Axon growth is enhanced by NCAM lacking the VASE exon when expressed in either the growth substrate or the growing axon

The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM ± VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM ? VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM ± VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM ? VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM ± VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM ? VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM ? VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a © Wiley-Liss, Inc. significant factor in the reduced axonation capabilities of mature neurons. © 1993 Wiley-Liss, Inc.     

Smooth muscle cells transiently express NCAM

NCAM (neural cell adhesion molecule) polypeptides were first detected on neuronal cells and were subsequently found to be expressed at least transiently by a number of other cell types including skeletal and cardiac but not smooth muscle. We report here that rat smooth muscle expresses NCAM in vitro and transiently in vivo. Using a monoclonal antibody 3F4 which reacts with most rat NCAM polypeptides, NCAM was found on the surface of cultured rat aortic smooth muscle lines A10 and A7r5 and mouse smooth muscle like line BC3H1 in abundances equal to or greater than those of cardiac muscle, skeletal muscle, and neuronal cell lines. The major NCAM polypeptide of muscle cells was Mr = 140 kDa with lesser amounts of the 120 kDa form. Consistent with these results, a major NCAM mRNA of 6.7 kb was detected in Northern analyses with lesser amounts of the 4.3 and 2.9 kb mRNA size classes. The relative abundance of NCAM mRNA was similar in RNA prepared from smooth muscle A7r5 cells, L6 skeletal muscle cells, and 9-day-old rat brain. NCAM was distributed across the entire surface of cultured smooth muscle cells in a highly punctate manner. Cryostat sections of rat aorta, intestine and bladder were examined by immunofluorescence to determine if NCAM is also expressed on smooth muscle in vivo. In each case NCAM was found to be transiently expressed by the smooth muscle cells of these tissues. Highest NCAM levels were observed at embryonic day 17 which then declined to undetectable levels in tissues from adults. These results extend previous observations to indicate all muscle types transiently express NCAM in development.     

Effect of experimental hyperphenylalaninemia on the postnatal rat brain

The molecular mechanism of the disturbance to brain development caused by phenylketonuria remains mostly unknown. We have studied three molecular markers that reflect the development of neurons, glia and the extracellular matrix of the postnatal rat brain in an animal model of hyperphenylalaninemia, in order to elucidate the possible mechanism by which increased phenylalanine influences brain development. The contents of NCAM, GFAP and hyaluronate-binding activity were compared in cerebellum and telencephalon of normal rats and those subjected to high phenylalanine. No statistically significant changeswere found in telencephalon when experimental animals were compared to controls. In the hyperphenylalaninemic cerebellum, the developmental dynamic of NCAM content (represented by two peaks at about postnatal days 5 and 22 during normal development) is dramatically altered. The GFAP content in the cerebellum of treated rats exceeded those in controls significantly during late developmental stages (postnatal days 28–35). Hyaluronate-binding activity in the extracellular protein fraction from treated rat cerebellum was increased compared to normal rat at the early stages of development only (postnatal day 7). These results suggest that high serum phenylalanine may lead to permanent brain dysfunction through a disturbance of a wide range of developmental events.     

VASE-encoded peptide modifies NCAM- and L1-mediated neurite outgrowth

Interactions between the neural cell adhesion molecule (NCAM) with NCAM-expressing neurons (trans-interaction) stimulate outgrowth of neurites. The extent of NCAM-triggered neurite outgrowth depends on the presence of 10 amino acids derived from the variable alternatively spliced exon (VASE or π-exon) in the fourth immunoglobulin-like domain of NCAM (Ig4): NCAM with VASE reduces and without VASE enhances neurite outgrowth in cis- or trans-interaction. We have investigated the role of VASE in neurite outgrowth by characterizing the receptors at the cell surface of cultured cerebellar neurons. Results from experiments with L1 and NCAM antibodies and with cerebellar neurons derived from wild-type or NCAM-deficient mice show that substrate-coated Ig4 with VASE (Ig4+) or without VASE (Ig4−) stimulates neurite outgrowth by a trans-interaction with L1 and that Ig4− promotes neurite outgrowth more strongly than Ig4+ by a transinteraction with NCAM. J. Neurosci. Res. 50:62–68, 1997. © 1997 Wiley-Liss, Inc.     

