ß amyloid protein precursor-like
The major component of senile plaques found in the brains of Alzheimer disease patients is the beta-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. Several cDNAs encoding an APP-related protein in the nematode Caenorhabditis elegans have been isolated and the corresponding gene has been designated as apl-1. The apl-1 transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-1 cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the beta-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-1 gene from C. elegans should be important for determining the normal function of human APP (Daigle, 1993).
Complimentary DNA clones have been isolated from Xenopus larva to delineate a protein highly homologous to the human beta-amyloid precursor protein (APP). Developmental change of Xenopus APP gene expression has been analyzed with molecular probes. From early oogenesis, there is a high accumulation of maternal APP. After fertilization, the mRNA is degraded, reaching a minimum level around the gastrula stage. Then zygotic transcription appears to be initiated, and this continues during the subsequent embryonic and larval stages. Splicing patterns differ between the maternal and zygotic transcripts. The ratio of mRNA including the protease inhibitor domain (PID) sequence is extremely low for the transcript of maternal origin as compared to that for the transcript of zygotic origin. These results suggest some roles for the APP molecule in Xenopus early development (Okado, 1992).
Mouse and human cDNA clones encoding amyloid precursor-like proteins (APLP1 and APLP2) have been identified and exhibit extensive sequence similarity to the Drosophila Appl and mammalian APP genes. To define the potential role of APLP in the mammalian brain, APLP1 localization was examined within the complex cortical synaptic structure. A focused was placed on the postsynaptic density (PSD), which appears to be central to synaptic function. The 90 kDa APLP1, the first known APLP, is localized to the PSD from rat and human cerebral cortex. APLP1 increases during cortical synaptic development, suggesting a role in synaptogenesis or synaptic maturation. In contrast, APP is predominantly expressed in the synaptic membrane fraction, but is barely detectable in the PSD, including the different subcellular locations of APP and APLP1. These observations raise the possibility that APLP1 participates in brain synaptic function in mammals (Kim, 1995).
The role of betaAPP gene transcription and promoter regulation in modifying amyloid beta-peptide (Abeta) levels is not well understood. Increased production of Abeta or changes in Abeta42/Abeta40 ratio by fibroblasts occurs in the presence of mutant presenilin or betaAPP alleles in familial Alzheimer's disease subjects. Both betaAPP mRNA and Abeta levels are increased in trisomy 21. The APP gene promoter is in a class of housekeeping genes and contains two putative consensus sites for the binding of transcription factor AP1. Electrophoretic mobility shift (EMSA) and DNase protection assays using human fibroblast and HeLa nuclear extract have identified specific protein binding with novel Sp1-like properties to both a near-upstream and a downstream domain of the betaAPP promoter. The upstream binding activity is localized to a putative AP1 consensus site and its immediate 5'-adjacent GC-rich element. However, c-Jun antibody and competition experiments have no effect on binding to this domain. A series of 5'-deleted betaAPP promoter-reporter gene transfections in HeLa and fibroblast cells shows that the domain-containing region, n.t. -383 to -348, exerts a 2.9-fold activating influence on basal pbetaAPP-reporter transcription. When subcloned to test enhancer function, the 5'-GC element/'AP1 site' tandem construct confers four-fold greater activity than either element alone and two-fold greater than the more 3'-situated HSE consensus sequence. Phorbol ester treatment has no effect in these reporter assays. This element shares homology and binding properties with a domain immediately 5' to the downstream E-box/USF element. An interaction model involving both domains and looping of interjacent DNA is proposed. It is concluded that this newly described binding protein-enhancer complex is required for full betaAPP promoter activation (Querfurth, 1999).
For information on gamma-secretase see the Presenilin overview and The role of presenilin in beta-amyloid precursor protein processing.
Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increases the amount of beta-secretase cleavage products: these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreases the amount of beta-secretase cleavage products, and purified BACE protein cleaves APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE are consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease (Vassar, 1999).
