Reduction of nerve growth factor level in the brain of genetically ataxic mice (weaver, reeler)

By a highly sensitive enzyme immunoassay we measured the level of nerve growth factor (NGF) in the cerebellum and cerebrum of the neurologically mutant mice, weaver, reeler and Purkinje cell degeneration (PCD). A significant decrease in NGF level was observed in both cerebellum and cerebrum of weaver and reeler mutants of either sex. However, there was no such difference between normals and mutants in the case of the PCD mice. These results show that weaver and reeler mice have abnormalities of NGF synthesis and/or degradation not only in the cerebellum but also in the cerebrum.     

Brain nucleic acids and protein in various neurological mutant mice

A study was made to compare alterations in the cerebral contents of nucleic acids and protein of several mouse strains affected by different neurological mutations: jimpy, msd, quaking, reeler, weaver, and dwarf. In normal and affected jimpy and msd mice the brain components analyzed were very similar. On the other hand, the cerebral hemispheres of quaking mice showed significant decreases in total RNA and DNA, when compared with those of normal littermates. In the affected reeler and weaver mice, total protein, RNA, and DNA in the cerebellum differed markedly from controls. Protein decreased slightly, whereas nucleic acids showed no significant variation in the cerebral hemispheres of the same mutants. The cerebella and cerebral hemispheres of affected dwarf mice had wet weights and total protein contents that were about 20% lower than those of their controls; DNA did not vary significantly in the various brain regions analyzed. The decrease of DNA we report in reeler and weaver mutant cerebellum in toto quantifies the lack of cell number, in contrast to histological studies which give only semiquantitative information.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.     

Neuronal expression of macrophage colony stimulating factor in Purkinje cells and olfactory mitral cells of wild-type and cerebellar-mutant mice

Macrophage colony stimulating factor (M-CSF) is known to be the most effective growth factor for macrophage and microglial proliferation. In the brain tissue system, M-CSF is mainly produced in astrocytes and microglia, but is not known to occur in neurons. In the present paper, we examined the distribution of neurons expressing M-CSF in the mouse brain by immuno-histochemistry and in situ hybridization. We observed M-CSF immunoreactivity in both the cerebellum and the olfactory bulb. These positive cells were found to be Purkinje cells in the cerebellum, and mitral cells in the olfactory bulb. M-CSF mRNA expression was also confirmed to occur in these cells. Purkinje cells of reeler and weaver mutants showed M-CSF expression as seen in wild-type mice; however, those in the staggerer mutant did not. This expression in wild-type mice first appeared at postnatal day 7 and continued stably thereafter. When Purkinje cells were deprived of their climbing fibre innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the expression of M-CSF remained unchanged. These data indicate that expression of M-CSF in Purkinje cells is controlled by an intrinsic mechanism and could, therefore, be a new marker of postnatal development in rodent cerebella. The absence of M-CSF expression in the staggerer mutant is possibly due to developmental arrest in the early postnatal period.     

Extent of multiple innervation of purkinje cells by climbing fibers in the olivocerebellar system of weaver,reeler, and staggerer mutant mice

Multiple innervation of cerebellar Purkinje cells (PCs) by climbing fibers (CFs) has been described recently in adult weaver, reeler, and staggerer mutant mice, instead of the monoinnervation found in normal adults. In the present study, the extent of this multiple innervation was estimated by two methods, using both evoked and spontaneous activity of the olivocerebellar system. Concordant values were obtained: the mean number of CF collaterals per PC was between 3.5 and 4 in weaver and staggerer and close to 3.2 for the multiply innervated PCs of reeler mice. These values are of the same order of magnitude as those for the transient multiple innervation in developing rats (Mariani and Changeux, 1981a, b).     

Reduced histone levels induced by "reeler" and "weaver" mutations in the mouse cerebellum.

