Quaking Mice Research Articles

An X-ray diffraction study of central and peripheral myelination in Jimpy and Quaking mice

An X-ray diffraction study of central and peripheral myelination in Jimpy and Quaking mice

Lipid and fatty acid composition of testes of quaking mice.

Testes of quaking mice (sterile mutants) and of controls were analyzed for major lipid classes and fatty acid composition. Of the main lipid classes, only cholesterol esters differed significantly in concentration between the two groups (1.01 for quakers vs 0.69 mg/g wet wt of tissue for controls). The concentration of triglycerides was 4.5-5.0 that of total phosphatides 18-19 and that of free cholesterol 1.9-2.0 mg/g for mutants and controls. The concentrations of phosphatidyl ethanolmanine and of sphingomyelin were both lower in quaking than in normal mice, but only the change in the former was statistically significant. Phosphatidyl choline was the major phosphatide (43-45% of total phosphatides) followed by phosphatidyl ethanolamine (24-26%) and sphingomyelin, phosphatidyl serine, and phosphatidyl inositol (all ca. 7% of total phosphatides). Minor differences between the mutants and controls were observed in concentrations of fatty acids of major lipid classes. The mutants, sterile because of faulty spermatid differentiation, had normal quantities of 22:6 w3 and 22:5 w6. These data are consistent with the hypothesis that the 22-carbon polyenes are associated with the formation of spermatids, rather than with their final differentiation into spermatozoa.

Hyperplasia of oligodendrocytes in quaking mice

The number of neuroglial cells in selected fiber tracts of 90-day-old quaking and normal mice was determined by a combination of light and electron microscopy. Oligodendrocytes of quaking mice are normal in number in the anterior commissure and corticospinal tract (in the cervical spinal cord) but are increased two- to fourfold in the optic nerve and the fasciculi cuneatus and gracilis (in the cervical spinal cord). The nuclei and perikarya are normal in size or smaller than normal. Those tracts with the greatest hyperplasia of oligodendrocytes also have the greatest content of myelin, suggesting that cell number influences content of myelin. However, the volume of myelin per oligodendrocyte also varies, between 2 and 11% of normal, in the different tracts of the mutant. The hyperplasia of oligodendrocytes in quaking mice may arise as compensation for their decreased production of myelin and reflect a normal plasticity in the processes of myelination. If so, the mutant may be a useful system for study of the regulation of myelogenesis.

Ceramide biosynthesis in mouse brain microsomes: Comparison between C57/BL controls and quaking mutants

The biosynthesis of ceramides by mouse brain microsomes from sphingosine and stearoyl or lignoceroyl CoA was investigated. Microsomes from Quaking and normal mice showed similar activities when the acyl donor was stearate, but the Quaking mice exhibited about 55% less activity than normals when the acyl donor was lignocerate. This is interpreted to support the previous conclusion that two acyl transferases are involved in the synthesis of the nonhydroxy ceramides. It would appear that the lack of the lignoceroyl transferase is a specific aspect of the Quaking disorder. The composition of the components of cerebrosides from the brains of normal and Quaking mice was determined. An increased proportion of dihydrosphingosine and glucocerebroside was observed in the lipid from the mutants. A relative lack of the very long chain nonhydroxy acids was also observed, in confirmation of previous observations. The acyltransferase catalyzing stearoyl sphingosine formation was found to be unstable when incubated without substrates for 30 min, but considerable stabilization by sphingosine was observed.

The accumulation of adenosine 3′,5′-monophosphate in cerebral cortical slices of the quaking mouse, a neurologic mutant

Norepinephrine, adenosine, veratridine, and adenosine-biogenic amine combinations elicit an accumulation of cyclic AMP in cerebral cortical slices from C57B1/6J control (+/+), quaking (qk/qk) and heterozygous (qk/+) mice. The percent conversion of radioactive adenine nucleotides to cyclic AMP in slices previously incubated with radioactive adenine or adenosine is markedly lower in slices from quaking and heterozygous mice compared to controls with all stimulatory agents except an adenosine-norepinephrine combination. Total incorporation of radioactive adenine or adenosine into adenine nucleotides is, however, significantly higher with slices of quaking and heterozygous mice. The absolute amount of radioactive cyclic AMP and the levels of endogenous cyclic AMP are nearly identical in the 3 groups of mice following incubation with all stimulatory agents except adenosine. The absolute accumulation of both radioactive and endogenous cyclic AMP was significantly lower in quaking and heterozygous mice after incubation with adenosine. The ratio of [ 14C]- to [ 3H]cyclic AMP in slices previously incubated with [ 14C]adenine and [ 3H]adenosine is dependent upon the stimulatory agent. The ratio with each agent is consistently lower in quaking mice compared to controls. The data provide evidence for biochemical alterations in nucleotide metabolism in cortical slices of both quaking and heterozygous mice. These effects would not appear to be directly associated with hypomyelination and tremor of the quaking mouse, since the heterozygous mouse, with one quaking gene does not show the latter gross abnormalities.

