- Research article
- Open Access
Viable mouse gene ablations that robustly alter brain Aβ levels are rare
© Toyn et al; licensee BioMed Central Ltd. 2010
- Received: 9 June 2010
- Accepted: 5 November 2010
- Published: 5 November 2010
Accumulation of amyloid-β (Aβ) peptide in the brain is thought to play a key pathological role in Alzheimer's disease. Many pharmacological targets have therefore been proposed based upon the biochemistry of Aβ, but not all are equally tractable for drug discovery.
To search for novel targets that affect brain Aβ without causing toxicity, we screened mouse brain samples from 1930 novel gene knock-out (KO) strains, representing 1926 genes, using Aβ ELISA assays. Although robust Aβ lowering was readily apparent in brains from a BACE1 KO strain, none of the novel strains exhibited robust decreases in brain Aβ, including a GPR3 KO strain, which had previously been proposed as an Aβ target. However, significantly increased Aβ was observed in brain samples from two KO strains, corresponding to genes encoding the glycosylphosphatidylinositol mannosyl transferase PIGZ and quinolinate phosphoribosyltransferase (QPRT).
Thus, gene ablations that are permissive for mouse survival and that also have a robust effect on Aβ levels in the brain are rare.
- Amyloid Precursor Protein
- Quinolinic Acid
- Kynurenine Pathway
- Tryptophan Degradation
- False Positive Error Rate
The amyloid hypothesis states that Alzheimer' disease (AD) is caused by accumulation of toxic forms of the amyloid-β (Aβ) peptide in the brain. Aβ is a secreted peptide formed through consecutive proteolytic cleavages of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE1), which releases the N-terminal end of Aβ, and γ-secretase, which releases a range of Aβ C-terminal ends resulting in Aβ peptides of typically 37-42 amino acids in length. The predominant form of Aβ has 40 amino acid residues, and is denoted Aβ40, whereas the disease-associated Aβ42 has two additional C-terminal residues . On the basis of the biochemistry and pathology of Aβ, many molecular targets have been proposed for inhibition of Aβ accumulation, aggregation, or the toxic effects of Aβ [2–5]. Thus, Aβ formation can be targeted directly via inhibition of BACE1 or γ-secretase, or indirectly via inhibition of pathways that regulate the activity of these proteases. Indirect regulation of BACE1 involves a particularly wide range of mechanisms, recently reviewed in detail by Hunt and Turner , and of potential pathological relevance because of the increased BACE1 activity observed in the AD brain [7–10]. In brief, BACE1 activity can be regulated through a variety of molecular targets involved in cytokine signaling [11, 12], hypoxia [13, 14], oxidative stress , energy deprivation , intracellular trafficking and maturation [17–19], and glycosylphosphatidylinositol (GPI) anchor metabolism [20–22]. Indirect targets have also been reported to regulate γ-secretase activity, including GSK3α , Rac1 GTPase , casein kinase I  and the G-protein coupled receptor GPR3 . In addition, competition between the proteasome and γ-secretase for the C-terminal BACE1-derived APP processing intermediate can affect Aβ levels in cell cultures .
APP itself is a direct target of small molecule modulators of Aβ production [28, 29], and can be targeted indirectly via the prolyl isomerase Pin1 , sphingolipid metabolism , reticulon/Nogo proteins [32, 33], Nogo-like LRRTM3 , sorLA  and membrane microdomain switching . APP-mediated changes in Aβ can also result from increased cleavage of the non-amyloidogenic α-site of APP, thus competing with BACE1 for the available APP substrate, as reviewed by Fahrenholz . In brief, metalloproteases such as ADAM10  carry out α-site cleavage, which can be activated via multiple targets, including retinoic acid receptor [39, 40] liver-X-receptor, muscarinic acytylcholine receptor M1 [41, 42], G protein coupled receptor PAC1 , protein kinase C [44, 45], and low cholesterol .
In addition to the regulation of Aβ biosynthesis, Aβ clearance is also regulated. Clearance of Aβ combines several mechanisms, including the LXR/ABCA1/APOE pathway , degradation by endoproteases, reviewed by Nalivaeva et al. 2008 , transport across the blood-brain barrier involving RAGE and LRP1 receptors, reviewed by Deane and Zlokovic , lymphatic drainage, reviewed by Weller et al.  and microglial uptake of Aβ [51, 52]. Indirect regulation of Aβ clearance has also been reported, for example, modulation of neprilysin endoprotease by somatostatin receptor signaling , and enhanced Aβ proteolysis dependent on the ApoE isoform . In addition, resveratrol, a polyphenol in red wine, has been proposed to enhance Aβ clearance via the proteasome .
