Myelinated, synapsing cultures of murine spinal cord--validation as an in vitro model of the central nervous system.

Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

Genetic background influences UPR but not PLP processing in the rumpshaker model of PMD/SPG2.

Mutations of the proteolipid protein gene (PLP1) cause Pelizaeus-Merzbacher disease (PMD) and Spastic paraplegia type 2 (SPG2). The rumpshaker mutation is associated with mild forms of PMD or SPG2 in man and the identical mutation occurs in mice, the phenotype depending on genetic background. The mild phenotype in C3H mice becomes a lethal disease when expressed on the C57BL/6 background. rumpshaker PLP is synthesised at a similar rate to wild type but is rapidly degraded by the proteasome. We show that the rates of synthesis, degradation and myelin incorporation of PLP/DM20 are similar in mutants on both backgrounds and therefore differences in PLP processing are unlikely to be the basis of the phenotypic variation. An unfolded protein response (UPR) is activated in rumpshaker. Whereas activation of CHOP correlates with phenotypic severity, we find no difference in the response of BiP and X-box protein1 (Xbp1) between the two strains.

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-mediated excitotoxic axonal damage is attenuated in the absence of myelin proteolipid protein.

In vivo and in vitro studies have shown that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated excitotoxicity causes cytoskeletal damage to axons. AMPA/kainate receptors are present on oligodendrocytes and myelin, but currently there is no evidence to suggest that axon cylinders contain AMPA receptors. Proteolipid protein (PLP) and DM20 are integral membrane proteins expressed by CNS oligodendrocytes and located in compact myelin. Humans and mice lacking normal PLP/DM20 develop axonal swellings and degeneration, suggesting that local interactions between axons and the oligodendrocyte/myelin unit are important for the normal functioning of axons and that PLP/DM20 is involved in this process. To determine whether perturbed glial-axonal interaction affects AMPA-receptor-mediated axonal damage, AMPA (1.5 nmol) was injected into the caudate nucleus of anesthetized Plp knockout and wild-type male mice (n = 13). Twenty-four hours later, axonal damage was detected by using neurofilament 200 (NF 200) immunohistochemistry and neuronal damage detected via histology. AMPA-induced axonal damage, assessed with NF 200 immunohistochemistry, was significantly reduced in Plp knockout mice compared with wild-type mice (P = 0.015). There was no significant difference in the levels of neuronal perikaryal damage between the Plp knockout and wild-type mice. In addition, there was no significant difference in the levels of glutamate receptor subunits GluR1-4 or KA2 in Plp knockout compared with wild-type littermates. The present study suggests that PLP-mediated interactions among oligodendrocytes, myelin, and axons may be involved in AMPA-mediated axonal damage.

Age-related axonal and myelin changes in the rumpshaker mutation of the Plp gene.

The PLP1/Plp gene encodes proteolipid protein (PLP) and DM20, the major central nervous system myelin proteins. Mutations in the PLP1/ Plp gene cause dysmyelinating disorders in man and animals. The rumpshaker mutation was first identified in mice and later linked to a family diagnosed with neurological deficits akin to spastic paraplegia. The dysmyelination in the young rumpshaker mouse is well characterised. Here we report evidence for an age-related increase in myelin due mainly to the myelination of small axons, many large axons remain dysmyelinated. Levels of PLP/DM20 and myelin basic protein are considerably greater in myelin fractions from older compared with younger mutants. Myelin in sheaths of larger axons remains poorly compacted and may account for levels of 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein being elevated over wild type in older mutant mice. A late-onset distal degeneration of the axons of the longest spinal tract, the fasciculus gracilis, is also noted. This is the first report of Wallerian-type degeneration in mice with spontaneous mutations of the Plp gene.

Genetic background determines phenotypic severity of the Plp rumpshaker mutation.

The rumpshaker mutation of the proteolipid protein (Plp) gene causes dysmyelination in man and mouse. We show that the phenotype in the mouse depends critically on the genetic background in which the mutation is expressed. On the C3H background there is normal longevity whereas changing to a C57BL/6 strain results in seizures and death at around postnatal day 30. The more severe phenotype is associated with less myelin and reduced levels of major myelin proteins. There are also more apoptotic cells, including oligodendrocytes, increased numbers of proliferating cells, increased numbers of NG2+ oligodendrocyte progenitors and increased microglia compared to the milder phenotype. The number of mature oligodendrocytes is similar to wild-type in both strains of mutant, however, suggesting that increased oligodendrocyte death is matched by increased generation from progenitors. The dichotomy of phenotype probably reflects the influence of modifying loci. The localization of these putative modifying genes and their mode of action remain to be determined.

Phenotypic analysis of mice deficient in the major myelin protein MOBP, and evidence for a novel Mobp isoform.

