Human leukodystrophies are hereditary and incurable dysmyelinating diseases. Their precise diagnosis and therapy must be studied using model animals. There is increasing interest in hereditary dysmyelinating mutant mice such as jimpy, quaking, twitcher, shiverer and myelin deficient (mld), as animal models for human leukodystrophies. Even though some of these mutants do not share the same enzymatic disturbances as human leukodystrophies, they are nice models of“dysmyelinating”diseases. Furthermore, they are also useful tools to analyze the genetic control and the intercellular (neurone-glial) recognition process of the myelination. We have intensely analyzing the dysmyelinating mutant mice designated as shiverer and myelin deficient (mld) . Both mutants have been revealed that they are disturbed in the expression of myelin basic protein (MBP) . MBP is one of major stractual proteins of the myelin. So shiverer and mld are good systems to analyze the relation between the regulation of MBP gene expression and the morphogenesis of myelin. Shiverer is an autosomal recessive mutant characterized by abnormal and poor myelin formation in the central nervous system accompanying severe tremor and tonic convulsions. Myelin of the shiverer has been proved to lack MBP by SDS-PAGE protein profile and immunohistochemical studies. We have used mice which have different strain specific isozyme pattern of GPI as wild type control. Brain tissue of chimeras examined is composed of both isozymes as well as coat color. By immunohistochemical staining of MBP, patches of MBP-negative and -positive sites are observed in the white matter of chimeras suggesting the absence of humoral factors to cause abnormal myelin formation. Shiverer-type myelin is observed adjacent to the normal myelin on the same axon, suggesting that oligodendrocyte itself would be responsible for the abnormal and poor myelination in the shiverer central nervous system. Kimura et al. have revealed shiverer has deletion mutation in MBP gene by Southern-blot analysis using MBP cDNA as a probe.
Mld displays similar findings to shiverer and shows the allelism to shiverer. In mld, too, remarkably reduced amount of MBP can be observed in SDS-PAGE protein profile. By immunohistochemical staining of MBP, scattered MBP-positive sites are slightly detectable in mld. This is the different point from shiverer. Molecular genetic analysis studies using MBP cDNA as a probe show the prominent decrease of myelin basic protein (MBP) in mld is based on the transcriptional level and MBP gene of mld has no significant changes compared with wild type control in the exons.

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Enzyme replacement with liposomes containing β-galactosidase obtained from charonia lumpas was carried out in murine globoid cell leukodystrophy (OLD) . Charonia lumpas β-galactosidase was able to hydrolyze galactocerebroside and trapped into liposomes prepared from lecithin, cholesterol and sulfatide (molar ratio; 7: 2: 1) . Liposomes containing charonia lumpas β-galactosidase were successfully incorporated into the mouse tissues, ³H-galactocerebroside labeled liposomes were also incorporated into mouse liver, spleen and other tissues. The accumulation rate of ³H-galactocerebroside into Twitcher mice liver and spleen was almost 40 to 100 times higher than those of controls and degraded to 70 to 80% of accumulated radioactivity of ³H-galactocere-broside by single injection of liposomes containing charonia lumpas β-galactosidase. Results suggest that exogeneous enzyme trapped in liposomes can be useful for the correction of accumulated compound.

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Jimpy mouse is a neurological mutant characterized by a defective myelination in the central nervous system and the spontaneous occurrence of severe intention tremor, unsteady gait and gen-eralized tonic-clonic seizures. The primary cause of the myelin deficiency has not yet been determined, although there have been numerous morphologic and biochemical studies.
In order to elucidate more clearly the disturbances of myelination in the central nervous system, we investigated the continuous administration of Forphenicinol, a small molecular weight im-munomodifier isolated from the culture of actinomycetes, to Jimpy mouse at early postnatal age to find the preventive effects of this substance on the development of clinical manifestation of the behavioral abnormalities and early death. The results obtained were summarized as follows:
1) Continuous administration of Forphenicinol at early postnatal age resulted in a striking recovery of myelin associated CNPase and CEHase activities and of total myelin concentration in the central nervous system of the Jimpy mouse.
2) A complete restration from clinical symptoms of intention tremor and convulsive seizures are appeared in about sixty percent members of Forphenicinol administrated Jimpy mice. The recovering mice also showed a marked prolongation of their life span.
3) Synthesis of galactocerebrosides, a specific lipid component of the myelin structure which is practically absent in the central nervous system of Jimpy mice, are regained to normal after administration of Forphenicinol.