Thus, it seems reasonable to anticipate numerous molecular targets capable of altering Aβ levels, and that at least some of the targets should be relevant to Aβ formation in the brain. The ideal target should have the potential for robust brain Aβ-lowering without toxicity, and characteristics that facilitate development of inhibitors. We therefore took the approach of direct screening of brain Aβ levels in novel mouse gene knock out (KO) strains, an approach that has the dual advantages of providing evidence both for target effectiveness in Aβ-lowering and for target safety. Given that even optimized drug molecules may not be capable of 100% ablation of target function, we were most interested in finding KO strains with 50% or more reduction in brain Aβ levels. A total of 1930 viable homozygous gene ablations, representing 1926 genes, were tested. Surprisingly, none of these gene ablations exhibited robust decreases in Aβ. In addition, we also evaluated GPR3 KO mice, recently proposed as an Aβ target , but found no overall effect on levels of brain Aβ. In contrast, significantly increased brain Aβ was detected in samples from two mouse KO strains corresponding to the proteins PIGZ and QPRT, respectively involved in GPI anchor biosynthesis and the kynurenine pathway of tryptophan degradation. Thus, while the mouse KO screen did not directly identify novel targets for lowering Aβ, it did suggest a limited number of biochemical pathways that might be significant for regulation of brain Aβ levels in vivo, and that gene ablations causing a robust effect on brain Aβ are rare.
A screen of mouse KO strains for altered brain Aβ levels
Summary of genes by class
G-Protein coupled receptor
Membrane and secreted
Nuclear hormone receptor
Receptor associated protein
Paired t-tests conducted on UBE2R2, ADRM1, PIGZ and QPRT KO strains
Left vs. right comparisons
t-value (degrees of freedom)
Ablation of GPR3 did not affect total brain Aβ levels
Comparisons between GPR3 KO and wild type animals using independent samples t-tests
t-value (degrees of freedom)
Aβ40 young -/- vs +/+
Aβ40 young +/- vs +/+
Aβ42 young -/- vs +/+
Aβ42 young +/- vs +/+
Aβ1-x young -/- vs +/+
Aβ1-x young +/- vs +/+
Aβ40 old -/- vs +/+
Aβ42 old -/- vs +/+
To identify novel molecular targets relevant to regulation of Aβ levels in the brain, we screened viable mouse KO strains for decreased brain Aβ40. A total of 1930 different gene ablations giving rise to viable homozygous mice were evaluated. Two of the strains, PIGZ KO and QPRT KO, showed an unequivocal increase of Aβ40 and Aβ42 in the screening samples, whereas changes in Aβ were less obvious for the other KO strains. The two KO strains showing the lowest values of Aβ40 and Aβ42 in the screen, UBE2R2 and ADRM1, were of uncertain significance given the small number of samples tested.
Given the wide variety of mechanisms and proteins that have been reported to affect Aβ, and the relatively large number of gene KO strains tested, it was surprising that we did not identify a single new gene KO strain that caused a robust (≥50%) decrease in Aβ. A combination of several reasons may account for this. First, the choice of gene KO strains entering the screen was based on the 'druggable genome', which mostly limited the KO strains to proteins in gene families known to interact with small molecules. Second, the gene KO strains entering the screen were limited to those strains that resulted in viable homozygous adult mice. Approximately one third of the gene KO constructs made were homozygous embryonic lethal, and were consequently not included in the brain Aβ screen. Therefore, effective Aβ-lowering targets such as presenilin 1, for which genetic ablation is deleterious, would not have been tested in this screen. Third, functional redundancy, e.g. the genes encoding the Aph1B and Aph1C subunits of γ-secretase , could have obscured any effects of single gene ablations. There is also the possibility of developmental compensation, in which alternative pathways functionally substitute for the missing gene, thus restoring Aβ levels in the adult. Fourth, inbred mice have been shown to express significantly different levels of brain Aβ , raising the possibility that genetic changes in these mice may have obscured the function of some genes (epistasis). Fifth, there is the possibility that some genes may affect Aβ levels only in older mice, and therefore the role of these putative genes would not have been apparent at the age of three months when our mice were harvested. Sixth the ability of the screen to detect changes in Aβ was limited by the intrinsic variability in Aβ combined with the small group size, which in most cases was equal to four homozygous KO animals. This limitation to four animals per KO strain was necessary because of the resource and time constraints of producing and maintaining multiple KO mouse colonies, and the use of most of the available KO mice for other phenotypic and biochemical tests not reported here. Nevertheless the statistical power of the screen was favorable. Based on the good fit of the data to the normal distribution (Shapiro-Wilk's test p > 0.05), the false negative error rate (Type II error) was found to be 1.7% for detection of a KO strain with 50% lowering of Aβ, and 5.99E-9 for a KO strain with 85% lowering of Aβ, as observed in the BACE1 KO samples. Thus, there was a high probability for detection of any KO strains robustly lowering Aβ. In addition, the most practical Aβ-lowering targets should have the potential to lower Aβ by a robust and substantial amount, and thus, for the purpose of identifying the most practical targets, a low group size could be tolerated. The false positive error rate (Type I error) for a KO strain more than 3 sd below the mean was 0.13%, and for 10 sd above the mean was negligible at less than 6.7E-16. Thus, the results for the two KO strains, UBE2R2 and ADRM1, which exhibited the lowest brain Aβ levels around three standard deviations below the mean, could have been due to chance, whereas the Aβ increases in the PIGZ and QPRT KO strains were very unlikely due to chance. Finally, despite the possible limitations discussed above, it is hard to escape the conclusion that molecular targets capable of robust Aβ lowering in the relevant context of brain are rare.
