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.

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.

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.

The early phenotype associated with the jimpy mutation of the proteolipid protein gene.

The jimpy mutation of the X-linked proteolipid protein (Plp) gene causes dysmyelination and premature death of the mice. The established phenotype is characterised by severe hypomyelination, increased numbers of dead oligodendrocytes and astrocytosis. The purpose of this study was to define the earliest cellular abnormalities in the cervical spinal cord. We find that on the first and third postnatal days the amount of myelin in jimpy spinal cord is approximately 20% of wild-type. However, the total glial cell density, the number of dead glial cells and the number and distribution of Plp-positive cells, as assessed by in situ hybridization, are similar to wild-type during the first week of life. Immunostaining of cryosections has identified that jimpy spinal cords express on schedule, a variety of antigens associated with mature oligodendrocytes. Dissociated oligodendrocytes, cultured for 18 hours to reflect their in vivo differentiation, express MBP and surface myelin-associated glycoprotein at the same frequency as wild-type. By comparison, the proportion of jimpy oligodendrocytes expressing surface myelin/oligodendrocyte glycoprotein is reduced by approximately 34%. In vivo, however, only a small minority of axons is surrounded by a collar of myelin-associated glycoprotein, suggesting that the majority of jimpy oligodendrocytes fail to make appropriate ensheathment of axons. Although the DM20 isoform is expressed in the embryonic CNS prior to myelin formation, the cellular abnormalities appear to correspond to the time at which the Plp isoform becomes predominant. The results suggest that the primary abnormality in jimpy is the inability of oligodendrocytes to properly associate with, and then ensheath, axons and that oligodendrocyte death compounds, rather than initiates, the established phenotype.

Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene.

Mutations of the proteolipid protein (Plp) gene cause a generalized central nervous system (CNS) myelin deficit in Pelizaeus-Merzbacher disease of man and various tremor syndromes in animal models. X-linked spastic paraplegia is also due to Plp gene mutations but has a different clinical profile and more restricted pathology involving specific tracts and regions. We have shown previously that PLP overexpression in mice homozygous for a Plp transgene results in premature arrest of CNS myelination and premature death. Here, we demonstrate that a low-level increase in Plp gene expression in transgenic mice causes significant axonal degeneration and demyelination with predilection for specific tracts. Following normal motor development, aged mice develop progressive myelin loss, axonal swellings with resultant Wallerian degeneration, and marked vacuolation of the neuropil associated with ataxia, tremor, and seizures. The age of onset and severity of the phenotype is a function of Plp gene dosage. The corticospinal tracts, optic nerve, fasciculus gracilis cerebellum, and brainstem are particularly involved. Although oligodendrocyte cell bodies show little abnormality, their inner adaxonal tongue is often abnormal, suggesting a perturbation of the axon/glial interface that may underlie the axonal changes. We conclude that abnormal expression of an oligodendrocyte-specific gene can cause axonal damage, a finding that is relevant to the pathogenesis of PLP-associated disorders and probably to other myelin-related diseases.

Hindshaker, a novel myelin mutant showing hypomyelination preferentially affecting the spinal cord.

Animals with spontaneous mutations affecting myelin formation have provided useful information about the genetic and cellular mechanisms regulating normal and abnormal myelination. In this paper we describe a novel murine mutation termed hindshaker (hsh), which is inherited in an autosomal recessive manner. Affected mice are characterised by a variable tremor of the hind end which commences at about 2 weeks of age and largely disappears in animals older than 6 weeks. There is hypomyelination affecting predominantly the spinal cord, although the optic nerves and brain are involved to a much lesser degree. The defect of thinly myelinated and naked axons is maximal at 20 days of age and largely resolves with time so that in the adult most axons are myelinated. The myelin structure appears normal and immunostains for the major proteins. Although the distribution of oligodendrocytes in the spinal cord is similar to normal during the period of hypomyelination, there are fewer mature cells. The hsh mutation appears to delay the maturation of oligodendrocytes, particularly in the spinal cord. Additionally, there is a considerable variation in phenotypic expression and in penetrance when the mutation is expressed on different genetic backgrounds, suggesting the hsh locus is subject to the influence of modifying gene(s). Identification of the hsh gene should identify a factor important in the development of oligodendrocytes, particularly those in the spinal cord.

In vitro studies of axonally-regulated Schwann cell genes during Wallerian degeneration.

