The structure and functions of the presenilins.

The presenilins (PSs) were new proteins discovered in 1995 to be involved, among other functions, in the molecular mechanisms leading to Alzheimer's disease. These proteins have been the subject of many investigations since then to elucidate their molecular structures and functions. Until now, the conclusions about PS structure have been discordant, but the 8-TM structure has been accepted by the Alzheimer's community, with the evidence for the 7-TM structure largely ignored. Here the evidence is reviewed for the 6-TM, 7-TM, 8-TM and other proposed models of PS topography and possibilities offered for the differences in interpretation of the various sets of data. The conclusion is that at this stage, the 7-TM model for cell surface PS is most likely the correct one.

Specific intercellular binding of the beta-amyloid precursor protein to the presenilins induces intercellular signaling: its significance for Alzheimer's disease.

Genetic evidence has implicated three proteins, the beta-amyloid precursor protein (beta-APP) and the two homologous presenilins (PS-1 and PS-2), in the etiology of Alzheimer's disease (AD). How these three proteins jointly contribute to AD, however, is not clear. Nor is any of their normal physiological functions known. Herein, we demonstrate, confirming a prediction made earlier, that beta-APP and either PS-1 or PS-2 act as a specific membrane-bound ligand binding intercellularly with either of its two membrane receptors. This results in a cell-cell adhesion, after which rapid transient increases in protein tyrosine kinase activity and protein tyrosine phosphorylation occur coordinately inside one or both of the two adherent cells. The spectrum of proteins modified by tyrosine phosphorylation differs depending on whether PS-1 or PS-2 is involved in the specific intercellular binding to beta-APP, which implies that PS-1 and PS-2 have distinct, rather than redundant, functions in normal physiology. The relevance of this intercellular interaction and signaling process to AD is discussed.

Cell surface expression of the Alzheimer disease-related presenilin proteins.

The presenilin proteins PS-1 and PS-2 are crucially involved in Alzheimer disease (AD), but their molecular functions are not known. They are integral membrane proteins, but whether they can be expressed at the surface of cells has been in dispute. Here we show by immunofluorescence experiments, using anti-peptide antibodies specific for either PS-1 or PS-2, that live cultured DAMI cells and differentiated human NT2N neuronal cells are specifically immunolabeled for their endogenous as well as transfected presenilins, although the cells cannot be immunolabeled for their intracellular tubulin, unless they are first fixed and permeabilized. These and other results establish that portions of the presenilins are indeed expressed at the surfaces of these cells. These findings support our previous proposal that the presenilins on the surface of a cell engage in intercellular interactions with the beta-amyloid precursor protein on the surface of a neighboring cell, as a critical step in the molecular and cellular mechanisms that lead to AD.

The seven-transmembrane spanning topography of the Alzheimer disease-related presenilin proteins in the plasma membranes of cultured cells.

To ascertain the membrane topography of the multi-transmembrane spanning presenilin proteins PS-1 and PS-2, anti-peptide antibodies were raised to several specific amino acid sequences in the two proteins, and, after their specificity was ascertained, the anti-peptide antibodies were used in immunofluorescent labeling of live PS-transfected, cultured DAMI cells, which are impermeable to the antibodies, as well as of their fixed and permeabilized counterparts. In such experiments, antibodies that specifically stain the intact live cells must label epitopes of the PS proteins that are on the exterior face of the plasma membrane whereas those antibodies that do not stain the live cells but do stain the fixed and permeabilized cells must label epitopes that face the cytoplasmic side of the membrane. The results obtained were entirely in accord with the predictions of the seven-transmembrane spanning topography (like that of rhodopsin and the beta-adrenergic receptor) and were totally inconsistent with the expectations for either the six- or eight-transmembrane topographies that have been proposed.

On the spurious endoproteolytic processing of the presenilin proteins in cultured cells and tissues.

