Hypomyelinated mutant mice. II. Myelination in vitro

Organotypic cultures of cerebellum from hypomyelinated mutant mice provide a powerful experimental system for studying the cell biology of the mutant diseases. We have examined the extent to which the culture system reproduces the diseases of three well-known mutants,qk,jp^msd, andjp.Quantitation of myelin profiles per sq. mm of section demonstrates that in vitro, as in situ,qk produces the most myelin,jp^msd an intermediate amount, andjp the least. Myelin inqk cultures is unique in being invisible by light microscopy of the living culture. Hypomyelination ofjp may be more severe in vitro than in situ. Cultures ofjp^msd exhibit many of the ultrastructural features of cerebellar abnormalities that occur in situ: degree of hypomyelination, clustering of myelin segments, scarcity of oligodendrocytes, absence of nodes of Ranvier but presence of heminodes, and apparent structural integrity of the myelin sheaths. Correspondence between in vitro and in situ ultrastructure is more difficult to assess forjp, because the available sample ofjp myelin in vitro is too small, and forqk, because the abnormalities observed in situ resemble nonspecific abnormalities of normal myelin in vitro.     

Myelination ofjp,jp andqk axons by normal glia in vitro: Ultrastructural and autoradiographic evidence

Normal optic nerve glia were ‘injected’ into hypomyelinated mutantjp,jp^msd, andqk cerebellum by co-culturing explants in direct physical contact. Quantitative light microscopic studies demonstrated that such glial injection significantly increased the number of myelin profiles counted in cultures, suggesting that axons in all 3 mutants can accept myelination from competent glia when they are made available. In each mutant, the observed increase in myelination was independent of the ages of donor optic nerves and recipient cultures, but absolutely required positioning of the optic nerve so that direct contact occurred with the mutant cerebellar explants. The additional myelin found near the zone of fusion with the optic nerve morphologically resembled normal, not mutant myelin. Autoradiographs made after [³H]thymidine-labeled normal optic nerve was injected intojp^msd cultures showed that labeled cells had colonized the nearby mutant tissue. Labeled cells identified as oligodendrocytes by ultrastructural criteria were found adjacent to myelin segments near the fusion zone, but direct continuity between processes of these oligodendrocytes and myelin sheaths was not demonstrated. The astrocytes and phagocytic cells which were also labeled had no obvious relationship to myelinated axons. These results provide experimental evidence that the primary abnormalities produced by the three mutationsjp,jp^msd, andqk are inherent in their glial cells, probably although not definitely in the oligodendrocytes.     

Hypomyelinated mutant mice IV: Peripheral myelin injp

This study compares peripheral myelination in a specific subdivision of the sciatic nerve ofjp^msd and unaffected littermate mice. No significant differences are found in numbers of myelinated and unmyelinated axons, diameters of axons, thickness of myelin sheaths relative to axon diameter, extent of unmyelinated axon segregation by Schwann cell processes, or in the ultrastructure of myelin and Schwann cells. By contrast,jp^msd mutant mice show severe CNS hypomyelination. This evidence, that thejp^msd mutation affects only oligodendrocytes, distinguishes mutations at this locus from others producing CNS hypomyelination in which PNS myelin is also affected.     

Cholesterol ester content and activities of the ester metabolizing enzymes in jp mouse brain

The free and esterified cholesterol content, and the activities of cholesterol esterifying and the ester hydrolyzing enzyme in brain tissue from myelin synthesis-deficient mutant jp^msd mice and normal littermates were determined. Results showed that cholesterol ester content was high in the brain of jp^msd-affected mice and that the esterification of cholesterol in vitro, with or without added fatty acids was also high in the brain tissue of the affected mutant mice. The data suggest that the increase in cholesterol ester concentrations in the brain of jp^msd-affected mutant mice occurs as a result of increased synthesis rather than decreased hydrolysis.     