Brain Aβ40, Aβ42 and Aβ1-x levels in GPR3 KO mice were evaluated more thoroughly by using a larger number of mice. No changes in brain Aβ42, Aβ40 or Aβ1-x were detected in sagittal brain halves from these mice. This contrasts with the results reported by Thathiah et al.  in which ca. 50% lowering of Aβ40 and Aβ42 was observed in APP-transgenic GPR3 KO mice. There are two noteworthy experimental differences between the two studies, first, we assayed endogenous mouse Aβ, not transgenic human Aβ, and second, we used sagittal brain halves not hippocampal sections. Unfortunately, our analysis did not extend to hippocampal sections, which constitutes only a small fraction of total brain. GPR3 is expressed at high levels in the cortex, which, like hippocampus, is relevant to AD. Thus, evaluation of Aβ in the hippocampus of non-APP GPR3 KO and in the cortex and/or whole brain of APP transgenic GPR3 KO would be interesting.
Two gene ablations, corresponding to the PIGZ and QPRT enzymes, exhibited significantly increased Aβ40 and Aβ42 in the screening samples. While further substantiation of the results for PIGZ and QPRT using larger groups of homozygous KO mice is clearly desirable, plausible molecular mechanisms for increased Aβ can be proposed. PIGZ, also known as SMP3, is a mannosyl transferase that catalyses addition of a fourth side chain mannose to the glycosylphosphatidylinositol (GPI) protein anchor precursor [58, 59]. In cell cultures, GPI anchored proteins are necessary for Aβ synthesis , and targeting of an artificial BACE1-GPI chimera to lipid rafts greatly increases Aβ production , although targeting of BACE1 to lipid rafts is not necessary for Aβ synthesis . Thus, in cell culture, a connection between GPI anchor metabolism and Aβ levels is well established. An effect of PIGZ on brain Aβ would extend these conclusions to a relevant organ in vivo, and further raises the possibility that the fourth mannose residue plays a specific role in Aβ metabolism. QPRT is the enzyme responsible for quinolinic acid turnover in the kynurenine pathway of tryptophan degradation , and therefore ablation of this gene would be expected to increase quinolinic acid levels. Increased quinolinic acid has been reported in AD brain [62, 63], and treatment of primary neuronal cultures with quinolinic acid has been reported to increase cell death and oxidative stress . The association of oxidative stress with increased BACE1 activity and Aβ production has been widely substantiated in AD [10, 15, 65–69], raising the possibility of a mechanistic connection between quinolinic acid and Aβ through activation of BACE1 by oxidative stress. In addition, quinolinic acid has been reported to increase APP expression in rat brain, which could contribute to increased Aβ production .
