Steven G. Younkin

Amyloid β Protein (Aβ) in Alzheimeri's Disease Brain BIOCHEMICAL AND IMMUNOCYTOCHEMICAL ANALYSIS WITH ANTIBODIES SPECIFIC FOR FORMS ENDING AT Aβ40 OR Aβ42(43)

Biochemical and immunocytochemical analyses were performed to evaluate the composition of the amyloid β protein (Aβ) deposited in the brains of patients with Alzheimers disease (AD). To quantitate all Aβs present, cerebral cortex was homogenized in 70% formic acid, and the supernatant was analyzed by sandwich enzyme-linked immunoabsorbent assays specific for various forms of Aβ. In 9 of 27 AD brains examined, there was minimal congophilic angiopathy and virtually all Aβ (96%) ended at Aβ42(43). The other 18 AD brains contained increasing amounts of Aβ ending at Aβ40. From this set, 6 brains with substantial congophilic angiopathy were separately analyzed. In these brains, the amount of Aβ ending at Aβ42(43) was much the same as in brains with minimal congophilic angiopathy, but a large amount of Aβ ending at Aβ40 (76% of total Aβ) was also present. Immunocytochemical analysis with monoclonal antibodies selective for Aβs ending at Aβ42(43) or Aβ40 confirmed that, in brains with minimal congophilic angiopathy, virtually all Aβ is Aβ ending at Aβ42(43) and showed that this Aβ is deposited in senile plaques of all types. In the remaining AD brains, Aβ42(43) was deposited in a similar fashion in plaques, but, in addition, widely varying amounts of Aβ ending at Aβ40 were deposited, primarily in blood vessel walls, where some Aβ ending at Aβ42(43) was also present. These observations indicate that Aβs ending at Aβ42(43), which are a minor component of the Aβ in human cerebrospinal fluid and plasma, are critically important in AD where they deposit selectively in plaques of all kinds.

Expression of β amyloid protein precursor mRNAs: Recognition of a novel alternatively spliced form and quantitation in alzheimer's disease using PCR

We have analyzed alternatively spliced beta amyloid protein precursor (beta APP) mRNAs by using the polymerase chain reaction to amplify beta APP cDNAs produced by reverse transcription. With this approach the three previously characterized beta APP mRNAs (beta APP695, beta APP751, and beta APP770) are readily detected and compared in RNA samples extracted from specimens as small as a single cryostat section. We show that the results obtained with this method are not affected by partial RNA degradation and use it to identify a novel alternatively spliced beta APP714 mRNA that is present at low abundance in each of the many human brain regions, peripheral tissues, and cell lines that we have examined; demonstrate that nonneuronal cells in the adult human brain and meninges produce appreciable beta APP695, beta APP751, and beta APP770 mRNA; and identify changes in beta APP gene expression in the AD brain and meninges that may contribute to amyloid deposition.

Amyloids and Are Generated Intracellularly in Cultured Human Neurons and Their Secretion Increases with Maturation

Previous studies have demonstrated the presence of amyloid β (Aβ) in neurons (NT2N) derived from a human embryonal carcinoma cell line (NT2) by steady state metabolic radiolabeling and immunoprecipitation. We show here that Aβ is present intracellularly since trypsin digestion of intact NT2N cells at 4°C did not eliminate the Aβ recovered in cell lysates. To determine whether both Aβ and Aβ are produced intracellularly, quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed using COOH-terminal end-specific anti-Aβ monoclonal antibodies. Sandwich ELISA detected intracellular Aβ and Aβ in NT2N cell lysates at a ratio of 3:1, whereas secreted Aβ and Aβ were recovered in medium conditioned by NT2N cells at a ratio of approximately 20:1. Metabolic steady state and pulse-chase labeling studies demonstrated a 2-h delay in the detection of cell-associated Aβ/Aβ in the medium, suggesting that Aβ is generated at a slow rate intracellularly prior to its secretion. Finally, as NT2N cells mature over time in culture, the secretion of Aβ and Aβ increases more than 5-fold over 7 weeks. This increase in the secretion of Aβ/Aβ in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain.

Amyloid β protein (Aβ) removal by neuroglial cells in culture

Abstract Because the mechanisms of Aβ degradation in normal and Alzheimers disease brain are poorly understood, we have examined whether various cortical cells are capable of processing this peptide. Rat microglia and astrocytes, as well as the human THP-1 monocyte cell line, degraded A β 1−42 added to culture medium. In contrast, neither rat cortical neurons or meningeal fibroblasts effectively catabolized this peptide. When Aβ fibrils were immobilized as plaque-like deposits on culture dishes, both microglia and THP-1 cells removed the peptide. Astrocytes were incapable of processing the Aβ deposits, but these cells released glycosaminoglycase-sensitive molecules that inhibited the subsequent removal of Aβ by microglia. This implied that astrocyte-derived proteoglycans associated with the amyloid peptide and slowed its degradation. The addition of purified proteoglycan to Aβ that was in medium or focally deposited also resulted in significant inhibition of peptide removal by microglia. These data suggest that Aβ can be catabolized by microglia and proteoglycans which co-localize with senile plaques may slow the degradation of Aβ within these pathologic bodies.

