Mld Mice Research Articles

Enzyme replacement in the CSF to treat metachromatic leukodystrophy in mouse model using single intracerebroventricular injection of self-complementary AAV1 vector.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency in human arylsulfatase A (hASA). We recently reported that ependymal cells and the choroid plexus are selectively transduced by intracerebroventricular (ICV) injection of adeno-associated virus serotype 1 (AAV1) vector and serve as a biological reservoir for the secretion of lysosomal enzymes into the cerebrospinal fluid (CSF). In the present study, we examined the feasibility of this AAV-mediated gene therapy to treat MLD model mice. Preliminary experiments showed that the hASA level in the CSF after ICV injection of self-complementary (sc) AAV1 was much higher than in mice injected with single-stranded AAV1 or scAAV9. However, when 18-week-old MLD mice were treated with ICV injection of scAAV1, the concentration of hASA in the CSF gradually decreased and was not detectable at 12 weeks after injection, probably due to the development of anti-hASA antibodies. As a result, the sulfatide levels in brain tissues of treated MLD mice were only slightly reduced compared with those of untreated MLD mice. These results suggest that this approach is potentially promising for treating MLD, but that controlling the immune response appears to be crucial for long-term expression of therapeutic proteins in the CSF.

97. Translational Studies toward Optimizing In Vivo Bone Marrow Stem Cell Gene Transfer Using Lentiviral Vector

strategy is hampered by low efficiency of gene transfer and expression in repopulating HSC. To overcome this problem, we analyze the effect of overexpression of HoxB4, which can expand HSC, in mouse BM cells by transplantation of MLD mice. 5FU treated GFP mouse BM cells were transduced with retroviral vector expressing HoxB4 (B4) or control retroviral vector expressing GFP (MIG). After expansion of these transduced cells in vitro for five days transplantation was performed into irradiated MLD mice. High level of engraftment was observed in B4 transplanted MLD mice (B4MLD) compared to MIG transplanted MLD mice (MIGMLD)(92.4 vs. 36.5 %). ASA enzyme activity in serum was also increased in B4MLD compared to MIGMLD (19.8 vs. 10.3 nm/mg/hr). To evaluate the contribution of bone marrow cells as carriers for gene therapy of neurological disorders, we studied the fate of transplanted BM cells in the adult mouse brain. Efficient GFP positive neuronal cells were detected in B4MLD brain compared to MIGMLD brain. Immunohistochemical staining showed that most of the GFP positive cells ware IbaI positive microglia cells as we and others reported. In addition, O4 positive oligodendocyte like cells were identified only in the B4MLD brain but not in the MIGMLD brain (O4 positive/GFP positive: 6/326 vs. 0/312). This is the first demonstration indicating that systemically infused HSCc could differentiate to oligodendrocytes in adult mouse brain. It was recently suggested that HoxB4 could play a role in oligodendrocyte maturation. Our data strongly support that HSCs transduced with HoxB4 expression vector are useful for ex vivo gene therapy of CNS disorders as carriers of production of therapeutic protein and for regeneration of oligodendrocytes to treat demyelinating disorders such as MLD, Krabbe disease, and multiple sclerosis.

426. Cure of Established Disease and Direct Evidence of Cellular Cross-Correction in the Nervous System of Metachromatic Leukodystrophy Mice after HSC-Based Gene Therapy

