Synergistic effects of the peptide fragment D-NAPVSIPQ on ethanol inhibition of synaptic plasticity and NMDA receptors in rat hippocampus.

The L1 cell adhesion molecule has been implicated in ethanol teratogenesis as well as NMDAR-dependent long-term potentiation (LTP) of synaptic transmission, a process thought to be critical for neural development. Ethanol inhibits LTP at least in part by interacting with NMDA receptors. Ethanol also inhibits L1-mediated cell adhesion in a manner that is prevented by an octapeptide, D-NAPVSIPQ (D-NAP), as well as long chain alcohols such as 1-octanol. Here we analyzed the effects of D-NAP and 1-octanol on ethanol modulation of LTP induced by theta burst stimulation in two subfields of the rat hippocampus, the dentate gyrus and area CA1. When theta burst stimulation was delivered in ethanol (50 mM), LTP was inhibited by about 50%. Surprisingly, when D-NAP (10(-7) M) and ethanol were co-applied or applied sequentially, LTP was completely absent. The effects of D-NAP were persistent, since delivery of a second theta burst stimulation following washout of D-NAP and ethanol elicited minimal plasticity. Application of D-NAP alone had no effect on LTP induction or expression. The synergistic effect of D-NAP on ethanol inhibition of LTP was concentration-dependent since D-NAP (10(-10) M) had an intermediate effect, while D-NAP (10(-13) M) had no effect on ethanol suppression of LTP. These observations were also replicated with a different ethanol antagonist, 1-octanol, in area CA1. To address the mechanisms underlying this long-lasting suppression of LTP, the sensitivity of pharmacologically isolated NMDAR extracellular field potentials to combinations of D-NAP and ethanol was determined. D-NAP (10(-7)M) alone had no effect on NMDA extracellular field potentials; however, the peptide significantly increased the inhibitory action of ethanol on NMDA extracellular field potential. The findings suggest that D-NAP and 1-octanol selectively interact with NMDA receptors in an ethanol-dependent manner, further implicating the L1 cell adhesion molecule in alcohol-related brain disorders.

Antagonists of alcohol inhibition of cell adhesion.

Increasing evidence suggests that alcohols act within specific binding pockets of selective neural proteins; however, antagonists at these sites have not been identified. 1-Alcohols from methanol through 1-butanol inhibit with increasing potency the cell-cell adhesion mediated by the immunoglobulin cell adhesion molecule L1. An abrupt cutoff exists after 1-butanol, with 1-pentanol and higher 1-alcohols showing no effect. Here, we demonstrate surprisingly strict structural requirements for alcohol inhibition of cell-cell adhesion in L1-transfected NIH 3T3 fibroblasts and in NG108-15 neuroblastoma x glioma hybrid cells treated with BMP-7, an inducer of L1 and neural cell adhesion molecule. The target site discriminates the tertiary structure of straight-chain and branched-chain alcohols and appears to comprise both a hydrophobic binding site and an adjacent hydrophilic allosteric site. Modifications to the 2- and 3-carbon positions of 1-butanol increased potency, whereas modifications that restrict movement about the 4-carbon abolished activity. The effects of ethanol and 1-butanol on cell-cell adhesion were antagonized by 1-pentanol (IC(50) = 715 microM) and 1-octanol (IC(50) = 3.6 microM). Antagonism by 1-octanol was complete, reversible, and noncompetitive. 1-Octanol also antagonized ethanol inhibition of BMP-7 morphogenesis in NG108-15 cells. 1-Octanol and related compounds may prove useful in dissecting the role of altered cell adhesion in ethanol-induced injury of the nervous system.

Alcohol inhibition of cell adhesion in BMP-treated NG108-15 cells.

BACKGROUND: The L1 cell adhesion molecule is expressed as alternatively spliced neuronal and nonneuronal isoforms. We have reported that in transfected fibroblasts, ethanol variably inhibits cell-cell adhesion mediated by the nonneuronal isoform of human L1. In contrast, ethanol consistently inhibits morphogenetic changes and cell-cell adhesion in NG108-15 cells treated with OP-1 (BMP-7), a powerful inducer of L1 and N-CAM gene expression. METHODS: All studies were performed by using NG108-15 cells cultured in serum-free medium. Cell morphology was assessed by a quantitative assay of cell clustering. Cell adhesion was measured by a short-term re-aggregation assay, and isoforms of L1 were characterized by RT-PCR and sequencing. RESULTS: We show that ethanol inhibits the morphogenetic effects of BMP-2, BMP-4, BMP-5, and BMP-6, each of which increases the expression of L1 and N-CAM. Pretreatment of NG108-15 cells with 25-100 mM ethanol did not induce tolerance to ethanol's inhibition of OP-1 morphogenesis or cell-cell adhesion. Ethanol or anti-L1 Fab fragments partially inhibited cell-cell adhesion in OP-1-treated NG108-15 cells. The combination of ethanol and Fab fragments did not inhibit cell-cell adhesion more than Fab fragments alone. As in L1-transfected fibroblasts, a series of n-alcohols displayed a cutoff between butanol and pentanol for inhibition of cell-cell adhesion in OP-1-treated NG108-15 cells. RT-PCR and direct sequencing revealed that the neuronal isoform was the sole or predominant L1 isoform in OP-1-treated NG108-15 cells. CONCLUSIONS: These data suggest that ethanol inhibits cell-cell adhesion in OP-1-treated NG108-15 cells by interacting directly or indirectly with the neuronal isoform of L1.