Five major protein bands present in the polyacrylamide gel electrophoresis pattern of normal cerebellum are apparently absent or decreased in amounts in both reeler and weaver mutant cerebellar tissues. All five bands were identified as histones and the deficiencies related to the decreased cerebellar cellularity produced by both mutations. These results, therefore, rule out an earlier suggestion that two of these proteins are granular cell specific proteins (1). Preliminary evidence for high levels of F1 histone in the nuclei of cerebellar cells, which appear to be reduced in the reeler syndrome, is presented.     

Interleukin-1 Hyperproduction by In Vitro Activated Peripheral Macrophages from Cerebellar Mutant Mice

Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin-1 (IL-1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL-1 cDNA probes, revealed a clear-cut hyperexpression of IL-1 mRNA in staggerer macrophages. An IL-1 bioassay using the IL-1-responsive D10.G4 cell line also revealed a sixfold increase of IL-1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3-week to 1-year-old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL-1 production by peripheral macrophages.     

Developmental expression and intracellular location of P400 protein characteristic of Purkinje cells in the mouse cerebellum

The developmental expression and intracellular localization of a cerebellum-characteristic 250-kDa glycoprotein, P400 protein, were studied by immunohistochemical and immunoblot methods using a monoclonal antibody against P400 protein. In the cerebellum of normal mouse, the expression of P400 protein increased from Postnatal Day 3 to Day 21. This enhancement of P400 protein expression occurred only in the Purkinje cells and proceeded with the growth of their dendritic arborization. Electron microscopic analysis indicated that P400 protein is present at the plasma membrane, the endoplasmic reticulum, and the postsynaptic densities of Purkinje cells. Immunohistochemistry of the cerebella of neurological mutant mice indicated that the Purkinje cells of reeler, weaver, and pcd mutant mice retain the ability to produce a large amount of P400 protein. However, the Purkinje cells of staggerer mutant mouse proved to be incapable of enhanced P400 protein expression. These results indicate that P400 protein is a Purkinje cell-characteristic plasma membrane-associated glycoprotein, which is also present at the postsynaptic density and endoplasmic reticulum and that the expression of P400 protein in Purkinje cells is closely associated with the growth of their dendritic arborization.     

In Vitro Release of Endogenous Dopamine from the Striatum of the Weaver Mutant Mouse

The weaver mutant mouse has a genetically determined defect in the nigrostriatal dopaminergic system. The present study was undertaken to test the hypothesis that in the weaver mutant mouse, striatal nerve terminals undergo compensatory changes in response to this deficiency. To test this hypothesis, we studied the basal and stimulated release of dopamine from striatal slices of weaver mutant mice and matched controls. By using a superfusion system and concentrating the superfusate by passage over alumina, resting dopamine release could be determined in the weaver mutant despite the fact that striatal tissue content of dopamine in these mice is reduced by greater than 75% compared with control mice. Fractional resting release of dopamine in weaver striatal slices was significantly elevated compared with that in controls, suggesting that the release mechanisms in the weaver may be adapting to overcome the dopamine deficit. Potassium-evoked release (24 and 48 mM potassium) was not significantly different between the two genotypes. In contrast, amphetamine-evoked release (1 microM) was significantly greater in the weaver mice than in controls. In both genotypes, release evoked by amphetamine was completely inhibited by cocaine, implicating the dopamine uptake carrier in this release process. These findings suggest that fundamental differences in dopamine release mechanisms exist between weaver and control mice and support the hypothesis that compensatory mechanisms may develop in neurons in response to dopamine deficits.     

