Shiverer*jimpy double mutant mice. I. Biochemical evidence for reciprocal intergenic suppression

Shiverer and jimpy are neurological mutations that cause hypomyelination in the mouse CNS. The 3 major protein components of CNS myelin are: myelin basic protein (MBP), proteolipid (PLP) and 2', 3'-cyclic nucleotide phosphohydrolase (CNP). Previous work has shown that in jimpy animals the CNS contains reduced levels of MBP and CNP while PLP is undetectable. In shiverer animals the major forms of MBP ae undetectable in either the CNS or PNS, but the level of CNP is unaffected by mutation. In this study we have measured MBP, PLP and CNP in both the CNS (cerebral hemispheres) and PNS (sciatic nerve) of mice carrying the jimpy and shiverer mutations, individually and in combination. The results indicate that in the double mutant the levels of all 3 myelin proteins in both the CNS and PNS are intermediate between the levels in jimpy and the levels in shiverer animals. This means that part of the biochemical phenotype of the jimpy mutation (reduced levels of CNP and absence of PLP) is suppressed by the shiverer mutation, and part of the biochemical phenotype of the shiverer mutation (absence of MBP) is suppressed by the jimpy mutation. Possible mechanisms for this reciprocal intergenic suppression are discussed.     

Low-level lead exposure attenuates the expression of three major isoforms of neural cell adhesion molecule

Toxic lead (Pb) exposure poses serious risks to human health, especially to children at developmental stages, even at low exposure levels. Neural cell adhesion molecule (NCAM) is considered to be a potential early target in the neurotoxicity of Pb due to its role in cell adhesion, neuronal migration, synaptic plasticity, and learning and memory. However, the effect of low-level Pb exposure on the specific expression of NCAM isoforms has not been reported. In the present study, we found that Pb could concentration-dependently (1-100 nM) inhibit the expression of three major NCAM isoforms (NCAM-180, -140, and -120) in primary cultured hippocampal neurons. Furthermore, it was verified that levels of all three major isoforms of NCAM were reduced by Pb exposure in human embryonic kidney (HEK)-293 cells transiently transfected with NCAM-120, -140, or -180 isoform cDNA constructs. In addition, low-level Pb exposure delayed the neurite outgrowth and reduced the survival rate of cultured hippocampal neurons at different time-points. Together, our results demonstrate that developmental low-level Pb exposure can attenuate the expression of all three major NCAM isoforms, which may contribute to the observed Pb-mediated neurotoxicity.     

Myelin basic protein-specific protein methylase I activity in shiverer mutant mouse brain

Myelin basic protein (MBP)-specific protein-arginine N-methyltransferase (protein methylase I) activity in homozygous shiverer (shi/shi) mutant mouse brain is significantly higher than in the normal littermate brain at the onset of myelination. While the enzyme activity (expressed as pmol of S-adenosyl-L-[methyl-14C]methionine used/min/mg enzyme protein) increases coincidently during the period of myelination in the normal brain (15-18 days of age), it decreases significantly in the mutant brain during this period of time. These results are in contrast to those found with another dysmyelinating mutant, jimpy (jp/Y) mice, in which the enzyme activity in the mutant brains is similar to that in the normal animals but remains unchanged during the myelination process. There is no difference in the weight and protein concentration of the normal and shiverer mutant brains with corresponding ages, and the histone-specific protein methylase I activity is also unaffected in the shiverer brain.     

Glial hyaluronate-binding protein in dysmyelinating mice mutants: jimpy,quaking and shiverer

Summary Cryostat sections of cerebral hemispheres, cerebellum and spinal cord from dysmyelinating mice mutants (quaking, jimpy and shiverer) and littermate controls were stained by indirect immunofluorescence with polyclonal antibodies to the glial hyaluronate-binding protein (GHAP), a brain-specific extracellular matrix glycoprotein produced by astrocytes. In normal mice, the distribution of GHAP was similar to that previously reported in human, calf, pig and dog. The antigen was mainly localized in white matter, the granular layer of the cerebellum being the main exception. No differences were observed between mutants and littermate controls, except that with both GHAP and glial fibrillary acidic protein antibodies the glial framework was denser in the mutants, probably due to the reduction in myelin. The findings suggest that GHAP expression by astrocytes is not induced by myelination and that white matter astrocytes constitute a distinct glial population.Supported by NIH grant NS 13034 and by the Veterans Administration     