The release of amyloidogenic amyloid-beta peptide (Abeta) from amyloid-beta precursor protein (APP) requires cleavage by beta- and gamma-secretases. alpha-secretase, in contrast, cleaves APP within the Abeta sequence and precludes amyloidogenesis. Regulated and unregulated alpha-secretase activities have been reported, and the fraction of cellular alpha-secretase activity regulated by protein kinase C (PKC) has been attributed to the ADAM (a disintegrin and metalloprotease) family members TACE and ADAM-10. Although unregulated alpha-secretase cleavage of APP has been shown to occur at the cell surface, attempts have been made to identify the intracellular site of PKC-regulated alpha-secretase APP cleavage. To accomplish this, levels of secreted ectodomains and C-terminal fragments of APP generated by alpha-secretase (sAPPalpha) (C83) versus beta-secretase (sAPPbeta) (C99) were measured and secreted Abeta was measured in cultured cells treated with PKC and inhibitors of TACE/ADAM-10. PKC stimulation increases sAPPalpha but decreases sAPPbeta levels by altering the competition between alpha- versus beta-secretase for APP within the same organelle rather than by perturbing APP trafficking. Moreover, data implicating the trans-Golgi network (TGN) as a major site for beta-secretase activity prompted the hypothesis that PKC-regulated alpha-secretase(s) also resides in this organelle. To test this hypothesis, studies were performed demonstrating proteolytically mature TACE intracellularly, and regulated alpha-secretase APP cleavage was shown to occur in the TGN using an APP mutant construct targeted specifically to the TGN. By detecting regulated alpha-secretase APP cleavage in the TGN by TACE/ADAM-10, ADAM activity was revealed in a novel location. Finally, the competition between TACE/ADAM-10 and beta-secretase for intracellular APP cleavage may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease (Skovronsky, 2000).
Cleavage of APP by unidentified proteases, referred to as beta- and gamma-secretases, generates the amyloid beta-peptide, the main component of the amyloid plaques found in Alzheimer's disease patients. The disease-causing mutations flank the protease cleavage sites in APP and facilitate APP cleavage. A new membrane-bound aspartyl protease (Asp2) with beta-secretase activity has been identified. The Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid beta-peptide production and blocks the accumulation of the carboxy-terminal APP fragment that is created by beta-secretase cleavage. Solubilized Asp2 protein cleaves a synthetic APP peptide substrate at the beta-secretase site, and the rate of cleavage is increased tenfold by a mutation associated with early-onset Alzheimer's disease in Sweden. Thus, Asp2 is a new protein target for drugs that are designed to block the production of amyloid beta-peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease (Yan, 1999).
Amyloid beta-protein (Abeta) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the beta-amyloid precursor protein (APP). The amyloidogenic processing of APP was examined using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3 gamma chain of the T cell antigen receptor (CD3gamma). CD3gamma contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface. The wild-type sequence of CD3gamma was used to create an APP chimera predicted to remain in the ER [gammaAPP(ER)]. Deletion of the RKK motif at the C-terminus directs the protein directly to the lysosomes [gammaAPP(LYS)]. A third chimera was created by removing both lysosomal targeting signals in addition to RKK [gammaAPP(DeltaDelta)]. This last construct does not contain known targeting signals and consequently accumulates at the cell surface. All three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. Abeta(1-40) is generated in the early secretory and endocytic pathways, whereas Abeta(1-42) is made mainly in the secretory pathway. More importantly, evidence is provided that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived Abeta forms can enter the secretable pool. Lysosomal processing is not involved in the generation or secretion of either Abeta(1-40) or Abeta(1-42) (Soriano, 1999).
