Amyloid-b Peptide Production: Alpha Secretase
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Published: Mon, 14 May 2018
Ab is generated by the proteolytic cleavage of APP by a group of enzymes known as secretases [Esler and Wolfe, 2001; Samabamurti et al., 2002]. APP is processed by alternative proteolytic pathways that generate different breakdown products. In the secretory pathway, at least three different secretases appear to be active. The majority of APP is cleaved at Ab16 by ‘‘a-secretase’’ to generate soluble amino-terminal derivatives of APP (sAPPa). This cleavage bisects the Ab peptide and thus prevents the formation of amyloidogenic fragments. Secreted derivatives of APP (sAPP) lacking the cytoplasmic tail, transmembrane domain and a small portion of the extracellular domain generated by the proteolytic processing of full-length APP, have been detected in the conditioned medium of several cell cultures, human plasma and in cerebrospinal fluid (CSF). An alternative secretase cleavage produces truncated sAPP containing a potentially amyloidogenic sequence. This product sAPPg presumably is a result of the cleavage by g-secretase. Secretion of APP cleaved at the amino-terminus of Ab (sAPPb) has also been seen in several systems, which is as a result of cleavage by b-secretase.
Beta Secretase Enzyme (BACE)
The enzymes involved in the generation of Ab are key components of APP metabolism that are targets for drug development [Hussein et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Dingwall, 2001]. Exciting developments in the last year have identified key candidate enzymes with these activities. The identification of these proteins should lead to an understanding of the agents that regulate these activities to overproduce Ab42 in AD, and serve as useful therapeutic targets. Two novel aspartyl proteases were identified by several approaches and were shown to be capable of bsecretase activity. The major activity appears to be mediated by the enzyme named BACE, Asp-2, or Memapsin by different workers. This enzyme fulfills several criteria for b-secretase activity including its substrate specificity and effects of modulating cellular expression.
TABLE 5. Selected Target/Products From Different Companies
Amgen Nerve growth and repair
Brsitol-Myers Squibb Gamma secretase inhibitor
Cephalon Gamma secretase inhibitor
Elan Beta amyloid inhibitor vaccine
Eli Lilly SERM; gamma secretase inhibitor
Glaxo Wellcome APOE modification, genetics
Merck Gamma secretase inhibitor
Pfizer Acetylcholinesterase and gamma secretase inhibitor
Schering-Plogh Presenilin gene function Vertex Nerve growth and repair
nSource: Lexis-Nexis, Inc.
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Gamma Secretase: Presenilin I
The presenilins have been implicated in AD by virtue of their key role in the proteolysis of APP [Haass and De Strooper, 1999]. It has been proposed that presenilins are g-secretases [Fraser et al., 2001]. Recent studies showing that g-secretase activity can be immunoprecipitated with presenilins in vitro strengthen this hypothesis. This activity is in a large multisubunit complex indicating that other proteins may also play an essential role in the g-secretase activity, and inhibitors of g-secretase activity have been reported that apparently do not affect Notch signaling; reported to be associated with presenilin activity. The processing of APP by different secretase enzymes and the resultant products are schematically shown in Fig. 1.
Amyloid-b Peptide Aggregation
Amyloid plaques are derived from soluble amyloid by aggregation into b-pleated sheet structures.
One hypothesis suggests that the plaque or aggregated amyloid is actually responsible for triggering the neurodegeneration in AD brain. If this hypothesis is correct, agents that break up the plaques or interfere in protein folding to allow their aggregation can serve as useful therapeutic agents [Soto et al., 1998, 2000; Cherny et al., 2001]. An endogenous melatonin-related indole structure, indole-3-propionic acid, has shown potent neuroprotective properties against Ab [Chyan et al., 1999] and, also, it appears that melatonin may reverse the profibrillogenic activity of apolipoprotein e4 on Ab peptide [Poeggeler et al., 2001].
Amyloid-b Peptide Turnover
The net yield of Ab for deposition of plaques is dependent both on its production and its turnover in the brain. Recent studies have identified several pathways for Ab turnover including the insulindegrading enzyme and neprilysin [Iwata et al., 2001].
