Chronic γ-secretase inhibition reduces amyloid plaque-associated instability of pre- and postsynaptic structures.

The loss of synapses is a strong histological correlate of the cognitive decline in Alzheimer's disease (AD). Amyloid ß-peptide (Aß), a cleavage product of the amyloid precursor protein (APP), exerts detrimental effects on synapses, a process thought to be causally related to the cognitive deficits in AD. Here, we used in vivo two-photon microscopy to characterize the dynamics of axonal boutons and dendritic spines in APP/Presenilin 1 (APP(swe)/PS1(L166P))-green fluorescent protein (GFP) transgenic mice. Time-lapse imaging over 4 weeks revealed a pronounced, concerted instability of pre- and postsynaptic structures within the vicinity of amyloid plaques. Treatment with a novel sulfonamide-type γ-secretase inhibitor (GSI) attenuated the formation and growth of new plaques and, most importantly, led to a normalization of the enhanced dynamics of synaptic structures close to plaques. GSI treatment did neither affect spines and boutons distant from plaques in amyloid precursor protein/presenilin 1-GFP (APPPS1-GFP) nor those in GFP-control mice, suggesting no obvious neuropathological side effects of the drug.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Cerebrospinal fluid levels of amyloid beta-protein in Alzheimer's disease: inverse correlation with severity of dementia and effect of apolipoprotein E genotype.

Alzheimer's disease (AD) is characterized by formation in brain of neurofibrillary tangles and of amyloid deposits. The major protein component of the former is tau, while the latter are composed of amyloid beta-peptides (A beta), which are derived by proteolytic cleavage of the amyloid beta-protein precursor (APP). Both tau and various secretory APP derivatives including A beta and APPS are present in human cerebrospinal fluid (CSF). To investigate whether clinical signs of AD are paralleled by changes in CSF levels of these proteins, we correlated quantitative measures of dementia severity with CSF concentrations of A beta, of APPS, and of tau. We found that levels of A beta in CSF of AD patients were inversely correlated both to cognitive and to functional measures of dementia severity. In contrast, levels of APPS and of tau did not correlate with dementia severity. Apolipoprotein E (apoE) genotype did not influence CSF levels of A beta, APPS, or tau, which were similar among AD patients with Apo E epsilon 3/3, epsilon 3/4, and epsilon 4/4 alleles. These data indicate that CSF levels of A beta decrease with advancing severity of dementia in AD and suggest that they are independent of a patient's Apo E genotype.

Labeling of cerebral amyloid in vivo with a monoclonal antibody.

We assessed the ability of a murine monoclonal antibody to bind selectively to beta-amyloid in the brains of living nonhuman primates. To circumvent the blood-brain barrier, we injected unlabeled antibody 10D5 (murine whole IgG1 and/or Fab fragments) into the cerebrospinal fluid of the cisterna magna in three aged monkeys. A control animal was given an intracisternal injection of nonimmune mouse whole IgG plus Fab. Twenty-four hours later, the animals were perfused and prepared for immunohistochemical detection of bound murine immunoglobulin in brain. All three experimental animals showed selective binding of 10D5 to approximately 5-15% of amyloid deposits in cerebral cortex, primarily near the cortical surface. There was no labeling in the control animal. In vivo-labeled deposits were confirmed to be beta-amyloid by electron microscopy and by in vitro immunohistochemistry in adjacent sections. The animals tolerated the injection well, although some polymorphonuclear leukocytes infiltrated portions of the subarachnoid space and superficial neocortex. These results provide the first demonstration that it may be feasible to selectively direct a tagged monoclonal antibody to beta-amyloid in the brain for therapeutic or diagnostic purposes. With enhancement of labeling efficiency, the method also may be useful for studying the progression of beta-amyloidosis in experimental animals using emission tomography.

Concentrations of amyloid-beta protein in cerebrospinal fluid increase with age in patients free from neurodegenerative disease.

