Amino acid sequence RERMS represents the active domain of amyloid beta/A4 protein precursor that promotes fibroblast growth.

The growth of A-1 fibroblasts depends on exogenous amyloid beta/A4 protein precursor (APP), providing a simple bioassay to study the function of APP. Our preliminary study, testing the activity of a series of fragments derived from the secreted form of APP-695 (sAPP-695) on this bioassay, has shown that at least one of the active sites of sAPP-695 was localized within a 40-mer sequence (APP296-335, Kang sequence; Roch, J.-M., I. P. Shapiro, M. P. Sundsmo, D. A. C. Otero, L. M. Refolo, N. K. Robakis, and T. Saitoh. 1992. J. Biol. Chem. 267:2214-2221). In the present study, to further characterize the growth-promoting activity of sAPP-695 on fibroblasts, we applied a battery of synthetic peptides on this bioassay and found that: (a) the sequence of five amino acids, RERMS (APP328-332), was uniquely required for the growth-promoting activity of sAPP-695; (b) the activity was sequence-specific because the reverse-sequence peptide of the active domain had no activity; and (c) the four-amino-acid peptide RMSQ (APP330-333), which partially overlaps the COOH-terminal side of the active sequence RERMS, could antagonize the activity of sAPP-695. Furthermore, a recombinant protein which lacks this active domain (APP20-591 without 306-335) did not promote fibroblast cell growth, suggesting that this domain is the only site of sAPP-695 involved in the growth stimulation. The availability of these biologically active, short peptides and their antagonists should prove to be an essential step for the elucidation of APP involvement in regulation of cellular homeostasis.

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice.

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.

Expression of myelin-associated glycoprotein by small neurons of the dorsal root ganglion in chickens.

Biochemical and immunocytochemical investigations have shown that myelin-associated glycoprotein (MAG) is exclusively related to myelin and myelin-forming cells in mammals. In the present study it was found that dorsal root ganglia in young chickens display MAG-immunoreactive material in most small sensory neurons. The presence of MAG at the surface of small sensory neurons raises the question of whether this glycoprotein acts as a cell adhesion molecule in lower vertebrates.

Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells.

1. The pharmacological properties of K(ATP) channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K(+) channel Kir6.2 were characterized in HEK-293 cells. 2. [(3)H]-Glyburide (glibenclamide) bound to transfected cells with a B(max) value of 18.5 pmol mg(-1) protein and with a K(D) value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3. Functional activity of K(ATP) channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 microM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC(50)=0.92 nM). 4. Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered (86)Rb(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC(50)=3.6 nM). 5. Diazoxide, but not levcromakalim, evoked concentration-dependen decreases in DiBAC(4)(3) fluorescence responses with an EC(50) value of 14.1 microM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. 6. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K(ATP) channels described in pancreatic and neuronal tissues.

Biologically active domain of the secreted form of the amyloid beta/A4 protein precursor.

The amyloid beta/A4 protein precursor (APP), a large transmembrane protein, is expressed ubiquitously in many organisms, as well as in a variety of cultured cells. Studies of the synthesis and processing of APP have revealed several intricate metabolic pathways for this protein. One of these pathways involves the cleavage of APP in the middle of the beta/A4 domain and results in the secretion of the large amino-terminal portion of the protein. The biological function of this secreted form of APP has been the subject of intense investigation by several groups and various activities have been described for the different domains of APP studied. Our initial approach was to create a fibroblast cell line in which APP expression is dramatically reduced. These fibroblasts, called A-1, have a very slow growth rate. Addition of exogenous APP in the medium of A-1 cells restores their growth to the level of normal parent fibroblasts, demonstrating a growth factor-like activity for the secreted form of APP. Using APP fragments made in bacteria as well as synthetic peptides, we have been able to locate the active site of APP within a domain of 17 amino-acids (Ala319-Met335). This domain of APP can stimulate neurite extension of cultured neuroblastoma cells and it is proposed that APP mediates this effect through binding to a cell surface receptor, triggering intracellular transduction mechanisms. Thus, the secreted form of APP can function as a growth and/or differentiation factor and the site involved in these activities is within a 17-mer domain in the middle of the molecule. Our current lines of research seek to further characterize the mechanisms of APP function as well as its activity in vivo.

