Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations.

The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer's disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N- and C-terminal fragments in association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1 proteins. Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin 1 complex mass, stability and association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1. Solubilization of mouse or human brain membranes with dodecyl-d-maltoside produced a 360-kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X-100 and SDS, but stable to reducing agent. Addition of 5 M urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and presenilin enhancer-2 were detected in the presenilin 1 complex following BN/PAGE, electroelution and second-dimension analysis. Anterior pharynx defective-1 was detected as an 18-kDa form and 9-kDa C-terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36-kDa band after presenilin 1 complex second-dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1-reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.     

Enrichment of Presenilin 1 Peptides in Neuronal Large Dense-Core and Somatodendritic Clathrin-Coated Vesicles

Abstract: Presenilin 1 is an integral membrane protein specifically cleaved to yield an N-terminal and a C-terminal fragment, both membrane-associated. More than 40 presenilin 1 mutations have been linked to early-onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function(s) of the wild-type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear-core synaptic vesicles, the large dense-core vesicles, and the somatodendritic and nerve terminal clathrin-coated vesicles. Both the N- and C-terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense-core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense-core vesicle-mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin-coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear-core synaptic vesicle or in the nerve terminal clathrin-coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full-length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full-length protein may not have a vesicular function.     

In vitro gamma-secretase cleavage of the Alzheimer's amyloid precursor protein correlates to a subset of presenilin complexes and is inhibited by zinc

The gamma-secretase complex mediates the final proteolytic event in Alzheimer's disease amyloid-beta biogenesis. This membrane complex of presenilin, anterior pharynx defective, nicastrin, and presenilin enhancer-2 cleaves the C-terminal 99-amino acid fragment of the amyloid precursor protein intramembranously at gamma-sites to form C-terminally heterogeneous amyloid-beta and cleaves at an epsilon-site to release the intracellular domain or epsilon-C-terminal fragment. In this work, two novel in vitro gamma-secretase assays are developed to further explore the biochemical characteristics of gamma-secretase activity. During development of a bacterial expression system for a substrate based on the amyloid precursor protein C-terminal 99-amino acid sequence, fragments similar to amyloid-beta and an epsilon-C-terminal fragment were observed. Upon purification this substrate was used in parallel with a transfected source of substrate to measure gamma-secretase activity from detergent extracted membranes. With these systems, it was determined that recovery of size-fractionated cellular and tissue-derived gamma-secretase activity is dependent upon detergent concentration and that activity correlates to a subset of high molecular mass presenilin complexes. We also show that by changing the solvent environment with dimethyl sulfoxide, detection of epsilon-C-terminal fragments can be elevated. Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein gamma-secretase substrate and inhibits its cleavage. These studies further refine our knowledge of the complexes and biochemical factors needed for gamma-secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer's disease pathogenesis.     

A 7-kDa prion protein (PrP) fragment, an integral component of the PrP region required for infectivity, is the major amyloid protein in Gerstmann-Sträussler-Scheinker disease A117V

Gerstmann-Str?ussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Str?ussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.     

SEL-10 interacts with presenilin 1, facilitates its ubiquitination,and alters A-beta peptide production

Mutations in the human presenilin genes (PS1 or PS2) have been linked to autosomal dominant, early onset Alzheimer's disease (AD). Presenilins, probably as an essential part of gamma-secretase, modulate gamma-cleavage of the amyloid protein precursor (APP) to the amyloid beta-peptide (Abeta). Mutations in sel-12, a Caenorhabditis elegans presenilin homologue, cause a defect in egg laying that can be suppressed by loss of function mutations in a second gene, SEL-10. SEL-10 protein is a homologue of yeast Cdc4, a member of the SCF (Skp1-Cdc53/CUL1-F-box protein) E2-E3 ubiquitin ligase family. In this study, we show that human SEL-10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N- and C-terminal fragments. Co-transfection of sel-10 and APP cDNAs in HEK293 cells leads to an alteration in the metabolism of APP and to an increase in the production of amyloid beta-peptide, the principal component of amyloid plaque in Alzheimer's disease.     

The presenilin proteins are components of multiple membrane-bound complexes that have different biological activities

Several lines of evidence have indicated that the presenilin proteins function within macromolecular complexes and are necessary for the regulated intramembranous proteolysis of certain type 1 transmembrane proteins, including the amyloid precursor protein, Notch, and p75. Data from multiple complementary experiments now suggest that there may be several distinct presenilin complexes. We show here that presenilin mutations and certain detergents affect the abundance and componentry of the presenilin complexes, and these structural effects correlate with their effects on gamma-secretase activity. Our data suggest that there are at least three complexes, including a approximately 150-kDa nicastrin-aph-1 complex (which is likely to be a precursor complex). There is a stable and abundant intermediate complex of approximately 440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. However, it is the very low abundance, high mass (>/=670 kDa) heteromeric complexes that are associated with the highest gamma-secretase-specific activity.     

Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3-cleavage of notch

The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.     

