Nervous system defects of AnkyrinB (-/-) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kD AnkyrinB in premyelinated axons

The L1 CAM family of cell adhesion molecules and the ankyrin family of spectrin-binding proteins are candidates to collaborate in transcellular complexes used in diverse contexts in nervous systems of vertebrates and invertebrates. This report presents evidence for functional coupling between L1 and 440-kD ankyrinB in premyelinated axons in the mouse nervous system. L1 and 440-kD ankyrinB are colocalized in premyelinated axon tracts in the developing nervous system and are both down-regulated after myelination. AnkyrinB (-/-) mice exhibit a phenotype similar to, but more severe, than L1 (-/-) mice and share features of human patients with L1 mutations. AnkyrinB (-/-) mice exhibit hypoplasia of the corpus callosum and pyramidal tracts, dilated ventricles, and extensive degeneration of the optic nerve, and they die by postnatal day 21. AnkyrinB (-/-) mice have reduced L1 in premyelinated axons of long fiber tracts, including the corpus callosum, fimbria, and internal capsule in the brain, and pyramidal tracts and lateral columns of the spinal cord. L1 was evident in the optic nerve at postnatal day 1 but disappeared by postnatal day 7 in mutant mice while NCAM was unchanged. Optic nerve axons of ankyrinB (-/-) mice become dilated with diameters up to eightfold greater than normal, and they degenerated by day 20. These findings provide the first evidence for a role of ankyrinB in the nervous system and support an interaction between 440-kD ankyrinB and L1 that is essential for maintenance of premyelinated axons in vivo.     

Choline availability modulates the expression of TGFbeta1 and cytoskeletal proteins in the hippocampus of developing rat brain

Choline availability influences long-term memory in concert with changes in the spatial organization and morphology of septal neurons, however little is known concerning the effects of choline on the hippocampus, a region of the brain also important for memory performance. Pregnant rats on gestational day 12 were fed a choline control (CT), choline supplemented (CS), or choline deficient (CD) diet for 6 days and fetal brain slices were prepared on embryonic day 18 (E18). The hippocampus in these brain slices was studied for the immunohistochemical localization of the growth-related proteins transforming growth factor beta type 1 (TGFbeta1) and GAP43, the cytoskeletal proteins vimentin and microtubule associated protein type 1 (MAP1), and the neuronal cell marker neuron specific enolase (NSE). In control hippocampus, there was weak expression of TGFbeta1 and vimentin proteins, but moderately intense expression of MAP1 protein. These proteins were not homogeneously distributed, but were preferentially localized to cells with large cell bodies located in the central (approximately CA1-CA3) region of the hippocampus, and to the filamentous processes of small cells in the fimbria region. Feeding a choline-supplemented diet decreased, whereas a choline-deficient diet increased the intensity of immunohistochemical labeling for these proteins in E18 hippocampus. GAP43 and NSE were localized to peripheral nervous tissue but not hippocampus, indicating that the maturation of axons and neurite outgrowth in embryonic hippocampus were unaffected by the availability of choline in the diet. These data suggest that the availability of choline affects the differentiation of specific regions of developing hippocampus.     

Molecular cloning and characterization of a novel form of neuropeptide gene as a developmentally regulated molecule

To examine the molecular basis controlling neuronal differentiation, subtraction library construction and differential screening were used to identify cDNAs whose mRNA levels are regulated in mouse NS20Y cells by dibutyryl cyclic AMP treatment. One of them, N27K, whose mRNA increases transiently during both neuronal differentiation in NS20Y cells and development in mouse brain. The deduced amino acid sequence of N27K comprises 212 amino acid residues and is a novel form of a precursor protein for a new neuropeptide nociceptin/orphanin FQ, which we independently cloned as N23K. That is, the putative protein encoded by N27K is 25 amino acids longer than that encoded by N23K. Using an antibody against a C-terminal peptide of the N27K protein that recognizes a 27-kDa protein in Western blot analysis, a punctate structure in the perinuclear region and areas near the tip of neurites is visualized in neurally differentiating NS20Y cells. The time of maximal expression correlates with periods of neurite extension, and expression decreases as the neuritic network develops. Immunohistochemistry of tissue sections of the mouse central nervous system revealed that reactivity for the anti-N27K protein antibody can detected in early generated neurons at embryonic day 14, in virtually all immature neurons at postnatal day 1, and in subsets of neurons of discrete brain regions such as the hypothalamus and spinal cord in adults. This remarkable redistribution suggests that N27K may be involved in a process in neurite outgrowth and nervous system development.     