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Homozygous reeler mice have action tremor, dystonic posture, and reeling atxic gait. The mutation at the reeler genetic locus (gene symbol: rl) causes cytoarchitectonic abnormalities to the cerebral and cerebellar cortices. Retrograde transport of horseradish peroxidase (HRP) was used to examine the cells of origin of the corticospinal tract (CST) and the callosal commissural fibers (CC) in the primary motor cortex of normal and reeler mice. In normal controls, CST neurons retrogradely labelled after. HRP injection into the lumbar cord were situated only in layer V. In the reeler, by contrast, the labelled CST neurons were scattered difusely all levels of the corresponding cortical area. In addition to the malpositioning of the somata, the labelled CST neurons in the cortex of the reeler could be divided into two major classes according to their dendritic pattern; typical pyramidal neurons and atypical ones. The typical pyramidal neuron had an atypical dendrite projecting from the superior pole of the soma and ascending toward the pia mater. The atypical pyramidal neurons consisted of six types: inverted, tumbled, bipolar, V-shaped, hook-shaped, and superficial polymorphic. The typical pyramidal neurons in the reeler tend to be situated relatively deep in the cortex and the atypical neurons tend to lie relatively superficially in the cortex.
In the normal mouse, CC neurons retrogradely labelled after the injection of HRP into the primary motor cortex are distributed in a bilaminar pattern such that the largest number of cells are located in supragranular layers II and III and infragranular layer V. In the reeler mutant, CC neurons are found in all cortical layers, but two-thirds are situated in the lower half of the cortex. On the basis of the cell shape and orientation of the apical dendrite, CC neurons of the reeler are classified into six morphological types: typical pyramidal, inverted pyramidal, tumbled, hook-shaped, polymorphic, and simple. The apical dendrites of the CC neurons in all layers of the cortex of the reeler mouse are randomly oriented: no direct relationship between the intracortical position of the soma and orientation of the apical dendrite was found. In contrast, CST neurons in the reeler mutant are concentrated in the outer third of the cortex, and there is a relationship between the laminar distribution of these cells and the alignment of their dendrites with respect to the pial surface : the apical dendrites of CST neurons lie in superficial layers tend to be oriented obliquely, whereas those of CST neurons lie in the deeper cortex most often are oriented vertically, i.e., toward the pial surface. Quantitative analysis revealed that the relative intracortical positions of the CST and CC neurons are reversed in the reeler mutant although both populations exhibited greater laminar disposition, and as a consequence, there is more intermingling of the two cell groups in the reeler than in the normal mouse. Thus, the present study suggests that the normal cytoarchitectonics of the primary motor cortex are inverted in the reeler mutant mouse.
Cerebella of the normal and reeler mutant mice at two months of age were studied by the Golgi impregnation method to elucidate the detailed configurations of the Golgi epithelial cells. On the basis of configuration of their Bergmann fibers, the Golgi epithelial cells of the reeler were relatively normal, or assumed inverted, swan neck, stellate, bipolar, small, or fan shapes, although their morphological changes were continuous rather than abrupt. A relatively normal type of Golgi epithelial cells which somewhat resemble those of normal controls was present in the reeler, although some differences between them in detailed appearance existed. It is important to note that the radially organized Bergmann fibers ascending from the somata of this type of Golgi epi-thelial cells gave rise to a glial architecture of normal appearance in small areas of

