic protein (Mbp) gene. In axon-free cultures, they produce membrane sheets with abnormally assembled microtubule and actin-based structures. This suggests that an Mbp gene product may have an important role in regulating the organization and stability of the wild-type oligodendrocyte cytoskeleton. We now present evidence extending these observations, using cultured oligodendrocytes that carry both the shiverer mutation and the Mbp1 transgene which partially corrects their deficit. Shiverer oligodendrocytes that carry one dose of the Mbp1 transgene abnormally express MBP along major cytoskeletal vein-like structures in processes and sheets. Shiverer oligodendrocytes that carry two doses of the Mbp1 transgene contain two types of membrane sheet regions, i.e. regions filled with aberrant punctate foci of MBP, and regions with normal domains of MBP. Immunocytochemical staining data show that the distribution of cytoskeleton and associated 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) is dependent upon how MBP is organized. Bundling of actin filaments occurs only around MBP domains, and the colocalization of CNPase along microtubular structures also appears to be regulated by MBP domains in sheets. Multinucleated oligodendrocytes are observed, a likely result of the inability of dividing pro-oligodendrocytes to bundle actin filaments. In addition, the ability of MBP to mediate extracellular signals that modulate cytoskeleton appears to be dependent upon MBP's organization. Transduction of the galactocerebroside signaling pathway, which results in the destabilization of microtubules but not actin filaments, occurs only in sheets containing MBP domains. The distribution of MBP, however, does not affect the myelin/oligodendrocyte-specific protein signaling pathway, which results in growth of microtubular structures and extensive destabilization of the actin cytoskeleton.

Cytoskeleton in myelin-basic-protein-deficient shiverer oligodendrocytes.

Production of CNS myelin by oligodendrocytes requires the regulated synthesis and assembly of cytoskeletal components. However, the molecular signals that mediate this process are not known. Here we use the shiverer mutant mouse, which is missing a large segment of the myelin basic protein (MBP) gene, to investigate the possible role in cytoskeletal assembly of an MBP gene product or of other myelin components whose expression may be linked to that of MBP. In axon-free cultures, we find that approximately half of shiverer oligodendrocytes have enlarged cell bodies, abnormal processes and fail to elaborate extensive membrane sheets. In those membrane sheets that are elaborated by shiverer oligodendrocytes, microtubular structures are abnormal in size and distribution. Additionally, 2',3'-cyclic nucleotide 3'-phosphohydrolase and microfilaments are not colocalized with microtubular structures as they would be in mature wild-type membrane sheets. These observations suggest that an MBP gene product has a direct or indirect role in regulating various aspects of cytoskeleton assembly in wild-type oligodendrocytes. In the absence of this signal, oligodendrocytes apparently do not normally assemble cytoskeleton; this may be one important basis for the abnormal morphology of intact shiverer CNS.

Jimpy 4J: a new X-linked mouse mutation producing severe CNS hypomyelination.

This study describes a new sex-linked myelin mutation in the mouse, jimpy 4J (Plpjp-4J), located in or very close to the proteolipid protein (Plp) gene. The Plpjp-4J/Y phenotype includes tremor, seizures, death during the 4th postnatal week, and the most severe central nervous system hypomyelination yet described in any mouse carrying a single myelin mutation. The few myelin sheaths are present in early myelinating areas where they form clusters of thin, usually loosely wrapped membranes which show several variations of morphology at their extracellular leaflets. Numbers of mature oligodendrocytes are sharply reduced; pycnotic glial nuclei and foamy cells are numerous. Astrocytosis is a prominent feature. No PLP protein is detected by immunoblotting in Plpjp-4J/Y brain but in spinal cord a faint band is present. Myelin basic protein and characteristic myelin lipids are also sharply reduced in both brain and spinal cord. Despite the qualitative similarity of the phenotypes reported in these and previous studies, DNA analysis demonstrate that Plpjp-4J is not a recurrence of the well known Plp mouse mutations jimpy (Plpjp) or myelin synthesis deficiency (Plpjp-msd).

Myelin basic protein mediates extracellular signals that regulate microtubule stability in oligodendrocyte membrane sheets.