Myelin-associated oligodendrocytic basic protein (MOBP) is a recently identified major component of central nervous system (CNS) myelin. We previously reported a detailed characterization of the genomic region encompassing the Mobp gene, elucidating the complex series of transcript splicing responsible for the generation of its diverse family of protein isoforms. These basic, positively charged polypeptides display spatial and temporal expression patterns consistent with a potential role in the compaction and maintenance of the myelin sheath. MOBP isoforms have also been localized to the nucleus and the microtubular network of oligodendrocytes; transcript corresponding to one isoform is present during embryonic development. Recent reports have identified a role for this protein family in the pathogenesis of multiple sclerosis, but a clear function for the wild-type protein has remained unclear. We report a detailed analysis of a targeted mutation of Mobp, which results in the deletion of the translational start site and most of the coding sequence of MOBP, and the deletion of the entire coding sequence corresponding to a novel, putative MOBP isoform. Our analyses clearly demonstrate that MOBP-deficient mice develop normally, generate intact compact CNS myelin, and demonstrate no obvious clinical phenotype. Furthermore, in contrast with another recent study, we find that Mobp null mice demonstrate no significant influence on the axonal diameter of myelinated axons. Although MOBP is not essential for myelination, it appears that its absence is not simply compensated for by increased expression of the "classic" myelin basic protein (MBP).

Survival of, and competition between, oligodendrocytes expressing different alleles of the Plp gene.

Mutations in the X-linked Plp gene lead to dysmyelinating phenotypes and oligodendrocyte cell death. Here, we exploit the X inactivation phenomenon to show that a hierarchy exists in the influence of different mutant Plp alleles on oligodendrocyte survival. We used compound heterozygote mice to study the long-term fate of oligodendrocytes expressing either the jimpy or rumpshaker allele against a background of cells expressing a Plp-null allele. Although mutant and null oligodendrocytes were generated in equal numbers, the proportion expressing the mutant allele subsequently declined, but whereas those expressing the rumpshaker allele formed a reduced but stable population, the number of jimpy cells fell progressively. The age of decline in the jimpy cells in different regions of the CNS correlated with the temporal sequence of myelination. In compound heterozygotes expressing rumpshaker and jimpy alleles, oligodendrocytes expressing the former predominated and were more abundant than when the rumpshaker and null alleles were in competition. Thus, oligodendrocyte survival is not determined solely by cell intrinsic factors, such as the conformation of the misfolded PLP, but is influenced by neighboring cells, possibly competing for cell survival factors.

Evidence for possible interactions between PLP and DM20 within the myelin sheath.

PLP and its smaller DM20 isoform constitute the major proteins of CNS myelin. Previous studies indicated a role for the proteins in maintaining the intraperiod line of the myelin sheath and the integrity of axons and suggested that both isoforms were necessary to provide these functions. The present study shows that each isoform is capable individually of inserting into compact myelin. Employing chromatographic extraction procedures designed to maintain the natural conformation of the proteins we found that most PLP and DM20 remained associated. Using an antibody specific to the PLP isoform, we were able to co-immunoprecipitate DM20 from the major fraction of the extracted equine myelin and from mouse native whole myelin. We suggest that PLP and DM20 may form a hetero-oligomeric complex within the myelin sheath, probably in association with specific lipids and that this arrangement is essential for the normal structure of myelin and axons.

Serum alpha-tocopherol concentrations in German shepherd dogs with chronic degenerative radiculomyelopathy.

The concentration of serum alpha-tocopherol was measured in German shepherd dogs with chronic degenerative radiculomyopathy, and in German shepherd dogs and dogs of other breeds unaffected by the condition. The mean concentration was significantly higher in German shepherd dogs with the condition than in other breeds of dog unaffected by it, but it was not significantly higher than in unaffected German shepherd dogs. Estimates of components of variance indicated that the concentration varied more widely in individual affected dogs than in unaffected dogs, irrespective of breed. These results suggest that chronic degenerative radiculomyopathy in German shepherd dogs is unlikely to be due to uncomplicated vitamin E deficiency.

Myelin proteolipid proteins promote the interaction of oligodendrocytes and axons.

Although proteolipid protein (PLP) and its DM20 isoform are the major membrane proteins of CNS myelin, their absence causes surprisingly few developmental defects. In comparison, missense mutations of the X-linked Plp gene cause severe dysmyelination. Previous studies have established roles for PLP/DM20 in the formation of the intraperiod line and in maintaining axonal integrity. We now show that a normal number of oligodendrocytes are present in mice lacking PLP/DM20. However, in heterozygous females, which are natural chimeras for X-linked genes, oligodendrocytes lacking PLP/DM20 are in direct competition with wild-type oligodendrocytes that have a distinct advantage. PLP+ oligodendrocytes and PLP+ myelin sheaths make up the greater majority, and this feature is generalised in the CNS throughout life. Moreover, in the absence of PLP/DM20, a proportion of small-diameter axons fails to myelinate, remaining ensheathed but lacking a compact sheath, or show delayed myelination. These findings suggest that PLP/DM20 is also involved in the early stages of axon-oligodendrocyte interaction and wrapping of the axon.

