Effects of taurine depletion on cell migration and NCAM expression in cultures of dissociated mouse cerebellum and N2A cells.

Cultures of dissociated cerebellum from 5- to 6-day-old mice as well as of the N2A neuronal cell line were exposed to guanidino ethane sulfonate (GES, 2-5 mM) to reduce the cellular taurine content. Control cultures were kept in culture medium or medium containing 2-5 mM GES plus 2-5 mM taurine to restore the intracellular taurine content. Taurine depletion led to changes in the expression of certain splice variants of NCAM mRNA such as the AAG and the VASE containing forms, while no differences were seen in the expression of the three forms of NCAM protein. In the N2A cells taurine depletion led to a decreased migration rate of the cells. The results suggest that the reduced migration rate of neurons caused by taurine depletion may be correlated to changes in expression of certain adhesion molecules such as NCAM. Moreover, taurine appears to be involved in regulation of transcription processes.

Cell proliferation, migration and CAM-dependent neurite outgrowth as developmental in vitro endpoints for screening teratogenic potential: Application to valproic acid and related analogues of varying potency.

The in vivo teratogenic potential of valproic acid (VPA) and related teratogenic and non-teratogenic analogues has been correlated with their effects on specific in vitro endpoints of cell proliferation, migration and CAM-dependent neurite outgrowth, as these events are common to crucial epochs of development. The (+/-)-2-n-propyl-4-pentynoic acid [(+/-)-4-yn-VPA] and S-2-n-propyl-4-pentynoic acid [S(-)-4-yn-VPA] analogues increased the incidence of neural tube defects in mouse embryos exposed to a single dose, whereas the E-2-n-propyl-2-pentenoic acid (E-2-en-VPA) analogue and R-2-n-propyl-4-pentynoic acid [R( + )-4-yn-VPA] enantiomer were without effect. VPA and related analogues tested exerted comparable G1 phase antiproliferative effects in C6 glioma and limb bud cells in a dose range of 0-3 mM; however, their relative potency did not correlate with in vivo teratogenicity. In contrast, VPA and all teratogenic analogues, at 3 mM, inhibited neuronal cell aggregation and limb bud chondrocyte differentiation in a manner that exhibited a reasonable correlation with their in vivo teratogenicity. The teratogenic S(-)-4-yn-VPA and non-teratogenic R( + )-4-yn-VPA enantiomers exhibited a differential inhibition of primary neurone outgrowth of neuntes stimulated by cell adhesion molecules [L1 and N-cadherin (NCAD)]. Half-maximal inhibition was observed at approximately 150 muM for the teratogenic S(-)-4-yn-VPA enantiomer, but not the non-teratogenic R( + )-4-yn-VPA form. These results suggest that in vitro perturbations of differentiation are likely to provide the greatest discriminatory power for in vivo teratogenicity.

The first immunoglobulin-like neural cell adhesion molecule (NCAM) domain is involved in double-reciprocal interaction with the second immunoglobulin-like NCAM domain and in heparin binding.

To study the function of the first immunoglobulin (Ig)-like domain of the neural cell adhesion molecule (NCAM), it was produced as a recombinant fusion protein in a bacterial expression system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris. For comparison, other NCAM domains were also produced as fusion proteins. By means of surface plasmon resonance analysis, it was shown that the first Ig-like NCAM domain binds the second Ig-like NCAM domain with a dissociation constant 5.5 +/- 1.6 x 10(-5) M. Furthermore, it was found that the first Ig-like domain binds heparin. It was also demonstrated that the second Ig-like NCAM domain binds heparin and that both domains bind collagen type I via heparin but not collagen type I directly.

Prediction of teratogenic potency of valproate analogues using cerebellar aggregation cultures.

The aim of this study was to develop a novel in vitro system suitable for preclinical testing for developmental toxicity of drugs. An assay system consisting of primary cultures of dissociated cerebella from 6-day-old mice was chosen, since it allowed quantification of neuronal aggregation and fasciculated neurites. A human teratogen, the antiepileptic drug valproic acid (VPA), as well as its structural analogues, ( +/- )-4-en-VPA and E-2-en-VPA, with varying teratogenic activities, were tested and found to affect aggregation and fiber formation of cerebellar neurons. Based on a dose-response study, the concentrations of compounds causing 50%, inhibition (IC50) of formation of thick and thin fibers were determined. The lowest IC50 values were found for VPA (52 +/- 7 and 86 +/- 11 microM for thick and thin fibers, respectively), which in vivo caused the highest rate of exencephaly among the three compounds tested, ( +/- )-4-en-VPA exhibited intermediate values (150 +/- 30 and 300 +/- 40 microM), whereas the highest IC50 values were found for E-2-en-VPA (260 +/- 42 and 430 +/- 40 microM). The latter compound does not induce neural tube defects, but has been shown to have neurobehavioral effects in prenatally exposed animals. Subsequently, the purified S- and R-enantiomers of 4-yn-VPA (teratogenic and non-teratogenic, respectively) were tested for their effects on aggregation and fiber formation of the cerebellar neurons. Treatment with S-4-yn-VPA resulted in pronounced changes in numbers of aggregates and fasciculated processes compared to the cultures treated with R-4-yn-VPA, indicating that the intrinsic stereoselective potency of the enantiomers may be correlated to the difference in their effects on cerebellar neurons in vitro. Thus, the teratogenic potency of VPA and its analogues correlated with their effects on aggregation of neural cells and formation of fasciculated neurites in primary cultures of dissociated cerebella, indicating that the in vitro assay system employed may be used as a pre-screening test for prediction of teratogenic potency of drugs.