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A mutant strain of Wistar rats with hereditary acholuric jaundice, now known as“Gunn rat, ”was first reported by C. K. Gunn in 1938. The trait of jaundice has been characterized as autosomal recessive inheritance and evidenced to be due to a virtually complete deficiency of an enzyme in hepatic bilirubin metabolism, bilirubin : UDP-glucuronyltransferase. From these points of view, homozygous Gunn rats are generally regarded as an animal model comparable to a human inborn error of bilirubin metabolism, Crigler-Najjar syndrome Type I, which is frequently accompanied by nuclear jaundice (kernicterus) in the neonatal period with rare exceptions.
In this communication, we describe our recent studies on the pathobiochemical aspect of bilirubin-induced cerebellar hypoplasia, another notable disorder of the central nervous system, in homozygous Gunn rats of Sprague-Dawley strain, which have been bred in our laboratory for several years. Heterozygous Gunn rats, which were apparently normal in every respect including cerebellar development, were used as a control. While the concentration of serum bilirubin in heterozygotes remained low throughout (0.5 mg/dl or less), its level in homozygotes was increased up to approximately 7 mg/dl as early as 24 hours after birth and reached maximum (17-18 mg/dl) at postnatal days 17-20, followed by a gradual decrease to about 8 mg/dl at day 30. The cerebellar underdevelopment of homozygotes began at day 5, and the increase of cerebellar wet weight virtually ceased after day 10. Photoirradiation of homozygous newborns from b to 10 days of life could effectively prevent their cerebellum from hypoplasia.
Analyses of cerebellar proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed characteristic changes of two proteins associated with cerebellar hypoplasia, i.e., marked decrease and increase of 250 k and 50 k-dalton proteins, respectively. The former protein called GR-250 showed very similar properties, except for its molecular weight, to P₄₀₀, a protein reportedly characteristic of cerebellar Purkinje cells. The latter protein designated GR-50 was unequivocally identified by using a newly developed immunoblotting method as glial fibrillary acidic protein, which was known to be localized in astrocytes.
It was found that in the hypoplastic cerebellum of homozygotes the number of lysosomes with different sizes was increased, and lysosomal enzymes were activated to various degrees, among which β-glucuronidase activity was most enhanced-more than 8 times compared with the control. From Percoll density gradient centrifugation analyses and the degree of activation of cerebellar lysosomal enzymes, it was strongly suggested that there were at least three types of lysosomes with functional heterogeneity, and also that a particular type of lysosomes or enzymes was specifically activated accompanied with bilirubin encephalopathy.

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A lack of thyroid hormone (T₄) during early postnatal life produces a delay or arrest of cell proliferation, migration, and differentiation in the brain, and reduced myelination. On the other hand, growth hormone (GH) deficiency also impairs cell migration and oligodendroglial differentiation, with subsequent failure of myelination. Both the above findings appear to act via a similar mechanism, a thyroid-pituitary linkage. Neonatal thyroid dysfunction produces a striking alteration of the pituitary gland, characterized mainly by degradation of eosinophil cells, which appear to synthesize or store GH. For this reason, the effect of GH on brain growth in neonatally thyroidectomized animals has been intensively studied, but the results are still controversial and inconclusive.
Pituitary-deficient Snell dwarf mice (dw) exhibit severe growth retardation shortly after birth, owing to defective anterior pituitary glands which secrete very low amounts of GH, prolactin, thyroid-stimulating hormone (TSH) and possibly corticotrophin. The Snell dwarf mouse, therefore, appears to be ideally suited for studying the effects of growth stimulating factors, not only somatic but also on cerebral development.
Differences in the brain weights of the dwarf and control mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity (myelinmarker enzyme) was found to be suppressed in the cerebrum and brain stem throughout the devel-opmental stage, but not in the other parts of the brain. Differences in DNA content per cerebrum from the dwarf and control mice first became apparent on the 10th day of age. Henceforth, the dwarf mice showed no further increase, although the normal controls continued to increase. [³H] -Thymidine incorporation into DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about half of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the dwarf cerebrum is a hypomyelination due to reduced oligodendroglial proliferation. Furthermore, we also demonstrated the faulty development of cortical neurons in the dwarf cerebrum.
On the next step, we investigated the effect of GH, TSH, and T₄ on myelinogenesis by monitoring the activity of the CNPase. The results obtained in this study suggest that acceleration of retarded myelinogenesis, possibly through the enhancement of glial cell division, depends essentially upon the GH level plus the synergistic effects of T₄. In addition, we also demonstrated that exogenous OH and T₄ will enhance neuronal growth in the Snell dwarf cerebrum, and retore neuronal arborization to normal.
To determine whether the two hormones, GH and T₄, canact independently, we used a prom-ising animal model, the inherited primary hypothyroid mouse (hyt), which has a hypoplastic thyroid gland because of an autosomal recessive mutation. CNPase activity in the cerebrum of this mutant is also reduced in comparison with the normal control. However, no differences were observed with regard to DNA and RNA contents and RNA/DNA ratio. These results indicate that hypomyelination in the hypothyroid mouse is restricted to the cerebrum, and is not related to arrest glial proliferation. In addition, no morphological abnormalities of cortical neurons were observed in the cerebrum, although hypomyelination was present.
To determine whether GH has an independent action on cerebral development, we examined the central nervous system of the Little mouse (lit), a promising model of isolated growth hormone deficiency Type I. Our findings are that; the weight of two parts of the lit brain, the cerebrum and

