Artifactual strain-specific signs of incipient brain amyloidosis in APP transgenic mice.

In an attempt to generate transgenic mice modeling Alzheimer-type amyloidogenesis, the COOH-terminal 103 residue human APP segment was expressed in brain regions known to be vulnerable in AD. Transfected cells overexpressing this transgene were previously shown to develop intracytoplasmic inclusions that were immunoreactive with antibodies to the APP COOH-terminus. Transgenic C57B6/SJL mice produced transgene-coded mRNA in their brains at levels up to sixfold above endogenous APP, most abundantly within cortical and hippocampal pyramidal neurons. Immunocytochemistry with anti-A beta antibodies revealed occasional structures that resembled diffuse amyloid, but which could not be detected on serial sections. Immunolabeling with antibodies to APP regions NH2-terminal to the transgene-coded domain revealed elevated immunoreactivity within perikarya and neurites in regions expressing the highest transgene and endogenous APP mRNA levels, similar to observations previously reported within vulnerable neurons in AD brain. However, subsequent breeding revealed that this phenotype segregated with the B6/SJL background rather than the transgene, thus emphasizing the importance of genetic background to observations of putative AD-type pathology in transgenic animals.

An interaction between basement membrane and Alzheimer amyloid precursor proteins suggests a role in the pathogenesis of Alzheimer's disease.

BACKGROUND: Extracellular matrix proteins (ECMPs) of the basement membrane type, such as the heparan sulfate proteoglycan perlecan, laminin, entactin, collagen IV, and fibronectin are present in and have been implicated in the genesis of amyloids. As in many forms of amyloid, perlecan, laminin, collagen IV, and fibronectin are present in Alzheimer deposits. We have previously demonstrated high-affinity interactions between Alzheimer amyloid precursor proteins (beta PP-695, -751, and -770), and perlecan or laminin. With a view to examining our hypothesis that beta PP:ECMP interactions are involved in Alzheimer's amyloidogenesis, additional studies have now been performed examining the interactions of the beta PPs with entactin, fibronectin, and collagen IV, the influence each of the ECMPs has on the binding of the others to beta PPs, and the effect of beta PPs on interactions among the various ECMPs. EXPERIMENTAL DESIGN: A modified solid-phase enzyme-linked immunosorbent assay was used to assess the binding of the various ECMPs to the beta PPs. One element was immobilized on plastic, and another element, operationally defined as a ligand, was incubated in solution at various concentrations over the immobilized protein. To evaluate the effect of one ECMP on the binding of other ECMPs to beta PP, the beta PP was immobilized and the binding of the "ligand" ECMP was assessed in the presence of a single concentration of a second "competitor" ECMP. Similarly, in evaluating the effect of beta PPs on the binding of ECMPs to each other, one ECMP was immobilized and the binding of a second ECMP "ligand" was assessed in the presence of a fixed concentration of beta PP "competitor." RESULTS: As in the case of perlecan and laminin, each of the ECMPs bound to the beta PPs with high affinity (Kd values in the nanomolar range). The binding of entactin to beta PPs was stimulated by collagen IV but was markedly inhibited by laminin, perlecan, and fibronectin. Conversely, the presence of entactin inhibited the binding of perlecan, laminin, and fibronectin to beta PPs. Moreover, the presence of beta PPs usually interfered with the binding of ECMPs to each other. Generally, in all binding assays, beta PP-751 and -770, behaved in similar ways, but beta PP-695, the brain-specific form, exhibited unique characteristics. CONCLUSIONS: These binding data may reflect the normal interactions of beta PPs with ECMPs. However, the fact that beta PPs interfere with the normal interactions between ECMPs themselves, a process that spontaneously generates a basement membrane, suggests that aspects of ECMP:beta PP binding may be a pathologic part of the amyloidogenic process in Alzheimer's disease.

Acceleration of GABA-dependent desensitization by mutating threonine 266 to alanine of the alpha 6 subunit of rat GABAA receptors.

Various GABAA receptor subunits share four highly homologous putative transmembrane domains (M1 to M4) and have been proposed to form an ion channel of pentameric structure with M2 lining the pore. The carboxyl terminal side of M2 contains three amino acid residues containing a hydroxyl group, which are Thr 265, 266 and serine 268 in the alpha 6 subunit. In order to study their functional role, we generated mutants of the alpha 6 subunit carrying a single point mutation of threonine 265 or 266 to alanine, or serine 268 to glycine. Co-expression of the mutants with beta 2 and gamma 2 subunits in human embryonic kidney cells produced functional receptors which are similar to the wild type in their sensitivity to a benzodiazepine agonist (U-92330), insensitivity to Zn, anion permeability, and GABA dose-response profiles as monitored with the whole cell patch clamp technique. Only in the alpha 6T266A beta 2 gamma 2 subtype, however, GABA-induced Cl- currents decayed much more rapidly than the wild type (about 10 times faster). Analysis of the GABA dependency of desensitization indicates that the T266A mutation enhanced the desensitization rate with little effect on the recovery rate from desensitization or on the half-maximal GABA concentration. We conclude that threonine 266 in the alpha 6 subunit plays a pivotal role in desensitization processes of GABAA receptors.