Top of pageAbstract Metachromatic Leukodystrophy (MLD) is an inborn lipidosis caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). It is characterized by storage of undegraded sulfatides in the CNS and PNS, leading to severe demyelination. We showed that transplantation of LV-transduced Hematopoietic Stem Cells (HSC) allowed: i) targeting therapeutic genes to CNS and PNS by turnover of microglia and endoneurial macrophages by HSC-derived cells; ii) preventing functional and neuropathological manifestations of MLD in the mouse model, when applied at pre-symptomatic stage (A. Biffi et al., J. Clin. Invest 2004, 113(8): 1118|[ndash]|29). Here we assessed whether this approach can be effective not only in preventing, but also in correcting already established MLD manifestations. By 5|[ndash]|6 months of age, MLD mice display motor conduction abnormalities in CNS and PNS, impaired motor coordination and learning, and sulfatide storage in the NS. We compared the outcome of transplant performed before disease onset with transplantation of ARSA-transduced HSC in 6|[ndash]|7 months old, already symptomatic, MLD recipients. We reached ARSA over-expression in the hematopoietic system of transplanted mice. Neurophysiological evaluations performed on 12 months old mice demonstrated normalization of motor stimuli conduction along CNS and PNS. Rotarod test showed, by comparison with age matched sham-treated MLD mice, protection from further worsening of motor coordination and learning abilities. Remarkably, 12 months old treated mice showed improved performance respect to 6 months old MLD controls and a similar performance to that of age-matched wild type mice. These data indicated that ex vivo gene therapy not only prevented disease progression, but also corrected already established functional deficits. In order to assess the mechanism responsible for the widespread correction of CNS pathology, and define the contribution of ARSA over-expression, we performed immunohistochemical studies of the brain of MLD mice transplanted with HSC expressing a HA-tagged ARSA. Confocal analysis showed the widespread occurrence of tagged enzyme within HSC-derived microglia and resident CNS neurons, oligodendrocytes and astrocytes. Importantly, ARSA-HA was correctly sorted to the lysosomal compartment in all cell types, proving the occurrence of in vivo enzyme transfer. Because these findings were associated to complete reversal of the neuronal degeneration which affects the hippocampus and cerebellum of aging MLD mice, in vivo enzyme transfer achieved robust metabolic cross-correction of the crucial CNS cell targets of the disease. Overall, these results demonstrate that transplantation of LV-transduced autologous HSC represents a promising therapeutic strategy for correcting MLD and possibly other neurodegenerative disorders in humans.

41. Ex Vivo Gene Therapy Provides a Unique Therapeutic Benefit and Corrects Metachromatic (MLD) and Globoid (GLD) Leukodystrophy in the Mouse Models

MLD and GLD are inborn lipidosis caused by the deficiency of the lysosomal enzymes arylsulfatase A (ARSA) and galactocerebrosidase (GALC). They are characterized by storage of undegraded sulfatides in the CNS and PNS, leading to progressive demyelination and early death. Using Lentiviral Vectors (LV), we efficiently transduced Hematopoietic Stem Cells (HSC) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice, and correct MLD and GLD phenotypes in the mouse models. We proved extensive transgene expression in the CNS and PNS of transplanted mice due to progressive turnover of microglia and endoneurial macrophages by HSC-derived cells. Recruitment of these cells was consistently observed independently from the conditioning protocol, and it was faster and more robust in MLD and GLD mice. By transplanting HSC transduced with the ARSA gene, we reconstituted enzyme activity in the hematopoietic system of MLD mice at supranormal levels and prevented all mayor disease manifestations. Transplanted mice were completely protected from the development of motor conduction impairment (normalization of motor conduction velocity and F wave latency, and of central conduction time), of learning and coordination deficits (normalization of the performance at rotarod test), and of neuropathological abnormalities (prevention of sulfatide accumulation and demyelination in CNS and PNS) typical of the disease. Remarkably, ex vivo gene therapy had a significantly better therapeutic impact than wild-type HSC transplantation on all examined parameters, highlighting the crucial role of enzyme over-expression and indicating the likely occurrence of in vivo cross-correction. Confocal analysis of the CNS of treated mice showed the presence of tagged ARSA enzyme within the lysosomes both of the transduced HSC-derived microglia cells and of resident neurons, demonstrating in vivo enzyme transfer and, most likely, cross-correction. To further evaluate the therapeutic impact of this approach we challenged it with the acute and devastating evolution of GLD in the mouse model. Neonate GLD mice were transplanted with HSC transduced with the GALC gene, supplemented with accessory Sca1- cells to reduce short-term toxicity of conditioning. We observed a dramatic improvement in survival of transplanted mice, which reached an average of 135 days, as compared to 40 days of untreated GLD mice and 80 days of mice transplanted with wild type bone marrow, providing striking evidence, in another disease setting, of the therapeutic advantage of ex vivo gene therapy versus allogeneic BMT. We observed a significant improvement in the GLD phenotype (absence of leg palsy and delayed, minor twitching) and prevention of mayor histopathological signs of the disease (demyelination, axonal loss). Overall, these results demonstrate that transplantation of LV-transduced autologous HSC represents a new, potentially efficacious therapeutic strategy for MLD, GLD and possibly other neurodegenerative disorders.

The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin‐Deficient (mld) Mutant Mice

The myelin-deficient (mld) mutation is autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mld. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin-associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low-molecular-mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.

