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D J Selkoe

Publications and source records attributed to D J Selkoe.

At least 19 recordsLinked to original sources
PubMed1992

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

Fibril formation by primate, rodent, and Dutch-hemorrhagic analogues of Alzheimer amyloid beta-protein.

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5-->Gly, Tyr10-->Phe, and His13-->Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22-->Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3-->Arg, Arg5-->Glu, Asp7-->Val, His13-->Lys, Lys16-->His, Val18-->Asp, Phe19-->Ser, Phe20-->Tyr, Ser26-->Pro, Ala30-->Val, Ile31-->Ala, Met35-->norLeu, Gly38-->Ile, Val39-->Ala, and Val40-->Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation of fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. beta-Sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Increased expression of beta-amyloid precursor protein during neuronal differentiation is not accompanied by secretory cleavage.

Despite increasing evidence for a pathogenetic role for the beta-amyloid precursor protein (beta APP) in Alzheimer disease, the physiological function of the protein remains unclear. The expression of the neural-specific isoform containing 695 amino acids, beta APP695, is consistent with a role for the protein in neuronal development. In this study, we analyzed the expression of beta APP during the retinoic acid-induced neuronal differentiation of P19 murine embryonal carcinoma cells. Northern blot and RNase protection analyses show a selective increase in beta APP695 expression, concomitant with the morphologic differentiation of P19-derived neurons. Moreover, the time course of increase observed for the beta APP695 mRNA is paralleled by other neuronal-specific transcripts. A similar increase in beta APP695 is observed at the protein level. Furthermore, we show that levels of beta APP695 protein progressively increase during the in vitro differentiation of primary hippocampal neurons. The finding that beta APP695 increases selectively and progressively during neuronal differentiation in two different cell culture systems suggests that this isoform has an important cellular function during this process in the brain. Unlike beta APP in most peripheral cell types, the increased levels of beta APP found in terminally differentiated neuronal cells are not processed in significant amounts by secretory cleavage. Thus, differentiation of neurons is accompanied by increased beta APP695 expression and membrane retention of the protein as intact, full-length molecules that could serve as potential substrates for amyloidogenesis.

Amyloid beta-Protein Precursor↗
PubMed1992

Mass spectrometry of purified amyloid beta protein in Alzheimer's disease.

The amyloid beta-protein (A beta) that is progressively deposited in Alzheimer's disease (AD) arises from proteolysis of the integral membrane protein, beta-amyloid precursor protein (beta APP). Although A beta formation appears to play a seminal role in AD, only a few studies have examined the chemical structure of A beta purified from brain, and there are discrepancies among the findings. We describe a new method for the rapid extraction and purification of A beta that minimizes artifactual proteolysis. A beta purified by two-dimensional reverse-phase HPLC was analyzed by combined amino acid sequencing and mass spectrometry after digestion with a lysylendopeptidase. The major A beta peptide in the cerebral cortex of all five AD brains examined was aspartic acid 1 to valine 40. A minor species beginning at glutamic acid 3 but blocked by conversion to pyroglutamate was also found in all cases. A species ending at threonine 43 was detected, varying from approximately 5 to 25% of total A beta COOH-terminal fragments. Peptides ending with valine 39, isoleucine 41, or alanine 42 were not detected, except for one brain with a minor peptide ending at valine 39. Our findings suggest that A beta 1-40 is the major species of beta-protein in AD cerebral cortex. A beta 1-40 and A beta 1-43 peptides could arise independently from beta APP, or A beta 1-43 could be the initial excised fragment, followed by digestion to yield A beta 1-40. These analyses of native A beta in AD brain recommend the use of synthetic A beta 1-40 peptide to model amyloid fibrillogenesis and toxicity in vitro.

Alzheimer Disease↗
PubMed1992

Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments.

Progressive cerebral deposition of the amyloid beta-peptide is an early and invariant feature of Alzheimer's disease. The beta-peptide is released by proteolytic cleavages from the beta-amyloid precursor protein (beta APP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of beta APP involves a cleavage in the beta-peptide region, releasing the soluble extramembranous portion and retaining a 10K C-terminal fragment in the membrane. Because this secretory pathway precludes beta-amyloid formation, we searched for an alternative proteolytic processing pathway that can generate beta-peptide-bearing fragments from full-length beta APP. Incubation of living human endothelial cells with a beta APP antibody revealed reinternalization of mature beta APP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated beta APP was recovered inside the cells. Purification of lysosomes directly demonstrated the presence of mature beta APP and an extensive array of beta-peptide-containing proteolytic products. Our results define a second processing pathway for beta APP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels.