APP1, a transmembrane protein with homology to glycosylated cell surface receptors, can reside at the cell surface and is reinternalized via clathrin-coated pits. Some internalized APP remains intact to be recycled to the cell surface plasma membrane. However, internalized APP can also be proteolytically processed into several distinct secreted fragments, which include the large secreted N-terminal APP ectodomain (APPs), and Abeta, the major protein component of senile plaques in Alzheimer's disease (AD). Because Abeta deposition may be central to AD pathogenesis, the mechanism by which Abeta is generated from the precursor is an important focus of AD research. At least two species of Abeta, differing by two amino acids at the C terminus (Abeta40 and Abeta42), are released from cells during normal cellular metabolism. Abeta42, which readily aggregates in vitro appears to be more pathogenic and may serve as a seed for plaque formation in individuals with AD, hereditary cerebral hemorrhage with amyloidosis Dutch type, and Down's syndrome. The source of Abeta deposited in brain tissues is still uncertain. However, cell lines expressing wild type APP can produce and release Abeta primarily after internalization of APP from the cell surface. Although familial mutations in APP can enhance Abeta secretion, almost all humans express wild type APP. Therefore, the major pathway for Abeta production appears to involve endocytic recycling of APP from the cell surface. To date, the specific contribution of APP endocytic processing to Abeta42 production in particular has not been established (Perez, 1999).
APP endocytosis relies on signals in the cytoplasmic C-terminal domain. An NPXY sequence similar to that found in the C terminus of the low density lipoprotein receptor and the LDLR-related protein is also found in APP. Because the tyrosine in NPXY is crucial for LDLR endocytosis, it has long been assumed that the C-terminal motif, NPXY, is the internalization signal for beta-amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr743 by APP751 numbering, Tyr682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737-743) were mutated. Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impair APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (DeltaC), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr743 regulates rapid APP turnover because half-life increases by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, Abeta, is tightly correlated with APP internalization, such that Abeta secretion is unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the Abeta42 isoform is also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic Abeta species (Perez, 1999).
The Alzheimer's beta-amyloid precursor protein (APP) is internalized from the axonal cell surface. Biochemical and cell biological methods have been used to characterize endocytotic compartments that participate in the trafficking of APP in central neurons. APP is present in presynaptic clathrin-coated vesicles purified from bovine brain, together with the recycling synaptic vesicle integral membrane proteins synaptophysin, synaptotagmin, and SV2. In contrast, APP is largely excluded from synaptic vesicles purified from rat brain. In primary cerebellar macroneurons, cell-surface APP is internalized with recycling synaptic vesicle integral membrane proteins but is subsequently sorted away from synaptic vesicles and transported retrogradely to the neuronal soma. Internalized APP partially co-localizes with rab5a-containing compartments in axons and with V-ATPase-containing compartments in both axons and neuronal soma. These results provide direct biochemical evidence that an obligate sorting compartment participates in the regeneration of synaptic vesicles during exo/endocytotic recycling at nerve terminals. Moreover, APP is now, the first demonstrated example of an axonal cell-surface protein that is internalized with recycling synaptic vesicle membrane proteins but is subsequently sorted away from synaptic vesicles (Marquez-Sterling, 1997).
The pheochromocytoma PC12 cell line has been used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increases neurite outgrowth, APP mRNA expression, and APP secretion in a dose-dependent fashion, with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolishes NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone does not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide (an analog of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase) mimics the effects of NGF on cell morphology and stimulates both APP mRNA levels and APP secretion. In contrast, specific stimulation of TrkA receptors by receptor cross-linking selectively stimulates neurite outgrowth and APP secretion but not APP mRNA levels, which are decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, these data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Specific activation of TrkA receptors, in contrast to p75NTR-associated signaling, stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels (Rossner, 1998).
Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite in the brain. The role of neprilysin, a candidate Abeta-degrading peptidase, in Abeta metabolism was examined using neprilysin gene-disrupted mice. Neprilysin deficiency results in defects both in the degradation of exogenously administered Abeta and in the metabolic suppression of the endogenous Abeta levels in a gene dose-dependent manner. The regional levels of Abeta in the neprilysin-deficient mouse brain were in the distinct order of hippocampus, cortex, thalamus/striatum, and cerebellum, where hippocampus has the highest level and cerebellum the lowest, correlating with the vulnerability to Abeta deposition in brains of humans with AD. These observations suggest that even partial down-regulation of neprilysin activity, which could be caused by aging, can contribute to AD development by promoting Abeta accumulation (Iwata, 2001).
Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Aß42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in C. elegans, Drosophila, and mammals. Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupts the HMW complex formation, and abolishes gamma-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescues the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolishes the gamma-secretase activity in S2 cells. Surprisingly, gamma-secretase activity dependent on wild-type Psn is associated with a drastic overproduction of Aß1-42 from human ßAPP in N2a cells, but not in S2 cells. These data suggest that the mechanism of gamma-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals (Takasugi, 2002).
The formation of the stabilized HMW complex of mammalian PS, that requires the integrity of the conserved PS C terminus, is essential to the acquisition of gamma-secretase activity, and an aspartate residue within 7th TMD (TMD7) is crucial to the gamma-secretase activity in mammalian PS. To verify the effects of missense mutations in Psn that cause Notch (i.e, loss-of-function) phenotype in Drosophila in vivo, on the metabolism of Psn polypeptides, the two types of amino acid substitutions (i.e., P507L or G516E) were introduced and stably expressed the mutant Psn in N2a NL/N cells. In addition, N2a NL/N cells, stably coexpressing Psn carrying D461A mutation that replaces the highly conserved aspartate residue in the TMD7 with alanine, were established to see if this mutation works as a dominant negative mutant on gamma-cleavage as in mammalian PS. Neither Psn/P507L, Psn/G516E nor Psn/D461A undergo endoproteolysis to give rise to NTF and CTF that normally occurs with wild type Psn. The replacement of endogenous PS1 did not occur in N2a NL/N cells coexpressing Psn/P507L or Psn/G516E. Upon CHX treatment of the N2a cells, the Psn/P507L or Psn/G516E holoproteins were rapidly degraded in a similar manner to wild type Psn holoprotein. In contrast, the overexpression of Psn/D461A results in a complete replacement of endogenous murine PS1 fragments, and a portion of Psn/D461A is stabilized as a holoprotein, as previously described in aspartate mutants of mammalian PS (i.e., PS1/D385A, PS2/D366A). The HMW complex formation of Psn and its derivatives was analyzed. The unstable Psn/P507L or Psn/G516E holoproteins were fractionated exclusively in the LMW range. In contrast, Psn/D461A, which was stabilized but not cleaved, was present as holoproteins broadly within LMW and HMW ranges in a similar manner to that of mammalian PS2/D366A (Takasugi, 2002).
Psn-dependent gamma-secretase activity in Drosophila has been shown to cleave Notch and other transmembrane proteins in vivo. The amino acid sequence of APPL, a Drosophila homolog of ßAPP, is not homologous to that of mammalian ßAPP especially within the TMD, and gamma-cleavage of APPL has not been documented. However, it has been shown that overexpression of the C-terminal 99 amino acid fragment of human ßAPP elicits the cleavage to generate Aß1-40 by a gamma-secretase-like activity in Drosophila SL-2 cells, although Drosophila cells lack ß-secretase activity. To evaluate the gamma-secretase-like activity for proteolytic processing of the TMD sequence of human ßAPP in Drosophila S2 cells, a cDNA encoding SC100, that corresponds to the C-terminal fragment of human ßAPP starting at the 1st residue of Aß preceded by a signal peptide, was transiently transfected and the conditioned media by ELISA was analyzed. Aß secretion was readily detectable in conditioned media of cells expressing SC100; surprisingly, however, percent Aß42 was ~15%, which was in sharp contrast to the robust Aß1-42 overproduction in mouse N2a cells, that is mediated by the same PS species, i.e., wild type Psn. To exclude the possibility that gamma-secretase-like activity in S2 cells is incapable of producing excessive amounts of Aß1-42, a cDNA encoding SC100 was constructed harboring an isoleucine to phenylalanine substitution at residue 716 of ßAPP (SC100/I716F); this substitution has been shown to cause robust increase in Aß1-42 secretion in COS cells. Transfection of SC100/I716F into S2 cells results in a dramatic increase in Aß1-42 secretion and simultaneous decrease in Aß40 secretion, suggesting that the endogenous gamma-secretase-like activity mediated by Psn normally cleaves the TMD sequence of human ßAPP predominantly at Aß40 position, but is capable of cleaving predominantly at position 42 under pathogenic conditions (e.g., ßAPP mutation) in S2 cells. Thus, Psn-dependent gamma-cleavage in S2 cells shows similar characteristics to those in mammalian cells, whereas it may be shifted to position 42 by some unknown mechanism in mouse N2a cells (Takasugi, 2002).