Quantitative Differences between Homozygous 'USA' and 'Swiss' <i>mld</i>Mutant Mice

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

In situ analysis of myelin basic protein gene expression in myelin-deficient oligodendrocytes: antisense hnRNA and readthrough transcription.

Mice homozygous for the recessive mutation myelin deficient (mld) exhibit a severe deficit in the synthesis of myelin basic protein (MBP). The primary defect in mld mice is an unusual rearrangement of the MBP gene. The mld MBP locus consists of two tandem MBP genes that span approximately 90 kb of DNA; the upstream gene contains an extensive inversion of its 3' region, while the downstream gene appears identical to the wild-type gene. In this report, the aberrant expression of the mld MBP locus was examined by in situ hybridization with MBP genomic and cDNA probes. In situ hybridization with a single-stranded genomic probe from the inverted region of the mld MBP gene revealed that primary transcripts initiating from the upstream MBP promoter elongate through the inverted region generating abundant antisense MBP heterogeneous nucleus RNA (hnRNA) transcripts in mld oligodendrocytes. Little or no antisense MBP RNA was detected in cytoplasmic regions in mld brain sections indicating that the antisense MBP hnRNA transcripts are not processed and transported out of the nucleus. Furthermore, we provide evidence that these abnormal transcripts elongate through the downstream MBP gene's promoter region, suggesting that transcription of the downstream mld MBP gene is inhibited by readthrough transcripts that originate at the upstream promoter.

Chimeric and Molecular Genetic Analysis of Myelin‐Deficient (Shiverer and Mld) Mutant Mice

The shiverer and myelin-deficient (mld) mutants are two allelic mutations. Both are characterized by hypomyelination in the CNS and deficient expression of the MBP gene. Chimeric analysis of the pathogenesis of shiverer showed that shiverer mutation acts intrinsic to the oligodendrocyte, which is the only cell type expressing the MBP gene in the CNS. Molecular genetic studies by several groups demonstrated that shiverer is a deletion mutation in the MBP gene. Consequently, no MBP is produced in the shiverer mutant. In mld, however, partial expression of the MBP gene was observed. Interestingly, MBP was expressed in a mosaic fashion in the CNS of mld mice. Molecular genetic studies revealed that the mld mutant has duplicated MBP genes in tandem on a distal part (E2-4) of chromosome 18q. The reduced expression was based on the level of mRNA. A large portion is inverted in the upstream copy, and an intact copy is located downstream. We showed in mld mutants that antisense RNA corresponding to the inverted segment is transcribed, and it forms RNA duplex, with the RNA transcribed from the normal gene located downstream.

Post-transcriptional events are responsible for low expression of myelin basic protein in myelin deficient mice: role of natural antisense RNA.

Myelin deficient mice (mld) are characterized by tandem duplication of the gene coding for myelin basic protein (MBP). The upstream gene contains a large inversion of the 3' region which includes exons 3-7, and therefore it cannot give rise to mature mRNA and functional protein. MBP and MBP mRNA concentrations in mld brains constitute only approximately 2% of the concentrations present in normal mice. The overall transcription rate of the Mbp gene is normal. In order to explain the discrepancy between mRNA concentration and transcription rate, we studied transcription of each individual gene. The two genes were transcribed independently, although some uninterrupted transcription could not be excluded. The rate of transcription of the upstream gene was higher than that of the downstream gene. This difference was reflected in the concentration of sense and antisense RNA found in nuclei. Our results indicate that the low concentration of the mature mRNA cannot be caused by transcriptional interference. High concentration of nuclear antisense RNA strongly suggests that post-transcriptional regulation occurs in mld mice through formation of double stranded RNA.

Myelin/Oligodendrocyte Glycoprotein Expression during Development in Normal and Myelin-Deficient Mice

The myelin/oligodendrocyte glycoprotein (MOG) is identified by monoclonal antibody 8-18C5. MOG is localized on the surface of myelin and oligodendrocyte processes. Recently, several studies have shown that MOG plays an important role as a target for antibody-induced demyelination. In the present study, we investigated MOG expression in the brains of normal and myelin-deficient (mld) mutant mice during development. By gel electrophoresis and immunoblotting, we observed the developmental pattern of two closely migrating bands, with apparent molecular masses of 26 and 28 kilodaltons. Their concentrations increased coordinately during the most active phase of myelin and myelin basic protein (MBP) synthesis. Between 20 and 25 days of age, the MOG developmental pattern superimposed that of MBP as well as myelin yields. In mld mutant mice, which are affected by a severe deficit of MBP synthesis, MOG was present at reduced levels (40% of controls at 60 days of age). At 85 days of age, mld mice exhibited increased concentrations of MBP, and myelin was better compacted. At this age, MOG concentrations increased and reached 70% of controls. These results suggest that MOG could play a role in the maintenance or completion of the myelin sheath. Its expression level may be modulated by the presence of compact myelin and/or MBP in the myelin sheath.