APP Regulatory Elements
From recent cellular, genetic, and clinical reports, there is strong support for the hypothesis that Ab42 production followed by its aggregation is the central process responsible for the neurodegeneration in AD.
It is thus important to examine the pathways involved not only in the regulation of Ab levels but also its precursor protein (APP). Moreover, studies of the human brain have suggested a higher APP geneexpression ratio than predicted between Down’s syndrome (DS) and normal controls [Tanzi et al., 1987]. The apparent overexpression of the APP gene in DS and in certain areas of the brain in AD patients suggests that overexpression might be an important factor in the neuropathology of AD [Johnson et al., 1990; Rumble et al., 1989] and that cell type-specific regulation of APP gene expression may be altered in AD. The structure and function of the APP regulatory regions, such as promoter and enhancer elements, have recently been investigated, and such studies indicate that these APP regulatory regions may play a pivotal role in the pathogenesis of AD [Lahiri and Robakis, 1991; Salbaum et al., 1988]. In this regard, the human, mouse, rat, and monkey APP promoters have been functionally characterized [Song and Lahiri, 1998].
These recent studies have unveiled the role of different cis-acting regulatory regions of the promoter on the transcriptional control of APP gene expression. Several growth factors and proinflammatory cytokines can increase the expression of APP and augment Ab deposition [Lahiri et al., 2000b]. For example, the coexpression of transforming growth factors (TGF-b1) in human APP transgenic mice have been shown to cause an increased production of APP and Ab deposition in cerebral blood vessels and meninges [Mattson et al., 1997]. Ab has also been shown to induce the production and secretion of interferon-g (IFN-g) and interleukin-1 (IL-1) in human vascular endothelial cells [Suo et al., 1998]. Studies with primary cultures [Goldgaber et al., 1989] and neuronal cells [Lahiri and Nall, 1995] have indicated that NGF, FGF, and IL- 1 increase APP mRNA and promoter levels. Moreover, Fig. 1. Major proteolytic events in APP processing. The full-length APP is a type-I integral membrane protein. It is cleaved in the extracellular domain of Ab by b-secretase and within Ab by a-secretase to generate secreted derivatives sAPPb and sAPPa and C-terminal fragments CTFb and CTFa, respectively. The enzyme g-secretase cleaves CTFb to Ab and CTFa to a smaller fragment P3.
Ab and P3 terminating at residue 40 are predominant but a small percentage is longer terminating at residues 42 and 43.
DRUG TARGETS FOR ALZHEIMER’S DISEASE 275
NGF treatment induces transcription of TGF-b1 through a specific promoter element on PC12 cells [Kim et al., 1994]. The regulation of a neural-specific gene is mediated by transcription factors that interact with NGF-responsive elements [Luc and Wagner, 1997]. These results suggest that Ab, acting in concert with proinflammatory cytokines, could potentially trigger a self-propagating cycle of APP overexpression resulting in an increased Ab deposition. Thus, factors that control both APP expression and processing are of key significance in AD pathogenesis.
In addition, it has recently been demonstrated that the cytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein, Fe65, and the histone acetyltransferase, Tip60 [Cao and Sudhof, 2001]. This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, and suggests that the release of the cytoplasmic tail of APP by gamma-cleavage may function in gene expression.
One can, therefore, postulate that it may be of therapeutic benefit to block this interaction, opening up further avenues for drug design and development.
APP, Ab, and Cholinesterase Inhibitors
Cholinesterase inhibitors (ChEIs), which, as described, are the only FDA approved drugs for the treatment of AD patients, can regulate APP processing in cell lines [Lahiri et al., 2000]. For example, the treatment of a human neuroblastoma cell line with tacrine or phenserine markedly suppressed the secretion of sAPPa and Ab [Lahiri et al., 1994; 1998; Shaw et al., 2001]. For phenserine, this action is noncholinergic as the antipode (the (þ)-enantiomer that lacks anticholinesterase action) similarly reduces sAPPa and Ab, and is allowing the development of a new class of agents that are cholinergically silent but possesses Ab lowering properties. The lowering of sAPP levels without a concomitant rise in Ab levels is another novel property of certain cholinesterase inhibitors, which can be further optimized for better clinical effects. Indeed, the effect of anticholinesterases on APP secretion is different from that of muscarinic agonists, such as carbachol, that stimulate sAPPa secretion after binding m1 receptors and activating PKC [Nitsch et al., 1992]. The dual mechanism of action of specific cholinesterase inhibitors and muscarinic agonists (i.e., cholinergic stimulation and Ab lowering) remains an attractive therapeutic strategy for AD treatment that requires further optimization.