The Alzheimer beta-amyloid protein precursor/protease nexin-II is cleaved by secretase in a trans-Golgi secretory compartment in human neuroglioma cells.

Alzheimer beta-amyloid protein precursor (beta APP) is expressed endogenously and abundantly by human neuroglioma (H4) cells. Its secretory processing has been shown to involve discrete proteolysis within the beta A4 region, thus preventing beta-amyloid formation, by an enzyme which has been referred to as 'beta APP secretase'. This cleavage results in secretion of a soluble N-terminal 135 kDa protein and retention of an integral membrane C-terminal fragment within the cell. The membrane-associated C-terminal fragment is sorted to lysosomes where it undergoes limited degradation. We show here that most newly synthesized beta APP is degraded via a non-lysosomal pathway before maturation in H4 cells, and most mature beta APP is processed predominantly by the so-called secretase. The rapid kinetics of appearance/disappearance of a cleaved 135 kDa protein within a microsomal fraction and the slow accumulation of this form in the extracellular medium indicated that secretase cleaves beta APP in an intracellular compartment. Low-temperature block (20 degrees C) was used to demonstrate that beta APP is cleaved within a late Golgi compartment after sulphation which occurs in the trans-Golgi network (TGN). This is consistent with (1) the immunolocalization of most of the beta APP within a Golgi compartment that reacts with wheat germ agglutinin, (2) the fact that less than 1.5% of the total mature full-length beta APP is present at the plasma membrane and (3) subcellular fractionation studies which showed that the mature full-length and intracellular cleaved beta APPs co-sediment with a membrane fraction that is slightly more dense than the plasma membrane. This study provides evidence that most of the beta APP secretase in H4 cells is intracellular, and confirms that the resulting C-terminal fragment is delivered to lysosomes immediately after cleavage. These results are discussed with regard to the possibility that mature full-length beta APP escapes secretase cleavage and is delivered directly from the TGN to the lysosome without passing through the plasma membrane. Either pathway will result in the generation of amyloidogenic fragments.

Expression of rat renal gamma-glutamyltranspeptidase in LLC-PK1 cells as a model for apical targeting.

In the rat, gamma-glutamyltranspeptidase (gamma GT) is transcribed into four unique mRNAs from a single gene by use of at least three different promoters and alternative splicing. For the first time, two distinct full-length cDNAs encoding the protein for rat renal gamma-glutamyltranspeptidase have now been isolated. Characterization by restriction enzyme mapping and nucleotide sequencing indicates that the two cDNAs, corresponding to transcripts I and II, differ only in the 5' noncoding region. However, transcription from promoter I, most proximal to the coding sequence, apparently began 20 bases upstream from the major transcription start previously reported. Since in vitro transcription and translation of these two new gamma GT cDNAs were found to produce a full-length peptide (M(r) approximately 62,000), both cDNAs were used to transfect LLC-PK1 (porcine) cells, a polarized cell line most representative of the renal proximal tubule. Rat gamma GT was expressed in transfected cells as judged by immunofluorescence analysis, direct immunoprecipitation after metabolic labeling with [35S]methionine, and an increase in gamma GT specific enzymatic activity (up to 5-fold). When clonal cell lines (I or II) were grown on Falcon filter inserts, the increased gamma GT activity was found only at the apical surface, consistent with polarized expression of the rat gamma GT. In contrast, transfection of the same cells with cDNA of human growth hormone resulted in both apical (70%) and basal lateral (30%) secretion of the expressed hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of high affinity binding between laminin and Alzheimer's disease amyloid precursor proteins.

BACKGROUND: In vivo amyloid formation apparently involves several extracellular matrix components that are usually found associated with basement membranes. These include laminin, heparan sulfate proteoglycan, collagen type IV, and entactin. These components have also been found in neuritic plaques. We have therefore been examining interactions between extracellular matrix components and the Alzheimer's amyloid precursors (AAPs). EXPERIMENTAL DESIGN: Binding interactions of laminin with AAP-695, -751, and -770 were examined using a solid phase enzyme-linked immunosorbent assay technique. RESULTS: Objective, quantitative analyses of the laminin AAP-695, -751, and -770 binding data reveal two binding sites for laminin, with Kd values of 1 x 10(-10) M and 1 x 10(-8) M. Zinc and dithiothreitol profoundly stimulate laminin binding to AAPs. Furthermore, zinc fingers were found in the laminin amino acid sequences. Previous binding studies of AAPs with the basement membrane heparan sulfate proteoglycan revealed similar affinities. A particular order of addition of laminin and heparan sulfate proteoglycan to AAPs can be demonstrated. CONCLUSIONS: These avid interactions with extracellular matrix proteins likely reflect normal functions of the AAPs and may be involved in nucleation events in Alzheimer-type amyloid formation.