Variable alternative spliced exon (VASE)-containing and VASE-lacking neural cell adhesion molecule in the dorsal and ventral hippocampus of SAMP8 mice

The neural cell adhesion molecule (NCAM) is involved in the development and synaptic plasticity of the brain. Differential splicing of the variable alternative spliced exon (VASE) in the fourth immunoglobulin domain can dramatically change the functional properties of NCAM. This paper discusses our analysis of the levels of different expression of VASE-containing NCAM (NCAM-VASE(+)) and VASE-lacking NCAM (NCAM-VASE(-)) mRNAs in the dorsal and ventral hippocampus of senescence-accelerated mice (SAM). We further investigated the individual level of NCAM-VASE(+) and NCAM-VASE(-) in relation to the capacity for spatial learning and memory as assessed by a Morris water maze task. The results showed that the levels of both NCAM-VASE(+) and NCAM-VASE(-) were increased significantly in dorsal but not ventral hippocampus in aged SAMP8 mice. The mean latencies to find the hidden platform of the learning task on the last day were positively correlated with the levels of NCAM-VASE(+) in the dorsal hippocampus of SAMP8, which reveals that the mice with high levels of NCAM-VASE(+) have poor learning performances. These results suggest that the up-regulation of NCAM-VASE(+) could be involved in the impairments of spatial learning and memory.     

VASE exon expression alters NCAM-mediated cell-cell interactions

The neural cell adhesion molecule (NCAM) is found on cells as several related polypeptides formed by alternative splicing of the single NCAM gene. The alternatively spliced 30-bp VASE exon in the fourth immunoglobulin-like domain is the structural variation nearest those portions of the polypeptide proposed to mediate cell-cell adhesion. To test the ability of distinct forms of the NCAM molecules to mediate cell adhesion, L cells were transfected with expression vectors encoding rat 140 kD NCAM ± the VASE exon. L cell lines which expressed these polypeptides were isolated and tested for self-aggregation in a low shear, rapid aggregation assay. Increased cellular aggregation of the transfectants was observed to be a function of the NCAM molecule expressed. These transfected cells showed segregation in a long term co-aggregation assay: cells expressing NCAM — VASE formed aggregates which tended to exclude cells expressing NCAM + VASE and vice versa. These results provide direct evidence that this small difference in NCAM structure is sufficient to allow segregation of cells. © 1994 Wiley-Liss, Inc.     

Cloning and developmental expression of the murine neurofilament gene family

DNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal alpha-helical region and the carboxy-terminal domains of the protein. These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene. This strongly suggests that the 3 neurofilament genes are organised in a cluster and derived by gene duplication of a common ancestral gene. RNA blot analyses using specific DNA probes for each of the genes indicate that NF mRNAs are differentially expressed during brain development. The NF-L and NF-M mRNAs are detected early in the embryonal brain, with a progressive increase in their levels during development, while the NF-H mRNA is barely detectable at embryonal stages and accumulates later in the postnatal brain.     

Soluble neural cell adhesion molecule in brain, cerebrospinal fluid and plasma in the developing rat

Neural cell adhesion molecule (NCAM) is a glycoprotein found in neurons, glial cells and muscle cells. In this report we describe the polypeptide composition and amount of soluble NCAM in brain, cerebrospinal fluid (CSF) and plasma in rats of various ages. One-two percent of total NCAM in rat brain was shown to be buffer-soluble. Soluble NCAM in brain, extracted at pH 7.2, constituted an increasing relative proportion of total NCAM during development, whereas the concentration of soluble NCAM in both CSF and plasma decreased in the same period. By radioiodination of brain extracts, CSF, and plasma, soluble NCAM was found to be composed of up to five polypeptides with molecular weights (Mr) of 200,000, 170,000, 150,000, 115,000 and 80,000. The relative composition did not vary significantly with age. The major forms in brain were the polypeptides with Mr of 200,000 and 115,000, whereas in CSF all forms seemed to appear in equal amounts. In plasma the 200,000 and the 170,000 Mr polypeptides were not observed and here the major forms were the 150,000 and 115,000 Mr polypeptides.     

NCAM expression induces neurogenesis in vivo

Neural cell adhesion molecule (NCAM) plays an important role during neural development and in the adult brain, whereby most functions of NCAM have been ascribed to its unique polysialic acid (PSA) modification. Recently we presented evidence suggesting that expression of NCAM in vivo interferes with the maintenance of forebrain neuronal stem cells. We here aimed at investigating the fate of cells generated from NCAM-overexpressing stem cells in postnatal mouse brain and at elucidating the functional domains of NCAM mediating this effect. We show that ectopic expression of the NCAM140 isoform in radial glia and type C cells induces an increase in cell proliferation and consequently the presence of additional neuronal type A cells in the rostral migratory stream. A mutant NCAM protein comprising only fibronectin type III repeats and immunoglobulin-like domain 5 was sufficient to induce this effect. Furthermore, we show that the neurogenic effect is independent of PSA, as transgenic NCAM is not polysialylated in radial glia and type C cells. These results suggest that heterophilic interactions of NCAM with other components of the cell membrane must be involved.     