Wallerian degeneration in vivo is associated with marked downregulation of myelin protein genes such as P(o) and upregulation of other genes such as nerve growth factor receptor (NGF-R), glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (N-CAM). This study examines the expression of these genes during Wallerian degeneration in vitro and how manipulating Ca2+ affects this response. Small explants of sciatic nerve from normal young adult rats cultured for five days show similar reversal of the myelinating phenotype as found in vivo. If Ca++ is removed from the culture medium through the addition of EGTA, expression of the nerve growth factor receptor and glial fibrillary acidic protein genes is inhibited but downregulation of the P(o) gene still occurs. Explants cultured in medium containing EGTA are still capable of expressing nerve growth factor receptor if the medium is replaced by one containing Ca2+. Supplementation of normal medium with drugs modulating Ca2+, such as Bepridil which blocks the Na+Ca2+ exchanger or compound 48/80 which inhibits calmodulin, also prevent the expression of the nerve growth factor receptor gene during Wallerian degeneration in vitro. Treatment of the cervical sympathetic trunk with Bepridil leads to loss of the nerve growth factor receptor immunoreactivity which is normally present. The results indicate that Ca2+ may play a role in the expression of the nerve growth factor receptor gene during Wallerian degeneration and provide some indication that this effect may be directly on the Schwann cell rather than operating indirectly via the axon.

Rumpshaker: an X-linked mutation causing hypomyelination: developmental differences in myelination and glial cells between the optic nerve and spinal cord.

The X-linked mutation rumpshaker (rsh), which is probably an allele of jimpy (jp), causes hypomyelination in the CNS of mice. This study examines the developmental expression of the morphology, glial cells, and immunostaining of myelin proteins in the optic nerve and spinal cord. The optic nerve contains varying numbers of amyelinated and myelinated fibres. The majority of such sheaths are of normal thickness whereas in the spinal cord most axons are associated with a disproportionately thin sheath which changes little in thickness during development. In the optic nerve glial cell numbers are elevated in mutants during early and peak myelination but then fall slightly below normal in adults. In contrast, the number of glial cells is consistently elevated after 16 days of age in the spinal cord. The majority of the alterations to total glial cells are due to corresponding changes in the oligodendrocyte population. Immunostaining intensity is somewhat reduced for myelin basic protein (MBP) and the C-terminal common to proteolipid protein (PLP) and DM-20 and profoundly decreased for the PLP-specific peptide. Glial fibrillary acidic protein (GFAP) is increased in rsh. It is probable that some of the variation in myelination between optic nerve and cord in rsh is related to the difference in axon diameter in the two locations, as there are adequate numbers of oligodendrocytes at the time of myelination. However, the effect of the mutation on cell development in the brain and the spinal cord may be different. The immunostaining indicates a marked deficiency in PLP in myelin but suggests that DM-20 levels may be relatively normal. rsh shows several major differences from jp and other X-linked myelin mutants, particularly in relation to oligodendrocyte numbers, and will be useful to elucidate the role of the PLP gene in influencing oligodendrocyte differentiation and survival.

Rumpshaker: an X-linked mutation affecting CNS myelination. A study of the female heterozygote.

This study examines the myelin deficits found in the spinal cord and optic nerves of female mice heterozygotes for rumpshaker (rsh), an X-linked mutation causing hypomyelination. No clinical abnormalities were detected but morphological changes were evident, particularly in the spinal cord, which showed no evidence of resolving with age. In the spinal cord, scattered hypomyelinated axons, occasionally grouped in twos or threes, were the major feature; oligodendrocyte numbers were slightly elevated at all ages compared to normal male littermates and the total amount of myelin was reduced. Myelin protein composition of the sheaths was examined by immunostaining for myelin basic protein (MBP) and two peptide regions of PLP/DM-20 molecule; one being proteolipid protein (PLP)-specific and the other recognizing the c-terminal common to PLP-DM-20. The majority of myelin sheaths immunostained for MBP and PLP. Occasional MBP-positive sheaths failed to stain with PLP/DM-20 or PLP-specific antiserum. Therefore, at least two types of immunocytochemically-defined myelin sheaths are present in the heterozygotes. Changes in the optic nerves were much less obvious; glial cell numbers were increased but thinly myelinated axons were not detected although the total amount of myelin was reduced compared to normal littermates. In no instance were mosaic, amyelinated/hypomyelinated patches detected. Heterozygotes for rsh, therefore, are considerably different from those for other X-linked myelin mutations like the jimpy mouse and the myelin-deficient rat, both in regard to the severity of the lesions and their failure to recover with age.