Characterization of microwell cultures of dissociated brain tissue for studies of cell-cell interactions.

Microwell cultures of dissociated tissue from prenatal rat hippocampus and cerebral cortex as well as from early postnatal cerebellum were used for quantification of neuronal aggregation, process extension, and fasciculation. It was shown that the cells in culture from these different brain regions developed differently with regard to both architecture and rate of differentiation. The effect of a polyclonal antibody against the neural cell adhesion molecule (NCAM), the excitatory amino acid receptor agonist N-methyl-D-aspartate (NMDA), and the neurotoxin acrylamide on aggregation and fiber formation was investigated. Exposure to the NCAM antibody led to formation of fewer but larger aggregates and stimulated the morphological development of the cultures. Acrylamide affected aggregate formation, leading to smaller but more numerous aggregates, and it inhibited process extension and fasciculation. Treatment with NMDA affected process formation and led to formation of more numerous but smaller aggregates. Some of these effects were strongly tissue-dependent. Thus, large differences were seen regarding the effect of the NCAM antibody on aggregation and process extension in cultures from the different brain areas. The culture systems appear to represent convenient and reliable screening tools to study the influence of putative morphoregulatory substances on cell-cell interactions during early neuronal development.

Functional characterization of NCAM fibronectin type III domains: demonstration of modulatory effects of the proline-rich sequence encoded by alternatively spliced exons a and AAG.

In order to characterize the functions of the two fibronectin type III (F3) homology domains of the neural cell adhesion molecule (NCAM), we investigated the effects of two variants, expressed as fusion proteins, of the NCAM-F3 domains on attachment and spreading of NCAM-expressing fibroblasts, cerebellar cell aggregation and fiber formation, and on growth cones. The two fusion proteins were different with regard to a short proline-rich insert of six amino acids between the two F3 domains. Immobilized NCAM-F3 fusion proteins were found to mediate attachment of both transmembrane and lipid-anchored NCAM expressing fibroblasts. Also NCAM-negative cells adhered to the NCAM-F3 substratum, although to a lesser extent, implying the possibility of a heterophilic ligand to NCAM-F3 domains on the surface of fibroblasts. Cellular spreading on NCAM-F3 substratum was selectively increased in fibroblasts expressing transmembrane NCAM, and only the NCAM-F3 fusion protein lacking the proline-rich insert was able to elicit this effect. Primary cultures of mouse cerebellum were strongly inhibited with regard to formation of cellular aggregates and fibers, when incubated in the presence of either of the two NCAM-F3 fusion proteins, the fusion protein with the proline-rich insert being the more effective one. Finally, the morphology of growth cones from rat cerebellar granule cells changed significantly when grown on NCAM-F3 substrata as revealed by computer-assisted image analysis. Thus, our data indicate that the NCAM-F3 domain are involved in cell-cell adhesion, and that insertion of the proline-rich sequence has a modulatory effect on NCAM-F3 domain functions.

Impaired cell volume regulation in taurine deficient cultured astrocytes.

Taurine concentration was reduced by 40 and 65%, respectively in rat cerebellar astrocytes grown in a chemically defined medium or in culture medium containing a blocker of taurine transport (GES). Cell volume in these taurine deficient cells was 10%-16% higher than in controls. When challenged by hyposmotic conditions, astrocytes release taurine and this efflux contributes to the volume regulatory decrease observed in these cells. Taurine deficient astrocytes showed a less efficient volume recovery as compared to controls with normal taurine levels. Exposed to 50% hyposmotic medium, astrocytes with normal taurine concentration recovered 60% of their original volume whereas taurine deficient cells recovered only 30-35%. Similarly, in 30% hyposmotic medium, taurine deficient astrocytes recovered only 40% as compared to 75% in controls. No compensatory increases in the efflux of other osmolytes (free amino acids or potassium) were observed during regulatory volume decrease in taurine deficient astrocytes.

Cell volume regulation in cultured cerebellar granule neurons.

Cultured rat cerebellar granule neurons exposed to solutions of reduced osmolarity, responded initially by swelling followed by a regulatory volume decrease (RVD) which is completed within 15 min. Increasing external osmolarity lead to cell shrinking but no evidence of volume regulation was observed within 1 hr. Replacing Na+ by choline did not affect RVD whereas N-methyl-D-glucamine accelerated the volume recovery and K+ suppressed it completely. The blockade of RVD in high extracellular K+ was only observed when chloride and nitrate but not sulfate or gluconate were the accompanying anions. Replacing intracellular Cl-, by long incubations with gluconate, markedly inhibited RVD. Removal of extracellular Ca2+ or addition of dantrolene which blocks Ca2+ released from intracellular stores had no effect on RVD. Increasing extracellular taurine prevented RVD. These results indicate that membrane permeability to K+, Cl-, and taurine is increased by hyposmolarity and suggest the involvement of these molecules in RVD in granule neurons.