Imprinting as a rapid technique for assessing the morphology of the central nervous system by immunofluorescence.

This paper describes a technique that has been developed to assess the in vivo morphology of central nervous system (CNS) tissue by immunofluorescence. This technique permits the study of tissue that is mainly just a monolayer of cells. Unlike routine cryosections that are much thicker (10-15 microm), imprinting does not section the cells, but can result in the detachment of whole cells onto a glass surface for subsequent staining. The imprinting technique is simple and rapid and does not require prior fixation or embedding of the tissue. It has been used to evaluate antigens expressed at the cell surface, in myelin and in the cytoskeleton in the studies of normal and myelin mutant mice. Using the imprinting/immunofluorescence technique one can now assay the genotype of mouse strains that differ in their expression of cell surface antigens within 2 h.

Peripheral demyelination and neuropathic pain behavior in periaxin-deficient mice.

The Prx gene in Schwann cells encodes L- and S-periaxin, two abundant PDZ domain proteins thought to have a role in the stabilization of myelin in the peripheral nervous system (PNS). Mice lacking a functional Prx gene assemble compact PNS myelin. However, the sheath is unstable, leading to demyelination and reflex behaviors that are associated with the painful conditions caused by peripheral nerve damage. Older Prx-/- animals display extensive peripheral demyelination and a severe clinical phenotype with mechanical allodynia and thermal hyperalgesia, which can be reversed by intrathecal administration of a selective NMDA receptor antagonist We conclude that the periaxins play an essential role in stabilizing the Schwann cell-axon unit and that the periaxin-deficient mouse will be an important model for studying neuropathic pain in late onset demyelinating disease.

Central nervous system pathology in 25 dogs with chronic degenerative radiculomyelopathy.

The neuropathology of 20 German shepherd dogs and five German shepherd dog crosses with chronic degenerative radiculomyelopathy were analysed by conventional techniques, immunocytochemistry and electron microscopy. There were previously unrecognised changes in brain nuclei. In the spinal cord, both motor and sensory tracts were involved, principally in their more distal regions. Wallerian degeneration affected the corticorubrospinal pathways in the lateral columns and the ventral funiculi, predominantly in the caudal thoracic and lumbar segments, although more cranial involvement was also observed. The dorsal columns were affected in the caudal lumbar region and the cervical fasciculus gracilis. The regional distribution was variable between cases. Within the brain, abnormalities, including chromatolysis, gliosis and neuronal loss were observed in the red nucleus, lateral vestibular nucleus and, occasionally, in the dentate nucleus. The changes in brain nuclei were compared with those found in dogs at various times after a focal spinal injury. The neuronal changes in the brain may be related to the primary site of damage, and possible aetiological mechanisms are discussed.

Uncoupling of myelin assembly and schwann cell differentiation by transgenic overexpression of peripheral myelin protein 22.

We have generated previously transgenic rats that overexpress peripheral myelin protein 22 (PMP22) in Schwann cells. In the nerves of these animals, Schwann cells have segregated with axons to the normal 1:1 ratio but remain arrested at the promyelinating stage, apparently unable to elaborate myelin sheaths. We have examined gene expression of these dysmyelinating Schwann cells using semiquantitative reverse transcription-PCR and immunofluorescence analysis. Unexpectedly, Schwann cell differentiation appears to proceed normally at the molecular level when monitored by the expression of mRNAs encoding major structural proteins of myelin. Furthermore, an aberrant coexpression of early and late Schwann cell markers was observed. PMP22 itself acquires complex glycosylation, suggesting that trafficking of the myelin protein through the endoplasmic reticulum is not significantly impaired. We suggest that PMP22, when overexpressed, accumulates in a late Golgi-cell membrane compartment and uncouples myelin assembly from the underlying program of Schwann cell differentiation.

A proteolipid protein-specific pre-mRNA (Ppm-1) contains intron 3 and is up-regulated during myelination in the CNS.