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

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

Neural cell adhesion molecule function is regulated by metalloproteinase-mediated ectodomain release

The neural cell adhesion molecule (NCAM) is involved in development of the nervous system, in brain plasticity associated with learning and memory, and in neuronal regeneration. NCAM regulates these processes by influencing cell adhesion, cell migration, and neurite outgrowth. NCAM activates intracellular signaling upon homophilic NCAM binding, and this is a prerequisite for NCAM-stimulated neurite outgrowth. NCAM is synthesized in three main membrane-bound isoforms, NCAM-120, NCAM-140, and NCAM-180. Soluble forms of NCAM in blood and cerebrospinal fluid have also been found, although the functional significance of these forms remains unclear. In this report, we demonstrate that NCAM can be released from primary hippocampal neurons in culture. The release was enhanced by application of ATP and inhibited by the metalloproteinase inhibitor BB-3103. ATP also induced metalloproteinase-dependent release of all three major NCAM isoforms from NCAM-transfected fibroblastoid L-cells. In this model system, the extracellular ATP-binding site of NCAM was shown not to be necessary for ATP-induced NCAM release. Furthermore, inhibition of serine, cysteine, and aspartic proteinases could not prevent ATP-induced down-regulation of NCAM in L-cells, suggesting that NCAM is cleaved directly by a metalloproteinase. Aggregation of hippocampal neurons in culture was increased in the presence of the metalloproteinase inhibitor GM 6001, consistent with a metalloproteinase-dependent shedding of NCAM occurring in these cells. Moreover, NCAM-dependent neurite outgrowth was significantly reduced by application of GM 6001. Taken together, these results suggest that membrane-bound NCAM can be cleaved extracellularly by a metalloproteinase and that metalloproteinase-dependent shedding of NCAM regulates NCAM-mediated neurite outgrowth.     

State-dependent changes in the expression levels of NCAM-140 and L1 in the peripheral blood cells of bipolar disorders, but not in the major depressive disorders

Recent postmortem brain and imaging studies provide evidence for disturbances of structural and synaptic plasticity in patients with mood disorders. Several lines of evidence suggest that the cell adhesion molecules (CAMs), neural cell adhesion molecules (NCAM) and L1, play important roles in both structural and synaptic plasticity. Although postmortem brain studies have indicated altered expression levels of NCAM and L1, it is still unclear whether these changes are state- or trait-dependent. In this study, the mRNA levels for various CAMs, including NCAM and L1, were measured using quantitative real-time PCR in peripheral blood cells of major depressive disorder patients, bipolar disorder patients and normal healthy subjects. Reduced expression levels of NCAM-140 mRNA were observed in bipolar disorder patients in a current depressive state. In contrast, L1 mRNA levels were increased in bipolar disorder patients in a current depressive state. NCAM-140 and L1 mRNA levels were not changed in bipolar disorder patients in a remissive state, or in major depressive disorder patients. In addition, there were no significant changes in the expression levels of intercellular adhesion molecule -1, vascular cell adhesion molecule -1, E-cadherin, or integrin alphaD among healthy controls, major depressive or bipolar disorder patients. Our results suggest that the reciprocal alteration in the expression of NCAM-140 and L1 mRNAs could be state-dependent and associated with the pathophysiology of bipolar disorder.     

Amphibian-specific regulation of polysialic acid and the neural cell adhesion molecule in development and regeneration of the retinotectal system of the salamander Pleurodeles waltl