It has been widely reported that the presenilin proteins PS-1 and PS-2 in extracts derived from a variety of cultured cells and from tissues are fragmented extensively by endoproteolytic processing events. It generally has been presumed that this endoproteolysis is a physiologically normal intracellular event following presenilin expression, which might play an important role in the still unknown functions of these molecules in connection with Alzheimer disease. We demonstrate herein, however, that, if a variety of cultured cells and several mouse tissues are examined under conditions minimizing cell trauma, the presenilin molecules in the extracts are found to be intact but that, if the cells and tissues are prepared under somewhat more stressful conditions, the endoproteolytic fragments are then observed. We conclude that these particular endoproteolytic events are not the result of physiologically normal processing of the presenilins but are rather artifacts occurring during the common procedures of specimen preparation.

Specific transcellular binding between membrane proteins crucial to Alzheimer disease.

Molecular genetic studies of families suffering from genetic forms of early onset Alzheimer disease (AD) have identified three genes and their protein products as being crucially involved in the etiology of AD. The three proteins are all integral membrane proteins. One of them is beta-APP, the precursor of the beta-amyloid found in the characteristic neuritic plaques present in the brains of AD patients. The other two, S182 and STM2, are homologous in amino acid sequence to one another but are unrelated to beta-APP. It is shown here, using cultured cells transfected for each of these proteins, that beta-APP binds specifically and transcellularly to either S182 or STM2. We propose that this transcellular binding may not only be important in normal neuronal physiology and development but may be directly involved in the process of formation of beta-amyloid from beta-APP.

Heat shock factor-1 mediates the transcriptional activation of Alzheimer's beta-amyloid precursor protein gene in response to stress.

Stress may be involved in the pathogenesis of Alzheimer's disease. There is a heat shock element located at position -317 bp on the beta-amyloid precursor protein (beta-APP) gene promoter. Recently we demonstrated [4] that stress, in the form of heat shock, ethanol and sodium arsenite treatment, transcriptionally activates the beta-APP gene. In this report we demonstrate that the nuclear factor that mediates this activation is heat shock factor-1.

Transcriptional activation of Alzheimer's beta-amyloid precursor protein gene by stress.

A neuropathological hallmark of Alzheimer's disease (AD) is the neuritic plaque, composed of an extracellular cluster of degenerating nerve terminals with a central core that is in part composed of deposits of a 4 kDa beta-amyloid peptide. Over-expression of the amyloid precursor protein (beta-APP) gene could be a contributing factor in the aberrant processing of the precursor protein, possibly leading to the formation of beta-amyloid. In AD the brain exhibits several features which indicate that neurons affected by AD exist under conditions of stress. Although the heat shock consensus sequence (CTCGACTTTTCTAG) located at position -317 bp is among the regulatory elements of the beta-APP gene, suggesting that this may act in the regulation of the beta-APP gene in response to stress, an induction of beta-APP as a result of interaction of this element with a heat shock factor has so far not been demonstrated. Moreover, there are conflicting reports in the literature regarding the up-regulation of beta-APP with stress. In this study we have used a fragment of the beta-APP promoter which includes the heat shock element, cloned into a luciferase expression vector pxP2 to transiently transfect cultured human NT2 and HeLa cells. Our findings directly demonstrate that transcription of the beta-APP gene is stimulated by various stresses--increase in temperature, treatment with ethanol and sodium arsenite. Gel mobility shift assays confirm the interaction of the heat shock element with a heat shock factor, induced as a result of stress.

A direct role for protein kinase C and the transcription factor Jun/AP-1 in the regulation of the Alzheimer's beta-amyloid precursor protein gene.

Overexpression of the beta-amyloid precursor protein gene (beta-APP) may contribute to the abnormal generation of beta-amyloid protein in Alzheimer's disease. We demonstrate using a human glial cell line (1321N1) that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) increases beta-APP mRNA levels, induces known components of the transcription factor activator protein-1 (AP-1), and increases protein-DNA binding activity to AP-1 sequences within the beta-APP promoter. A beta-APP promoter-luciferase reporter gene is transcriptionally activated by PMA, as well as by expression of constitutively activated PKC or by expression of c-Jun. Further characterization suggests that the distal but not the proximal AP-1 recognition site binds nuclear proteins regulated by PKC, and that the AP-1 binding activity is likely to be composed of Jun-Jun homodimers rather than Jun-Fos heterodimers. Additional studies demonstrate that a single copy of the distal AP-1 site fused to a heterologous promoter is sufficient to confer a response to PMA. Mutagenesis of this site in the beta-APP promoter renders it unresponsive to c-Jun and attenuates transcriptional activation by PMA. We suggest that cellular mediators that activate PKC, particularly those that induce significant increases in c-Jun, may up-regulate expression of the beta-APP gene and consequently affect production and processing of this protein.