Retinal FABP principally localizes to neurons and not to glial cells

The presence of fatty acid-binding protein (FABP) in the embryonic chick retina may be linked to the demand for polyunsaturated fatty acids in this developing neural tissue. There is a decline in the overall level of FABP as the retina matures, suggesting a role for FABP in cellular differentiation. However, this pattern is not present in the chick brain, indicating a unique function for FABP in the retina. Immunohistochemical staining of paraffin sections of chick retina from embryonic day 21 revealed immunopositive photoreceptor inner segments, outer nuclear layer, 'radial processes' in the inner nuclear layer, a subpopulation of cells in the ganglion cell layer, and inner limiting membrane. This pattern suggested that FABP positive cells were photoreceptors, Müller (glial) cells, and possibly ganglion cells. Staining of sections for glutamine synthetase, an enzyme specific for Müller cells, was similar but not identical to the pattern observed with FABP; thus identification of these cells as FABP-positive was not conclusive. However, in retinal cells dissociated from day E14 embryos and cultured for one week, staining with FABP was more intense in the neurons than in the 'flat' cells (presumed to be derived from the Müller cells). Retinal FABP thus appears to be localized predominantly in neurons, and may serve to sequester fatty acids in preparation for neurite outgrowth as the retinal cells differentiate.     

Generation and regulation of beta-amyloid peptide variants by neurons

Studies of processing of the Alzheimer beta-amyloid precursor protein (betaAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "beta-secretase" pathway, which generates beta-amyloid (A beta(1-40/42); approximately 4 kDa), and the "alpha-secretase" pathway, which generates a smaller fragment, the "p3" peptide (A beta(17-40/42); approximately 3 kDa). To determine whether similar processing events underlie betaAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [35S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa A beta-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional A beta beginning at position A beta(Asp1), whereas both radiosequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with A beta(Glu11) at the N terminus, rather than A beta(Leu17) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble betaAPP(alpha) release and decreased generation of both the 4-kDa A beta and the 3-kDa N-truncated A beta. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing A beta secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant A beta variant peptides and emphasize the role of protein phosphatases in modulating neuronal A beta generation.     

Calretinin immunoreactivity in the developing thalamus of the rat: a marker of early generated thalamic cells

The present work was aimed to study the immunocytochemical localization of the calcium-binding protein, calretinin, in the rat thalamus from embryonic day 14 to the third postnatal week. In the adult rat thalamus, calretinin immunoreactivity is intensely expressed in some intralaminar and midline nuclei, as well as in selected regions of the reticular nucleus. At embryonic day 14, calretinin was expressed by immature and migrating neurons and fibres laterally to the neuroepithelium of the diencephalic vesicle in the region identified as reticular neuroepithelium. At embryonic day 16, immunoreactive neurons were present in the primordium of the reticular nucleus and in the region of the reticular thalamic migration, where neurons showed the morphology of migratory cells. At the end of embryonic development and in the first postnatal week, calretinin-positive neurons were observed in selected region of the reticular nucleus and it was intensely expressed in some intralaminar and midline nuclei. Bands of immunopositive fibres were also observed crossing the thalamus. During the second postnatal week, the immunolabelling in the reuniens, rhomboid, paraventricular and central medial thalamic nuclei remains very intense while a decrease of immunoreactivity in mediodorsal, centrolateral and laterodorsal nuclei was observed. The immunostaining of fibres, particularly evident in the perinatal period, progressively decreased and it was no longer visible by the end of the second postnatal week when the distribution and intensity of calretinin immunostaining was similar to that observed in the adult rat thalamus. The present findings indicate that the immunolocalization of calretinin can be used to identify subsets of thalamic neuronal population during pre- and postnatal maturation allowing also the detection of the migratory pattern of early generated reticular thalamic neurons.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.     

Neurotoxins in neurobiology: from basic research to clinical application

Recently, we reported that in rat, cyclosporine A (CsA) markedly decreases the levels of calbindin-D (CABP-D) 28 kDa in kidney. CABP-D 28 kDa is a calcium-binding protein which is highly expressed in calcium-transporting tissues such as kidney or brain. In this study, we investigated whether, in addition to the kidney, CsA also has an effect on CABP-D 28 kDa in rat brain. Three groups of male Wistar rats received 15 mg/kg/day or 50 mg/kg/day of CsA orally for 12 days, whereas controls received vehicle solution for the same period. CABP-D 28-kDa protein and CsA were quantified in homogenates of kidney, cerebral cortex and cerebellum, and the localization of CABP-D 28 kDa was assessed in the different tissue sections by immunohistochemistry. In kidney, CABP-D 28 kDa was strongly and dose dependently decreased, and was located in tubular epithelial cells. In brain, CABP-D 28 kDa was not changed and was mainly located in pyramidal cells of the cortex and in cerebellum exclusively in Purkinje cells. High CsA concentrations were measured in kidney, more than 17-fold greater than those found in cortex. In cerebellum, CsA was below the limit of detection. These data suggest that at clinically relevant doses, CsA may not affect CABP-D 28-kDa levels in brain.     