Expression of only one myelin basic protein allele in mouse is compatible with normal myelination.

Myelin deficiency (mld) is an autosomal recessive mutation in mice characterized by a severe myelin deficit in the central nervous system (CNS). The primary defect in mld is a reduction of the synthesis of the myelin basic protein (MBP) and probably lies in a regulatory element of the MBP gene. In young mld heterozygotes, the MBP mRNA and MBP levels are intermediate. In order to study whether reduced levels of MBP gene expression affect myelination, we determined the levels of MBP mRNA and MBP itself in mld heterozygous and control brains, at different ages during development. Total proteins and MBP were also measured in myelin isolated at 25 and 85 days of age. Myelin proteins were analyzed by SDS-PAGE. In addition, we carried out a morphometric analysis on 25- and 85-day-old optic nerves. Our results indicate that in spite of a roughly 50% reduction of MBP gene expression (compared to controls), the amounts of myelin isolated and the concentration of MBP in myelin were normal in heterozygous brains. Nevertheless, morphometric analyses of optic nerves, which myelinate later than the brainstem, showed thinner myelin sheaths in 25-day-old heterozygotes when compared to controls. This difference disappeared at 85 days of age. These results indicate that normal mice synthesize MBP in excess. The synthesis of this extramyelinic pool of MBP represents a safety factor allowing normal myelination to proceed even when MBP synthesis is severely reduced. In mld heterozygotes, a 30-50% reduction of this rate of synthesis can represent a limiting factor and locally delay myelin deposition without affecting the overall myelin content or myelin composition in heterozygous adult brains.

The duplicated myelin basic protein gene in mld mutant mice does not impair transcription.

Myelin basic protein (MBP) gene organization and expression were analyzed in wild type and myelin deficient (mld) mutant mice. Southern analysis demonstrated MBP gene duplication in mld mice. In addition, we present evidence that one MBP gene in mld mice is normal for at least 14 kilobases (kb) upstream from exon I, whereas the second gene is normal for at least 3.5 kb but not more than 7 kb upstream from exon I. Run-on experiments showed that the rate of MBP gene transcription in mld mice is similar to that seen in normal mice. Detailed analysis of the transcriptional activity of various regions of the gene led us to conclude that all portions of the MBP gene are transcribed in mld mice. Consequently, we propose that the low levels of MBP mRNA observed in these mice (2-5% of the wild-type level) are not due to deficient transcriptional activity.

Mice heterozygous for the mld mutation have intermediate levels of myelin basic protein mRNA and its translation products.

Myelin-deficiency (mld) is an autosomal recessive mutation in mice exhibiting a severe deficit in the synthesis of myelin basic protein (MBP). In order to understand the mechanisms involved in the regulation of MBP synthesis in the mld mutation, we examined the amount of MBP and MBP-specific mRNA in control, heterozygous and homozygous mld brains. In vitro translation of poly(A)+ RNA in a cell-free system, in situ hybridization, and filter hybridization with a radiolabelled probe pMBP-1 after dot or Northern blotting were used in this study. The levels of MBP and MBP-specific mRNA were very low but detectable in mld homozygotes, and intermediate in heterozygotes. MBP specific mRNA from mutants, and its translation products, were of normal size. These results show that the mld mutation is expressed co-dominantly in heterozygotes and affects a cis-acting regulatory element controlling the MBP gene.

Gain of function mutation of the alpha7 nicotinic receptor: distinct pharmacology of the human alpha7V274T variant.