To examine whether Psn plays an essential role in Aß generation by a gamma-secretase-like activity in S2 cells, an S2 cell line was generated stably expressing SC100 (S2-SC100) and the expression of endogenous Psn gene was suppressed by double-stranded RNA (dsRNA)-mediated interference (RNAi). After a 48-h transfection of Psn dsRNA, the expression of Psn polypeptide in the form of fragments was completely and specifically abolished in S2-SC100 cells. After incubation in fresh media for additional 24 h, the cell lysates and conditioned media were analyzed. Immunoblot analysis has revealed an accumulation of SC100 as well as of a ~10-kDa polypeptide comigrating with C83 of mammalian cells. The latter band presumably represents the SC100 derivative cleaved by an alpha-secretase-like activity, that has been reported in Drosophila and SL-2 cells. No Aß secretion was observed in conditioned media, suggesting that the total suppression of the expression of Psn by RNAi results in a complete loss of gamma-secretase activity. Thus, Psn-dependent gamma-secretase activity is required for Aß generation from a human ßAPP derivative (i.e., SC100) in Drosophila S2 cells (Takasugi, 2002).
Alzheimer's disease is associated with increased production and aggregation of amyloid-ß (Aß) peptides. Aß peptides are derived from the amyloid precursor protein (APP) by sequential proteolysis, catalysed by the aspartyl protease BACE, followed by presenilin-dependent gamma-secretase cleavage. Presenilin interacts with nicastrin (see Drosophila nicastrin) , APH-1 and PEN-2, all of which are required for gamma-secretase function. Presenilins also interact with alpha-catenin, ß-catenin and glycogen synthase kinase-3ß (GSK-3ß), but a functional role for these proteins in gamma-secretase activity has not been established. Therapeutic concentrations of lithium, a GSK-3 inhibitor, block the production of Aß peptides by interfering with APP cleavage at the gamma-secretase step, but do not inhibit Notch processing. Importantly, lithium also blocks the accumulation of Aß peptides in the brains of mice that overproduce APP. The target of lithium in this setting is GSK-3alpha, which is required for maximal processing of APP. Since GSK-3 also phosphorylates tau protein, the principal component of neurofibrillary tangles, inhibition of GSK-3alpha offers a new approach to reduce the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimer's disease (Phiel, 2003).
In summary, this study shows that GSK-3alpha facilitates APP processing and that lithium inhibits the generation of Aß peptides through inhibition of GSK-3alpha. In support of this conclusion: (1) lithium reduces Aß production in cultured cells and in the brains of mice that overproduce Aß peptides; (2) kenpaullone, an alternative GSK-3alpha inhibitor, also inhibits Aß production; (3) RNAi-mediated depletion of GSK-3alpha reduces Aß production, and (4) moderate overexpression of GSK-3alpha increases Aß production. Lithium inhibits the GSK-3-mediated phosphorylation of tau, which, in its hyperphosphorylated state, is the main component of neurofibrillary tangles. Thus, GSK-3alpha offers an attractive target for pharmacological agents aimed at reducing the formation of amyloid plaques and neurofibrillary tangles, the pathological hallmarks of Alzheimer's disease. Lithium also protects neurons from proapoptotic stimuli and could therefore reduce neuronal cell death associated with Alzheimer's disease. Lithium has been used for more than 50 years to treat bipolar disorder, but has a narrow therapeutic window and a higher frequency of side effects in older patients. Thus, although lithium might be considered for the prevention of Alzheimer's disease, especially in younger patients with FAD mutations or Down's syndrome, new agents that specifically target GSK-3alpha may prove to be valuable in the treatment of Alzheimer's disease (Phiel, 2003).