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Rolling mouse Nagoya (rolling) is a neurological mutant mouse that shows an incoordination of the hind limbs and gait disturbance. Morphological studies of the brain have not revealed any definite abnormalities in rolling. Mainly due to resemblance of the gait disturbance to cerebellar ataxia, rolling has been regarded as an animal model of hereditary cerebellar ataxia.
In order to elucidate the neural mechanism of the motor disturbance of rolling, local cerebral glucose utilization (LCGU) was studied in rolling (genotype rol/rol), its heterozygote (+/rol) and normal mice (+/+), as well as in weaver mouse (weaver) that has a definite degeneration of the granule cells of cerebellar cortex. Pharmacological effects of dopamine agonist, apomorphine, and antagonist, haloperidol, were also studied. LCGU has been proved to be an excellent parameter for observing local brain activity.
Rolling showed a markedly increased LCGU in the globus pallidus, entopeduncular nucleus, subthalamic nucleus and substantia nigra pars reticulata, bilaterally, compared to +/rol and +/+. A part of the cerebellar cortex showed a tendency to reduction of LCGU when compared with +/+, but not with +/rol. Weaver showed a decreased LCGU in the granular layer of the cerebellar cortex, a consistent finding with the morphological studies. The basal ganglia of weaver, however, did not show any abnormalities. These results suggested that rolling has a primary dysfunction in the basal ganglia, not secondary to disturbance of the cerebellum. Clinical observation also supported that the motor disturbance of rolling is probably due to extrapyramidal dysfunction.
LCGU studies with apomorphine and haloperidol in rolling and its control mice suggested that the effect of apomorphine on LCGU was reduced or abolished in the basal ganglia of rolling, and this alteration was brought about by a dysfunction of the striatum, consequently affecting the functions of the globus pallidus, entopeduncular nucleus, subthalamic nucleus and substantia nigra pars reticulata.
These observations indicated a possibility that rolling is an animal model of hereditary extrapyramidal diseases.

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A new autosomal recessive gene causing sphingomyelinosis in mice is described. The disease syndrome caused by this gene has been diagnosed as an analogue of Niemann-Pick disease in humans. Affected mice cannot breed. They show neurological symptoms and weight loss beginning from around 7 weeks of age, and die at 12-14 weeks. By 8 weeks of age striking hepatosplenomegaly and marked enlargement of lymph nodes are present. Large areas of the liver and spleen are replaced by clusters of foam cells. The contents of sphingomyelin and free cholesterol in the liver and spleen are markedly elevated. But the brain shows no obvious changes in lipid concentration. Sphingomyelinase activity is reduced to about 30 percent that of the homozygous normal controls in the liver, 50 percent in the spleen and 70-80 percent in the brain. Heterozygotes are normal in both lipid concentrations and sphingomyelinase activity.
The isoelectric focusing pattern of sphingomyelinase in the liver and the brain from both a control and an affected mouse was studied. In the control liver, 2 major peaks of sphingomyelinase activity were seen. One peak with a pI of 4.2, indicates acid sphingomyelinase. Other peak with a pI of 3.0 shows neutral sphingomyelinase. In the affected liver, peak with pI of 3.0 was quite similar to that of control, whereas a significant decline of peak with pI of 4.2 was noticed. On the other hand, in the control brain, the 2 major peaks of sphingomyelinase activity were also observed. However, in contrast to the liver, one peak with pI of 3.0, which indicated neutral sphingomyelinase, was the most prominent peak, and the pI of acid sphingomyelinase was different from that of the liver. In the affected brain, 2 peaks of sphingomyelinase activity were also seen. This pattern did not differ from the control brain. Subsequently, the subcellular distribution of the brain sphingomyelinases was studied. In the whole homogenate fraction, sphingomyelinase activity of the affected brain showed approximately 70% of the control activity. However, sphingomyelinase activity in the mitochondria-lysosome fraction of affected brain had only 19% of the normal control. The deficiency of lysosomal acid sphingomyelinase in the brain probably is responsible for the development of neurological manifestations in the affected mice.
The Niemann-Pick mice which received bone marrow transplants showed a decreased ac-cumulation of sphingomyelin and cholesterol quantitatively and a increased activity of sphingomyelinase in their spleen and liver. The sphingomyelin deposit in the bone marrow was also reduced histochemically and foam cells in the liver and spleen were significantly decreased. However, the neurological manifestations were not improved by the bone marrow graft. Our result has encouraged us to apply bone marrow transplantation to the patients with Niemann-Pick disease except the neurological manifestation type. It also might warn of unqualified application of bone marrow transplants to humans with lysosomal storage disese showing neurologiacl involvement.

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[in Japanese]

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[in Japanese]

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