The two KO strains for which the lowest values of Aβ40 and Aβ42 (ca. 30% lowering) were observed in our screen corresponded to the UPS proteins Adrm1 and Ube2R2. Adrm1, also known as hRpn13, associates with the proteasome 19S regulatory particle, and is required for recruitment of the Uch37 deubiquitinating enzyme to the proteasome [71, 72]. Ube2R2 (sequence NM-017811) is a ubiquitin conjugating enzyme. Decreased expression of several other ubiquitin conjugating enzymes has been reported to decrease Aβ production in cell culture . The UPS has multiple potential roles in AD in addition to possible regulation of Aβ levels, as recently reviewed in detail by Upadhya and Hegde . Possible mechanisms of proteasomal regulation of Aβ include resveratrol-activated clearance of Aβ , and competition with γ-secretase for APP processing . Thus, an intriguing possibility is that selective inhibition of specific sub-pathways of the UPS might decrease brain Aβ levels by both biosynthetic and clearance mechanisms. However, from a drug discovery perspective, this would carry the risk of further exacerbating the already defective proteasome activity prevalent in AD thought to result from the accumulation of toxic Aβ and tau aggregates. Furthermore, assuming that maximal inhibition of Adrm1 or Ube2R2 would elicit only a 30% decrease in brain Aβ, even the effect of an inhibitor with ideal drug properties would be limited, and the expected small changes in Aβ difficult to quantify.
Gene ablations that have a robust effect on brain Aβ appear to be rare, at a rate of approximately one in a thousand of the genes reported here. However, several pathways including GPI anchor metabolism, the kynurenine pathway of tryptophan degradation, and the UPS may be worth further evaluation for their roles in brain Aβ regulation.
Mouse KO strains and brain samples
Experimental procedures with mice were authorized by, and in compliance with, the Lexicon Pharmaceuticals Animal Care and Use Committee, and the Bristol-Myers Squibb Animal Care and Use Committee. The BACE1, BACE2 and GPR3 KO strains were provided by Lexicon Pharmaceuticals under the terms of the LexVision® Database and Collaboration Agreement. The BACE1/BACE2 double KO obtained by intercrossing the BACE1 and BACE2 KO strains has been described previously . The GPR3 KO was made by targeted homologous recombination in mouse strain 129SvEvBrd-derived embryonic stem cells, using a targeting construct containing a bGeo/puromycin selection cassette to remove 1,023 nucleotides encompassing the entire amino acid coding region in the single exon of this gene (see NCBI nucleotide reference sequence NM_008154 for GPR3). Recombination in ES cells was confirmed by Southern analysis. Chimeric mice were bred with C57BL/6J albino mice to generate F1 heterozygous animals. The F1 mice were intercrossed, and the genotypes of F2 progeny were determined by Southern analysis. The observed genotype frequencies, 20 wild type, 33 heterozygous and 15 homozygous mice, were not significantly different from the Mendelian segregation ratios expected for a viable allele. The genotypes of further F2 progeny were determined by PCR of tail or ear DNA using the DNA primer 5'-GAATTAAGCCCTGGTGGACCTA, corresponding to sequence adjacent to the GPR3 deletion, in combination with the primer 5'-GTTGCCCTTCACTGTCTACTGC, corresponding to deleted GPR3 sequence, to detect a 286 nucleotide product from the wild type GPR3 allele, or in combination with the primer 5'-GCAGCGCATCGCCTTCTATC, derived from the neo marker gene, to detect a 208 nucleotide product from the GPR3 KO allele. For the GPR3 KO studies, animals were either 3 months old 'young' or 1 year old 'aged' at the time of harvesting. The other 1930 KO strains, corresponding to 1926 different genes, including the PIGZ, QPRT, and ADRM1 KO strains derived from the OmniBank® gene trap library, and the UBE2R2 KO strain made by targeted homologous recombination, were made by Lexicon Pharmaceuticals as part of its Genome5000™ program . A summary of the 1926 genes by gene class is shown in Table 1.
Preparation and storage of brain samples
For the primary screen of brain Aβ levels, four mice from each KO strain were euthanized at three months of age by CO2 asphyxiation and sagittal brain halves lacking cerebellum were frozen and stored at -80°C. Samples were shipped on dry ice from Lexicon Pharmaceuticals to Bristol-Myers Squibb as they became available over a period of four years. Because the availability of individual mice occurred with varied timing, and because multiple different KO strains were in production simultaneously, the order of acquisition of brain samples was variable. This resulted in the four samples from any given KO strain arriving at different times, and thus the Aβ ELISA being carried out usually on different assay plates and different assay dates for each sample of a given KO strain. The Aβ primary screen values obtained even for a given gene KO therefore represented essentially all of the sources of variability to which the data were subject.