Cholesterol ester content and activities of the ester metabolizing enzymes in jpmsd mouse brain

The free and esterified cholesterol content, and the activities of cholesterol esterifying and the ester hydrolyzing enzyme in brain tissue from myelin synthesis-deficient mutant jp^msd mice and normal littermates were determined. Results showed that cholesterol ester content was high in the brain of jp^msd-affected mice and that the esterification of cholesterol in vitro, with or without added fatty acids was also high in the brain tissue of the affected mutant mice. The data suggest that the increase in cholesterol ester concentrations in the brain of jp^msd-affected mutant mice occurs as a result of increased synthesis rather than decreased hydrolysis.     

Quaking shiverer double mutant mice: morphological phenotypes support possible dual actions of the shiverer locus

Mice doubly homozygous for the two different hypomyelination mutations, quaking (qk) and shiverer (shi) or shiverer myelin-deficient (shimld) (abbreviations: qk*shi and qk*shimld), both have much less myelin than either single mutant ancestor, myelin morphology resembling shi or shimld rather than qk, and abundant shi-type oligodendrocytic microprocesses. The qk*shimld double mutant differs from qk*shi only in having small amounts of normal or abnormal major dense line, in keeping with the morphologic difference between the shi and shimld single mutants. By contrast, shi*jp and shimld*jp have clearly different morphological phenotypes; unexpectedly the major dense line is present in the CNS myelin of shi*jp but not shimld*jp. When shi and shimld act alone, their different DNA abnormalities produce similar protein abnormalities. We speculate that the two mutations interact with qk at a different, later step of DNA expression than they interact with jp. In the interaction with qk, the similar proteins produce similar morphologies. In the interaction with jp, the different DNAs are somehow caused to produce protein differences that are reflected in different morphologies. In this study we have observed for the first time a morphological effect of these mutant genes in heterozygous animals. Of particular importance, animals whose genomes combine shi/+ or shimld/+ with qk/qk produce qk-type, compacted myelin but abundant shi-type oligodendrocyte microprocesses. We consider this as evidence that both shi and shimld have two effects: non-production of a normal structural protein, myelin basic protein, and production of an abnormal protein which perturbs the cytogogic function we postulate to be normally exercised by the myelin basic protein gene.     

Shiverer*jimpy double mutant mice. II. Morphological evidence supports reciprocal intergenic suppression

Mice which carry both the shiverer (shi) and the jimpy (jp) mutations have a morphological phenotype with features of each single mutation by itself but in milder form: the number of myelin sheaths is increased relative to jp, the thickness of sheaths and amount of major dense line is increased relative to shi, and the abnormal, lipid-filled cells characteristic of jp are not seen. However, the abnormal bundles of oligodendrocyte microprocesses and errors in the targeting of myelination which characterize shi are not altered by the presence of the jp mutation. This morphological evidence suggests partial reciprocal intergenic suppression in shiverer*jimpy double mutant mice and therefore agrees with conclusions based on biochemical data presented by Kerner and Carson ( Brain Research, 374 (1986) 45–53).     

Quaking*jimpy double mutant mice: additional evidence for independence of primary deficits in jimpy.

Mice which have the genotype qk/qk*Tajp/Y, and therefore simultaneously express both the quaking (qk) and jimpy (jp) mutations, have CNS white matter morphology intermediate between qk and jp with respect to amount of myelin, myelin structure, and oligodendrocyte number. The level of myelin basic protein in the CNS is also intermediate; however, myelin proteolipid protein (PLP) is virtually absent. Thus in the qk/qk*Tajp/Y double mutant mouse the PLP deficit is as severe as in jp alone but the oligodendrocyte survival deficit (reflected in number and myelin production) of jp alone is rendered less severe. The observation that these two cardinal deficits of the jp mutation can be independently altered in double mutant combinations is consistent with our previous suggestion that the PLP genetic locus may encode at least two independently regulated primary gene functions: a structural protein and signal influencing oligodendrocyte behavior.     