The abnormal accumulation of beta-amyloid (Abeta) in the brain is an early and invariant feature in Alzheimer's disease (AD) and is believed to play a pivotal role in the etiology and pathogenesis of the disease. As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Abeta. As with any peptide, however, the degree of Abeta accumulation is dependent not only on its production but also on its removal. In cell-based and in vitro models endothelin-converting enzyme-1 (ECE-1) has been characterized as an Abeta-degrading enzyme that appears to act intracellularly, thus limiting the amount of Abeta available for secretion. To determine the physiological significance of this activity, Abeta levels were analyzed in the brains of mice deficient for ECE-1 and a closely related enzyme, ECE-2. Significant increases in the levels of both Abeta40 and Abeta42 were found in the brains of these animals when compared with age-matched littermate controls. The increase in Abeta levels in the ECE-deficient mice provides the first direct evidence for a physiological role for both ECE-1 and ECE-2 in limiting Abeta accumulation in the brain and also provides further insight into the factors involved in Abeta clearance in vivo (Echman, 2003).
Two substrates of insulin-degrading enzyme (IDE), amyloid beta-protein (Abeta) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. IDE has been identified as a principal regulator of Abeta levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Abeta and insulin degradation in vivo and chronically increases their levels, mice with homozygous deletions of the IDE gene were characterized. IDE deficiency resulted in a >50% decrease in Abeta degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE minus mice showed increased cerebral accumulation of endogenous Abeta, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the beta-amyloid precursor protein, which is degraded by IDE in vitro. Together with emerging genetic evidence, these in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD (Farris, 2003).
Factors that elevate Abeta peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in Abeta degradation based on its hydrolysis of Abeta peptides in vitro. In this study, in vivo levels of brain Abeta40 and Abeta42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the gamma-secretase-generated C-terminal fragment of the Abeta precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased approximately 50%, brain Abeta peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Abeta peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease (Miller, 2003).
Converging evidence suggests that the accumulation of cerebral amyloid ß-protein (Aß) in Alzheimer's disease (AD) reflects an imbalance between the production and degradation of this self-aggregating peptide. Upregulation of proteases that degrade Aß thus represents a novel therapeutic approach to lowering steady-state Aß levels, but the consequences of sustained upregulation in vivo have not been studied. Transgenic overexpression of insulin-degrading enzyme (IDE) or neprilysin (NEP) in neurons has been shown to significantly reduce brain Aß levels, retard or completely prevent amyloid plaque formation and its associated cytopathology, and rescue the premature lethality present in amyloid precursor protein (APP) transgenic mice. These findings demonstrate that chronic upregulation of Aß-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo (Leissring, 2004).
gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. This study reports the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147 (see Drosophila Basigin), a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression has no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides (Zhou, 2005; full text of article).
Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. A genetic screen was set up based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. Clones were selected deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. Two of these clones were examined and previously undescribed mutations were identified in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired (Olry, 2005).
Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Aβ homeostasis. ApoE plays a role in facilitating the proteolytic clearance of soluble Aβ from the brain. The endolytic degradation of Aβ peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Aβ degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Aβ degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Aβ degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Aβ load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Aβ from the brain and suggest that LXR agonists may represent a novel therapy for AD (Jiang, 2008).
