A substrate-based difluoro ketone selectively inhibits Alzheimer's gamma-secretase activity.
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Alzheimer Disease↗
Biomedical subjects
D J Selkoe
Publications and source records attributed to D J Selkoe.
Additive effects of PS1 and APP mutations on secretion of the 42-residue amyloid beta-protein.
Humans harboring missense mutations in the presenilin 1 (PS1) gene undergo progressive cerebral deposition of the 42-residue amyloid beta-protein (A beta 42) at an early age and develop severe Alzheimer's disease. A beta 42 is selectively elevated in the conditioned media of cells expressing mutant but not wild-type PS1, indicating that presenilin mutations alter APP processing. Here we analyze the effects of various PS1 mutant constructs on the cellular production of A beta 42. A construct expressing only the PS1 N-terminal endoproteolytic fragment with the mutation Y115H causes no significant increase in A beta 42, whereas a full-length PS1 construct with the same mutation does. This result suggests that the pathogenic effect of mutant presenilins is produced by the full-length molecule even though only a minor proportion of total PS1 occurs as holoprotein in tissues and cell lines. We demonstrate that the effects of two different PS1 mutations are additive when engineered into the same PS1 molecule. Therefore, two mutations alter gamma-secretase processing of APP more than one and the PS1 mutations described to date do not cause the maximum possible PS1-mediated rise in A beta 42. When a PS1 mutation was expressed in cells carrying the APPV717I mutation, A beta 42 rose dramatically to become the predominant secreted A beta species, an observation of interest for transgenic modeling of AD. Our results are consistent with the hypothesis that presenilin is a major regulator of the proteolytic processing of APP by gamma-secretases.
Age of Onset↗
The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease.
It is a truism of modern biomedical science that the development of therapies expected to slow or arrest the progression of a disease requires as detailed an understanding of its molecular and cellular pathogenesis as possible. In turn, the cloning of novel gene products implicated in a disease often leads to new insights about fundamental features of protein structure and function. A particularly compelling example of this beneficial interplay between basic and applied cell biology arises from the exciting recent progress in deciphering Alzheimer's disease (AD). This review discusses the current understanding of the cell biology of two proteins crucial for the pathogenesis of AD, the beta-amyloid precursor protein and presenilin.
Alzheimer Disease↗
Heparin-binding properties of the amyloidogenic peptides Abeta and amylin. Dependence on aggregation state and inhibition by Congo red.
Aggregation and deposition of the 40-42-residue amyloid beta-protein (Abeta) are early and necessary neuropathological events in Alzheimer's disease. An understanding of the molecular interactions that trigger these events is important for therapeutic strategies aimed at blocking Abeta plaque formation at the earliest stages. Heparan sulfate proteoglycans may play a fundamental role since they are invariably associated with Abeta and other amyloid deposits at all stages. However, the nature of the Abeta-heparan sulfate proteoglycan binding has been difficult to elucidate because of the strong tendency of Abeta to self-aggregate. Affinity co-electrophoresis can measure the binding of proteoglycans or glycosaminoglycans to proteins without altering the physical state of the protein during the assay. We used affinity co-electrophoresis to study the interaction between Abeta and the glycosaminoglycan heparin and found that the aggregation state of Abeta governs its heparin-binding properties: heparin binds to fibrillar but not nonfibrillar Abeta. The amyloid binding dye, Congo red, inhibited the interaction in a specific and dose-dependent manner. The "Dutch" mutant AbetaE22Q peptide formed fibrils more readily than wild type Abeta and it also attained a heparin-binding state more readily, but, once formed, mutant and wild type fibrils bound heparin with similar affinities. The heparin-binding ability of aggregated AbetaE22Q was reversible with incubation in a solvent that promotes alpha-helical conformation, further suggesting that conformation of the peptide is important. Studies with another human amyloidogenic protein, amylin, suggested that its heparin-binding properties were also dependent on aggregation state. These results demonstrate the dependence of the Abeta-heparin interaction on the conformation and aggregation state of Abeta rather than primary sequence alone, and suggest methods of interfering with this association.
Amino Acid Sequence↗
Interaction between amyloid precursor protein and presenilins in mammalian cells: implications for the pathogenesis of Alzheimer disease.
Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes increase the production of the highly amyloidogenic 42-residue form of amyloid beta-protein (Abeta42) in a variety of cell lines and transgenic mice. To elucidate the molecular mechanism of this effect, wild-type (wt) or mutant PS1 and PS2 genes were stably transfected into Chinese hamster ovary cells expressing endogenous or transfected beta-amyloid precursor protein (APP). By immunoprecipitation/Western blot analysis, APP was consistently found to coimmunoprecipitate with PS1 or PS2 proteins. Several distinct PS1, PS2, or APP antibodies precipitated PS-APP complexes that were detectable by blotting with either APP or PS antibodies. Importantly, complex formation could be detected at endogenous protein levels in nontransfected cells. In various Chinese hamster ovary cell lines, the amounts of APP coprecipitated by PS antibodies were proportional to the expression levels of both APP and PS. APP-PS complexes also were recovered from human 293 and HS683 cells. Full maturation of APP was not required for the interaction; most APP molecules complexed with PS were solely N-glycosylated. Treatment of cells with brefeldin A or incubation at 20 degrees C did not block complex formation, suggesting that the association between APP and PS occurs in part in the endoplasmic reticulum. Complex formation was detected for both wt and mutant PS and APP proteins. Deletion of the APP C-terminal domain did not abrogate complex formation, suggesting that the interaction does not occur in the cytoplasmic domains of the proteins. Our results demonstrate that wt and mutant PS1 and PS2 proteins form complexes with APP in living cells, strongly supporting the hypothesis that mutant PS interacts with APP in a way that enhances the intramembranous proteolysis of the latter by a gamma-secretase cleaving at Abeta42.
Alzheimer Disease↗
Skeletal and CNS defects in Presenilin-1-deficient mice.
Presenilin-1 (PS1) is the major gene responsible for early-onset familial Alzheimer's disease (FAD). To understand the normal function of PS1, we have generated a targeted null mutation in the murine homolog of PS1. We report that PS1-/- mice die shortly after natural birth or Caesarean section. The skeleton of homozygous mutants is grossly deformed. Hemorrhages occur in the CNS of PS1 null mutants with varying location, severity, and time of onset. The ventricular zone of PS1-/- brains is markedly thinner by embryonic day 14.5, indicating an impairment in neurogenesis. Bilateral cerebral cavitation caused by massive neuronal loss in specific subregions of the mutant brain is prominent after embryonic day 16.5. These results show that PS1 is required for proper formation of the axial skeleton, normal neurogenesis, and neuronal survival.
Alzheimer Disease↗
Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C.
The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of approximately 20-kDa C-terminal fragments (CTF) and approximately 30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the approximately 20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181-190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.
Alkaline Phosphatase↗
Enhanced production and oligomerization of the 42-residue amyloid beta-protein by Chinese hamster ovary cells stably expressing mutant presenilins.
Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of early onset familial Alzheimer's disease. To elucidate their pathogenic mechanism, wild-type (wt) or mutant (M146L, C410Y) PS1 and wt or mutant (M239V) PS2 genes were stably transfected into Chinese hamster ovary cells that overexpress the beta-amyloid precursor protein (APP). The identity of the 43-45-kDa PS1 holoproteins was confirmed by N-terminal radiosequencing. PS1 was rapidly processed (t1/2 = 40 min) in the endoplasmic reticulum into stable fragments. Wild-type and mutant PS2 holoproteins exhibited similar half lives (1.5 h); however, their endoproteolytic fragments showed both mutation-specific and cell type-specific differences. Mutant PS1 or PS2 consistently induced a 1.4-2.5-fold increase (p < 0.001) in the relative production of the highly amyloidogenic 42-residue form of amyloid beta-protein (Abeta42) as determined by quantitative immunoprecipitation and by enzyme-linked immunosorbent assay. In mutant PS1 and PS2 cell lines with high increases in Abeta42/Abetatotal ratios, spontaneous formation of low molecular weight oligomers of Abeta42 was observed in media, suggesting enhanced Abeta aggregation from the elevation of Abeta42. We conclude that mutant PS1 and PS2 proteins enhance the proteolysis of beta-amyloid precursor protein by the gamma-secretase cleaving at Abeta residue 42, thereby promoting amyloidogenesis.
Amyloid beta-Peptides↗
Degradation of amyloid beta-protein by a metalloprotease secreted by microglia and other neural and non-neural cells.