Expression of P0 mRNA in myelinating Schwann cells is related to fibre size.

This study examines whether there is a relationship between the abundance of expression for P0 mRNA in myelinated Schwann cells and fibre diameter. Individual teased sciatic nerve fibres from young adult rats were hybridized with radiolabelled probe for P0 mRNA which is expressed in the perinuclear cytoplasm of the mid-internode. Signal intensity was measured as optical density of the developed autoradiograms. A highly significant positive linear correlation was present between signal intensity and fibre diameter. In a companion study, individual fibres were mounted in Araldite resin and transversely serially sectioned at 4 microns for autoradiography. Grain densities were determined for fibres of different diameters. Again, larger diameter fibres were associated with higher grain densities. The results indicate that the abundance of P0 mRNA expressed by a myelin-producing Schwann cell is related to fibre diameter with axonal size probably being the critical determinant. Axons may regulate P0 expression through the number of signalling molecules exposed on or released from the axolemma.

Rumpshaker mouse: a new X-linked mutation affecting myelination: evidence for a defect in PLP expression.

This report describes a new X-linked mutation in mice, named rumpshaker (rsh) which is associated with hypomyelination of the central nervous system. Myelination commences appropriately but the majority of sheaths fail to develop normally. Oligodendrocytes are increased in number and have prominent Golgi apparatus, rough endoplasmic reticulum and free ribosomes. Occasional cisternae of rough endoplasmic reticulum are distended. Some dense lamellar inclusions occur in oligodendrocytes but overall, degenerative changes and cell death are uncommon. Immunostaining demonstrates a major defect in expression of PLP DM-20. Using site-specific antisera directed at different portions of the PLP/DM-20 molecule, the major defect appears to be with PLP where virtually no myelin sheaths are positive. Antiserum against the C-terminal common to PLP and DM-20 shows reduced but definite myelin staining. Genetic analysis indicates a locus at or close to the PLP/jimpy (jp) locus. The rsh mutation, however, differs from jp in that affected mice have normal longevity, can breed, produce substantially more myelin and have increased numbers of oligodendrocytes.

Progressive axonopathy: an inherited neuropathy of boxer dogs. 4. Myelin sheath and Schwann cell changes in the nerve roots.

Changes in the myelin sheath have been studied in the nerve roots of dogs with Progressive axonopathy, an autosomal recessive inherited neuropathy. The earliest changes were attenuation of the sheath at the proximal paranode and adjacent internode, probably in response to the axonal swelling which occurs in this area. Myelin bubbles were frequently observed along internodes. As the disease developed, progressively more fibres demonstrated short internodes of irregular length and thin myelin sheaths suggesting extensive remyelination and remodelling of the sheath. Short lengths of axons devoid of myelin, and occasional macrophages were also encountered. Sheaths of both original and newly formed internodes were highly irregular in outline. Occasional intra-axonal projections of adaxonal Schwann cell cytoplasm were observed, but complex interdigitations were unusual. A moderately electron-dense, granular material accumulated within the myelin sheath, becoming more obvious in the advanced disease. This material of unknown origin and composition was located predominantly at the intraperiod line principally between the adaxonal cytoplasm and the inner major dense line, but also at Schmidt-Lanterman incisures and between paranodal loops. Xenografts of the canine nerves into athymic mice failed to demonstrate any of the myelin sheath changes. The temporal and spatial relationship of the myelin sheath and axonal changes and the failure to reproduce the natural lesion in grafts suggest that Schwann cell alterations probably occur in response to the axonal changes.

Progressive axonopathy: an inherited neuropathy of boxer dogs. 3. The peripheral axon lesion with special reference to the nerve roots.

Progressive axonopathy is an autosomal recessive inherited neuropathy of Boxer dogs with lesions in the CNS and PNS. This paper describes the axonal changes in the lumbar and cervical nerve roots and tibial nerve. By 2 months of age the proximal paranodal areas of many larger diameter fibres show small axonal swellings, sometimes with attenuation or loss of the associated myelin sheath. Axoplasmic changes within swollen and non-swollen fibres include disorganization of the peripheral neurofilaments and small accumulations of vesicles and vesiculo-tubular profiles, particularly in the sub-axolemmal area. Occasional fibres, more often in the cervical roots, are massively distended with disorganized neurofilaments. The frequency of the membranous accumulations decreases with progression of the disease. Many axons show a markedly irregular or corrugated outline and are surrounded by an attenuated sheath. The peripheral axonal cytoskeleton is disorganized and misaligned, whereas the central structures maintain a more normal arrangement. Regenerating axonal clusters are common in the cervical ventral roots but occur infrequently in the lumbar roots. Similar axonal changes occur in the peripheral nerves but at a much lower frequency. Any membranous accumulations or cytoskeletal disorganization are more probable in the proximal tibial nerves, while the frequency of axonal degeneration and regeneration increases distally. The morphological appearances indicate gross disturbances in axon-sheath cell relationships and suggest that abnormalities in the transport of various axoplasmic organelles may be involved in the pathogenesis of the axonal lesion.