Fine structure of the myelinated nerves of the iris with special reference to wallerian degeneration after denervation of the ophthalmic division of the trigeminal nerve

The cationic ion binding affinity of peripheral nervous system fibers in quaking and littermate control mice was investigated with the technique described by Langley & Landon (1969). In many fibers from controls, the electron dense material was localized on the nodal axolemma as well as within the axoplasm. In fibers from quaking mice, the electron dense material was similarly localized at the well-formed node although it was less intense and less consistent. In immature nodes and non-myelinated segments of fibers from quaking mice, there was no recognizable additional electron dense material on the axolemma.Such a difference in the localization of the electron dense material on the axolemma may be due to possible differences in the concentration of sodium channels in the axolemma of certain peripheral fibers in quaking mice. However, the presence of the electron dense product in the Schmidt-Lanterman incisures and periaxonal spaces of some fibers may suggest some diffusible chemical substances as a sodium reservoir in these regions.

Etude “ in vitro” des acides gras synthétisés dans les microsomes de cerveaux de souris normales et “quaking”

Radiogas chromatographic studies of the products of fatty acid biosynthesis in mice brain microsomes confirm the existence of a « de novo system from acetyl-CoA and malonyl-CoA and of a least two elongating systems for long chain fatty acids, involving malonyl-CoA. The possibility of an intermediary system leading from C 18 to C 20 fatty acids has been evoked. Comparison between non mutant and quaking mice indicates that all the microsomal fatty acid biosynthetic systems are depressed. The biosynthetic system elongating fatty acids from C 18 is the one which is the most modified quantitatively and qualitatively in quaking. Microsomal and soluble « de novo systems are qualitatively intact. L'étude par chromatographie en phase gazeuse des produits de la biosynthèse des acides gras dans les microsomes de cerveaux de souris confirme l'existence d'un système de synthèse de type « de novo à partir d'acétyl-CoA et de malonyl-CoA et d'au moins deux systèmes d'allongement des acides gras à longue chaîne par le malonyl-CoA. La possibilité d'un système intermédiaire conduisant des acides en C 18 aux acides en C 20 est envisagée. La comparaison entre les souris non mutantes et les souris «quaking indique que chez ces dernières l'activité de tous les systèmes microsomaux de synthèse des acides gras est diminuée. Le système de biosynthèse des acides gras au-delà du C 18 est qualitativement et quantitativement le plus perturbé chez les «quaking. Le système de type « de novo microsomal et «soluble n'est qualitativement pas atteint.

PROTEOLIPIDS IN THE DEVELOPING BRAINS OF NORMAL MICE AND MYELIN DEFICIENT MUTANTS1

Abstract— A developmental study of proteolipids from brains of normal mice and two myelin deficient mutants, jimpy and quaking, was performed. The proteolipids were obtained by diethyl ether precipitation of washed total lipid extracts from whole brains and were analysed on polyacrylamide gels containing sodium dodecyl sulphate. The amount of ether precipitable material extractable from normal brains increased almost six‐fold between 12 and 21 days posr partum. This increase was not observed with the mutant mice. Polyacrylamide gel electrophoretic analysis of the proteolipid fraction showed it to be heterogeneous, with eight major protein bands. Two of these proteins increased rapidly in quantity in normal mice between 13 and 21 days. These two proteins were present, in severely reduced quantities in the brains of jimpy and quaking mice at all ages examined. One of these proteolipids was the major species present in proteolipid extracts from the brains of normal mature mice. This protein coelectrophoresed with proteolipid isolated from purified myelin and has been tentatively identified as the myelin proteolipid. The other proteolipid which was deficient in jimpy and quaking brains was not characterized, but it appeared to be of extra‐myelin origin, and suggests that parts of the brain other than the myelin sheath may be involved in the jimpy and quaking disorders.

Glial fibrillary acidic protein in mutant mice with deficiency of myelination: quaking and jimpy.

Fibrous neuroglia has been studied by immunofluorescence with GFA protein antibodies in the spinal cord of mutant mice with deficient myelination: quaking and jimpy. In both these mutants a well formed astrocytic framework was observed in the non-myelinated white matter, indicating that the arrest of myelination in quaking and jimpy mice is associated with defects of the oligodendroglial cell line, rather than with an abnormality of glioblast differentiation.