TrkA, TrkB and p75 mRNA expression is developmentally regulated in the rat retina

We examined the cellular distribution of mRNAs coding for the neurotrophin receptors TrkA, TrkB and p75 in the rat retina during early postnatal development. At PO (postnatal day 0), mRNAs coding for each of the three receptors were detected in the ganglion cell layer (GCL) and in the inner plexiform layer (IPL), the latter structure essentially containing retinal ganglion cell processes at this developmental stage. At P5, the innermost part of the inner nuclear layer (INL) also expressed TrkA, TrkB and p75 mRNAs. Finally, the GCL, IPL and the whole INL of P10 retinae were labeled by the three probes. The developmentally regulated expression of these receptors underlies a possible role for neurotrophins in the differentiation and survival of retinal cells.     

Postnatal changes in muscarinic receptor subtype mRNAs in rat brain and heart

Expression of mRNAs for the muscarinic acetylcholine receptor subtypes in various brain regions and in the heart of male rats was examined during postnatal development. The mRNAs for the four subtypes displayed different developmental patterns in the different regions, depending both on the cell type composition and on the age of the brain region examined.     

Regulation of the glial fibrillary acidic protein, beta actin and prion protein mRNAs during brain development in mouse.

Developmental regulation in mRNAs of three brain proteins has been investigated by Northern blot evaluation in C57BL/6 mice. The mRNAs of two cytoskeletal components, glial fibrillary acidic protein (GFAP) and beta actin, varied significantly, and differently, during brain development (0-56 days postnatal). The beta actin mRNAs peaked at day 1 after a slight increase, then dropped rapidly during the first 15 days postnatal, and thereafter remained at a level which was strictly maintained throughout development and adulthood. Conversely, the GFAP mRNAs increased during the first two weeks after birth (astroglial proliferation), and then slightly declined until the adult stage (astroglial cell differentiation). The prion protein (PrP) mRNAs were detectable as soon as birth, and increased 4-fold during brain maturation. Then, during the adult life, the GFAP and PrP mRNAs did not change markedly. Nevertheless, slight but significant increases in the mRNA levels of both GFAP and PrP were observed at older stages (360 days). These results are analysed in the light of the implications of PrP and GFAP in scrapie infection models.     

Developmental profile and differential localization of mRNAs of myelin proteins (MBP and PLP) in oligodendrocytes in the brain and in culture

We applied the in situ hybridization technique to localize the mRNAs for two myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). In the oligodendrocyte in primary culture, PLP mRNA was located exclusively in the cell body throughout development. However, MBP mRNA was first located in the cell body and was rapidly distributed to the processes but not to the membranous sheets formed from the tips and lengths of the processes. Expression of PLP and MBP genes progressed in the caudo-cranial direction in the brain as far as we examined it in the tissue sections up to the 30th postnatal day: mRNAs of both genes were first detected in the pons and the medulla oblongata on the 3rd postnatal day and then in the cerebellum and the anterior part of the brain. PLP mRNA was located exclusively in the cell body throughout development. The number of PLP mRNA-positive cells reached a plateau in the posterior part of the brain on the 18th postnatal day, whereas it continued to increase in the anterior part of the brain by the 30th day. MBP mRNA was first expressed in the cell body, but later, it was found along the myelin sheath.     

Cloning of chicken choline acetyltransferase and its expression in early embryonic retina

The enzyme choline acetyltransferase [EC 2.3.1.6] (ChAT) synthesizes the neurotransmitter acetylcholine that plays a key morphogenic role in vertebrate retina development. As the embryonic avian retina is particularly useful for morphogenetic studies, we cloned the complete coding region of chicken ChAT cDNA. At the deduced amino acid level, chicken ChAT is approximately 76% identical to mammalian ChAT proteins. We also report here the cloning of the complete 5' end of the complex cholinergic locus. This locus contains both the ChAT gene and the nested intronless gene for the vesicular acetylcholine transporter (VAChT). The genomic organization of the 5' end of the chicken cholinergic locus is similar to that reported in other vertebrate species. A 5.7 kb mRNA corresponding to the ChAT message was detected in both embryonic retina and post-hatch brain. An analysis of the ChAT mRNA in embryonic chick retina shows that the message can be detected by E6 and its level increased during early retinal development. Vertebrate ChAT mRNAs can contain one or more of three non-coding exons, M, N or R and by RT-PCR we demonstrate, at least, a chicken ChAT mRNA containing exon M.