The duplicated myelin basic protein gene in mld mutant mice does not impair transcription

Myelin basic protein (MBP) gene organization and expression were analyzed in wild type and myelin deficient ( mld) mutant mice. Southern analysis demonstrated MBP gene duplication in mld mice. In addition, we present evidence that one MBP gene in mld mice is normal for at least 14 kilobases (kb) upstream from exon I, whereas the second gene is normal for at least 3.5 kb but not more than 7 kb upstream from exon I. Run-on experiments showed that the rate of MBP gene transcription in mld mice is similar to that seen in normal mice. Detailed analysis of the transcriptional activity of various regions of the gene led us to conclude that all portions of the MBP gene are transcribed in mld mice. Consequently, we propose that the low levels of MBP mRNA observed in these mice (2–5% of the wild-type level) are not due to deficient transcriptional activity.

Immunocytochemical localization by electron microscopy of 2'3'-cyclic nucleotide 3'-phosphodiesterase in developing oligodendrocytes of normal and mutant brain

Oligodendrocytes or their putative progenitors were the only cells found to be immunoreactive to polyclonal antisera against the enzyme 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in developing and mature brains of rats and mice, as visualized by light and electron microscopy. Prior to myelination (day 6), oligodendrocytes of the corpus callosum have reticular networks of CNP-containing filopodia, in addition to abundant CNP throughout the cytoplasm. Some glioblast-like cells of the subventricular zone are also immunoreactive to anti-CNP, suggesting that, as progenitors of oligodendroglia, they express this myelination-related protein as one of the earliest events in myelinogenesis. Following the commencement of myelination (day 15), many oligodendrocytes lose much of their lacelike network of fine projections, possessing, instead, larger CNP-filled processes that extend to myelin-bearing fibers. CNP was always found only in the cytoplasm-containing compartments of the cells and myelin sheaths; neither lamellae nor cellular membranes were immunostained. These data support our contention that CNP is not an intrinsic membrane protein, despite its strong interaction with membrane components when cells are disrupted. In mutant (mld) mice (day 25), the many distended and uncompacted oligodendroglial processes that invest axons with only a few turns of membrane contained cytoplasmic CNP, accounting for the elevated levels of CNP activity previously noted in tissue fractions.

A novel mutation in myelin-deficient mice results in unstable myelin basic protein gene transcripts

Mice homozygous for the myelin-deficient (mld) mutation have an unusual phenotype in which the gene encoding myelin basic protein (MBP) is expressed at low levels and on an abnormal developmental schedule. In this report we describe the organization of the mld MBP locus, which results in this alteration of MBP expression. The mld MBP locus consists of two tandem MBP genes, with the upstream gene containing an inversion of its 3′ region. We also demonstrate that although there are low steady-state levels of MBP RNA in mld mice, the mid MBP locus is transcribed at a rate comparable to that of the wild-type MBP gene, indicating that the MBP transcripts are abnormally unstable.

Immunohistochemical, biochemical and electron microscopic analysis of myelin formation in the central nervous system of myelin deficient (mld) mutant mice

Myelin deficiency (mld) is an autosomal recessive mutation in mice and is considered to be allelic to the shiverer (shi) mutation. Mld mice are characterized by hypomyelination of the central nervous system (CNS). They show typical symptoms such as tremor, tonic convulsion and ataxic movement. Subcellular fractionation of the CNS revealed that the MBP bands were greatly decreased in the P2A (myelin) fraction and the total content of myelin basic protein (MBP) was much lower than that in the control in all parts of the CNS. Sections from mld mice were examined by immunohistochemical tests with MBP antiserum, and a mosaic expression of MBP was found in the myelin of the mld mice. Since the major dense line is considered to be composed mainly of MBP, we investigated the myelin of mld mice by electron microscopy and found that there were 3 types of myelin: (1) a normal type compact myelin with a major dense line, (2) a shiverer-type myelin with no major dense line, and (3) a mixed-type myelin, in which within a myelin lamella the major dense line abruptly changes to cytoplasm of oligodendrocytes.