Cerebral deposition of amyloid-beta protein (A beta) is central to the pathogenesis of Alzheimer's disease (AD). Increasing age is one of the few definitively established risk factors for this disease. The concentration of A beta was measured in cerebrospinal fluid (CSF) with a sensitive enzyme-linked immunosorbent assay in 18 adult neurological patients free from neurodegenerative disease. CSF A beta increased with age, yielding a significant correlation of 0.84. This observation suggests that increased levels of A beta in CSF may be an index of age-related changes in the processing of the amyloid-beta precursor protein resulting in an increased risk for AD.

Identification of secreted beta-amyloid precursor protein binding sites on intact human fibroblasts.

Based upon recent evidence that the secreted form of APP can cause the release of cytokines and elicit other biological activities, we sought to identify whether a receptor could be identified on the surface of cells. The secreted amyloid precursor protein containing the Kunitz domain (scAPP751) is identical to protease nexin II, a protease inhibitor which has been shown to form complexes with labeled EGF binding protein that subsequently binds to cells. Results of [125I]scAPP751-trypsin complex incubated with intact fibroblast cells show that the complex appears to bind in a saturable time-dependent and reversible manner. The kinetic constants from the binding studies demonstrate a k1 = 2.5 x 10(7) M-1 s-1 and k2 = 4.7 x 10(-4) s-1 and thus a KD (= k2/k1) = 20 pM. Furthermore, the complex formation of [125I]scAPP751 with a protease appears to be a requirement for optimal binding. The binding affinity of secreted APP demonstrated in this study is consistent with its potency in eliminating a range of biological efforts that have been documented.

Characterization of beta-amyloid peptide from human cerebrospinal fluid.

beta-Amyloid peptide (A beta) is one of the main components of senile plaques in the brain tissue of Alzheimer's disease (AD) patients. A beta is proteolytically cleaved from the amyloid precursor protein (APP), an integral membrane protein possessing a large extracellular N-terminal domain followed by a single membrane-spanning region and a short cytoplasmic C-terminal tail. A beta has been isolated from senile plaques and cerebral vascular tissue of AD brain and characterized as a heterogeneous peptide containing 28-43 amino acids whose sequence begins in the extracellular domain of APP and extends into the putative transmembrane sequence. It has long been speculated that A beta may also be present in body fluids, such as CSF, that contact neuritic plaques. Recently using a specific enzyme-linked immunosorbent assay we were able to quantify one form of A beta in CSF. In this report, using one of these antibodies covalently bound as an affinity matrix, multiple complex forms of A beta have been isolated and characterized from CSF derived from patients with either meningitis or other neurological disorders. Amino acid sequencing reveals A beta species with N-termini of Asp1, Glu3, His6, Glu11, and Val12, although on a molar basis, Asp1 represents the predominant aminoterminus. Laser desorption mass spectrometry confirmed the presence in CSF of A beta species containing 27, 28, 30, 34, 35, 40, 42, and 43 amino acids, all beginning at Asp1; two stable trimers, (Asp1-Met35)3 and (His6-Ala42)3; and one stable dimer containing (Asp1-Val40)2.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of baculovirus-derived secreted and full-length beta-amyloid precursor protein.

We have expressed two forms of the Alzheimer's beta-amyloid precursor protein (beta APP), the 695-amino acid form (695 beta APP), and the 751-amino acid form (751 beta APP) in a baculovirus system. Both forms were expressed as full-length precursor, and were subsequently processed in vivo to release extracellular secreted proteins. The secreted forms were cleaved from the full-length beta APP in a manner analogous to the cleavage of beta APP during constitutive secretion in mammalian cells (Weidemann, A., König, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., Beyreuther, K. (1989) Cell 57, 115-126; Oltersdorf, T., Ward, P. J., Henriksson, T., Beattie, E. C., Neve, R., Lieberburg, I., and Fritz, L. J. (1990) J. Biol. Chem. 265, 4492-4497). High levels of expression of 20-50 mg/liter were achieved. Both full-length and secreted forms of the beta-amyloid precursor proteins were purified using a combination of ion-exchange and immunoaffinity chromatography using a monoclonal antibody directed against beta APP. The 751 beta APP-derived full-length and secreted forms, which contain the Kunitz protease inhibitor domain, were shown to be as active in the inhibition of trypsin as is mammalian-derived secreted beta APP. The availability of purified full-length beta APP from the baculovirus system will be valuable for biochemical and cell biological analyses that may elucidate the mechanism of the inappropriate processing that leads to beta-amyloid formation in Alzheimer's disease.