Phenotypic severity of murine Plp mutants reflects in vivo and in vitro variatioans in transport of PLP isoproteins

Mutations of the major myelin gene, proteolipid protein (Plp), cause Pelizaeus-Merzbacher disease and some forms of spastic paraplegia in man and dysmyelinating phenotypes in animals. The clinical severity is markedly heterogeneous, ranging from relatively mild to severe and fatal. Point mutations, or frame shifts, which are predicted to result in translation of structurally altered proteins account for many of these cases, including 3 of the allelic murine conditions. Plp^jp-rsh, Plp^jp-msd, and Plp^jp represent an increasing severity of clinical and pathological phenotypes, respectively. In this study we determined whether there was any correlation between the severity of phenotype and the transport of the predicted abnormal protein. We examined the ability of the two products of the Plp gene, PLP and DM20, to insert into the plasma membrane of transfected BHK or COS-7 cells, and into the myelin sheath of oligodendrocytes. With these complementary in vitro and in vivo approaches we find that proteins of Plp^jp-rsh, associated with the mildest phenotype, have a far greater ability to insert into the cell membrane or myelin than those associated with the severe phenotypes. Additionally, altered DM20 is more readily transported to the cell surface and to myelin than the PLP isoprotein. Interestingly, the two clonal cell lines chosen for transient transfection differ in their ability to fold DM20 from Plp^jp-rsh and Plp^jp-msd mice correctly, as inferred by staining for the conformation-sensitive O10 epitope. In the case of Plp^jp, which is associated with the most severe phenotype, no PLP or O10 staining is present at the cell surface or in myelin. The perturbation in trafficking observed for altered Plp^jp PLP and DM20 in oligodendrocytes does not extend to other myelin membrane proteins, such as MAG and MOG, nor to wild type PLP co-expressed in the same cell, all of which are correctly inserted into myelin. As Plp-knockout mice do not have a dysmyelinating phenotype it seems unlikely that absence of PLP and/or DM20 in the membrane is responsible for the pathology. It remains to be determined whether the perturbation in protein trafficking is associated with the dysmyelination, or if the altered product of the mutant alleles acquire a novel function which is deleterious to myelin production by oligodendrocytes. GLIA 20:322–332, 1997. © 1997 Wiley-Liss, Inc.     

Spatial distribution of myelin basic protein mRNA and polypeptide in quaking oligodendrocytes in culture.

In the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of oligodendrocytes were established from affected and control animals. The cytoarchitecture of the oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP polypeptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture.     

Normal proliferation rate of galactocerebroside positive oligodendrocytes in brain cell cultures of the hypomyelinated mouse mutant jimpy

Proliferation of oligodendrocytes from the jimpy (jp) hypomyelinated mouse mutant was studied in dissociated brain cell cultures. This was done by combining anti-galactocerebroside (GC) immunostaining (for identifying oligodendrocytes) with [³H]thymidine autoradiography (for identifying proliferating cells). Previously we showed that the expression of GC in culture by jp oligodendrocytes is not altered by the jp mutation. Present results show that in 7-, 14- and 21-day-old jp cultures oligodendrocytes proliferate at a rate similar to that of normal GC⁺ oligodendrocytes. This indicates that, in jp brain cell cultures, oligodendrocytes which are not affected by mutation in their capability to express GC are also unaffected with regard to their proliferation rate.     