In the human alpha7 nicotinic receptor, valine-274 in the pore-lining transmembrane-2 region was mutated to threonine to produce the variant human alpha7V274T, which was evaluated electrophysiologically following expression in Xenopus laevis oocytes. Inward current rectification was strong in human alpha7V274T as in the human alpha7 wild type nicotinic receptor. However, human alpha7V274T was 100-fold more sensitive to the nicotinic receptor agonists acetylcholine, (-)-nicotine and 1,1-dimethyl-4-phenylpiperazinium. Choline also activated human alpha7V274T (EC50 = 12 microM) and was 82-fold more potent than at human alpha7 wild type nicotinic receptor. (-)-Cotinine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine (ABT-089), weak partial agonists at human alpha7 wild type, were much stronger agonists at human alpha7V274T with EC50 values of 70 microM, 4 microM and 28 microM and fractional activation values of 93%, 96% and 40%, respectively. However, (-)-lobeline, a human alpha7 wild type nicotinic receptor antagonist, and dihydro-beta-erythroidine, which activates chick mutagenized alpha7 nicotinic receptors, had only weak agonist-like activity at human alpha7V274T (< or = 4% of the maximal acetylcholine response). Methyllycaconitine, mecamylamine, d-tubocurarine and dihydro-beta-erythroidine retained antagonist activity and, indeed, appeared to be at least as potent at human alpha7V274T as at human alpha7 wild type. These results support and extend the concept that human nicotinic receptor pharmacology can be profoundly altered by single amino acid changes in the pore-lining segment.

Myelin basic protein transcriptional activity in myelin-deficient mutant mice.

Myelin-deficient (mld) mice present two tandem myelin basic protein genes, with the upstream gene containing an inversion (Popko et al., Neuron 1: 221, 1988). Detailed analysis of the transcriptional activity of various regions of the gene showed that all portions of the MBP gene are transcribed in mld mice. Consequently, we propose that the low levels of MPB mRNA observed in mld mice are due to post-transcriptional events.

Variants in Apaf-1 segregating with major depression promote apoptosome function.

APAF1, encoding the protein apoptosis protease activating factor 1 (Apaf-1), has recently been established as a chromosome 12 gene conferring predisposition to major depression in humans. The molecular phenotypes of Apaf-1 variants were determined by in vitro reconstruction of the apoptosome complex in which Apaf-1 activates caspase 9 and thus initiates a cascade of proteolytic events leading to apoptotic destruction of the cell. Cellular phenotypes were measured using a yeast heterologous expression assay in which human Apaf-1 and other proteins necessary to constitute a functional apoptotic pathway were overexpressed. Apaf-1 variants encoded by APAF1 alleles that segregate with major depression in families linked to chromosome 12 shared a common gain-of-function phenotype in both assay systems. In contrast, other Apaf-1 variants showed neutral or loss-of-function phenotypes. The depression-associated alleles thus have a common phenotype that is distinct from that of non-associated variants. This result suggests an etiologic role for enhanced apoptosis in major depression.

Secreted form of amyloid beta/A4 protein precursor (APP) binds to two distinct APP binding sites on rat B103 neuron-like cells through two different domains, but only one site is involved in neuritotropic activity.

Recent studies have identified the Alzheimer's disease amyloid beta/A4 protein precursor (APP) as a trophic and/or tropic protein on several types of cells, including fibroblasts, primary culture neurons, PC12 cells, and B103 neuron-like cells. Many trophic proteins bind heparin, and it is believed that the heparin-binding domain is crucial for the trophic activity of these proteins. APP also binds heparin. The current studies were undertaken to examine the hypothesis that the neuritotropic activity of APP requires heparin binding. It was found that APP produced in E. coli bound B103 cells through detergent-extractable molecules. Approximately 50% of the binding sites were heparinase-sensitive, and heparin and heparan sulfate competed for APP binding to these sites. The heparinase-insensitive sites were recognized by a stretch of 17 amino acids of APP (residues 319-335) that contains the neuritotropic activity of APP. A mutant APP with a deletion at this site was capable of binding to the heparinase-sensitive sites, although this molecule was not neuritotropic to B103 neuron-like cells. Therefore, the neuritotropic site and the heparin-binding site are distinct in APP, and the neuritotropic effect of APP is produced through its binding to detergent-extractable and heparinase-insensitive sites.