The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II.

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.

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.

Characterization of the atrial natriuretic peptide clearance receptor using a vaccinia virus expression vector.

A recombinant vaccinia virus has been used to direct the expression of the atrial natriuretic peptide clearance receptor (ANP C-receptor) in mammalian cell lines normally deficient in this protein. The recombinant receptor binds 125I-ANP-(102-126) in a specific and saturable manner and carboxyl-terminal truncated and internal-deleted ANP analogs bind to this site with high affinity. Following the covalent attachment of 125I-ANP-(102-126) to the recombinant ANP C-receptor, the protein exhibits an electrophoretic mobility identical to that of the native ANP C-receptor of cultured vascular cells. Expression of the ANP C-receptor in heterologous cells does not affect ANP-stimulated cyclic GMP accumulation, confirming previous reports that this novel ANP receptor subpopulation is not coupled to cyclic GMP metabolism. Furthermore, specific antisera, generated by inoculating rabbits with living recombinant virus, block 125I-ANP binding to the ANP C-receptor but do not inhibit ANP stimulation of cyclic GMP, supporting the existence of two receptor subpopulations that are functionally and immunologically distinct.

Atrial natriuretic peptide clearance receptor. Complete sequence and functional expression of cDNA clones.

The major class of atrial natriuretic peptide (ANP) receptors was isolated from cultured vascular smooth muscle cells, and a partial amino acid sequence was obtained. This allowed the isolation of cDNA clones from which the entire amino acid sequence was established. The smooth muscle cell ANP receptor appears to be synthesized as a 537-amino acid precursor with an N-terminal membrane translocation signal. The mature form consists of 496 amino acids with a single potential transmembrane domain predicting a 37-amino acid cytoplasmic domain and a large, acidic, extracellular domain low in cysteine and probably containing attached carbohydrate. The receptor is therefore similar in structure to the growth factor receptors but notably lacks repetitive cysteine-rich domains and has a relatively small intracellular domain. Expression of the cloned receptor in Xenopus oocytes elicited high affinity, membrane-associated binding sites for ANP and for truncated and internally deleted analogs of ANP. These results reflect the ligand binding specificity found for the major class of ANP receptors on smooth muscle cells and thus provide additional evidence that two distinct ANP receptors exist since ANP receptor-coupled guanylate cyclase activity exhibits a very different ANP analog specificity.

Monoclonal antibodies to surfactant proteins SP28-36 label canine type II and nonciliated bronchiolar cells by immunofluorescence.

The major 28,000- to 36,000-dalton proteins of pulmonary surfactant (SP28-36) have been shown by various techniques to be synthetic and secretory products of alveolar type II cells. Surfactant lipids are also secreted by these cells. Immunocytochemical studies of human and rodent lungs have indicated that nonciliated epithelial cells of small bronchioles appear to contain SP28-36 in their synthetic organelles and secretory granules. Because these observations were obtained with polyclonal antibodies against SP28-36, it was possible that bronchiolar cell staining was due to contaminant antibodies not detected by biochemical analyses. To clarify the role of bronchiolar cells in the metabolism of SP28-36, we have prepared 5 monoclonal antibodies against canine SP28-36. Electrophoresis and immunoblots of surfactant showed that each antibody reacted with SP32 and 36, as well as SP28, the nonglycosylated species. This indicates that the antibodies are directed against the protein rather than carbohydrate moieties of SP28-36. Immunoblot analysis of collagenase-treated SP28-36 showed that the antibodies DS-3 and DS-1 were directed against the noncollagen region of the protein. Immunoblot analysis of whole canine lung homogenates showed that a single protein species was recognized by the antibodies. Immunofluorescence studies of cryostat sections of canine lung showed that both type II and nonciliated bronchiolar cells were specifically labeled with each antibody. These and previous data are consistent with and support the idea that bronchiolar cells synthesize and secrete SP28-36.