Lysosomal storage of subunit c of mitochondrial ATP synthase in Batten's disease (ceroid-lipofuscinosis).

Immunochemical studies demonstrate that subunit c of mitochondrial ATP synthase is stored in the late-infantile, juvenile and adult forms of Batten's disease. It does not accumulate in the infantile form, or in other conditions involving lysosomal hypertrophy. These results suggest that the defective metabolism of subunit c is central to the pathogenesis of these three forms of Batten's disease.

Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.

The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4- to 5-kDa amyloid beta-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of beta-amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyclonal antibodies to a synthetic peptide of the beta-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. Regulated expression of beta-amyloid precursor protein may be an important determinant of growth and metabolic responses to serum and growth factors under physiological as well as pathological conditions.

Processing of Alzheimer's disease-associated beta-amyloid precursor protein.

Studies were undertaken on the processing of Alzheimer's disease-associated beta-amyloid precursor protein in normal cultured human fibroblasts and a human neuroblastoma cell line. Major differences in processing between the secreted and intracellular forms of the precursor were found. The intracellular form appears to undergo amino-terminal processing yielding many smaller fragments, whereas the secreted form does not show any further proteolytic cleavage after its release from the cell surface. In pulse-chase experiments, antibodies to the A4 region immunoprecipitated bands of Mr = 92,000-128,000, which represent the intact precursor; several smaller intracellular fragments of Mr = 70,000-72,000, 55,000, 33,000 and 6,000 also immunoprecipitated with this antibody. The Mr = 6,000 band cleared from the cell very quickly and is postulated to be the A4-carrying remnant of the secreted protein. The data show that a fragment of Mr = 33,000, which includes the A4 region, is one stable processed end-product of the intracellular precursor protein. It is possible that different posttranslational modifications are the signals responsible for the differences in processing between the secreted and intracellular amyloid precursor protein.

Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor.

Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a lambda gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of approximately 2 kilobases (lambda-S-1.2 and lambda-S-1.3) and both were both homologous with a previously isolated clone (lambda-S-1.1) for mature SAP-1. We report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is approximately 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide (23 amino acids) mature SAP-1 (78 amino acids) is generated by removing an additional 7 amino acids from the amino terminus and approximately 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which we designate as P-2. The molecular mass of glycosylated pro-SAP-1 is estimated at approximately 69 kDa, assuming glycosylation of all four sites. The value is close to the reported 70-kDa value for glycosylated pro-SAP-1. A computer search failed to reveal homology between P-2 and the sequence of any other protein; its function is uncertain. The 3' untranslated region is composed of 90 base pairs and is incomplete, since it does not contain a polyadenylylation site or a poly(A) tail.

Purification and characterization of beta-N-acetylhexosaminidase I2 from human liver.

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

Chromogenic immunodetection of human serum albumin and alpha-L-fucosidase clones in a human hepatoma cDNA expression library.

A human hepatoma cDNA library was constructed in lambda gt11, a bacteriophage vector that was designed to express cDNA-encoded antigenic determinants in Escherichia coli. The cDNA expression library contained approximately 8 X 10(6) recombinant phages with an average insert size of 780 bp. The feasibility of using a chromogenic immunodetection procedure to isolate cDNA clones was proved by isolating a human serum albumin (HSA) cDNA clone. An approximately 1.0-kb cDNA clone was then isolated by screening the library with rabbit anti-human alpha-L-fucosidase antibodies. The identity of the alpha-L-fucosidase cDNA clone was confirmed by DNA sequence analysis and a comparison of the predicted amino acid sequence to the amino acid sequences of human alpha-L-fucosidase tryptic peptides.