Characterization of ethanol-sensitive and insensitive fibroblast cell lines expressing human L1.

Ethanol inhibits L1-mediated cell-cell adhesion in fibroblast cell lines stably transfected with human L1. Here we show that this action of ethanol is present in only a subset of transfected NIH/3T3 and L cell clonal cell lines. All L1-expressing cell lines had higher levels of cell adhesion than cell lines transfected with empty vector. In all ethanol-sensitive cell lines, L1-mediated adhesion was inhibited by ethanol (IC50 5-10 mM), 2 mM butanol, but not 5 mM pentanol. In contrast, ethanol-insensitive cell lines were not inhibited by up to 200 mM ethanol, 2 mM butanol, or 5 mM pentanol. Ethanol sensitivity or insensitivity was a stable property of each cell line and was not associated with differences in electrophoretic mobility, abundance, or cell surface localization of L1. Fab fragments prepared from anti-L1 polyclonal antisera inhibited cell adhesion only in the ethanol-sensitive cell lines. These data suggest that L1 may exist in an alcohol-sensitive or an alcohol-insensitive state that may be governed by host cell factors.

Alcohol inhibits cell-cell adhesion mediated by human L1.

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector-transfected controls. Ethanol potently and completely inhibited L1-mediated adhesion both in transfected L cells and NIH/3T3 cells. Half-maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n-butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1-mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N-CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.

Addition of tetrodotoxin alters the morphology of thalamocortical axons in organotypic cocultures.

Living organotypic cocultures of rat thalamic and cortical explants were used to examine the effects of blocking action potential activity on the morphological development of axons in the mammalian neocortex. Studies in vivo have suggested that blocking sodium channel-dependent activity influences the growth characteristics of thalamocortical axons during development. We have extended these observations by using an in vitro system that affords more direct observational analysis of the early events of axonal growth in an accessible cellular environment DiI-labeled thalamocortical axons grow exuberantly into the target cortex and establish axonal connections that reflect the events of early thalamocortical afferent development. Within these cocultures, the morphological features of DiI-labeled axons can be readily distinguished. Tracings of thalamocortical axons were quantitated with respect to number, length, and termination pattern of axonal branches, as well as number of varicosities. Addition of the voltage-dependent sodium channel blocker, tetrodotoxin, to cocultures did not change the general pattern of thalamocortical axonal ingrowth or the average length of collateral branches of these axons. However, in the presence of tetrodotoxin, axons were more highly branched, with an increased number of varicosities as compared to untreated cocultures. This pattern of axonal growth and branching may reflect the activity-dependent fine-tuning and trimming of collaterals that occur as thalamic afferents begin to refine their cortical territory. Our observations in thalamocortical cocultures are consistent with the view that neuronal activity modulates the pattern of axonal growth and development.

Adenovirus-mediated gene transfer into dissociated and explant cultures of rat hippocampal neurons.

Genetic manipulation offers great potential for studying the molecular and cellular processes which control or regulate the complex developmental properties of neurons. Gene transfer into neurons, however, is notoriously difficult. In this study we have used a replication-defective adenovirus (Adv/RSV beta gal), expressing beta-galactosidase (beta-gal) as a reporter gene, to infect dissociated cultures of rat hippocampal neurons and hippocampal slice cultures. Because future studies will require either long-term (e.g., developmental) or short-term (e.g., electrophysiological) expression of recombinant genes in neuronal cultures, we have optimized infection conditions for each situation. The Adv/RSV beta gal construct infects neurons and glial cells equally well, with no apparent alterations in cellular morphology. In slice cultures, the same efficiency and temporal control of beta-gal expression following Adv/RSV beta gal infection was achieved. Focal application of the adenoviruses, by microinjection, permitted infection of discrete subregions within the hippocampal explants. Whole cell recordings of dissociated hippocampal neurons and field recordings from the explant cultures, infected with Adv/RSV beta gal at low multiplicities of infection, indicated no significant alteration in the electrophysiological profiles of neurons in these cultures. The results demonstrate the utility of adenoviruses as gene transfer vectors for primary cultures of neurons. Adenovirus-mediated gene transfer into slice cultures also provides an opportunity to study development or plasticity in an environment where the circuitry and cytoarchitecture of the tissue are preserved and the areas of genetic manipulation can be spatially isolated.