Treatment of cultured oligodendrocytes with a monoclonal antibody to galactocerebroside (GalC) triggers a cascade of events including the redistribution of membrane surface GalC over internal domains of MBP and loss of microtubular structures within the sheets (Dyer and Benjamins: J Neurosci 8:4307-4318, 1988; Dyer and Benjamins: J Neurosci Res 24:212-221, 1989). In this report, wild type and myelin basic protein (MBP)-deficient shiverer oligodendrocytes were used to study the possible relationships between these events, and specifically to determine if MBP mediates signals which destabilize microtubular assemblies in cultured oligodendrocytes. We now show that MBP and GalC, which are both initially Triton X-100 soluble, become Triton X-100 insoluble following anti-GalC binding and anti-GalC:GalC complex redistribution, suggesting that the surface anti-GalC: GalC complexes become associated with cytoplasmic MBP. Mediation of the signaling event by MBP is further demonstrated by 1) a decreased phosphorylation of MBP in wild type oligodendrocytes after antibody binding, and 2) the absence of responses, such as GalC redistribution and microtubule loss, in MBP-deficient shiverer oligodendrocytes treated with anti-GalC. Continuous activation of the GalC/MBP pathway for 7 days in wild type oligodendrocytes results in enlarged cell bodies and production of numerous microprocesses, a morphology that is similar to MBP-deficient shiverer oligodendrocytes. A second signaling pathway which produces an opposite effect, i.e., the stabilization and apparent up-regulation of microtubular structures in cultured oligodendrocyte membrane sheets, remains functional in shiverer oligodendrocytes. Thus, MBP appears to be important for mediating extracellular signals that cause a loss of microtubular structures in oligodendrocyte membrane sheets and abnormal morphology.

Trembler mouse carries a point mutation in a myelin gene.

The autosomal dominant trembler mutation (Tr), maps to mouse chromosome 11 (ref. 2) and manifests as a Schwann-cell defect characterized by severe hypomyelination and continuing Schwann-cell proliferation throughout life. Affected animals move clumsily and develop tremor and transient seizures at a young age. We have recently described a potentially growth-regulating myelin protein, peripheral myelin protein-22 (PMP-22; refs 7, 8), which is expressed by Schwann cells and found in peripheral myelin. We now report the assignment of the gene for PMP-22 to mouse chromosome 11. Cloning and sequencing of PMP-22 complementary DNAs from inbred Tr mice reveals a point mutation that substitutes an aspartic acid residue for a glycine in a putative membrane-associated domain of the PMP-22 protein. Our results identify the PMP-22 gene as a likely candidate for the mouse trembler locus and will encourage the search for mutations in the corresponding human gene in pedigrees with hypertrophic neuropathies such as Charcot-Marie-Tooth and Dejerine-Sottas diseases (hereditary motor and sensory neuropathies I and III).

Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization.

We have localized myosin heavy chain (MHC) mRNAs in cells of intact embryonic chick muscle using high resolution in situ hybridization. Blocks of muscle were aldehyde-fixed prior to detergent treatment and hybridized with a biotinated cDNA probe, followed by colloidal gold-labeled antibodies, before embedment. Labeling was determined to represent MHC mRNA by extensive quantitative comparisons of electron micrographs from experimental and four different types of control samples. MHC mRNA was localized primarily to peripheral regions of 14-day chick pectoral muscle cells, where the majority of developing myofibrils were found. MHC mRNAs were consistently associated with the nonmyofibrillar cytoskeletal filaments which had diameters ranging from 4 to 10 nm. They were often oriented parallel to the longitudinal axis of the cell. The resolution of the ultrastructural approach allowed us to demonstrate that the mRNA molecules visualized were not directly associated with myofilaments, suggesting that nascent chains read from those messages do not assemble directly into myofilaments simultaneous with translation.

Shiverer jimpy double mutant mice. V. Correlation of genotype and myelin proteins.

We have reexamined the levels of myelin basic protein (MBP) and proteolipid protein (PLP) in the brains of mice bred to carry both the shi/shi and jp/Y hypomyelination defects. The genotype of each putative double mutant was confirmed by direct DNA analysis: shi/shi by Southern blot analysis, and jp/Y by restriction enzyme analysis of polymerase chain reaction-amplified fragments. MBP and PLP levels were assessed by immunoblotting. All putative double mutants were found to be shi/shi. However, examination of the PLP locus revealed both jp and wild-type genotypes, the latter produced by an expected crossover. Animals proven to be shi/shi*jp/Y had no detectable MBP or PLP; those proven to be shi/shi*+/Y (the crossover) had no MBP but had PLP. These results differ from an earlier report of both MBP and PLP in the brains of presumed shi*jp animals.

Quantitative differences between homozygous 'USA' and 'Swiss' mld mutant mice.

Parallel developmental studies of central nervous system myelin proteins and morphology (postnatal days 15-118; P15-118) confirm qualitative similarities but substantial quantitative differences between homozygous mld mice with Billings-Gagliardi and Wolf's 'USA' versus Matthieu's 'Swiss' genetic backgrounds. The USA mld/mld have fewer convulsions and significantly longer life span. While whole-brain homogenates from both Swiss and USA mld/mld show increases in myelin basic protein (MBP) and in 2',3'-cyclic nucleotide 3'-phosphohydrolase specific activity with age, at P50 and older the levels of both proteins are approximately twice as high in the Swiss. The number of optic nerve axons myelinated is always greater in Swiss mld/mld, and they have approximately twice as many myelin sheaths showing any apposition of cytoplasmic membrane faces (the location of the major dense line in normal myelin), except at the youngest age. Evidence is presented which suggests that these quantitative differences between Swiss and USA mld stocks most likely reflect different regulatory genes influencing the expression of the same (mld) allele, rather than the presence of a different allele at the MBP locus.