Completion of myelin compaction, but not the attachment of oligodendroglial processes triggers K(+) channel clustering

The characteristic localization of ion channels is crucial for the propagation of saltatory conduction in myelinated nerves. Voltage-gated Na(+) channels are located at nodes of Ranvier while voltage-gated K(+) channels are mainly found at juxtaparanodal regions. Recently, a humoral factor secreted by oligodendrocytes has been reported to induce clustering of Na(+) channels in CNS axons. However, the molecular mechanisms for K(+) channel clustering as well as the role of oligodendrocytes are still uncertain. To clarify whether myelin sheath itself can induce the distinct distribution of K(+) channels, we have investigated the localization of K(+) channels in adult and developing mouse optic nerves. The CNS axons from chronic demyelinating and hypomyelinating mice were also examined to determine if myelin sheaths were required for the maintenance of clusters. In all cases, the K(+) channel clustering correlated well with compact myelin, but not with the presence of oligodendrocytes, suggesting that, in contrast to Na(+) channel clustering, the formation of compact myelin is required for initiation as well as maintenance of K(+) channel clustering. In addition, postsynaptic density protein-95 (PSD-95) or its highly related protein was found colocalized with K(+) channels, suggesting that it may interact with K(+) channels to form clusters at juxtaparanodal regions.     

Ultrastructural identification of HRP-injected oligodendrocytes in the intact rat optic nerve

Glial cells in the rat optic nerve were visualized by intracellular injections of horseradish peroxidase (HRP). A novel class of cell was encountered that was presumed to be an oligodendrocyte on the basis of arguments related to its light microscopic appearance after intracellular staining (Butt and Ransom, Glia 1989;2:470-475). These cells had 10-20 parallel processes 200-300 microns long that were oriented exclusively along the long axis of the optic nerve; the parallel processes were connected to the cell body by thin branches 15-30 microns long. To determine if these HRP-filled cells were oligodendrocytes, they were examined ultrastructurally; all cells examined in this way were unequivocally found to be myelin-producing oligodendrocytes. The oligodendrocytes contained intracellular organelles that were characteristic of this cell type, including abundant Golgi profiles and microtubules. In addition, HRP was found to fill the inner and outer tongue processes of myelin sheaths and the paranodal loops at nodes of Ranvier, proving that the entire cytoplasmic border surrounding the myelin sheath rapidly communicates by intracellular diffusion with the cell body. This electron microscopic study demonstrates that oligodendrocytes in the rat optic nerve can be positively identified by their distinctive light microscopic appearance after intracellular dye injection, and provides light microscopic criteria for establishing the number, distribution, and dimensions of the myelin segments provided by individual oligodendrocytes.     

Abnormal Ca regulation in oligodendrocytes from the dysmyelinating jimpy mouse

Jimpy (jp) is a point mutation in the gene on the X chromosome which codes for the major myelin proteolipid protein. Most oligodendrocytes (OLs) in the jp mouse undergo cell death at the time when they should be actively myelinating. Loss of mature OLs results in severe CNS dysmyelination. Dying jp OLs have the morphology of apoptotic cells but it is not clear how the mutation activates biochemical pathways which lead to programmed death of OLs in jp CNS. There is compelling evidence from a number of systems that high levels of intracellular Ca²⁺ ([Ca²⁺]_i) can activate downstream processes which result in both apoptotic and necrotic cell death. To determine whether [Ca²⁺]_i dysregulation might be involved in the death of jp OLs, we used ratiometric imaging to determine levels of [Ca²⁺]_i in OLs cultured from jp and normal CNS and in immortalized cell lines derived from jp and normal OLs. Immortalized jp OLs and OLs isolated directly from jp brain both showed a similar elevation in [Ca²⁺]_i ranging from 60% to 150% over control values. A higher baseline [Ca²⁺]_i in jp OLs might increase their vulnerability to other insults due to abnormal protein processing or changes in signaling pathways which act as a final trigger for cell death.     

Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis

P0, the major peripheral nervous system (PNS) myelin protein, is a member of the immunoglobulin supergene family of membrane proteins and can mediate homotypic adhesion. P0 is an essential structural component of PNS myelin; mice in which P0 expression has been eliminated by homologous recombination (P0-/-) develop a severe dysmyelinating neuropathy with predominantly uncompacted myelin. Although P0 is thought to play a role in myelin compaction by promoting adhesion between adjacent extracellular myelin wraps, as an adhesion molecule it could also have a regulatory function. Consistent with this hypothesis, Schwann cells in adult P0-/- mice display a novel molecular phenotype: PMP22 expression is down-regulated, MAG and PLP expression are up-regulated, and MBP expression is unchanged. As in quaking viable mutant mice (qk(v)), which have uncompacted myelin morphologically similar to that found in P0-/- mice, neither the qKI-6 or qKI-7 proteins are expressed in P0-/- peripheral nerve. In addition to these changes in gene expression in the P0 knockout, PLP/DM-20 accumulates in the endoplasmic reticulum of P0-/- Schwann cells, whereas MAG accumulates in redundant loops of uncompacted myelin, not at nodes of Ranvier or Schmidt-Lantermann incisures. Taken together, these results demonstrate that P0 is involved, either directly or indirectly, in the regulation of both myelin gene expression and myelin morphogenesis.     

Astrocytes and neurons regulate the expression of the neural recognition molecule janusin by cultured oligodendrocytes

Janusin (formerly designated J1–160/180) is an extracellular matrix glycoprotein highly homologous to tenascin, consisting of two major molecular forms of 160 and 180 kD expressed by oligodendrocytes and in myelin. Janusin expression is upregulated during myelination and in the adult it remains expressed at lower levels. It is also present at the node of Ranvier, where myelin, axon, and astrocytic process are in close contact. To gain an understanding of the regulatory mechanisms which may under-lie expression of janusin, the differentiation stage-dependent expression of janusin was studied in cultures enriched in mouse oligodendrocytes and their precursor cells. Expression of janusin by these cells was highest on both A2B5⁺ and O4⁺/O1^? oligodendroglial precursor cells and a subset of myelin associated glycoprotein-positive (MAG⁺) oligodendrocytes. Hardly any of the more differentiated O1⁺ or O10⁺ oligodendrocytes expressed janusin. Expression of janusin was influenced by co-culture with astrocytes or neurons. Astrocytes or astrocytic-conditioned culture supernatants elevated the expression of janusin by the more differentiated oligodendrocytes (O1⁺ or MAG⁺ cells), while its expression by oligodendroglial precursor cells was relatively unchanged. Platelet-derived growth factor, but not basic fibroblast growth factor, also elevated the expression of janusin by O1⁺ or O10⁺ oligodendrocytes. In contrast, co-culture with neurons originating from dorsal root ganglia or spinal cord decreased the expression of cell-bound janusin by oligodendrocytes and their precursor cells. These observations indicate that expression of janusin on these cells in culture is susceptible to opposing regulatory influences from astrocytes and neurons. Such influences may modulate the temporal and spatial distribution of janusin in the developing and adult central nervous system. © 1993 Wiley-Liss, Inc.     

Decrease in newly generated oligodendrocytes leads to motor dysfunctions and changed myelin structures that can be rescued by transplanted cells

NG2‐glia in the adult brain are known to proliferate and differentiate into mature and myelinating oligodendrocytes throughout lifetime. However, the role of these newly generated oligodendrocytes in the adult brain still remains little understood. Here we took advantage of the Sox10‐iCreER^T2 x CAG‐eGFP x Esco2^fl/fl mouse line in which we can specifically ablate proliferating NG2‐glia in adult animals. Surprisingly, we observed that the generation of new oligodendrocytes in the adult brain was severely affected, although the number of NG2‐glia remained stable due to the enhanced proliferation of non‐recombined cells. This lack of oligodendrogenesis led to the elongation of the nodes of Ranvier as well as the associated paranodes, which could be locally rescued by myelinating oligodendrocytes differentiated from transplanted NG2‐glia deriving from wildtype mice. Repetitive measurements of conduction velocity in the corpus callosum of awake animals revealed a progressive deceleration specifically in the mice lacking adult oligodendrogenesis that resulted in progressive motor deficits. In summary, here we demonstrated for the first time that axon function is not only controlled by the reliable organization of myelin, but also requires a dynamic and continuous generation of new oligodendrocytes in the adult brain. GLIA 2016;64:2201–2218     

Death of individual oligodendrocytes in jimpy brain precedes expression of proteolipid protein.