Altered proteolysis of the beta-amyloid precursor protein (beta APP) resulting in release of the approximately 40-residue amyloid beta-protein (A beta P) may be a seminal pathogenetic event in Alzheimer disease. Using region-specific beta APP antibodies, we searched for stable proteolytic intermediates containing the intact A beta P region in brain tissue. A 22-kDa beta APP fragment was selectively detected in microvessels purified from cerebral cortex and other brain regions. On immunoblots, the 22-kDa band is labeled by five distinct antisera to beta APP carboxyl-terminal peptides and by affinity-purified antibodies to the recombinant proteins beta APP444-592 and beta APP592-695, which flank the A beta P region. The protein is virtually undetectable in whole-brain homogenates or microvessel-free fractions of brain. The protein is extractable from microvessels in Triton X-100 and other detergents, indicating its membrane association. In comparison with cortical microvessels, microvessels purified from white matter, cerebellum, and nonneural tissues contain lower amounts of the 22-kDa protein. The protein is found in microvessels of both normal and Alzheimer disease brains and occurs in low amounts in microvessels from fresh bovine brain. The size and specific immunoreactivity of the 22-kDa protein indicate that it is a stable fragment of beta APP containing the intact A beta P. The occurrence of this potentially amyloidogenic intermediate in microvessels is consistent with a vascular or hematogenous origin for some A beta P deposits in Alzheimer disease.

Age Factors↗
PubMed1992

Processing of the amyloid protein precursor to potentially amyloidogenic derivatives.

The approximately 120-kilodalton amyloid beta protein precursor (beta APP) is processed into a complex set of 8- to 12-kilodalton carboxyl-terminal derivatives that includes potentially amyloidogenic forms with the approximately 4-kilodalton amyloid beta protein (beta AP) at or near their amino terminus. In order to determine if these derivatives are processed in a secretory pathway or by the endosomal-lysosomal system, (i) deletion mutants that produce the normal set of carboxyl-terminal derivatives and shortened secreted derivatives were analyzed and (ii) the effect of inhibitors of endosomal-lysosomal processing was examined. In the secretory pathway, cleavage of the beta APP occurs at a single site within the beta AP to generate one secreted derivative and one nonamyloidogenic carboxyl-terminal fragment, whereas, in the endosomal-lysosomal system, a complex set of carboxyl-terminal derivatives is produced that includes the potentially amyloidogenic forms.

Ammonium Chloride↗
PubMed1992

The seminal role of beta-amyloid in the pathogenesis of Alzheimer disease.

New molecular information about Alzheimer disease (AD) is appearing at an unprecedented rate. Much interest centers on the beta A4 amyloid protein, which is progressively deposited in senile plaques and blood vessels in AD brain tissue. The discovery that some kindreds with familial AD have a mutation in the gene coding for the beta A4 amyloid precursor protein (APP) suggests that this mutation alone may be sufficient to cause the full spectrum of clinical and pathological changes that characterize AD. Although APP point mutations may turn out to be relatively rare causes of AD, the idea that accelerated beta A4 deposition is an early and critical event in many patients continues to gain support from studies in humans, animals, and cultured cells. Identification of the biochemical steps leading to production of the beta A4 peptide from APP is now a critical issue. Recent reports indicate that normal lysosomal processing pathways can produce carboxyl-terminal fragments of APP that contain the entire beta A4 sequence, and are therefore potentially amyloidogenic. The mechanisms by which such intermediate forms are further processed and released, resulting in extracellular beta A4 deposits in plaques and vessels, are yet to be determined. It is likely that full elucidation of the beta A4-producing pathways will ultimately yield new therapeutic approaches to this complex and tragic disorder.

Alzheimer Disease↗
PubMed1992

Beta amyloid is focally deposited within the outer basement membrane in the amyloid angiopathy of Alzheimer's disease. An immunoelectron microscopic study.

The fine structure of cerebral amyloid angiopathy, especially in small and presumably early deposits, was examined by immunolabeling of the beta/A4 protein in semithin and ultrathin sections from brains with Alzheimer's disease. The following findings emerged: 1) in large leptomeningeal arteries, small, focal amyloid deposits appear to consist of clusters of delicate (approximately 8 nm diameter) amyloid fibrils, not previously described, in the outermost part of the basement membrane (BM) at the media-adventitia junction; 2) in small leptomeningeal arteries and perforating cortical arterioles, small foci of delicate amyloid fibrils were observed within the BM. They appeared mostly in the outer portion of the BM, around intact smooth muscle cells, rather than in the subendothelial region. In larger and presumably more advanced deposits, coarse amyloid fibrils (approximately 10 nm) occupied the abluminal BM, and adjacent smooth muscle cells showed degeneration; and 3) in capillaries, small amounts of delicate (approximately 8 nm) amyloid fibrils, not previously described, were seen within the BM in the smallest discernible deposits. The BM at these sites was abnormally folded and layered. In larger deposits, amyloid fibrils appeared to extravasate from the outer BM of the capillary into the neuropil and were surrounded by astrocytic foot processes and/or microglia. Our results suggest that vascular amyloid fibrils may first be formed within the abluminal vascular BM, that is, outside of cells. The BM may trap degradative intermediates of the amyloid precursor protein that contain the beta/A4 region, and local proteases may then cleave them further to yield amyloidogenic fragments.