Adenovirus-mediated expression of a reporter gene in thalamocortical cocultures.

Organotypic cocultures of thalamic and cortical explants have recently been used to study the development of the thalamocortical axonal network in the mammalian neocortex. To explore the possibility of genetically manipulating organotypic explants, rat thalamocortical (TC) cocultures were infected with the recombinant adenovirus, Adv/RSV beta gal. Infection of the cortical explants resulted in long-term expression (2 weeks) of the reporter gene (beta-galactosidase) with no significant alterations to the structural integrity of the explants. By micro-injecting the adenoviruses into cortical explants a significant degree of spatial control over reporter gene expression was obtained. DiI-labeled axonal projections from thalamic explants into infected (n = 116) and control cortical (n = 120) explants were also analyzed. There was no significant difference in the extent or degree of TC ingrowth into infected or control cortical explants. Thalamic explants were also efficiently infected with the Adv/RSV beta gal virus. While the pattern and extent of TC ingrowth from infected thalamic explants was similar to controls, the percentage of viable, infected thalamic explants was decreased. These experiments were necessary precursors for future studies using recombinant adenoviruses and organotypic cocultures. Genetic manipulation of these cocultures should enable the dissection of proteins involved in the development of axonal networks in the mammalian neocortex, using a system amenable to direct manipulation and observation.

Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity.

Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3' untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.

Differential diagnosis of mut and cbl methylmalonic aciduria by DNA-mediated gene transfer in primary fibroblasts.

Methylmalonic aciduria can be caused by mutations in the gene encoding the methylmalonyl coenzyme A mutase apoenzyme (mut) or genes required for the provision of cofactor B12 (cbl). The mut and cbl forms are classically differentiated by somatic cell complementation. We describe a novel method for differential diagnosis of mut and cbl methylmalonic aciduria using DNA-mediated gene transfer of a methylmalonyl CoA mutase cDNA clone. Gene transfer of a functional methylmalonyl CoA mutase cDNA clone into mut fibroblasts reconstitutes holoenzyme activity measured by metabolism of [14C]-propionate in culture. Identical gene transfers into cbl fibroblasts have no effect. This method is used for the differential diagnosis of mut and cbl genotypes in cells from patients with a clinical diagnosis of methylmalonic aciduria and is shown to be a facile, sensitive, and specific method for genetic diagnosis. This work establishes the principle of using DNA-mediated gene transfer to identify the genotype of diseases which can result from mutations at several different genetic loci. This type of differential genotypic diagnosis will be particularly important for establishing the applicability of somatic gene therapy in individual patients.

Structure of the human methylmalonyl-CoA mutase (MUT) locus.

The MUT locus encoding the enzyme methylmalonyl-CoA mutase is defective in mut forms of methylmalonic acidemia. This locus has been mapped to chromosome 6p12-21.1. We report cloning and characterization of this locus which comprises 13 exons spanning greater than 35 kb of the genome. The MUT locus exhibits consensus sequences for transcription, splicing, and polyadenylation. The putative promoter region was localized in a CG island 5' to exon I and was shown to direct expression of a beta-galactosidase reporter gene in cultured cells. Of interest is the observation that the first intron occurs within the 5' untranslated region, and no introns separate the mitochondrial targeting sequences and the mature apoenzyme. An informative HindIII polymorphism was localized within the coding sequence and can be assayed using the polymerase chain reaction. These studies describe the structure of the MUTlocus and provide a foundation for characterization of mutations in mut methylmalonic acidemia.

Primary structure and activity of mouse methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase (MCM) is an adenosylcobalamin-dependent enzyme that catalyses isomerization between methylmalonyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA). Genetic deficiency of this enzyme in man causes an often fatal disorder of organic acid metabolism termed mut methylmalonicacidaemia. We report cloning of a mouse MCM cDNA and the characterization of its primary structure and biological function. Mouse MCM in fibroblasts and crude liver extracts exhibits activity and reaction kinetics similar to those of the human enzyme. The predicted amino acid sequence of mouse MCM exhibits 94% identity with its human homologue and considerable identity with a prokaryotic MCM. Transfection of the mouse cDNA into cultured cells constitutes an active apoenzyme and can complement genetic deficiency of the apoenzyme in cells from patients with mut methylmalonicacidaemia. These results establish that mouse MCM is homologous to human MCM in structure and function and provides a basis for using the mouse as a model for studying this enzyme and its deficiency state.