Fatty acyl-coenzyme A elongation in brain of normal and quaking mice.

Microsomal enzyme systems from mouse brain that catalyze, respectively, the elongation of palmitoyl-coenzyme A (palmitoyl-CoA), stearoyl-CoA, or arachidyl-CoA appear and reach maximal activity at different times after birth of the animal. A specific C(20)-CoA elongating system exists in mouse brain in addition to the previously recognized C(16)-CoA and C(l8)-CoA elongating enzymes. The C(20)-CoA elongation system is severely reduced in the mutant quaking mouse.

Improved rapidity and precision in the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase

A rapid and precise method for the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) activity has been developed. Total brain homogenates were treated with deoxycholate, and CNP activity was measured as inorganic phosphate (phosphomolybdic acid, 410 nm) released from the product, 2′-AMP, by alkaline phosphatase. Measurements were carried out under optimal conditions of temperature (30°C) and pH (6.2) using the whole brain of the rat, chicken, and quaking mouse. The entire assay was applicable to multiple samples and could be completed in less than 1 hr.

Brain proteolipids in neurological mutant mice

The proteolipid content of brains of control and neurological mutant mice was followed during development. Total brain proteolipids, dissolved in 0.01 M Tris-HCl buffer (pH 7.4) containing 1% sodium dodecylsulphate (SDS) (w/v) were separated on 12% polyacrylamide disc gels into 11 protein bands. Mitochondria, synaptosomes and myelin were the only subcellular fractions containing proteolipids. Some proteolipid bands could be attributed specifically to myelin and mitochondria. Before active myelination, band P7, identified as the major proteolipid of myelin, accounted for only a small percentage of the total proteolipid of normal animals. The level of this band increased very rapidly from 15 to 25 days whereas the proteolipid distribution in 15–25-day-old Jimpy, MSD and Quaking mice was similar to that of a 10-day-old normal mouse. These findings are in aggrement with the severe reduction of the myelin proteins in mutants observed when myelin was isolated. Besides the main myelin proteolipid (P7), a second proteolipid (P8M) was detected. Some evidence for a specific localization in myelin is presented.

Turnover of brain adrenergic transmitters in Quaking mice.

Quaking mice, neurological mutants of the C57 BL/6J strain have a markedly deficient myelination of the CNS(Sidman, Dickie and Appel, 1964). Many studies have been carried out on their lipids (Jacque, Harpin and Baumann, 1969; Neskovic, Nussbaum and Mandel, 1969, 1970; Kurihara, Nussbaum and Mandel, 1970; Sarlieve, Neskovic and Mandel, 1971; Singh, Spritz and Geyer, 1971) for this reason, but little attention has been paid to other aspects of their aminergic transmitters metabolism. During the course of our work, Tillement, Debarle, Simon and Boissier (1970) reported on the transmitter levels in Quaking mice. The reasons for thinking that the monoamine metabolism of these mice might be altered are as follows. The trembling might be associated with a change in the activity and therefore metabolism of dopaminergic structures. Chronic stress due to permanent tremor might induce changes in norepinephrine metabolism. Since there are changes in the sleep patterns of Quaking mice (Valatx and Jouvet, 1971), there might be changes in brain serotonin metabolism. This study of the turnover of norepinephrine, dopamine and serotonin in the whole brains of control and Quaking mice is the preliminary to detailed examination of the turnover of the transmitters in well‐defined brain regions.

Biosynthesis of psychosine and levels of cerebrosides in the central and peripheral nervous systems of quaking mice.

Abstract— The levels of cerebrosides in neural tissues of adult mice were determined by densitometry of cerebroside spots on charred thin‐layer chromatograms of washed total lipid extracts. Values for brain, spinal cord and peripheral nerves were 9·2, 33·0 and 36·9 mg/g of tissue, respectively. In adult Quaking mice these values were 6·4, 24 and 35 % of normal, respectively. Normal levels in brain, spinal cord and peripheral nerve of 21‐day‐old mice were 3·10, 13·5 and 17·8 mg/g, respectively. In 21‐day‐old Quaking mice the levels were reduced to 16,21 and 57% of normal, respectively. Biosynthesis of psychosine (galacto‐sylsphingosine) by homogenates of Quaking brain, spinal cord and peripheral nerve, respectively, was 18, 24 and 42% of the normal rates at 21 days after birth and 16, 66 and 60% of the normal rates at 94 days. Our results suggest a quantitative relationship between the rate of formation of psychosine in vitro and the rate of accumulation of cerebrosides. in vivo. Biosynthesis of lactosylceramide was not reduced in homogenates of brain and spinal cord from Quaking mice. Cerebroside levels in normal and Quaking spinal cord and in normal brain increased 2‐ to 3‐fold after 21 days of age, but in Quaking brain there was little or no increase.