Galactosyltransferase Defects in Reeler Mouse Brains

Galactosyltransferase activities were examined in the cerebellum, cerebral cortex, and brain stem of reeler and wild-type mice. Galactosyltransferase assays were optimal for all required substrates, linear with incubation time, and proportional to protein concentration. In brain areas affected by the reeler mutation (i.e., cerebral cortex and cerebellum), galactosylation of both endogenous and exogenous glycoprotein acceptors was greatly reduced in reeler relative to controls. On the other hand, glycosylation of endogenous glycolipids was low, and equal between reeler and wild-type. Galactosyltransferase activities were similar, though not identical, in reeler and wild-type brain stems, which are phenotypically normal in reeler mice. Glucosyltransferase, beta-galactosidase, beta-N-acetylglucosaminidase, acid phosphatase, and lactate dehydrogenase specific activities were all unaffected in reeler cerebella, while galactosyltransferase activity was 52% of control. Inhibition of either UDPgalactose hydrolysis or beta-galactosidase had no effect on galactosyltransferase activity. The spectrum or galactosyltransferase deficiencies in reeler suggests that this enzyme is associated with the development of young granule cells.     

Reelin regulates male mouse reproductive capacity via the sertoli cells

Reelin plays important roles in brain development. Reeler mutant mice that lack the protein reelin (RELN) suffer from cell type- and region-dependent changes in their neocortical layers, and adult reeler mutant mice have dilated seminiferous tubules. Meanwhile, the mechanism by which Reelin regulates the spermatogenic cell development in mice and their reproductive abilities remains unclear. In the present study, we used reeler mutant mice to investigate the effects of Reelin on reproduction in mice. The results indicated variations in sex hormone expression among the reeler mice, indicating that they produce few offspring and their spermatogenic cells are irregularly developed. Moreover, glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha 1, Ras/extracellular regulated protein kinases (ERK), and promyelocytic leukemia zinc finger (PLZF)/chemokine (C-X-C motif) receptor 4 (CXCR4) serve as potential regulatory pathways that respond to the changes in sertoli cells and the niche of male germ cells. Our findings provided valuable insights into the role of reeler in the reproductive abilities of male mice and development of their spermatogonia stem cells.     

Coenzyme Q10 inhibits mitochondrial complex-1 down-regulation and nuclear factor-kappa B activation

We have used control-homozygous weaver mutant, and -heterozygous weaver mutant mice in order to explore the basic molecular mechanism of neurodegeneration and the neuroprotective potential of coenzyme Q(10). Homozygous weaver mutant mice exhibited progressive neurodegeneration in the hippocampus, striatum, and cerebellum, and a reduction in the striatal levels of dopamine and coenzyme Qs (Q(9) and Q(10)) without any significant changes in norepinephrine and serotonin. Mitochondrial complex-1 was down regulated; whereas nuclear factor-kappa B was up regulated in homozygous weaver mutant mice. Rotenone inhibited complex-1, enhanced nuclear factor-kappa B, and caused apoptosis in human dopaminergic (SK-N-SH) neurons; whereas nuclear factor-kappa B antibody suppressed rotenone-induced apoptosis, suggesting that enhancing coenzyme Q(10) synthesis and suppressing the induction of NF-kappa B, may provide neuroprotection.     

Contents of several amino acids in the cerebellum, brain stem and cerebrum of the 'staggerer', 'weaver' and 'nervous' neurologically mutant mice.

The content of glutamate, GABA, aspartate, glycine and alanine was determined in the cerebellum, brain stem and cerebrum of three different mutant mice which have been named ‘staggerer’, ‘weaver’ and ‘nervous’ on the basis of neurological symptoms. In the ‘staggerer’ and ‘weaver’ mutants there is an almost complete absence of granule cells in the cerebellar cortex while in the ‘nervous’ mutant there is a loss of Purkinje cells (and to a lesser extent a loss of granule cells) in the cerebellar cortex. In the cerebellum of the ‘weaver’ mutant, the content of glutamate was signficantly lower (P < 0.025) than control values (8.77 ± 0.76 vs 12.0 ± 1.3 μmol/g tissue wet wt) and the contents of GABA and glycine were significantly greater than normal levels. In the cerebellum of the ‘staggerer’ mutant, the content of glutamate was significantly lower (6.62 ± 0.70 μmol/g) and the contents of glycine and alanine significantly higher than control values. In the cerebrum and brain stem regions of the staggerer mutant, weaver mutant and the normals the contents of the five amino acids were the same. The contents of glycine and alanine in the cerebellum, GARA and glycine in the brain stem and GABA and alanine in the cerebrum of the nervous mutants were higher than control values. The data are discussed in terms of a possible role for glutamate functioning as an excitatory transmitter when released from the cerebellar granule cells.     