Alternative splicing of the precursor for messenger RNA (pre-mRNA) is a common process utilised by higher eukaryotes to modulate gene expression. A single primary transcript may generate several proteins with distinct functions, expressed in tissue-specific, developmental patterns. This article describes an oligodendrocyte-specific pre-mRNA product of proteolipid protein gene (P/p) transcription, which is the precursor for P/p but not Dm20 mRNA in the CNS. This P/p-specific pre-mRNA (Ppm-1) includes the intact intron 3 of the P/p gene. It is first expressed during active myelination, and it localises to the nucleus of oligodendrocytes, in both normal and jimpy (jp) murine CNS. In addition to mouse, Ppm-1 is found also in rat and dog, but not toad or trout. Our work suggests that alternative splicing of the P/p gene primary transcript follows a branching pattern, resulting in the presence of at least one P/p isoform-specific pre-mRNA molecule, Ppm-1. Therefore, Dm20 mRNA may be the product of a divergent set of pre-mRNA splicing events.

Distinct phenotypes associated with increasing dosage of the PLP gene: implications for CMT1A due to PMP22 gene duplication.

Increased dosage of the proteolipid protein (Plp) gene causes CNS disease (Pelizaeus-Merzbacher disease [PMD]), which has many similarities to disorders of the PNS associated with duplication of the peripheral myelin protein-22 (PMP22) gene locus. Transgenic mice carrying extra copies of the wild-type Plp gene provide a valid model of PMD. Variations in gene dosage can cause a wide range of phenotypes from severe, lethal dysmyelination through late-onset demyelination. A predilection for different fiber diameters may occur within the various phenotypes with dysmyelination being more obvious in large fibers and late-onset degeneration predominantly affecting small fibers. Although the frequency of apoptotic oligodendrocytes is increased with high gene dosage, the number of mature oligodendrocytes appears adequate. Oligodendrocytes in the dysmyelinated CNS express a range of genes typical of mature cells, yet are unable to assemble sufficient myelin. Oligodendrocytes contain abnormal vacuoles and stain intensely for PLP and other proteins such as MAG. The findings suggest that with high gene dosage much of the PLP, and possibly other proteins, is missorted and degraded in the lysosomal system.

The "CMT rat": peripheral neuropathy and dysmyelination caused by transgenic overexpression of PMP22.

We have generated a transgenic rat model of Charcot-Marie-Tooth disease type 1A (CMT1A) providing formal proof that this neuropathy can be caused by increased expression of peripheral myelin protein-22 (PMP22). Heterozygous PMP22-transgenic rats develop muscle weakness and gait abnormalities as well as reduced nerve conduction velocities and EMG abnormalities, which closely resemble recordings in patients with CMT1A. Dys- and demyelination, Schwann cell hypertrophy, and "onion bulb" formation are also similar to findings in humans. When bred to homozygosity, transgenic rats completely fail to elaborate myelin, but all myelin-forming Schwann cells segregate with axons in the normal one-to-one ratio. Although arrested at this "promyelin" stage, differentiation proceeds in homozygous rats at the molecular level, as demonstrated by high-level expression of myelin structural genes. Intracellular trafficking of the wild-type protein is not visibly impaired, even when strongly overexpressed, suggesting that PMP22 blocks myelin assembly in a late Golgi/cell membrane compartment of the affected Schwann cell.

Subdural injection of contrast medium as a complication of myelography.

In a retrospective evaluation of 654 canine and feline myelograms, 58 were found to have been complicated by injection of the contrast medium into the subdural space. The medium was present predominantly dorsal to the spinal cord, with a sharp dorsal border and an undulating ventral border. Confirmation that this myelographic appearance was due to subdural localisation was achieved using fresh cadavers injected with contrast medium labelled with Indian ink. It was further showed that the dorsal accumulation of contrast medium was due to the denticulate ligaments which restricted the ventral extension of the contrast medium.

Splicing pattern, transcript start distribution, and DNA sequence of the mouse gene (Mobp) encoding myelin-associated oligodendrocytic basic protein.

We have cloned the mouse gene Mobp, encoding the family of myelin-associated oligodendrocytic basic proteins (MOBP), to facilitate elucidation of its genomic organization and regulation. We report near complete sequence analysis of the Mobp gene (>11 kb), including complete sequence of all exons and their associated splice junctions. The Mobp gene comprises eight discrete exons and encompasses a genomic region in excess of 15 kb. We provide a definitive analysis of the alternative splicing events and exon usage required in the generation of the reported splice variants of Mobp transcripts. We identify sequences corresponding to the coding regions of all reported protein isoforms. Consequently, we demonstrate that sequence regions, predicted to encode unique portions of two putative protein isoforms in the rat (MOBP 71 and MOBP 99), are not fully conserved between the rat and the mouse: we predict that the mouse equivalents are two distinct polypeptides of 73 amino acids, MOBP73A and MOBP73B, respectively. We have analyzed sequence from 63 oligo-capped, cloned cDNA fragments and identify six transcription start points associated with the Mobp gene at postnatal day 26. This study provides the platform for a more detailed analysis of the function of the Mobp gene product and subsequent evaluation of its possible involvement in known neuropathies.