Presenilin-1 and -2 are molecular targets for gamma-secretase inhibitors

Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.     

The Role of γ-Secretase Activating Protein (GSAP) and Imatinib in the Regulation of γ-Secretase Activity and Amyloid-β Generation

γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-β production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-β production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-β levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-β generation. Likewise, purified recombinant GSAP had no effect on amyloid-β generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-β levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-β levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-β generation are uncertain.     

Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. Possible relationship of this protein to physiologic glutamate receptors

Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approximately 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger Mr protein in synaptic membranes. Protease activity during protein purification was prevented by introducing five protease inhibitors, and a three-step purification procedure was developed that yielded a high degree of purification of glutamate-binding proteins. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 microM, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L-[3H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.     

The proteolytic processing of the amyloid precursor protein gene family members APLP-1 and APLP-2 involves alpha-, beta-, gamma-, and epsilon-like cleavages: modulation of APLP-1 processing by n-glycosylation

Amyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages by beta- and gamma-secretase lead to the formation of the 4-kDa amyloid Abeta protein peptide that accumulates in Alzheimer's disease brain. The processing of APP involves proteolytic conversion by different secretases leading to alpha-, beta-, gamma-, delta-, and epsilon-cleavages. Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are processed by alpha- and gamma-secretase-like cleavages, and that their intracellular domains can be released by cleavage at epsilon-sites. APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and Abeta-like fragments of APLP-2 in the media of stably transfected SH-SY5Y cells.     

Cellular Expression and Proteolytic Processing of Presenilin Proteins Is Developmentally Regulated During Neuronal Differentiation

Abstract: We have determined the expression of the Alzheimer's disease-associated proteins presenilin-1 and presenilin-2 in primary cultures of rat hippocampal neurons. Neurons highly express presenilin-1 and presenilin-2, whereas both proteins were not detected in astrocytes. Further, we have analyzed the subcellular localization and expression in rat hippocampal neurons during development. Although presenilin proteins were localized predominantly to the endoplasmic reticulum in nonneuronal cells transfected with presenilin cDNAs, in neurons, presenilin proteins were also found in compartments not staining with antibodies to grp78(BiP). Presenilin-1 and presenilin-2 were predominantly detected in vesicular structures within the somatodendritic compartment with much less expression in axons. Polarized distribution of presenilin-1 and presenilin-2 differs slightly, with more presenilin-2 expressed in axons compared with presenilin-1. Presenilin expression was found to be developmentally regulated. Presenilin expression strongly increased during neuronal differentiation until full morphological polarization and then declined. No full-length presenilin-1 or presenilin-2 could be detected within cell lysates. At early developmental stages the expected ～34-kDa N-terminal proteolytic fragment of presenilin-1 and the ～38-kDa fragment of presenilin-2 were detected. Later during differentiation we predominantly detected a ～38-kDa fragment for presenilin-1 and a ～42-kDa fragment for presenilin-2. By epitope mapping, we show that these slower migrating peptides represent N-terminal proteolytic fragments, cleaved C-terminal to the conventional site of processing. It is noteworthy that both presenilin-1 and presenilin-2 undergo alternative proteolytic cleavage at the same stage of neuronal differentiation. Regulation of presenilin expression and proteolytic processing might have implications for the pathological as well as the biological function of presenilins during aging in the human brain.     

Partial purification and characterization of gamma-secretase from post-mortem human brain

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.     

Distinct intramembrane cleavage of the beta-amyloid precursor protein family resembling gamma-secretase-like cleavage of Notch

The intramembrane cleavage of beta-amyloid precursor protein by gamma-secretase is the final step in the generation of amyloid beta-protein. A 59- or 57-residue C-terminal fragment called CTFgamma is produced concomitantly. Putative CTFgamma generated in rat brain membrane preparations was purified and sequenced. Instead of CTFgamma, shorter 50- and 49-residue fragments were identified. In addition, we found similar C-terminal fragments of beta-amyloid precursor-like proteins 1 and 2; these were also cleaved at corresponding sites. This newly identified cleavage occurs at a site two to five residues inside the cytoplasmic membrane boundary, which is very similar to gamma-secretase-like cleavage of Notch 1.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

Nicastrin expression in mouse peripheral tissues is not co-ordinated with presenilin and is high in muscle

Nicastrin was the first binding partner of presenilin (PS) shown to be a critical component of the presenilin/gamma-secretase complex essential in development and differentiation, and in generation of Alzheimer's disease Abeta amyloid peptide. To investigate the function of this glycoprotein, we compared nicastrin and presenilin protein expression in various mouse tissues. Western blot analysis of PS1, PS2 and nicastrin indicates their expression levels are not coordinated. In adult mouse, nicastrin is highly expressed in muscle membranes, whereas presenilin levels are very low. By Blue Native electrophoresis, a PS1 complex of 400 kDa was detected in lung, brain, thymus and heart; nicastrin was also detected as a 400-kDa complex in brain but in muscle it was detected with a complex mobility of 240 and 290 kDa, suggesting association with alternate protein complexes. Immunocytochemistry confirms strong intracellular expression of nicastrin in skeletal muscle and blood vessel smooth muscle. These findings suggest a function for nicastrin in muscle other than participation in the gamma-secretase complex.     