Amyloid beta-protein (Abeta) is the major component of neuritic (amyloid) plaques in Alzheimer's disease, and its deposition is an early and constant event in the complex pathogenetic cascade of the disease. Although many studies have focused on the biosynthetic processing of the beta-amyloid precursor protein and on the production and polymerization of Abeta, understanding the degradation and clearance of Abeta has received very little attention. By incubating the conditioned medium of metabolically labeled Abeta-secreting cells with media of various cultured cell lines, we observed a time-dependent decrease in the amount of Abeta in the mixed media. The factor principally responsible for this decrease was a secreted metalloprotease released by both neural and non-neural cells. Among the cells examined, the microglial cell line, BV-2, produced the most Abeta-degrading activity. The protease was completely blocked by the metalloprotease inhibitor, 1,10-phenanthroline, and partially inhibited by EDTA, whereas inhibitors of other protease classes produced little or no inhibition. Substrate analysis suggests that the enzyme was a non-matrix metalloprotease. The protease cleaved both Abeta1-40 and Abeta1-42 peptides secreted by beta-amyloid precursor protein-transfected cells but failed to degrade low molecular weight oligomers of Abeta that form in the culture medium. Lipopolysaccharide, a stimulator of macrophages/microglia, activated BV-2 cells to increase their Abeta-degrading metalloprotease activity. We conclude that secreted Abeta1-40 and Abeta1-42 peptides are constitutively degraded by a metalloprotease released by microglia and other neural cells, providing a potential mechanism for the clearance of Abeta in brain tissue.
Amyloid beta-Peptides↗
Cell surface amyloid beta-protein precursor colocalizes with beta 1 integrins at substrate contact sites in neural cells.
Amyloid beta-protein (A beta), the principal constituent of the senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The distribution and trafficking of cell surface beta PP are of particular interest because some of these molecules are direct precursors of secreted A beta and because the localization of beta PP at the cell surface may be related directly to its physiological functions. Recently, we reported that, in cultured hippocampal neurons, cell surface beta PP is preferentially expressed on axons in a striking discontinuous pattern. In this study, we describe the colocalization of cell surface beta PP and integrins in primary cultured cells. In rat hippocampal neurons, cell surface beta PP was colocalized selectively with alpha 1 beta 1 and alpha 5 beta 1 integrin heterodimers at these characteristic segmental locations. In rat cortical astrocytes, both cell surface beta PP and beta 1 integrin were located at the cell periphery in the "spreading" stage shortly after plating. In "flattened" astrocytes cultured for several days, beta PP was found in punctate deposits called point contacts. In these sites, beta PP was colocalized with alpha 1 beta 1, but not with alpha 5 beta 1 integrin heterodimers, the latter of which were situated at focal contact sites. In both neurons and astrocytes examined after shearing, clathrin and alpha-adaptin were colocalized with beta PP on the surface that directly contacts the substratum. These results are consistent with the putative role of beta PP in cell adhesion and suggests that beta PP either interacts with selected integrins or shares similar cellular machinery to promote cell adhesion.
Adaptor Protein Complex alpha Subunits↗
Alzheimer's disease: genotypes, phenotypes, and treatments.
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Alzheimer Disease↗
Ectodomain phosphorylation of beta-amyloid precursor protein at two distinct cellular locations.
The beta-amyloid precursor protein (betaAPP) is a transmembrane protein that is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of betaAPP phosphorylation, we took advantage of an antibody that specifically detects the free C terminus of beta-secretase-cleaved betaAPP containing the Swedish missense mutation (APPssw-beta). This antibody previously established the cellular location of the beta-secretase cleavage of Swedish betaAPP as a post-Golgi secretory compartment (Haass, C., Lemere, C., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nature Med. 1, 1291-1296). We have now localized the selective ectodomain phosphorylation of betaAPP to the same compartment. Moreover, the phosphorylation sites of betaAPP were identified at Ser198 and Ser206 of betaAPP695 by tryptic peptide mapping, mass spectrometry, and site-directed mutagenesis. Intracellular phosphorylation of betaAPP was inhibited by Brefeldin A and by incubating cells at 20 degrees C, thus excluding phosphorylation in the endoplasmic reticulum or trans-Golgi network. Ectodomain phosphorylation within a post-Golgi compartment occurred not only with mutant Swedish betaAPP, but also with wild type betaAPP. In addition to phosphorylation within a post-Golgi compartment, betaAPP was also found to undergo phosphorylation at the cell surface by an ectoprotein kinase. Therefore, this study revealed two distinct cellular locations for betaAPP phosphorylation.