Efficacy and safety of cetirizine therapy in perennial allergic rhinitis.

A double-blind, placebo-controlled trial was undertaken to assess the safety and efficacy of once daily cetirizine in alleviating the symptoms of perennial allergic rhinitis. Subjects were adults with perennial allergic rhinitis, characterized by nasal congestion, postnasal discharge, sneezing, rhinorrhea, nasal itching, lacrimation, ocular itching, and itching of the roof of the mouth, and a total pretreatment symptom severity score of greater than or equal to 8. Patients were randomized to treatment with 10 mg cetirizine, 20 mg cetirizine, or placebo for 4 weeks. Efficacy was assessed in 215 patients and safety in 216. Cetirizine in once daily dosages of 10 or 20 mg proved to be effective in relieving the overall symptoms of perennial allergic rhinitis and particularly postnasal discharge and sneezing. The 10-mg dose afforded optimal symptomatic relief, and the 20-mg dose provided little or no additional benefit. Cetirizine was well tolerated, and the frequency of somnolence was not significantly greater in patients receiving this drug than in those given placebo.

Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties.

The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms. Insect cells infected with each of these baculoviruses produced both secreted and cell-associated AAPs. Full-length constructs produced secreted derivatives which were COOH-terminally cleaved within the beta-protein domain at Gln15 or Lys16, essentially identical to previous reports utilizing mammalian cell systems. Rare secreted forms (less than 5%) appeared to extend to Lys28. Secretion constructs produced these same forms, but in different ratios. Most (approximately 60%) terminated at Gln15 or Lys16, while the remainder apparently extended to Lys28. AAPs containing the Kunitz-type serine protease inhibitory domain (AAP-751 and -770) were shown to be active inhibitors. No differences were observed in the inhibitors activities of these two forms. The similarities in AAP processing by insect and mammalian systems, together with the large amounts of recombinant protein produced by baculovirus expression, make this an attractive system for studies of AAP processing and biochemical properties.

The identification of two subcellular sites for cleavage of gamma-glutamyltranspeptidase propeptide.

In order to determine the subcellular site(s) of rat renal gamma-glutamyltranspeptidase propeptide cleavage labeled immunoprecipitates were obtained from preparations of either intracellular membranes or brush border membrane vesicles. Heterodimer accounts for 25% of the label associated with transpeptidase in intracellular membranes from 5 to 40 min postinjection of [35S]methionine, consistent with a cotranslational cleavage of propeptide in the endoplasmic reticulum. Labeled propeptide and heterodimer appear in the brush border membrane fraction between 20-30 min postinjection and accumulate for 1 h and 4h, respectively. Subsequently, the propeptide disappears with a half-life of 1 h while the heterodimer is relatively stable. These results confirm our previous proposal for two distinct subcellular sites for transpeptidase propeptide cleavage (Capraro, M.A. and Hughey, R.P. (1983) FEBS Lett. 157, 139-143).

Evidence for stable homodimers and heterodimers of gamma-glutamyltranspeptidase subunits under protein-denaturing conditions.

gamma-Glutamyltranspeptidase is synthesized as a core glycosylated propeptide (Mr 75,000) which is subsequently cleaved to yield a stable heterodimeric structure (subunit Mr 50,000 and 30,000). The propeptide represents an insignificant mass of the transpeptidase but higher molecular weight bands designated H1 (Mr 85,000) and H2 (Mr 100,000) are readily observed by protein staining or immunoblot analysis of the enzyme or crude membranes after SDS-polyacrylamide gel electrophoresis. Although H1 and H2 represent the predominant antigenic forms of transpeptidase in tissues which exhibit relatively low specific enzyme activity, neither their structure nor their physiological function is known. In order to determine the relationship between H1 and H2, and the large (L) and small (S) subunits of the transpeptidase, individual bands (H1, H2, L and S) of the purified renal enzyme were cut from a Coomassie-stained SDS gel, eluted and re-electrophoresed. Isolated S produced S and dimers of S (Mr 60,000), while isolated L produced L and dimers of L corresponding to H2. Equivalent mixtures of L and S also produced H1. Utilizing IgG affinity-purified against either L or S, immunoblot analysis confirmed that H2 is a dimer of L, and H1 is a heterodimer of L and S. However, monoclonal IgG which recognizes both transpeptidase propeptide and native heterodimer did not react with H1. Thus, it is clear that isolated L and S can form and maintain unique dimeric structures during SDS-polyacrylamide gel electrophoresis. With this information it should now be possible to ascertain the basis for the apparent predominance of H1 and H2 in non-renal tissues.