Secretory processing of the Alzheimer amyloid βA4 protein precursor is increased by protein phosphorylation

The 39-43 residue polypeptide (amyloid beta protein, beta A4) deposited as amyloid in Alzheimers disease (AD) is derived from a set of 695-770 residue precursors referred to as the amyloid beta A4 protein precursor (beta APP). In each of the 695, 751, and 770 residue precursors, the 43 residue beta A4 is an internal peptide that begins 99 residues from the COOH-terminus of the beta APP. Each holoform is normally cleaved within the beta A4 to produce a large secreted derivative as well as a small membrane associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire beta A4 peptide. In this study, we employ cells stably transfected with full length beta APP695, beta APP751, or beta APP770 expression constructs to show that phorbol ester activation of protein kinase C substantially increases the production of secreted forms from each isoform. By increasing processing of beta APP in the secretory pathway, PKC phosphorylation may help to prevent amyloid deposition.

Soluble derivatives of the β amyloid protein precursor of Alzheimer's disease are labeled by antisera to the β amyloid protein

Abstract The amyloid deposited in Alzheimers disease (AD) is composed primarily of a 39–42 residue polypeptide (βAP) that is derived from a larger β amyloid protein precursor (βAPP). In previous studies, we and others identified full-length, membrane-associated forms of the βAPP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble ∼125 and ∼105 kDa forms of the βAPP found in human cerebrospinal fluid are specifically labeled by several different antisera to the βAP. This finding indicates that both soluble derivatives contain all or part of the βAP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.

Accumulation of β-Amyloid fibrils in pancreas of transgenic mice

Some forms of familial Alzheimers disease are caused by mutations in the amyloid beta protein precursor (beta APP), and there is excellent evidence that these mutations foster amyloid deposition by increasing secretion of total amyloid beta protein (A beta) or the highly amyloidogenic A beta 1-42 form. These observations provide a powerful rationale for developing an animal model of AD by generating transgenic mice in which cerebral amyloid deposition is induced by A beta overproduction. To produce substantial A beta in vivo, we generated mice expressing the transgene of signal peptide and 99 residues of carboxyl-terminal fragment (CTF) of beta APP under control of the cytomegalovirus enhancer/chicken beta-actin promoter. The transgenic mRNA was detected in many tissues of these mice, but the levels of transgenic mRNA, CTF, and A beta did not correlate well indicating that tissue-specific posttranslational processing may play an important role in determining the amount of A beta that accumulates in various tissues. A beta was detected biochemically in brain, kidney, and pancreas with the largest amount present in pancreas. In transgenic plasma, there was a marked accumulation of human A beta 1-40 and A beta 1-42(43) to levels over 30-times those observed in normal human plasma. Thus, the transgenic mice produce and secrete considerable A beta. Despite this increase in A beta secretion and the elevated A beta in brain, immunohistochemistry revealed no consistent cerebral A beta deposition. In pancreas, however, intracellular A beta deposits were detected immunohistochemically in acinar cells and interstitial macrophages, some of which showed severe degeneration. In addition, examination of these cells by immunoelectron microscopy revealed many putative amyloid fibrils (7-12 nm) that were stained by anti-A beta antibodies. Overall, our findings indicate that tissue-specific posttranslational processing may play a pivotal role in A beta production and amyloid fibril formation in vivo. By carefully analyzing the changes that occur in the transgenic mice described here as compared to the transgenic line that has recently been shown to form extracellular amyloid plaques in brain, it may be possible to gain considerable insight into the factors that determine the location and amount of A beta that accumulates as amyloid.

Detection of soluble forms of the β-amyloid precursor protein in human plasma

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimers disease (AD), Downs syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.

Antisera to an amino-terminal peptide detect the amyloid protein precursor of alzheimer's disease and recognize senile plaques

The cerebral amyloid deposited in Alzheimers disease (AD) contains a 4.2 kDa beta amyloid polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). Three beta APP mRNAs encoding proteins of 695, 751, and 770 amino acids have previously been identified. In each of these, there is a single membrane-spanning domain close to the carboxyl-terminus of the beta APP, and the 42 amino acid beta AP sequence extends from within the membrane-spanning domain into the large extracellular region of the beta APP. We raised rabbit antisera to a peptide corresponding to amino acids 45-62 near the amino-terminus of the beta APP. We show that these antisera detect the beta APP by demonstrating that they (i) label a set of approximately 120 kDa membrane-associated proteins in human brain previously detected by antisera to the carboxyl-terminus of beta APP and (ii) label a set of approximately 120 kDa membrane-associated proteins that are selectively overexpressed in cells transfected with a full length beta APP expression construct. The beta APP45-62 antisera specifically stain senile plaques in AD brains. This finding, along with the previous demonstration that antisera to the carboxyl-terminus of the beta APP label senile plaques, indicates that both near amino-terminal and carboxyl-terminal domains of the beta APP are present in senile plaques and suggests that proteolytic processing of the full length beta APP molecule into insoluble amyloid fibrils occurs in a highly localized fashion at the sites of amyloid deposition in AD brains.

Co-expression of β-amyloid precursor protein (βAPP) and apolipoprotein E in cell culture: analysis of βAPP processing

Abstract Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimers disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Aβ), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Aβ production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Aβ, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of βAPP and the secretion of Aβ in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Aβ at concentrations like those in human cerebrospinal fluid. βAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on βAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of βAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Aβ aggregation or clearance under physiologically relevant conditions.