Differential sensitivity in the survival of oligodendrocyte cell lines to overexpression of myelin proteolipid protein gene products.

The proteolipid (PLP) gene encodes at least four proteins, including the classic PLP and DM20, which are important components of the myelin sheath, and the recently identified soma-restricted (sr) isoforms, srPLP and srDM20. The classic PLP and DM20 gene products have been implicated in oligodendrocyte survival by overexpression studies in vitro and in vivo. The classic and sr proteolipids are targeted to different cellular compartments in the oligodendrocyte, suggesting different cellular functions. Accordingly, we examined the effects of in vitro overexpression of the sr-PLP/DM20 isoforms on the survival of stably transfected, conditionally immortalized, oligodendroglial cell lines and compared this to overexpression of the classic and the jimpy-mutated proteolipids. The results indicate that overexpression of either normal or jimpy classic PLP/DM20 resulted in a dramatic reduction in the survival of the oligodendrocyte cell lines at the nonpermissive temperature, but not the COS-7 cell line, a cell line expressing the same oncogene constitutively. Survival of the oligodendrocyte cell lines was significantly less affected when either the sr-PLP/DM20 or the dopamine D-2 receptor, another cell membrane protein, was overexpressed in the cell lines. These results suggest that overexpression of the "classic" PLP or DM20 can compromise the survival of oligodendrocytes whether or not they are mutated. Furthermore, they suggest that the internal mechanisms for normal targeting of the PLP/DM20 isoforms of either the "classic" or the "sr" types influence the oligodendrocyte's ability to survive when these proteolipids are overexpressed.

Connatal Pelizaeus-Merzbacher disease associated with the jimpy(msd) mice mutation.

In a patient with connatal Pelizaeus-Merzbacher disease with the same mutation in the proteolipid protein gene as in jimpy(msd) mice the immunohistochemical study of the brain demonstrated deficiencies of myelin and proteolipid protein despite good expression of myelin basic protein. The mechanism of myelination is partly disturbed by the mutation; therefore jimpy(msd) mice can be used as a suitable model for further studies in connatal Pelizaeus-Merzbacher disease.

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.

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.

Understanding glial abnormalities associated with myelin deficiency in the jimpy mutant mouse.

Jimpy is a shortened life-span murine mutant showing recessive sex-linked inheritance. The genetic defect consists of a point mutation in the PLP gene and produces a severe CNS myelin deficiency that is associated with a variety of complex abnormalities affecting all glial populations. The myelin deficiency is primarily due to a failure to produce the normal amount of myelin during development. However, myelin destruction and oligodendrocyte death also account for the drastic myelin deficit observed in jimpy. The oligodendroglial cell line shows complex abnormalities in its differentiation pattern, including the degeneration of oligodendrocytes through an apoptotic mechanism. Oligodendrocytes seem to be the most likely candidate to be primarily altered in a disorder affecting myelination, but disturbances affecting astrocytes and microglia are also remarkable and may have a crucial significance in the development of the jimpy disorder. In fact, the jimpy phenotype may not be attributed to a defect in a single cell but rather to a deficiency in the normal relations between glial cells. Evidences from a variety of sources indicate that the jimpy mutant could be a model for disturbed glial development in the CNS. The accurate knowledge of the significance of PLP and its regulation during development must be of vital importance in order to understand glial abnormalities in jimpy.

In situ expression of PLP/DM-20, MBP, and CNP during embryonic and postnatal development of the jimpy mutant and of transgenic mice overexpressing PLP.

We analyzed by in situ hybridization the spatiotemporal expression of dm-20, myelin basic protein (MBP) and 2'-3' cyclic nucleotide phosphodiesterase (CNP) during embryonic and postnatal development of the normal mouse and two plp/dm-20 mutants: the jimpy mouse and a transgenic mouse overexpressing the plp gene. In the central nervous system (CNS) of the normal mouse, dm-20 mRNA was detected at embryonic day (E)9.5 in the laterobasal plate of the diencephalon. The pattern of expression of CNP transcript was superimposable on that of dm-20, but appeared slightly later, at E12.5. MBP mRNA was detected even later (E14.5), and, in addition, only in the caudal (rhombencephalon and spinal cord) territories of expression of dm-20 and CNP. These observations support our previous proposals: (1) dm-20-expressing cells in the germinative neuroepithelium are precursors of oligodendrocytes, and (2) oligodendrocytes emerge from distinct pools of precursors along the neural tube (Timsit et al., 1995). In the jimpy mutant, despite the mutation in the plp gene, cells of the oligodendrocyte lineage developed normally. It is only at the time of myelin deposition that oligodendrocytes die. During embryonic development of the transgenic mutant overexpressing plp, there were no alterations in the spatiotemporal pattern or the level of expression of dm-20 in the CNS, in contrast to the higher levels of dm-20 observed in the peripheral nervous system (PNS).