Biosynthesis of glycolipids in myelin deficient mutants: Brain glycosyl transferases in Jimpy and Quaking mice

Two glycosyl transferases involved in the biosynthesis of glycolipids were investigated in the brain of myelin deficient mutants, Jimpy and Quaking mice. The first, UDP-galactose-ceramide galactosyl transferase, which catalyzes the synthesis of cerebroside from UDP-galactose and ceramide, was markedly reduced in Jimpy mouse and its activity did not change during the myelination period. In Quaking mouse the activity of this enzyme was also reduced but showed characteristic developmental changes, similar to those observed in normal mouse brain. The second glycosyl transferase, UDP-glucose-ceramide glucosyl transferase, involved in the biosynthesis of gangliosides, had a different pattern of developmental changes, and normal levels of activity were found in mutant brain. These data, as well as others discussed in the paper, indicate the existence in the postnatal brain development of two different glycosyl transferase systems, presumably with independent genetic controls.

Reversal learning and RNA labeling in neurological mutant mice and normal littermates

Incorporation of radioactive uridine into five brain areas, following reversal learning, was explored for neurological mutant mice with a deficit of myelin in the brain and spinal cord (quaking, qkqk) and normal littermates (?/+) from strain C57BL/6-qk (Jackson Laboratory). Both groups mastered the reversal task, although the mutants performed at a lower level. There was a Genotype × Test Conditions × Brain Area interaction with littermates exhibiting increased labeling of RNA in the amygdala and the hippocampus, and the quaking mice exhibiting no significant changes in these two areas following learning. Findings suggest that a genetic defect in the integrity of the CNS, a deficiency of myelin, affects reversal learning, and that this effect may be reflected in changes in RNA synthesis, or specific activity, for structures which are critical to reversal learning ability.

The composition of myelin from the mutant mouse 'quaking'.

Abstract— Myelin was isolated from the brains of adult Quaking mice, a mutant showing a deficiency of myelin in the central nervous system, and normal controls. The mutant myelin was found to have a higher flotation density than that of the control and showed marked differences in lipid composition. The myelin from Quaking mice was found to be deficient in cerebroside and ethanolamine phospholipid. Acrylamide gel electrophoresis of total myelin protein demonstrated a pronounced deficiency of proteolipid protein. The activity of cyclic 2′,3′‐AMP phosphohydrolase was normal.

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

AbstractA new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4‐ to 6‐month‐old) quaking mice, littermate controls and young (10‐day‐old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5‐10 per cent of that from adult controls. After deli‐pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high‐resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA‐linked gels permitted direct chemical determination of protein in specific bands.Myelin from brains of normal adult mice contained, as major components, one proteo‐lipid and two basic proteins. There were also a number of high‐molecular‐weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high‐molecular‐weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10‐day‐old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.

Studies of brain myelin in the “quaking mouse”

Myelin was isolated from the brains of "quaking" and littermate control animals and its composition was determined. The brains of quaking animals contained approximately one-fourth as much myelin as the control animals. There were qualitative as well as quantitative differences between the myelin from the two groups. By continuous cesium chloride gradient flotation it was shown that the myelin from the quaking animals consisted solely of a band corresponding to the heavier and smaller of the two bands found in normal controls. Cholesterol and glycolipids were lower and phospholipids (mainly phosphatidylcholine) and protein were higher in quaking animals than in controls. Also, phosphatidal-ethanolamine was decreased, and several consistent differences in the fatty acids (both unsubstituted and hydroxy) and aldehydes of the component lipids were found. In general there were smaller amounts of monounsaturated fatty acids in quaking animals. We suggest from these findings that myelin in the quaking mouse has certain compositional similarities with juvenile myelin, but it may be an abnormal type of myelin.

Polyphosphoinositide levels and biosynthesis in quaking mouse brain

Levels of polyphosphoinositides are considerably lower in brains of quaking mutant mice, which are characterized by inadequate myelination, than in normal mouse brains. The deficiency of triphosphoinositide is greater than that of diphosphoinositide, but less than the lack of cerebrosides, indicating that, although the major portion of polyphosphoinositides is located in myelin, extra-myelin pools of these lipids exist. The ability to incorporate 32P i in vivo into polyphosphoinositides is substantially reduced in the quaking animals, probably as the result of an as yet unknown biochemical lesion related to normal myelination.