Aged↗
PubMed1991

Processing of beta-amyloid precursor protein in microglia and astrocytes favors an internal localization over constitutive secretion.

Microglial cells and astrocytes are closely associated with nearly all compact deposits of the amyloid beta-protein found in the senile plaques characteristic of Alzheimer's disease and trisomy 21. The biosynthesis and metabolic fate of the beta-amyloid precursor protein (beta APP) in astrocytes has not been characterized, and its identification in microglia has not been described. Here, we report the expression of beta APP by astrocytes and microglia in primary cultures of cerebral cortex from newborn rats. Using metabolic labeling followed by immunoprecipitation, we show that both astrocytes and microglia express substantial amounts of the major isoforms of beta APP. This is confirmed by PCR-mediated amplification of the corresponding mRNAs, showing that all three major transcripts (beta APP695, beta APP751, and beta APP770) are present in relatively equal amounts. Despite rapid turnover of the precursor, astrocytes and microglia show a reduced production of soluble fragments of beta APP compared to cells transfected with beta APP cDNAs. The relative amount of soluble beta APP molecules generated is both cell type and isoform specific. Immunocytochemistry reveals that full-length beta APP is located in internal membranous vesicles, with only very little insertion at the cell surface. The latter data are in agreement with the reduced ability of microglia and astrocytes to cleave the beta APP into soluble derivatives. Our findings indicate that both astrocytes and microglia strongly express all three major forms of beta APP but apparently process these molecules by an alternative pathway that generates very small amounts of soluble beta APP. The immunocytochemical localization and the biochemical data lead to the suggestion that beta APP may not function principally as a cell surface or secreted protein in vivo but may have an important intracellular function.

Amyloid beta-Protein Precursor↗
PubMed1991

Homology of the amyloid beta protein precursor in monkey and human supports a primate model for beta amyloidosis in Alzheimer's disease.

Progressive cerebral deposition of the amyloid beta-protein (A beta P) occurs in Alzheimer's disease and during aging of certain mammals (eg, human, monkey, dog) but not others (eg, mouse, rat). The authors cloned and sequenced a full-length cDNA encoding the beta-protein precursor (beta APP) of cynomolgus monkey. The predicted amino acid sequence of the 695-residue protein is completely homologous to that of human. The alternatively transcribed exons encoding the Kunitz protease inhibitor region in monkey were cloned, showing only a single conservative amino acid substitution in the 751-residue form of beta APP and four substitutions in beta APP770. Immunoblots of cerebral cortex with antibodies to various beta APP domains showed highly similar beta APP polypeptides in human and monkey, in contrast to those of mouse and rat. The latter differences reflect sequence substitutions, transcriptional regulation, and possibly post-translational modifications that may decrease the amyloidogenic potential of rodent beta APP. Immunocytochemistry of aged cynomolgus brain showed A beta P deposited in blood vessels and diffuse and compacted plaques closely resembling those of humans, and the presence of beta-amyloid-associated proteins (alpha 1-antichymotrypsin; complements C1q and C3c) characteristic of A beta P deposits in Alzheimer's disease. The authors' findings demonstrate that cynomolgus monkey and perhaps other primates provide a close animal model for examining the early transcriptional and post-translational processing of beta APP that precedes A beta P deposition during aging and in Alzheimer's disease.

Aging↗
PubMed1990

Transforming growth factor-beta bound to soluble derivatives of the beta amyloid precursor protein of Alzheimer's disease.

Transforming growth factors beta (TGF beta) are multifunctional polypeptides that participate in regulation of growth, differentiation and function of many cell types. The mature TGF beta molecule is a 25 kDa protein composed of two 12.5 kDa monomers linked by disulfide bonds. Human glioblastoma cells secrete biologically active TGF beta 2. Here we report that in addition to the free form of TGF beta 2, a stable complex between a approximately 110 kDa binding protein and TGF beta 2 was isolated from glioblastoma cell supernatant. This binding protein was purified and was found to show sequence identity to part of the beta amyloid precursor protein (beta APP), to be specifically labeled by several different antisera to beta APP, and to be affinity labeled with TGF beta by crosslinking. The complex formation between TGF beta and beta APP may have important implications in regulation of biological activity of the two proteins and in delivery or clearance of TGF beta and beta APP in the brain and other compartments.

Alzheimer Disease↗
PubMed1990

Detection of soluble forms of the beta-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 Alzheimer's disease (AD), Down's 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.

Aged↗