Amyloid beta-Protein Precursor↗
Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue.
Humans inheriting missense mutations in the presenilin (PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid beta-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimer's disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T1/2 approximately 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.
Alzheimer Disease↗
Permeability and residual plasma volume of human, Dutch variant, and rat amyloid beta-protein 1-40 at the blood-brain barrier.
The permeability of normal human, the human Dutch variant, and the rat A beta 1-40 proteins at the blood-brain barrier (BBB) was determined in the normal adult rat by quantifying the permeability coefficient-surface area (PS) product for each protein after correction for the residual plasma volume (Vp) occupied by the protein in the blood vessels of different brain regions. The PS for normal and Dutch A beta ranged from 13 x 10(-6) to 22 x 10(-6) ml/g/s in different brain regions, which is 130 to 220 times greater than albumin. These high PS values compare to that of insulin, whose uptake is decidedly by a receptor-mediated transport process, and suggest a similar mechanism for A beta. Remarkably, the PS for rat A beta was 4 times higher and ranged from 54 x 10(-6) to 82 x 10(-6) ml/g/s for different brain regions, suggesting a distinctive species specificity. While the Vp values of human and rat A beta were comparable, the Dutch variant was 2 to 3 times higher, indicating adherence to the vessel walls in different brain regions, consistent with the heavy A beta deposition that has been described in intracerebral vessel walls with this variant. The high PS values observed for A beta at the BBB suggest that sources outside the nervous system could contribute, at least in part, to the cerebral A beta deposits seen in Alzheimer's disease. SDS-PAGE of 125I-labeled human A beta after 60 min of uptake revealed intact protein in plasma and in different brain regions. In addition, 125I-labeled human A beta binding to a protein of 67,000 in both plasma and brain tissue regions was observed with SDS-PAGE. This protein was tentatively identified as albumin, and it was not detectable in the brain regions of animals that had undergone intracardiac perfusion; hence, a portion of A beta binds tightly to and is likely transported by albumin in plasma. The absence of this A beta-albumin complex in brain regions after perfusion and the low permeability of albumin at the BBB imply that A beta itself is efficiently transported at the BBB to account for the high PS values, although presentation of A beta to the capillary endothelial cell by albumin or other plasma proteins cannot be excluded.
Amino Acid Sequence↗
Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice.
The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.
Alzheimer Disease↗
Caffeine stimulates amyloid beta-peptide release from beta-amyloid precursor protein-transfected HEK293 cells.
Extracellular amyloid beta-peptide (A beta) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular A beta accumulation is observed in the human muscle disease, inclusion body myositis. A beta has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of A beta in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of A beta from its precursor protein (betaAPP). A receptor-based mechanism for the increase in A beta production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of A beta. In cultured HEK293 cells transfected with betaAPP cDNA, caffeine (5-10 mM) significantly increased the release of A beta fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated A beta release. NH4Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular betaAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.
Amyloid beta-Peptides↗
Images in neuroscience. Alzheimer's disease: from genes to pathogenesis.
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Alzheimer Disease↗
The "nonamyloidogenic" p3 fragment (amyloid beta17-42) is a major constituent of Down's syndrome cerebellar preamyloid.
Down's syndrome (DS) patients show accelerated Alzheimer's disease (AD) neuropathology, which consists of preamyloid lesions followed by the development of neuritic plaques and neurofibrillary tangles. The major constituents of preamyloid and neuritic plaques are amyloid beta (Abeta) peptides. Preamyloid lesions are defined as being Abeta immunoreactive lesions, which unlike neuritic plaque amyloid are Congo red-negative and largely nonfibrillar ultrastructurally. DS patients can develop extensive preamyloid deposits in the cerebellum, without neuritic plaques; hence, DS cerebellums are a source of relatively pure preamyloid. We biochemically characterized the composition of DS preamyloid and compared it to amyloid in the neuritic plaques and leptomeninges in the same patients. We found that Abeta17-42 or p3 is a major Abeta peptide of DS cerebellar preamyloid. This 26-residue peptide is also present in low quantities in neuritic plaques. We suggest that preamyloid can now be defined biochemically as lesions in which a major Abeta peptide is p3.
Alzheimer Disease↗