50-UTR of APPmRNA: A Novel Target
As discussed previously, studies with primary cultures and neuronal cells have shown that NGF, FGF, and IL-1 increase both APP mRNA and promoter levels [Goldgaber et al., 1989; Lahiri and Nall, 1995]. In addition, some factors can also regulate protein levels by their effects at the post transcriptional level via the 50-untranslated region (UTR). For example, evidence for the regulation of translation of the APP mRNA by an IL-1 responsive element has been reported [Rogers et al., 1999]. Notably, a cholinesterase inhibitor, phenserine, has recently been shown to reduce the translation of APP by taking advantage of its translational regulation via the 50-UTR [Greig et al., 2000a; Shaw et al., 2001]. This 50-UTR thus represents a novel target for AD drug development.
However, our current understanding of the cellular and molecular mechanisms that connect inflammatory cytokines to APP gene regulation is limited. Similarly, whether or not the large 30-UTRAPP mRNA can control protein levels and to what extent the 30-UTR can be used as a potential drug target remains to be fully explored.
The phosphorylated form of tau has been shown to be functionally inactive [Goedert and Spillantini, 2001]. Recently, mutations in a splice site of tau have been shown to result in an increase in the ratio of fourrepeat tau. This mutation is linked to a form of dementia with NFT formation in the absence of amyloid deposition. The finding that mutations in the tau protein, an important component of NFT, can also lead to neurodegeneration suggests that NFT formation may be the cause and not the consequence of neurodegeneration. Secretion of abnormally glycosylated forms of tau into the CSF is reported to be an early event in the AD brain and may eventually be developed as an early diagnostic marker in AD.
Although phosphorylated tau is known to be present in NFT, this cannot explain the deposition of tau as tangles. It has been reported that the four-repeat tau that is generated by mutations in the tau gene associated with dementia is due to the tau protein’s capacity to aggregate rapidly.
Recent studies indicate a link between heart disease, cholesterol, and AD [Simons et al., 2001; Sparks et al., 2000]. Notably, cholesterol has been shown to accumulate in senile plaques of AD patients and in transgenic APP(SW) mice [Mori et al., 2001].
This observation has strong therapeutic implications.
For example, a cholesterol-lowering statin drug has been shown to reduce beta-amyloid pathology in a transgenic mouse model of AD [Refolo et al., 2001].
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Epidemiological studies have suggested that the taking of HMG-Coreductase inhibitors reduces the risk of developing AD in the normal elderly population.
Finally, an independent study has reported that Simvastatin strongly reduces levels of AD the betaamyloid peptides, Ab-42 and Ab-40, both in vitro and in vivo [Fassbender et al., 2001].
NONSTEROIDAL ANTI-INFLAMMATORY DRUGS AND APP
Several recent studies suggest that the use of nonsteroidal anti-inflammatory (NSAIDs) drugs are associated with a reduced risk for developing AD [In t’ Veld et al., 2001]. Moreover there is a report of using celastrol, which is a potent antioxidant and antiinflammatory drug, as a possible treatment for AD [Allison et al., 2001]. Unfortunately, recent studies with Prednisone, Celebrex and Vioxx have not been beneficial in patients with established AD. Interestingly, other connections between NSAIDs and the amyloid pathways have been made. For example, it has been recently shown that a subset of NSAIDs lower amyloidogenic Ab-42 independently of cyclooxygenase activity [Weggen et al., 2001]. These recent studies have rekindled some enthusiasm for an anti-inflammatory drug to treat AD [De Strooper and Konig, 2001].
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