Structure and Expression of Myelin Basic Protein Gene Sequences in the mld Mutant Mouse: Reiteration and Rearrangement of the MBP Gene

The mld mutation on chromosome 18 in the mouse is a putative allele of the shiverer (shi) mutation. We have analyzed the structure of myelin basic protein (MBP) gene sequences in mld DNA by restriction mapping of genomic DNA. The results indicate that the mld chromosome carries two copies of the MBP structural gene, one of which is intact and one of which is interrupted. Genetic analysis indicates that the interrupted gene is close to the intact MBP structural gene and cosegregates with the mld mutation. We have also analyzed the levels of MBP polypeptides and MBP-specific mRNA in wild-type, homozygous and heterozygous shiverer and mld mice and in mice carrying both mutations. The results indicate that both shi and mld are cis-acting codominant mutations that cause severely reduced steady state levels of MBP-specific mRNA and MBP polypeptides in the brain. We have analyzed the total number of oligodendrocytes and the number of MBP-positive oligodendrocytes in mld and shi brain primary cultures. In shi cultures, none of the oligodendrocytes expresses MBP. However, in mld cultures, approximately 5% of the oligodendrocytes express MBP. The nature of the "revertant" mld oligodendrocytes is not known.

Myelin deficient mice: Expression of myelin basic protein and generation of mice with varying levels of myelin

Mice homozygous for the mutation myelin deficient ( mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mid mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice.

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.

Myelin deficient ( shi mld) mutant allele: Morphological comparison with shiverer ( shi) allele on a B6C3 mouse stock

A new B6C3 stock of shi mld mutant mice is compared in terms of behavior and CNS morphology with both a B6C3 shi stock and reports on other shi mld animals. Defects of B6C3 shi mld myelination seen at postnatal day 21 (P-21) are comparable to those in B6C3 shi with respect to % axons myelinated, sheath thickness, errors in the wrapping and targeting of myelin and abnormal oligodendrocyte shape. The two mutations are similarly expressed in cerebellar organotypic cultures. However, the major dense line (MDL) is present in a few shi mld myelin sheaths at P-12 and a few sheaths show myelin basic protein by immunocytochemistry, while neither phenomenon is seen shi at this age in the same CNS regions. Shi mld mice survive their disease significantly better than shi. The Shi mld stock currently under study elsewhere differs from this B6C3 stock in that MDL was reported only in older animals, and behavior and survival were severely compromised.

Anticonvulsive treatment of myelin-deficient (mld) mice improves survival and confirms the delayed increase of myelin basic protein.

Myelin-deficient (mld) mutant mice were treated with phenobarbital between 60 and 90 d of age. The survival rate at 90 d increased from 1.4% in untreated mutants to 46% in those who received phenobarbital. This is evidence that apneic spells during tonic seizures are a major cause of death in mld mice. Myelin basic protein (MBP) content of brain homogenates from treated mld mice increased significantly between 30 and 90 d. MBP was present in myelin purified from the 90-d-old treated mld mice. These results demonstrate that the MBP increase, which occurs after the active phase of myelin formation is completed, is a general phenomenon and is not caused by the selection of a small and mildly affected subpopulation of mutants.

Myelination in the CNS of mld mutant mice: Comparison between composition and structure

Myelination was studied between 15 and 135 days postnatally in the brain and optic nerves of myelin deficient ( mld) mutant mice. Between 15 and 30 days almost no myelin basic protein (MBP) could be detected in mld myelin. The axons were loosely wrapped by membranes which only fused at the extracellular sites forming the intraperiod line. At this age the major dense line was absent. At 25–30 days, purified myelin contained extremely high 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNP) (EC 3.1.4.37) activities which could be related to the redundant paranodal-like structures observed at this age in mld CNS. Therefore, it can be suggested that CNP is probably localized in such paranodal loops. After the active phase of myelin deposition was completed in controls, mld mutants showed important increases of MBP concentration in myelin with the concomitant appearance of the major electron dense line and better compaction of the myelin lamellae. The yield of myelin increased from 5 to 14% of control values during the period of 30 to 135 days. Since the recovery phase occurred at the time when myelin lipid synthesizing enzymes are at low residual activities, the myelin deficit could only be partially corrected. This study indicates that there is a delay of MBP synthesis in mld mice and the decrease of other myelin proteins could be secondary to the assumed primary defect involving MBP.