Expression of glial antigens C1 and M1 in developing and adult neurologically mutant mice

The distribution of two glial antigens (C1 and M1) has been studied by indi-rect immunofluorescence during postnatal development of the cerebella of normal and neurologically mutant mice (weaver, staggerer, reeler, Purkinje cell degeneration, and wobbler). During the first postnatal week of normal development, C1 antigen is expressed in ependyma, Bergmann glial fibers (BG), and astrocytes of the internal granular layer and white matter. After day 10, C1 antigen is restricted to BG and ependymal cells. During the sec-ond and third week, BG undergo a transient loss of C1 antigen that starts in medioventral areas and spreads in a gradient dorsally and laterally. In reeler, weaver, and staggerer, C1 antigen expression is normal during the first postnatal week, and subsides in BG in a similar spatial gra- dient as described for the normal littermates. However, the loss of C1 anti-gen in BG occurs earlier (first in reeler, then in weaver, and last in staggerer) and is not reversible as it is in normal mice. In Purkinje cell de-generation, C1 antigen expression is diminished in BG after the onset of be-havioral abnormalities. Wobbler is normal with respect to C1 antigen ex-pression at adult ages. M1 antigen is detectable in white matter astrocytes from postnatal day 7 on, and persists in these cells into adulthood. Astrocytes of the internal granular layer and BG express M1 antigen only transiently in normal mice during the second and third weeks. The appearance of M1 antigen in BG occurs in a spatiotemporal gradient, matching the one in which C1 antigen disappears. M1 antigen expression is abnormally maintained in BG of reeler, staggerer, and weaver. In Purkinje cell degeneration, M1 antigen is ex-pressed abnormally at the onset of behavioral abnormalities first in.astro-cytes of the internal granular layer and, with growing age, increasingly also in BG. In wobbler, BG do not express M1 antigen. However, astrocytes of the granular layer are abnormally M1 antigen-positive.     

Glutamic acid: A strong candidate as the neurotransmitter of the cerebellar granule cell

Free amino acids and cholinergic enzymes were investigated in the cerebellum of reeler and weaver mice in an attempt to identify the neurotransmitter characteristic of the granule cell population and to clarify any neurotransmitter abnormalities of their pre- and postsynaptic neurons induced by their depletion. The data indicate that glutamic acid may be the neurotransmitter of the granule cells. Pre- and postsynaptic neurotransmitter activity seemed not to be markedly altered in cerebellar granule cell dysgenesis.     

Glial Cell Markers in the Reeler Mutant Mouse: A Biochemical and Immunohistological Study

The glial cell contents of S100 protein, 2',3'-cyclic AMP, 3'-phosphohydrolase (CNP), isoenzyme II of carbonic anhydrase (CAII) and butyrylcholinesterase (BuChE) were biochemically determined in the cerebellum and cerebrum of the reeler mutant mouse. Astrocytes and oligodendrocytes, shown by this study, contain abnormal amounts of these components. The CAII concentration was significantly increased in the particulate fraction of the reeler cerebellum and cerebrum (by 50% and 89%, respectively). The BuChE specific activity was greatly increased in the reeler, by 120% for cerebellum and by 40% in cerebrum. In contrast, the S100 protein concentration was reduced in the reeler cerebellum by 40% and by 25% in cerebrum, while the CNP specific activity increased by 30% in the reeler cerebellum. In addition, the glial cell distribution was studied by immunohistological techniques with antibodies directed against S100 protein, glial fibrillary acidic protein (GFA) and CAII. Apparently the density of glial cells is not significantly affected. However, the Golgi epithelial cells were usually abnormally placed and their Bergmann fibres were less well developed.     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