An isoform of apolipoprotein E, ApoE4, has been shown to confer dramatically increased risk for late-onset AD (LOAD); however, the basis for this remains one of the major unanswered questions of disease pathogenesis. ApoE plays critical roles in regulating brain Aβ peptide levels, as well as their deposition and clearance. Thus, processes that regulate ApoE expression and functional state could affect its ability to influence brain Aβ homeostasis. ApoE is the predominant apolipoprotein in the brain and is synthesized and secreted mainly by astrocytes (but also by microglia) within unilamellar HDL-like particles. ApoE is lipidated principally through the action of the ATP-binding cassette transporter ABCA1 (and related transporters), which acts in a variety of cell types to transfer both phospholipids and cholesterol to ApoE, and, in this way, ApoE acts to traffic lipids throughout the brain. The lipidation status of ApoE is an important functional parameter, governing its conformation, intrinsic stability, and interactions with membrane receptors. Importantly, ApoE binds to Aβ, and this, too, is influenced by its lipidation status. Studies with APP transgenic mice have demonstrated ApoE isoform-specific effects on the propensity of Aβ to be deposited in the brain (E4 > E3 > E2), the nature of the deposits, and a gene-dosage-related influence on the magnitude of these effects. ApoE lipidation status is also a significant determinant of whether its interaction with Aβ leads to efflux of the peptides from the brain or, alternatively, to the formation of fibrils and their deposition into plaques. Recently, three independent studies have reported that inactivation of the Abca1 gene in APP-expressing transgenic mice resulted in reduced levels of ApoE. Remarkably, these mice exhibited a seemingly paradoxical elevation of brain Aβ peptide levels and a doubling of Aβ plaque burden. The outcomes of these studies strongly suggested that lipidated forms of ApoE act to enhance the clearance of Aβ peptides from the brain. The aim of the present study was to establish the mechanism by which ApoE and its lipidation affect Aβ homeostasis (Jiang, 2008).
Liver X receptors (LXRs) are ligand-activated transcription factors that induce the expression of Apoe, Abca1, and other genes of lipid metabolism. LXRs act physiologically as cellular cholesterol sensors and are activated by oxysterols. There are two LXR isoforms, LXRα and LXRβ (encoded by Nrlh3 and Nrlh2, respectively), both of which are expressed in the brain, and their activation results in the rapid and robust increase in the levels of lipidated forms of ApoE. Thus, regulation of LXR transcriptional activity provides a mechanism to regulate brain ApoE levels and its lipidation status (Jiang, 2008).
The brain possesses robust intrinsic Aβ clearance mechanisms. Aβ peptides are proteolytically degraded within the brain principally by neprilysin (NEP) and insulin-degrading enzyme (IDE, insulysin). Genetic inactivation of these genes or administration of inhibitors of these proteinases into the brain results in substantial elevation of Aβ levels in the brain and induction of plaque deposition. Conversely, overexpression of IDE or neprilysin lowered brain Aβ levels and reduced plaque formation. It has been argued that the predominant mode of Aβ42 clearance from the brain is through its proteolytic degradation because this peptide is not efficiently exported through the vasculature. Microglia, the brain's resident macrophages, play an essential role in Aβ clearance through their ability to take up and degrade soluble and fibrillar forms of Aβ. Moreover, both microglia and astrocytes secrete proteinases, including IDE, that mediate the degradation of Aβ peptides in the extracellular milieu (Jiang, 2008).
Despite considerable effort, the cellular mechanisms through which ApoE influences Aβ clearance remain unresolved. This paper reports that ApoE acts to facilitate the proteolytic degradation of Aβ, a previously unappreciated action of this apolipoprotein. Moreover, the lipidation status of ApoE is a critical determinant of its ability to stimulate Aβ degradation, and this finding provides a mechanistic explanation of the increased Aβ levels and deposition observed in APP-expressing mice lacking the Abca1 gene. Importantly, it was demonstrated that elevation of lipidated forms of ApoE, through activation of LXRs, results in reduced Aβ peptide and plaque levels in an animal model of AD and is associated with improved contextual memory. Therapeutic agents that increase the abundance of highly lipidated forms of ApoE, including LXR agonists, may attenuate disease pathogenesis and represent a promising strategy for the treatment of AD (Jiang, 2008).
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