The stability of bacteriophage T4 gene 32 mRNA: a 5' leader sequence that can stabilize mRNA transcripts.

In T4-infected cells, the gene 32 monocistronic mRNA is very stable. To study the molecular basis for this stability, we have constructed chimeric plasmids containing the monocistronic promoter and the gene 32 translation initiation sequence fused to either most of the E. coli lac operon or only a segment of the lacZ gene, followed by the gene 32 transcription terminator. The resulting hybrid transcripts are unstable in uninfected cells. In phage-infected cells, however, the hybrid mRNAs are at least as stable as gene 32 mRNA itself. Analysis of other plasmid constructs indicates that the sequences on the gene 32 mRNA from its 5' end to slightly beyond the initiation codon suffice to stabilize these hybrids. Studies with a series of deletions of the gene 32 leader sequence suggest that an RNA sequence near the gene 32 initiation codon is involved. Various models to explain this mRNA stabilization are discussed.

Differential expression of alternative splice variants of beta-arrestin-1 and -2 in rat central nervous system and peripheral tissues.

Members of arrestin/beta-arrestin protein family are thought to participate in agonist-mediated desensitization of G-protein-coupled receptors, including rhodopsin and beta2-adrenergic receptor. Unlike in human and cow, splice variants of this protein family in rat have not been studied extensively, and there has been no report on their existence at protein level. Hence, a previous report by others on the localization of both beta-arrestin-1 and -2 in a wide range of innervated rat tissues could imply their broad receptor specificity. In this report we show the presence of two alternatively spliced forms of beta-arrestin-1 in several rat tissues using both reverse transcription-polymerase chain reaction and Western immunoblot. Splicing of beta-arrestin-1 pre-mRNA appears to be subject to differential regulation between the rat CNS and peripheral tissues. In contrast, we detected no splice variants of beta-arrestin-2 in rat. A comparison of the genomic DNA sequences of bovine and rat beta-arrestin-2, where the splicing of bovine beta-arrestin-2 mRNA has been reported, revealed a high degree of homology in their organization of exons and introns as well as certain differences that might be responsible for the different processing of beta-arrestin-2 mRNA in the two species. Our two-dimensional isoelectric focusing gels using rat spinal cord and heart tissues demonstrate isoelectric heterogeneity of rat beta-arrestin-1, suggesting that beta-arrestin-1 is subject to post-translational modification unlike beta-arrestin-2.

Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension.

Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.

Defective neurite extension is caused by a mutation in amyloid beta/A4 (A beta) protein precursor found in familial Alzheimer's disease.

Clonal central nervous system neuronal cells, B103, do not synthesize detectable endogenous APP or APLP. B103 cells transfected with both wild-type (B103/APP) and mutant APP construct (B103/APP delta NL) secreted comparable amounts of soluble forms of APP (sAPP). B103/APP cells produced sAPP and cleaved at amyloid beta/A4 (A beta) 16, the alpha-secretase site, and B103/APP delta NL cells produced sAPP beta cleaved at A beta 1, the beta-secretase site. B103/APP delta NL cells developed fewer neurites than B103/APP cells in a serum-free defined medium. Neurite numbers of parent B103 cells were increased by the 50% conditioned medium (CM) from B103/APP cells but reduced by the CM from B103/APP delta NL cells. Chemically synthesized A beta at concentration levels higher than 1 nM reduced numbers of neurites from B103 or B103/APP delta NL cells. However, A beta at 1-100 nM could not reduce the neurite number of B103/APP cells. The protective activity against A beta's deleterious effect to reduce neurite numbers was attributed to sAPP alpha in the CM. Although sAPP alpha could block the effect of A beta, sAPP beta could not do so under the identical condition, suggesting the importance of the C-terminal 15-amino acid sequence in sAPP alpha. Nevertheless, sAPP alpha's protective activity required the N-terminal sequence around RERMS, previously identified to be the active domain of sAPP beta. The overall effect of APP mutation which overproduced A beta and sAPP beta and underproduced sAPP alpha was a marked decline in the neurotrophic effect of APP. We suggest that the disruption of balance between the detrimental effect of A beta and the trophic effect of sAPP may be important in the pathogenesis of AD caused by this pathogenic APP mutation.