Dopamine Transporter-Dependent and -Independent Endogenous Dopamine Release from Weaver Mouse Striatum In Vitro

Abstract: The weaver mutant mouse (wv/wv) has an ～70% loss of nigrostriatal dopamine (DA) neurons, but the fractional DA release evoked by amphetamine (but not a high potassium level) has been shown to be greater from striatal slices of the weaver compared with +/+ mice. In the present work we tested the hypothesis that fractional DA release from weaver striatum would be greater when release was mediated by the DA transporter. Serotonin (5-HT)-stimulated fractional DA release was greater from weaver than from +/+ striatum. The release evoked by 5-HT in the presence of 10 µM nomifensine (an antagonist of the DA transporter) was less than in its absence, but the difference between weaver and +/+ striatum remained. In the presence of nomifensine, 1-(m-chlorophenyl)biguanide, classified as a 5-HT₃ agonist, also induced a greater fractional release from weaver compared with +/+ striatum. When veratridine was used at a low concentration (1 µM), the fractional evoked release of DA was higher from the weaver in the presence and absence of nomifensine. These findings suggest that the reason for the difference in the responsiveness of the two genotypes to these release-inducing agents is not related to DA transporter function.     

Altered Expression of the D1.1 Ganglioside in the Cerebellum of the Weaver Mouse

Expression of the D1.1 ganglioside was studied immunohistochemically in developing cerebella from normal and weaver mutant mice. In the normal cerebellum at postnatal day 7 (P7), D1.1 expression was restricted to the external granule-cell layer (EGL). At later ages, D1.1 disappeared as the developing granule neurons ceased mitosis and began migrating toward the internal granule-cell layer. In the weaver cerebellum, D1.1 was expressed in the EGL in apparently normal fashion at P7, but failed to disappear at later ages. As late as P35, D1.1 immunoreactivity was observed throughout the weaver cerebellar cortex. The relative amounts of D1.1 ganglioside in weaver and normal cerebella were compared by thin layer chromatography of total gangliosides, followed by overlay of the chromatogram with anti-D1.1 and 125I-labelled second antibody. Autoradiograms showed that at P12 and P35 the weaver tissue contains six- to tenfold more D1.1 than normal tissue. These findings suggest that one result of the weaver mutation is prolonged expression of D1.1. We speculate that the D1.1 ganglioside might be involved in adhesive interactions that regulate the timing of granule-cell migration from the EGL. The prolonged expression of D1.1 could be responsible, in part, for the failure of granule-cell migration in the weaver cerebellum.     

Recognition of Bergmann glial and ependymal cells in the mouse nervous system by monoclonal antibody

A monoclonal antibody designated anti-Cl was obtained from a hybridoma clone isolated from a fusion of NS1 myeloma with spleen cells from BALB/c mice injected with homogenate of white matter from bovine corpus callosum. In the adult mouse neuroectoderm, C1 antigen is detectable by indirect immunohistology in the processes of Bergmann glial cells (also called Golgi epithelial cells) in the cerebellum and of Muller cells in the retina, whereas other astrocytes that express glial fibrillary acidic protein in these brain areas are negative for C1. In addition, C1 antigen is expressed in most, if not all, ependymal cells and in large blood vessels, but not capillaries. In the developing, early postnatal cerebellum, C1 antigen is not confined to Bergmann glial and ependymal cells but is additionally present in astrocytes of presumptive white matter and Purkinje cell layer. In the embryonic neuroectoderm, C1 antigen is already expressed at day 10, the earliest stage tested so far. The antigen is distinguished in radially oriented structures in telencephalon, pons, pituitary anlage, and retina. Ventricular cells are not labeled by C1 antibody at this stage. C1 antigen is not detectable in astrocytes of adult or nearly adult cerebella from the neurological mutant mice staggerer, reeler, and weaver, but is present in ependymal cells and large blood vessels. C1 antigen is expressed not only in the intact animal but also in cultured cerebellar astrocytes and fibroblastlike cells. It is localized intracellularly.