Antibodies specific to the neural cell adhesion molecule (NCAM-total), the 180 × 10³ My component of NCAM (NCAM-180) and polysialic acid (PSA) were used in immunohistochemistry and Western blots to detect the spatiotemporal dynamics of these molecules in development and regeneration of the retinotectal system of Pleurodeles waltl. NCAM-total and NCAM-180 are continuously expressed in the retina, optic nerve, and tectum of the developing and adult salamander. This is also found for the 140 × 10³ My component of NCAM in Western blots of the retina. In the larval retina, PSA is present in the inner plexiform layer (IPL) and a few cells in all nuclear layers. At metamorphosis, PSA expression in the retina strongly increases in the layer of cone photoreceptor somata. Several cells in the inner nuclear layer and Muller cell processes also begin to express PSA. This pattern persists into adulthood. The optic nerve and the tectum are strongly PSA-immunoreactive throughout development. In the adult optic nerve and optic fiber pathway in the brain, PSA expression is selectively downregulated. In the crush-lesioned adult optic nerve, regenerating fibers are NCAM-180-positive but PSA-negative. This demonstrates a molecular difference between growing nerve fibers of Pleurodeles in development and in regeneration. PSA regulation is closely correlated with metamorphosis, thus suggesting that PSA expression may be under hormonal control. Some aspects of PSA and NCAM isoform expression patterns in the retinotectal system of salamanders differ considerably from that of other vertebrates. The substained expression of NCAM isoforms in adult salamanders might be due to secondary simplification (paedomorphosis). © 1993 Wiley-Liss, Inc.     

Connatal Pelizaeus-Merzbacher disease associated with the jimpy(msd) mice mutation.

In a patient with connatal Pelizaeus-Merzbacher disease with the same mutation in the proteolipid protein gene as in jimpy(msd) mice the immunohistochemical study of the brain demonstrated deficiencies of myelin and proteolipid protein despite good expression of myelin basic protein. The mechanism of myelination is partly disturbed by the mutation; therefore jimpy(msd) mice can be used as a suitable model for further studies in connatal Pelizaeus-Merzbacher disease.     

Localization of glial cell antigens in the brains of young normal mice and the dysmyelinating mutant mice, jimpy and shiverer

Tissue sections from the brains of normal, jimpy, and shiverer mice were immunostained by the peroxidase antiperoxidase method for carbonic anhydrase (CA) and the putative astrocytic "markers" glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). The cells in normal gray matter that immunostained with anti-CA and anti-GS were similar to one another in size and process elaboration. In the normal gray matter there were relatively few GFAP-positive astrocytes. When present, these cells resembled the CA- and GS-positive cells; however, the GFAP appeared to be concentrated in the astroglial processes, as distinguished from the cell bodies. Glial cell processes, immunostained for CA or GS, surrounded blood vessels and unstained neurons in the normal gray matter. The glial cells in shiverer gray matter were similar to those in the normal gray matter. When stained for GS or GFAP, the glial cells in the jimpy gray matter appeared to be somewhat hypertrophied, and when the glial cells in this mutant were stained for CA, the nuclei appeared to be swollen. It was concluded that some of the CA-positive cells in the gray matter of the normal and of each mutant mouse brain could be astrocytes. The patterns of immunostaining in the white matter emphasized the different complements of glial cells in the mutants. In the normal and shiverer mouse corpus callosum, CA, in particular, was detected only in the oligodendrocytes, their processes, and myelin. However, the data concerning the jimpy mouse suggested that the few CA-positive cells in the corpus callosum of that mutant could be astrocytes.     

Migrating and myelinating potential of neural precursors engineered to overexpress PSA-NCAM

Polysialic acid (PSA) on NCAM is an important modulator of cell-cell interactions during development and regeneration. Here we investigated whether PSA overexpression influences neural cell migration and myelination. We stably expressed a GFP-tagged polysialytransferase, PSTGFP, in mouse neurospheres and induced prolonged PSA synthesis. Using a chick xenograft assay for migration, we show that PSA can instruct precursor migration along the ventral pathway. PSA persistence did not change neural precursor multipotentiality in vitro but induced a delay in oligodendrocyte differentiation. PSTGFP+ precursors showed widespread engraftment in shiverer brain, closely similar to that observed with control precursors expressing a fluorescent protein. Initially, myelination by oligodendrocytes was delayed but, eventually, down-regulation of PSTGFP occurred, allowing myelination to proceed. Thus down-regulation of polysialyltransferases takes place even in cells where its RNA is under the control of a heterologous promoter and engineering PSA overexpression in neural precursors does not cause irreversible unphysiological effects.     