Purification and subunit composition of atrial natriuretic peptide receptor.

A receptor for atrial natriuretic peptide (ANP) was purified 2700-fold, to apparent homogeneity, from cultured bovine aortic smooth muscle cells by affinity chromatography. The native ANP receptor has a molecular weight of 125,000 as determined by both metrizamide gradient centrifugation and nonreducing NaDodSO4/polyacrylamide gel electrophoresis. With 125I-labeled ANP as ligand, the purified receptor bound a maximum of 5.70 nmol of ligand per mg of protein and the dissociation constant was 4.0 X 10(-10)M. Upon treatment with 10 mM dithiothreitol, the purified receptor migrated as a single band at Mr 60,500 in NaDodSO4/polyacrylamide gel electrophoresis. These findings show that the holoreceptor for ANP in vascular tissue is composed of two subunits of identical apparent molecular weight, presumably linked by a disulfide bridge(s).

Truncated atrial natriuretic peptide analogs. Comparison between receptor binding and stimulation of cyclic GMP accumulation in cultured vascular smooth muscle cells.

A series of truncated atrial natriuretic peptide analogs were examined as a means of defining the structural requirements for receptor occupancy and stimulation of cyclic GMP accumulation in bovine aortic smooth muscle cells. It was determined that deletion of amino acids from the carboxyl and/or amino termini of the peptides diminished their ability to increase cyclic GMP levels. Deletion of amino acids from the carboxyl terminus had the greatest effect, and atrial natriuretic peptide analogs lacking the carboxyl-terminal phenylalanyl-arginyl-tyrosine tripeptide were 100-1000-fold less active than parent compounds in stimulating intracellular cyclic GMP accumulation. In marked contrast to the cyclic GMP effects, deletion of amino- and/or carboxyl-terminal amino acids had only minor effects on the affinity of the peptides for specific smooth muscle cell-associated receptors. Peptide analogs lacking the phenylalanyl-arginyl-tyrosine tripeptide bound to receptors with an affinity only 1.1-5-fold weaker than the parent compounds. Thus, there was no correlation between apparent receptor binding affinity of atrial natriuretic peptide analogs and potency of these same peptides for stimulating intracellular cyclic GMP accumulation. Furthermore, analogs that bound to receptors and failed to elicit significant cyclic GMP responses did not antagonize or modulate increases in cyclic GMP induced by parent compounds. These data are most consistent with the existence of multiple subpopulations of atrial natriuretic peptide receptors on aortic smooth muscle cells.

Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping.

The catalytic alpha-subunit of rat hepatic (Na+, K+)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody ("9-A5") covalently linked to Sepharose 4B that specifically blocks phosphorylation of the sodium pump's alpha-subunit from [gamma-32P]ATP [Schenk, D. B., Hubert, J.J., & Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951]. The hepatic subunit is virtually identical with purified rat, dog, and human renal alpha-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Mr 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal beta-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic "beta"-subunits (Mr 50K and 55K) eluted from 9-A5-Sepharose. Additional studies of ouabain-sensitive 86Rb+ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points (ID50 = 0.1 mM ouabain) in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two known structurally conserved forms of catalytic subunit (the renallike alpha form) and, if at all, structurally divergent forms of the sodium pump's beta-subunit. In addition, immunoaffinity chromatography with 9-A5-Sepharose facilitates the isolation of (Na+, K+)-ATPases from nonrenal tissues with low levels of sodium pumps.

Identification of the receptor for atrial natriuretic factor on cultured vascular cells.

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.