A novel gamma -secretase assay based on detection of the putative C-terminal fragment-gamma of amyloid beta protein precursor

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.     

22R-hydroxycholesterol and 9-cis-retinoic acid induce ATP-binding cassette transporter A1 expression and cholesterol efflux in brain cells and decrease amyloid beta secretion

The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of peripheral cholesterol efflux and plasma high density lipoprotein metabolism. In adult rat brain we found high expression of ABCA1 in neurons in the hypothalamus, thalamus, amygdala, cholinergic basal forebrain, and hippocampus. Large neurons of the cholinergic nucleus basalis together with CA1 and CA3 pyramidal neurons were among the most abundantly immunolabeled neurons. Glia cells were largely negative. Because cholesterol homeostasis may have an essential role in central nervous system function and neurodegeneration, we examined ABCA1 expression and function in different brain cell types using cultures of primary neurons, astrocytes, and microglia isolated from embryonic rat brain. The basal ABCA1 mRNA and protein levels detected in these cell types were increased markedly after exposure to oxysterols and 9-cis-retinoic acid, which are ligands for the nuclear hormone liver X receptors and retinoic X receptors, respectively. Functionally, the increased ABCA1 expression caused by these ligands was followed by elevated apoA-I- and apoE-specific cholesterol efflux in neurons and glia. In non-neuronal and neuronal cells overexpressing a human Swedish variant of amyloid precursor protein, 22R-hydroxycholesterol and 9-cis-retinoic acid induced ABCA1 expression and increased apoA-I-mediated cholesterol efflux consequently decreasing cellular cholesterol content. More importantly, we demonstrated that these ligands alone or in combination with apoA-I caused a substantial reduction in the stability of amyloid precursor protein C-terminal fragments and decreased amyloid beta production. These effects of 22R-hydroxycholesterol may provide a novel strategy to decrease amyloid beta secretion and consequently reduce the amyloid burden in the brain.     

Protein expression of BACE1, BACE2 and APP in Down syndrome brains

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21. The phenotype of DS is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. Several reports have shown that the neuropathology of DS comprises developmental abnormalities and Alzheimer-like lesions such as senile plaques. A key component of senile plaques is amyloid beta-peptide which is generated from the amyloid precursor protein (APP) by sequential action of beta-secretases (BACE1 and BACE2) and gamma-secretase. While BACE1 maps to chromosome 11, APP and BACE2 are located on chromosome 21. To challenge the gene dosage effect and gain insight into the expressional relation between beta-secretases and APP in DS brain, we evaluated protein expression levels of BACE1, BACE2 and APP in fetal and adult DS brain compared to controls. In fetal brain, protein expression levels of BACE2 and APP were comparable between DS and controls. BACE1 was increased, but did not reach statistical significance. In adult brain, BACE1 and BACE2 were comparable between DS and controls, but APP was significantly increased. We conclude that APP overexpression seems to be absent during the development of DS brain up to 18-19 weeks of gestational age. However, its overexpression in adult DS brain could lead to disturbance of normal function of APP contributing to neurodegeneration. Comparable expression of BACE1 and BACE2 speaks against the hypothesis that increased beta-secretase results in (or even underlies) increased production of amyloidogenic A beta fragments. Furthermore, current data indicate that the DS phenotype cannot be fully explained by simple gene dosage effect.     

Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid β-Protein

Abstract: Cleavage after Met⁵⁹⁶ of the β-amyloid precursor protein to generate the N-terminus of β-protein indicates the activity of a protease having chymotrypsin-like specificity. A chymotrypsin-like protease is further implicated in Alzheimer's disease by the increased synthesis of the protease inhibitor α₁-antichymotrypsin in pathologically affected brain regions and by the presence in the amyloid deposits of inactivated forms of α₁-antichymotrypsin (indicating irreversible binding to a target chymotrypsin-like protease). In the present report, we have purified from rat brain a chymotrypsin-like protease that (a) binds with high affinity to human α₁-antichymotrypsin, (b) proteolytically generates a β-protein-containing C-terminal fragment from full-length recombinant human β-amyloid precursor protein, and (c) selectively cleaves methoxysuccinyl-Glu-Val-Lys-Met-p-nitroanilide (a substrate modeling the protease recognition domain for the β-protein N-terminal cleavage site). Amino acid sequences of tryptic fragments of the purified rat brain chymotrypsin-like protease indicate an identity with rat mast cell protease I. Moreover, the ontogeny and compartmentalization of rat brain chymotrypsin-like protease are consistent with those of connective tissue-type mast cells in the meningeal and intracortical perivasculature. Because these areas in human brain form extensive β-amyloid deposits in Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, the present findings suggest that a brain mast cell chymotrypsin-like protease may participate in generating perivascular β-protein, which ultimately aggregates into β-amyloid deposits.