Spatial learning impairment induced by chronic stress is related to individual differences in novelty reactivity: search for neurobiological correlates

Although chronic stress has been reported to induce deleterious effects on hippocampal structure and function, the possible existence of individual differences in the vulnerability to develop stress-induced cognitive alterations was hypothesized. This study was designed to evaluate (i) whether individual variability in behavioural reactivity to novelty could be related to a differential vulnerability to show spatial learning deficits after chronic stress in young adult rats, and (ii) to what extent, could individual differences in stress-induced cognitive alterations be related to alterations in specific neurobiological substrates. Four month-old Wistar male rats were classified according to their locomotor reactivity to a novel environment, as either low (LR) or highly (HR) reactive, and then either submitted to psychosocial stress for 21-days (consisting of the daily cohabitation of each young adult rat with a new middle-aged rat) or left undisturbed. The results showed that psychosocial stress induced a marked deficit in spatial learning in the water maze in HR, but not in LR, rats. Then, a second experiment investigated the possible differential expression of corticosteroid receptors (MR and GR) and cell adhesion molecules (NCAM and L1) in the hippocampus of HR and LR rats, both under basal conditions and after exposure to chronic social stress. Although chronic stress induced a reduction on the hippocampal expression of MRs and the NCAM-140 isoform, the levels of these molecules did not differ between stressed rats with and without spatial learning impairments; i.e., between HR- and LR-stressed rats, respectively. Nevertheless, it should be noted that the reduction of the hippocampal expression of NCAM-140 induced by psychosocial stress was particularly marked in HR stressed rats. However, the expression of GRs, NCAM-120 and NCAM-180 isoforms, and L1, was not affected by stress, regardless of the reactivity of the animals. Therefore, although we failed to find a neurobiological substrate that specifically correlated with the differential cognitive vulnerability to chronic stress shown by animals with a different novelty reactivity, this study confirms the hypothesis that rats differ in their susceptibility to display stress-induced impairments in hippocampus-dependent spatial learning tasks. In addition, it provides a model to further search for the neurobiological substrate(s) involved in the differential susceptibility to develop stress-induced cognitive impairments.     

Assessment of Neural Cell Adhesion Molecule (NCAM) in Autistic Serum and Postmortem Brain

Studies have identified structural abnormalities in areas of the autistic brain, with a pattern suggesting that a neurodevelopmental anomaly took place. Neural cell adhesion molecule (NCAM), which is involved in development of the central nervous system, was previously shown to be decreased in the serum of autistic individuals. In the present study, we measured NCAM protein in the sera from controls, patients with autism, siblings of autistic patients, and individuals with other neurologic disorders, but found no significant differences. We also measured NCAM protein in autistic postmortem brain samples and found the longest isoform, NCAM-180, to be significantly decreased. In addition, we investigated the mRNA expression of NCAM in these brain samples using cDNA microarrays and RT-PCR. Results show that NCAM mRNA levels are not altered in autism.     

Increased NCAM-180 immunoreactivity and maintenance of L1 immunoreactivity in injured optic fibers of adult mice

The injury related expression of two axon-growth promoting cell adhesion molecules (CAMs), NCAM-180 which is developmentally downregulated and L1 which is regionally restricted, were compared in optic fibers in the adult mouse. The neuron-specific isoform of NCAM (NCAM-180) is present at very low levels in unlesioned adult optic axons. At 7 days after nerve crush, immunoreactivity was strongly and uniformly increased in optic axons within the nerve and throughout retina. Reactivity in surviving axons had returned to control levels at 4 weeks. To induce regrowth of adult retinal ganglion cell axons retinal explants were placed in culture. Strong NCAM-180 staining was observed on these regenerating optic axons. The neuronal cell adhesion molecule L1 is restricted to retina and to the unmyelinated segment of the optic nerve near the optic nerve head in unlesioned adult animals. Following nerve crush, L1 immunoreactivity was retained within retina and proximal nerve and novel staining was detected in the more distal segment of the optic nerve up to the lesion site where it persisted for at least eight months. The capacity of optic fibers to show increased NCAM-180 immunoreactivity and maintain L1 expression after a lesion may explain why these fibers exhibit relatively good potential for regeneration.     