Effects of rumpshaker mutation on CNS myelin composition and structure.

Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jprsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jprsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jprsh myelin, further substantiating that PLP is not a component of this junctional complex. X-ray diffraction measurements on unfixed optic nerves showed that the jprsh period is 5-10 A larger than normal. Moreover, jprsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jprsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jprsh mutant correlate with a wider periodicity and less stable packing of the myelin.

Selective retrieval of jimpy mutant pups over normal male littermates by lactating female B6CBACa-Aw-J/A-Ta jp mice.

The jimpy sex-linked mutation results in dysmyelination throughout the CNS although myelination in the peripheral nervous system is unaffected. Jimpy pups (jp/Y) weigh less, produce fewer ultrasonic vocalizations, and spend less time engaged in coordinated activity than their normal littermates and rarely live past 30 days of age. Both jp/Y and normal male pups occur within the same litter, making it possible to examine maternal responsiveness to the two types of pups. Using maternal retrieval as a measure of maternal responsiveness, we predicted, in agreement with parental investment theory, that mothers would retrieve normal littermates in preference to mutant pups. When tested for pup retrieval preferences in a T-maze at 2-day intervals from day 3 to day 15 postpartum, mothers retrieved the mutant mouse first on 78 to 100% of the trials and carried him back to the stem section of the T-maze before returning for the normal littermate. We are thus left with the paradox that the mother appears to "prefer" the mutant pup to the normal littermate, despite its smaller size, reduced ultrasonic vocalization rate, and compromised fitness.

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).

Fate of jimpy-type oligodendrocytes in jimpy heterozygote.

In the jimpy mutant mouse, as well as in many other animals with mutations in the myelin proteolipid protein (PLP) gene, oligodendrocytes degenerate before their maturation. To analyze whether this degeneration is caused by the loss of function of PLP gene products related to oligodendrocyte maturation/survival acting extrinsically, expression of the PLP gene was investigated in the jimpy heterozygote, in which one-half of the cells are jimpy type and the other half are wild type due to random X-chromosome inactivation. We first showed that jimpy PLP gene expression is normally regulated at the early stages of development in brains of jimpy hemizygotes and heterozygotes, at least to day 2 after birth. However, the great increase in the level of PLP gene transcripts observed in wild-type mouse brain is suppressed in jimpy mouse brain. This increase was also suppressed in the jimpy heterozygote, and by 2 months after birth, very few jimpy-type PLP gene transcripts were detected in heterozygotes. These results indicate that jimpy-type oligodendrocytes cannot survive or are still in the immature stage in the brain of jimpy heterozygotes. Thus, degeneration of jimpy oligodendrocytes is not caused merely by the lack of trophic factors.

Many naturally occurring mutations of myelin proteolipid protein impair its intracellular transport.

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

Oligodendrocyte development and differentiation in the rumpshaker mutation.

The jimpy rumpshaker (jprsh) mutation is an amino acid substitution in exon 4 (Ile186-->Thr) of the proteolipid protein (PLP) gene on the X chromosome. Affected mice show moderate hypomyelination of the central nervous system (CNS) with increased numbers of oligodendrocytes in the white matter of the spinal cord, a feature distinguishing them from other PLP mutations such as jp, in which premature cell death occurs with reduced numbers of oligodendrocytes. Myelin sheaths of jprsh immunostain for myelin basic protein (MBP) and DM-20, but very few contain PLP. This study examines the differentiation of oligodendrocytes cultured from the spinal cords of young mutant and wild type mice using various surface and cytoplasmic antigenic markers to define the stage of development. The majority of oligodendrocytes from mutant mice progress normally to express MBP; approximately 30%, relative to wild type, contain DM-20 at the in vivo age of 16 days, but very few immunostain for PLP or the O10 and O11 markers. The morphology of mutant cells in respect to membrane sheets and processes appears similar to normal. The jprsh oligodendrocyte is, therefore, characterized by a failure to express the markers indicative of the most mature cell; however, it is probably able to achieve a normal period of survival. These data, taken in conjunction with previous results, suggest that the PLP gene has at least two functions; one, probably involving PLP, is concerned with a structural role in normal myelin compaction; the other, perhaps related to DM-20 (or another lower molecular weight proteolipid), is essential for cell survival. The mutation in jprsh at residue 186 suggests that this region, which is common to PLP and DM-20, is not critical for this latter function.

Oligodendrocytes of the jimpy phenotype can be partially restored by environmental factors in vivo.