Immunocytochemistry and thymidine autoradiography were combined to determine the time elapsed between cell division and the expression of proteolipid protein (PLP) in individual oligodendrocytes in normal mouse brain. In jimpy (jp) brains, autoradiography was used to determine the time elapsed between cell division in an individual oligodendrocyte and evidence of cell death. Oligodendrocytes in normal mouse brain do not express PLP until 72 h after a single injection of [3H]-thymidine. In contrast, oligodendrocytes in jp brains begin to die within 9-11 h after an injection of thymidine. The jp mouse is one of several X-linked, hypomyelinated mutants in which a defect has been demonstrated in the gene coding for PLP. It has been presumed that the lack of this protein in the myelin sheath is responsible for the jp phenotype. However, the present study shows that individual jp oligodendrocytes begin to die long before they would normally have synthesized detectable levels of PLP. Therefore, it seems unlikely that the death of jp oligodendrocytes is due to the absence of PLP in myelin sheaths. Oligodendrocyte death and other early jp abnormalities may be due to the presence of abnormal PLP message which may interfere with glial differentiation. Alternatively, the PLP message may code for another protein which is important for normal development of neuroglia.     

Shiverer jimpy double mutant mice. III. Comparison of shimld*jpmsd and shi*jp phenotypes demonstrates dissimilar interactions of allelic mutations

Double mutant mice, which are of the genotype shimld/shimld*jpmsd/Y and therefore express both the shimld and jpmsd mutations, have a CNS myelin protein composition which resembles shimld/shimld alone but not jpmsd/Y alone. The double mutant CNS white matter morphology shows much less myelin and major dense line than either shimld/shimld or jpmsd/Y, but has other features which resemble jpmsd/Y but not shimld/shimld. In contrast, the parallel double mutant shi/shi*jp/Y, which expresses the alleles shi and jp rather than shimld and jpmsd, has already been shown to have biochemical and morphological phenotypes which are consistent with each other, both being intermediate between shi/shi and jp/Y and therefore suggesting partial reciprocal intergenic suppression (Brain Research, 374 (1986) 45-53 and 54-62). To assist in explaining the apparent inconsistencies between the biochemical and morphological phenotypes of the shimld/shimld*jpmsd/Y double mutant and between interactions of allelic mutations at the shi and jp loci, a hypothesis of multiple primary gene functions at these two loci is proposed.     

Oligodendrocytes from jimpy and normal mature tissue can be 'activated' when transplanted in a newborn environment.

Fragments of corpus callosum from P13 normal and jimpy (jp) mutant mice (containing only postmigrating precursors and differentiated oligodendrocytes (ODCs, some of them myelinating soon) have been transplanted into the thalamus of newborn shiverer (shi) mutant mice. The behaviour of transplanted ODCs has been assayed by immunohistochemistry of their myelin basic protein (MBP)-positive myelin synthesized in the host shi brain whose myelin is deprived of this component. ODCs and postmigrating precursors contained in P13 normal corpus callosum survived, migrated out of the graft and myelinated in the shi host parenchyma. The high ratio of positive cases observed was comparable to the one observed in previous experiments using fragments of newborn or embryonic normal tissue. When fragments of jp tissue were used as donors, postmigrating jp ODCs or precursors migrated on long distances out of the graft and synthesized large amounts of myelin as estimated by the size of the MBP-positive myelin patches present in the host shi brain. The extent of migration and the size of these myelin patches were more important than those observed in previous experiments using fragments of newborn or embryonic jp CNS as donors. By contrast, the low ratio of positive cases observed suggested that the survival of P13 jp ODCs or their postmigrating precursors cannot be restored by the newborn shi environment.(ABSTRACT TRUNCATED AT 250 WORDS)