The neural cell adhesion molecule is necessary for normal adult retinal ganglion cell number and survival

Retinal ganglion cells (RGCs) undergo apoptotic death in predictable time-dependant manners during development and as a consequence of injury. Recently, synthetic neural cell adhesion molecule (NCAM) agonists have been shown to provide neuroprotective support. Within the adult mouse retina, NCAM has been localized on all neurons and glia; however, no functional role has been determined. Using adult NCAM-/- mice, we directly tested the potential influence of NCAM on neuron survival in vivo and observed that, in NCAM-/- retinas, RGC densities are greater, RGC loss after injury is earlier and target tissue significantly influences adult RGC survival, all in contrast to wild-type retinas. Collectively, our results indicate that NCAM may play a vital role in regulating the developmental change in the effectiveness of local versus target-derived RGC trophic support and that, in the adult, endogenous NCAM influences the total number of CNS neurons and their survival following injury.     

Selective interaction of amyloid precursor protein with different isoforms of neural cell adhesion molecule

Compare to the thoroughly studied beta-amyloid, the physiological function of amyloid precursor protein (APP) is not well understood. We now had identified neural cell adhesion molecule (NCAM)-140 as a potential interaction partner of APP. Our data indicated that NCAM-140, but not NCAM-180, binds to the conserved central extracellular domain of APP. We also found that the phosphorylation levels of ERK1 and ERK2 were increased when cells were co-transfected with NCAM-140 and APP indicate that the interaction between NCAM-140 and APP may involve the MAPK pathway. These findings demonstrated that NCAM-140 interacts with APP, potentially playing a role in neurite outgrowth and neural development.     

Transplantation of CNS fragments into the brain of shiverer mutant mice: extensive myelination by implanted oligodendrocytes. I. Immunohistochemical studies

Solid fragments of olfactory bulb from new-born normal (B6CBA and C57BL6) mice were implanted into new-born shiverer (shi/shi) brains. The shiverer mouse being characterized by the absence of myelin basic protein (MBP), myelination due to implanted oligodendrocytes can be detected in the shiverer brain using an antiserum anti-MBP. Observation of sagittal sections of the host brains revealed very extensive areas of normal myelination from the level of the graft (rostral thalamus) up to the caudal brain (diencephalon, cerebellum, pons). Thus, oligodendrocytes contained in the implant migrate out of the graft over long distances in the host brain, before they differentiate and synthesize myelin. These results raise the question of the behaviour of oligodendrocytes in normal development.     

Brain-specific ceramide synthesis activity: change during brain maturation and in jimpy mouse brain

The synthesis of ceramide from lignoceric acid was studied in normally developing rat brain and jimpy mouse brain. The developmental pattern of the synthesis of both non-hydroxy and hydroxyceramide from free lignoceric acid in a brain-specific pathway requiring NADPH and cytosolic factors was similar to the synthesis from lignoceroyl CoA and both showed increasing activity during myelination. There were no differences in non-hydroxyceramide synthesis between jimpy and normal littermate control mouse brains by both pathways. HPLC analysis showed the same non-hydroxyceramide level in both types of brains.     

Endogenous cerebellar soluble lectin and its ligands in central nervous system myelin of quaking and jimpy mutant mice

The presence of an endogenous 'cerebellar soluble lectin' (CSL) involved in myelin compaction and myelination was analyzed in the dysmyelinating mutant mice quaking and jimpy. The primary defect in these mutations with severe hypomyelination is still unknown in the quaking mutant but results from a single mutation in the proteolipid protein gene in the jimpy mutant. Both immunocytochemical and immunoblotting techniques showed that CSL was not considerably reduced in its expression in the myelin fraction purified from adult quaking mutants. Furthermore, the myelin-associated glycoprotein and an axonal glycoprotein with a relative molecular weight (Mr) of 31 kilodaltons (kDa) were not decreased in quaking mice. This contrasted with several glycoproteins of Mr 23, 18, 16 and 12 kDa which were absent from the purified quaking myelin. In myelin preparations obtained from the jimpy mutant the CSL level was considerably reduced. This defect did not result from a deficient synthesis of CSL. However, as in the quaking mutation low-Mr glycoproteins were lacking. The nature of the low-Mr glycoproteins absent in quaking and jimpy mice is discussed in relation to previous reports on myelin glycoproteins. In the various mutants, due to different primary mutations, a similar absence of myelin compaction was observed, which could be associated with a deficient level of low-Mr glycoproteins. It is thus postulated that these molecules are essential for ensuring myelin compaction as ligands for the endogenous CSL.