Cross-transplantations of neural tissue between jimpy (jp) shiverer (shi) and normal mice have been performed under heterochronic conditions. In all series, fragments of E14-E15 embryonic neural tissue from the different donors have been transplanted into newborn host brain in order to study environmental influences by differentiated tissue on transplanted embryonic cell lines. Large patches of proteolipid protein (PLP)-positive myelin have been observed in the jp brain after transplantation of shi or normal embryonic tissue into the newborn jp brain, suggesting that the jp parenchyma did not inhibit the differentiation of other oligodendrocytes (ODCs). Jp embryonic tissue had the same mitotic potential as normal tissue, as demonstrated by the larger size of myelin patches observed when jp embryonic tissue was used instead of newborn jp tissue. By contrast, whatever the conditions, jp myelin patches were always obviously smaller than normal or shi myelin patches, suggesting that the myelinating capacity of jp ODCs was not enhanced by environmental factors. Finally, comparison of the ratio of successful outcomes observed following embryonic vs. newborn jp donor tissue, strongly suggests a partial or total normalization of jp embryonic ODCs survival by a more mature shi environment.

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.

The dysmyelinating mouse mutations shiverer (shi) and myelin deficient (shimld).

Shiverer (shi/shi) is an autosomal recessive mouse mutation that produces a shivering phenotype in affected mice. A shivering gait can be seen from a few weeks after birth until their early death, which occurs between 50 and 100 days. The central nervous system of the mutant mouse is hypomyelinated but the peripheral nervous system appears normal. The myelin of the CNS, wherever present, is not well compacted and lacks the major dense line. Myelin basic protein (MBP), which is associated with the major dense line, is absent, and this is due to a deletion of the major part of the gene encoding MBP. Transgenic shiverer mice that have integrated and express the wild-type mouse MBP transgene no longer shiver and have normal life spans. Conversely, normal mice that have integrated an antisense MBP transgene, shiver. Myelin deficient shimld/shimld is allelic to shiverer (shi/shi) but the mutant mouse is less severely affected. Although MBP is present in the CNS, it is low in quantity and is not developmentally regulated. The gene encoding MBP has been both duplicated and inverted. Transgenic shimld/shimld mice with the wild-type MBP transgene have normal phenotypes.

Patchy myelination pattern in the jimpy mouse brain: immunohistochemical study.

The jimpy (jp) mutation of the mouse leads to a dramatic decrease of myelination in the hemizygous mutant central nervous system (CNS). Several descriptions based on classical histology, immunohistochemistry, and electron microscopy (EM) have demonstrated the scarcity of myelin formation in the different parts of the CNS. The immunohistochemical study presented here showed a very singular patchy pattern of myelin distribution in the different areas of the whole mutant brain. The myelin patches are randomly dispersed without bilateral symmetry, and their density and location vary from one animal to another. No reproducible pattern of myelination could be found among the population observed. This distribution has been compared with observations on young heterozygotes and wild-type homozygotes from the same strain. A similar patchy and random distribution of myelin could be observed in heterozygotes, which present an intermediate level of myelination. This strongly suggests that a migration of precursors or immature oligodendrocytes (ODCs) from the periventricular zone followed by local multiplication of colonies of ODCs before myelination is a general feature in normal as well as pathological conditions.

Gene expression in the jimpy mutant: evidence for fewer oligodendrocytes expressing myelin protein genes and impaired translocation of myelin basic protein mRNA.

Myelin basic protein (MBP) and proteolipid protein (PLP) gene expression was investigated in the murine dysmyelinating mutant, jimpy and age-matched normal mice. MBP and PLP mRNA expression was examined in several brain regions by in situ hybridization histochemistry between 10 and 20 days postpartum. The results showed a general reduction in both PLP and MBP mRNA expression throughout in jimpy brain due primarily to fewer numbers of jimpy oligodendrocytes expressing these genes. A less severe, but significant, reduction in the level of PLP expression per oligodendrocyte was also observed in the mutant brain. Because of the diffuse pattern of MBP mRNA distribution in the controls it was not possible to determine whether MBP expression was reduced on a per cell basis in the jimpy mutant. Silver grains representing MBP mRNAs were diffusely distributed over myelinating regions in normal mice but, in contrast, the grains were primarily clustered over oligodendrocyte cell bodies in the jimpy brains. No significant differences in jimpy oligodendrocyte morphology were evident by galactocerebroside immunohistochemistry. These results suggested that the apparent MBP mRNA clustering was not due to truncated or reduced numbers of oligodendrocyte processes. Rather, they suggest that the inability of jimpy mice to incorporate MBP into myelin may be due, in part, to a disruption in the translocation of MBP mRNAs within the oligodendrocyte.