Biochemical studies of chick brain development and maturation: II. Alterations in the mechanisms of cell-free protein synthesis

The molecular mechanisms responsible for the decreased cell-free protein synthetic activity of chicken brain (cerebrum, cerebellum, and optic lobes) from the late embryonic stage to the adult stage were investigated. The changes in polyribosome content closely paralleled changes in cell-free protein synthetic activity; both increased during late embryonic development, reached a maximum around hatching,and thereafter decreased to the level found in the adult. Both cell sap and microsomal or ribosomal fractions from the adult brain tissue were less active in protein synthesis; however, the microsomal or ribosomal fractions contributed more to the decreased protein synthesis than did the cell sap. The lower activity of adult cell sap in protein synthesis was primarily due to a decreased activity in the aminoacylation of tRNA with no apparent change in the ability of the cell sap to catalyze the elongation of polyphenylalanine synthesis. Ribosomal particles (80 S) from adult and embryonic brain tissue had similar biological activities and fidelity in the translation of polyuridylic acid; however, the cell-free protein synthetic activity of the embryonic post-mitochondrial supernatant preparation was more sensitive to inhibitors of the initiation of protein synthesis (aurintricarboxylic acid and polyinosinic acid) than adult post-mitochondrial supernatant, indicating a decreased initiation capacity in adult brain post-mitochondrial supernatant compared to embryonic brain post-mitochondrial supernatant.     

NGF binding to p75 enhances the sensitivity of sensory and sympathetic neurons to NGF at different stages of development

To clarify the role of the common neurotrophin receptor p75 in modulating the survival response of sensory and sympathetic neurons to NGF at different stages of development, we compared the actions of wild-type NGF with a mutated NGF protein that binds normally to TrkA, the NGF receptor tyrosine kinase, but has greatly reduced binding to p75. At saturating concentrations, the NGF mutant promoted the survival of similar numbers of trigeminal sensory and sympathetic neurons as NGF. At subsaturating concentrations, the NGF mutant was less effective than wild-type NGF in promoting the survival of embryonic sensory neurons and postnatal sympathetic neurons but was equally effective as wild-type NGF in promoting the survival of embryonic sympathetic neurons. Whereas the levels of trkA and p75 were similar in embryonic sensory neurons and postnatal sympathetic neurons, the level of p75 was significantly lower than that of trkA in embryonic sympathetic neurons. These results indicate that binding of NGF to p75 enhances the sensitivity of NGF-dependent neurons to NGF at stages in their development when the levels of p75 and TrkA are similar.     

Melanocortin and MCH precursor-derived NEI effects on striatum-midbrain co-cultures

The possibility of developmental effects of POMC-derived melanocortins and analogs on neurons of fetal rat brain regions exhibiting marked developmental melanocortin receptor expression, was studied in serum-free co-cultures of gestational day 18 striatal and mesencephalic cells, and compared with NEI and NGE. These two peptide fragments of the melanin concentrating hormone precursor, occurring in brain areas devoid of POMC terminals, cross-react with alpha-MSH antibodies; NEI elicits grooming similar to alpha-MSH. Neurofilament protein (NF), growth-associated protein (GAP-43) and synaptophysin of the synaptosomal fraction were determined by ELISA as markers for neuritogenesis, growth cones, and nerve terminal differentiation. Cell survival was analyzed by MTT assay, proportions of major cell types by immunocytochemistry. alpha-Melanocyte-stimulating hormone (alpha-MSH, effective concentration 250-2500 nM), the analog Nle4-, D-Phe7-alpha-MSH (NDP, 3.1-750 nM), and NEI (250 nM) increased NF in 3 day cultures by 11%, 17%, and 22%, respectively, whereas ACTH(1-24) and ACTH(1-39) (25 2500 nM) were ineffective. In 11 day cultures, alpha-MSH (250-750 nM), but not NDP, ACTH(1-24) or ACTH(1-39), increased synaptosomal synaptophysin by 11%. GAP-43 and cell survival remained unaffected. These data indicate that selected melanocortins as well as NEI can influence differentiation of neural processes in brain neurons.     