Secreted form of amyloid beta protein precursor is involved in the growth regulation of fibroblasts.

Fibroblasts that harbor an antisense construct of amyloid beta protein precursor (ABPP) cDNA, A-1, produced less ABPP mRNA and ABPP and grew poorly. Normal growth was restored when either parent cell conditioned medium (CM) or purified ABPP was provided. The capacity of the CM to restore cell growth was abolished by passage through an anti-ABPP immunoaffinity column; the activity was in the bound fraction. A Mr 90,000 protein recognized by the anti-ABPP antibody was diminished in the CM of A-1. CM from ABPP cDNA-transfected cells expressing high levels of ABPP was more potent than that from non-transfected parent cells in restoring A-1 growth. These results indicate that ABPP is released from cells into the medium and has an autocrine function in growth regulation.

The survival of rat cerebral cortical neurons in the presence of trophic APP peptides.

One function of Alzheimer amyloid protein precursor (APP) is the regulation of growth and differentiation in several types of cells, including fibroblasts, PC12 cells, and neurons. This activity is represented by a small stretch of amino acids in the center of the molecule around RERMS. The APP 17-mer peptide containing the RERMS domain supported survival and neurite extension of rat cortical neurons in a dose-dependent and sequence-specific manner. The APP fragment synthesized in Escherichia coli supported the survival and neurite extension of rat cortical neurons, whereas the mutant APP fragment lacking the 30 amino acids around the RERMS domain had drastically reduced activity to support the survival and neurite extension. The current study established APP as a neuron survival factor and determined that the sequence around RERMS is important for this function.

Increase of synaptic density and memory retention by a peptide representing the trophic domain of the amyloid beta/A4 protein precursor.

The secreted form (sAPP) of the Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in the in vitro regulation of fibroblast growth and neurite extension from neuronal cells. The active site of sAPP responsible for these functions is within a small domain just C-terminal to the Kunitz-type protease inhibitor (KPI) insertion site. We report here that a 17-mer peptide, containing this active domain of sAPP, can induce cellular and behavioral changes when infused into rat brains. After 2 weeks of APP 17-mer peptide infusion, the animals were tested for reversal learning and memory retention and were sacrificed for morphological examination of brains. We found that administration of the APP 17-mer peptide resulted in an 18% increase in the number of presynaptic terminals in the frontoparietal cortex. At the behavioral level, 17-mer-infused animals with nonimpaired learning capability showed an increased memory retention that seemed to interfere with reversal learning performance. This APP 17-mer effect on memory retention was not observed in animals with impaired initial learning capacity. These results suggest that APP is involved in memory retention through its effect on synaptic structure.

Bacterial expression, purification, and functional mapping of the amyloid beta/A4 protein precursor.

The secreted form of Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in cell growth regulation (Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T., and Schenk, D.B. (1989) Cell 58, 615-622). Using a strong prokaryotic expression system, we expressed, in Escherichia coli, peptide fragments covering different regions of the secreted form of APP-695. The longest of these fragments (KB75, 572 amino acids from Val-20 to Ile-591), which contained neither the Kunitz-type protease inhibitor (KPI) domain nor the amyloid beta/A4-protein domain, was purified and shown to be biologically active in terms of growth regulation. Two other APP fragments (KB48, 316 amino acids from Val-20 to Met-335; and RB17, 150 amino acids from Thr-296 to Pro-445), overlapping by only 40 amino acids at a close site C-terminal to the KPI insertion site, were also active. Furthermore, a chemically synthesized 40-residue peptide corresponding to this region of overlap also stimulated the growth of A-1 fibroblasts. These results establish the presence of growth-promoting activity in the secreted form of APP-695 and suggest that the site of this activity of APP-695 lies within a 40-amino acid domain next to the KPI insertion site.