Progressive axonopathy: an inherited neuropathy of boxer dogs. 2. The nature and distribution of the pathological changes.

This report describes the neuropathology of progressive axonopathy (PA), an autosomal recessive inherited neuropathy of Boxer dogs, which affects CNS and PNS. The nerve roots contain numerous myelin bubbles and proximal paranodal axonal swellings containing vesicles, vesiculo-tubular profiles and disorganized neurofilaments. The myelin sheath overlying such swellings is often attenuated. As the disease develops there are progressive changes in the myelin sheath with thinning at paranodal and internodal locations, loss of myelin from lengths of axon and the formation of short internodes with disproportionately thin sheaths. The abnormalities show a very definite selectivity for nerve roots and proximal nerves. Conversely, the frequency of degeneration and regeneration is greater distally except in the cervical ventral roots which contain numerous regenerating clusters. In the CNS numerous axonal spheroids are found in the lateral and ventral columns of the spinal cord and in various brain stem nuclei, particularly the superior olives, accessory cuneate nuclei and lateral lemniscus and its nucleus. Axonal degeneration which occurs mainly in the cord shows no obvious tract or proximal/distal selectivity. The optic pathways are also involved, predominantly adjacent to the chiasma. The autonomic nervous system is affected and distal limb muscles show varying, but usually minor, degrees of neurogenic atrophy. The condition, which has no obvious direct parallel in human or veterinary medicine, shows gross disturbances of axon-glial inter-relationships in both CNS and PNS.

Feline dysautonomia (the Key-Gaskell syndrome): an ultra structural study of autonomic ganglia and nerves.

Recently a feline dysautonomia of unknown aetiology, the Key-Gaskell syndrome, has caused widespread morbidity in the UK. This report describes the ultrastructural appearances of the autonomic ganglia and axons of the sympathetic chain in this condition. Nuclei of affected neurones were eccentric and abnormally crenated. Nucleolar abnormalities such as increased electron density (due to loss of the intranucleolar vacuoles), nucleolar segregation and ring nucleoli were observed in a proportion of neurones. There was marked loss of ribosomes, both bound and unbound, and cisternae of the rough endoplasmic reticulum were distended with a floccular electron dense material. Numerous smooth-walled cisternae were also present and complex stacks of smooth semi-parallel membranes were observed, probably derived from the smooth endoplasmic reticulum or Golgi apparatus. No normal Golgi formations were seen. Frequent autophagic vacuoles and membranous dense bodies were present in some cells. Many unmyelinated fibres in the sympathetic chain were swollen and contained vesiculo-tubular profiles, disordered neurotubules and filaments and various degenerating membranous organelles. Myelinated fibres within the sympathetic chain were also degenerating. These studies indicate that the organelles involved with protein biosynthesis are severely affected by the disease.

Nerve fibres in spinal cord impact injuries. Part 1. Changes in the myelin sheath during the initial 5 weeks.

The spinal cords of cats were subjected to an impact injury using a "weight dropping" technique and sequential changes in the sheaths of non-degenerate myelinated fibres studied over a 3-week period. By 1 1/2 h after impact fibres showed retraction of some lateral loops from one paranode. The extent and severity of this change increased over the first week so that partial and full thickness demyelination were seen frequently. Partial demyelination most commonly resulted from the internodal termination of the innermost lamellae at an internodal location often associated with a Schmidt-Lantermann incisure. Remyelination by both Schwann cells and oligodendroglia occurred at the end of the second week. Oligodendroglial myelin showed many features of immaturity, similar to those found during development. It is suggested that the very earliest myelin damage is mechanical but is aggravated by other factor(s) one of which is probably ischaemia. Within the most severely injured areas there is death of oligodendroglia and any surviving axons are remyelinated principally by Schwann cells. In intermediate and minimally damaged areas of white matter oligodendroglial remyelination predominates.