AMPA receptor protein in developing rat brain: glutamate receptor-1 expression and localization change at regional, cellular, and subcellular levels with maturation

We tested the hypothesis that the regional, cellular, and synaptic localizations of the glutamate receptor 1 (GluR 1) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor are regulated developmentally in rat brain. By immunoblotting, GluR1 was first detected in whole brain at embryonic day E15.5, and levels increased progressively during late embryonic (E20) and early postnatal (P2-P11) days. Regionally, GluR1 increased in cerebral cortex but decreased in striatum with postnatal maturation. These changes occurred in the presence of increased presynaptic maturation, as determined by synaptophysin detection. By immunocytochemistry, distinct cellular populations showed different temporal profiles of GluR1 expression during postnatal maturation. The neocortex and hippocampus showed a progressive maturation-related enrichment of GluR1, whereas the striatum showed a gradual reduction in GluR1 during maturation. In cerebellum, GluR1 protein was expressed transiently at restricted times postnatally by granule cells (P0-P11) and Purkinje cells (P13-P19), but by P21 and thereafter these neurons had sparse GluR1 immunoreactivity. By immunoelectron microscopy. GluR1 was found in neurites, specifically in both dendritic and axon terminal components of developing synapses. GluR1 was clustered at the plasma membrane of apparent growth cone appositions, neuronal cell bodies, and dendrites of developing neurons. The presence of GluR1 at presynaptic sites dissipated with synaptic maturation, as GluR1 became confined to the somatodendritic compartment as maturation progressed. We conclude that the regional expression as well as the cellular and synaptic localizations of the GluR1 are developmentally regulated and are different in immature and mature brain. Differences in glutamate receptor expression and synaptic localization in immature and mature brain may be relevant to the phenomenon that the perinatal and adult brain differ in their regional vulnerability to hypoxia-ischemia and excitotoxicity.     

Molecular cloning of a new unc-33-like cDNA from rat brain and its relation to paraneoplastic neurological syndromes

Anti-CV2-autoantibodies from patients with paraneoplastic neurological syndromes were used to purify protein(s) related to this disease. A novel cDNA, c-22, was obtained by PCR with primers based on amino-acid sequence of peptides obtained from this protein and rat brain cDNA as template. The deduced amino-acid sequence of c-22 shows homology to the Unc-33 gene from C. elegans in which mutations lead to defects in neuritic outgrowth and axonal guidance and cause uncoordinated movements of the nematode. Several consensus sites for putative protein kinase C phosphorylation were found, suggesting that the c-22 gene product may be a phosphoprotein. Northern hybridizations show that the apparently unique 3.8-kb mRNA of c-22 is present in rat brain tissue and its expression is developmentally regulated: the levels of C-22 mRNA, detectable in brain at embryonic day 17 (E17), increase up to post-natal day 7 (P7) and decline rapidly to an almost undetectable level in adult.     

ADP-ribosylation of brain neuronal proteins is altered by in vitro and in vivo exposure to inorganic mercury

ADP-ribosylation is an essential process in the metabolism of brain neuronal proteins, including the regulation of assembly and disassembly of biological polymers. Here, we examine the effect of HgCl2 exposure on the ADP-ribosylation of tubulin and actin, both cytoskeletal proteins also found in neurons, and B-50/43-kDa growth-associated protein (B-50/GAP-43), a neuronal tissue-specific phosphoprotein. In rats we demonstrate, with both in vitro and in vivo experiments, that HgCl2 markedly inhibits the ADP-ribosylation of tubulin and actin. This is direct quantitative evidence that HgCl2, a toxic xenobiotic, alters specific neurochemical reactions involved in maintaining brain neuron structure.     

Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan

Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.     

The protein tyrosine phosphatase SHP-2 is expressed in glial and neuronal progenitor cells, postmitotic neurons and reactive astrocytes

In this study we examined the distribution and developmental profile of the src homology 2 (SH2) domain-containing protein tyrosine phosphatase SHP-2 in the mouse brain. We found that SHP-2 is present in both mitotically active and postmitotic cells in the forebrains of embryonic day 12 (E12) mice. In a developmental study extending from embryonic day 12 to adulthood, Western blotting analysis demonstrated equivalent levels of SHP-2 protein at all of the ages examined. Expression of SHP-2 paralleled the level of enzymatic activity at the different developmental periods. In the adult brain SHP-2 was restricted to diverse classes of neurons, while the majority of glial cells did not express detectable levels of protein. However, reactive astrocytes in response to an ischemic brain injury showed SHP-2 immunolabelling. Our data suggest that SHP-2 may play a role in pathways of neuronal and glial progenitor cells, in a broad spectrum of neuronal responses in the adult brain and in the gliotic response to the injury.