Aβ sample preparation and ELISA assays
The assay used for the primary screen of all brain samples has been partly described previously . Frozen left brain halves were thawed and homogenized at a concentration of 4 ml/g in ice cold 2% CHAPS, 20 mM Tris-Cl pH 7.7, in the presence of complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Homogenization was carried out by agitation in 2.0 ml polypropylene tubes containing a steel bead (5 mm diameter) for 4 min. at maximum power using the TissueLyzer (Qiagen). Homogenates were centrifuged at 21,000 × g at 4°C for 30 min. Supernatant from the centrifugation was stored frozen at -80°C, and thawed immediately before use in the ELISA. ELISA to quantify Aβ was carried out in 96-well format using three wells per sample. The capture antibody was the Aβ40 C-terminal end-specific monoclonal antibody TSD9S3.2 (Bristol-Myers Squibb), and detection was carried out using peroxidase conjugated monoclonal antibody 252Q6 (Invitrogen), specific for the N-terminal region of mouse Aβ. To confirm results for selected brain samples, the corresponding frozen right brain halves were thawed and homogenized in CHAPS buffer for assay of Aβ40, as described above. Alternatively, right brain halves were homogenized at a concentration of 10 ml/g in 6M guanidine by agitation with a steel bead, as described above, centrifuged at 21,000 × g at 4°C for 60 min., and Aβ was concentrated from the supernatant by solid phase extraction using Oasis HLB cartridges (Waters) on a vacuum manifold as described . Eluates in methanol/0.1% NH4OH containing Aβ from the solid phase extraction were dried in a rotary evaporator under vacuum and resuspended in phosphate buffered saline pH 7.4 containing 0.1% bovine serum albumin (Sigma cat. #A7030) and 0.1% Triton X-100 immediately before use in ELISA. Aβ40 was quantified by ELISA in triplicate wells as described above, and Aβ42 was quantified by ELISA using the monoclonal 252Q6 (Invitrogen) as capture antibody, and Aβ42 C-terminal end-specific monoclonal 565 peroxidase conjugate (Bristol-Myers Squibb) as the detection antibody. To monitor assay performance, the first 109 assay plates contained control wells of wild type and BACE dKO extracts. The value of z' ranged from 0.5 to 0.74 for these first plates, indicating that any samples containing decreased Aβ should be readily detectable . To monitor assay performance without the need for BACE dKO animals, subsequent plates contained wild type extract control wells in which the detecting antibody incubation contained rat Aβ1-14 synthetic peptide (Anaspec) at a concentration of 1 μg/ml. All reagents, unless otherwise stated, were obtained from Sigma. The guanidine/solid phase extraction method, as described above, was also used for determination of Aβ42 and Aβ40 in the aged GPR3 KO study. For the young GPR3 KO animals, brain samples were homogenized in 0.2% diethanolamine, 50 mM NaCl and protease inhibitor cocktail (Roche) for Aβ42 and Aβ40 ELISAs as described above. The Aβ1-x assay utilized the combination of monoclonals 252Q6 and 4G8 (Covance). Assay results were calibrated using synthetic Aβ42 or Aβ40 peptides (Anaspec) and expressed as average pM concentration present in brain tissue prior to homogenization.
Evaluation of Aβ results
To maintain consistency in the Aβ40 determinations between assay plates, the baseline value of Aβ40 was set equal to the mean value of all wells containing brain samples, excluding the BACE1/2 double KO or Aβ1-14 inhibited wells. Thus, the plate baseline generally depended on the mean value of 72 wells per plate. Alternative baseline values determined from known concentrations of synthetic Aβ40 peptides, or determined from pools of brain extracts from wild type mice, were found to be less consistent over time, and were therefore not used for evaluation of Aβ40 baseline levels. Median values of Aβ40 were calculated to avoid the potentially disproportionate effect of unusually high or low individual samples that could occur if using mean values, however, in the final analysis, use of mean or median values yielded the same outcome. Initially, calculations were carried out using data from a limited number of KO strains, and therefore some initial follow up assays were carried out for KO strains for which the median Aβ40 value was less than 2 sd from baseline.
We thank Camellia Symonowicz, BMS, and Doreen Hewston, BMS, for management of the GPR3 KO colony, Jinwen Huang, BMS, for PCR genotyping of mice in the GPR3 KO colony, Timur Güngör, BMS, and J.D. Wallace, Lexicon Pharmaceuticals, for help during preparation of the manuscript.
- Younkin SG: Evidence that Aβ42 is the real culprit in Alzheimer's disease. Ann Neurol. 1995, 37: 287-288. 10.1002/ana.410370303.View ArticlePubMedGoogle Scholar
- Hardy J, Selkoe DJ: The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics. Science. 2002, 297: 353-356. 10.1126/science.1072994.View ArticlePubMedGoogle Scholar
- Sambamurti K, Hardy J, Refolo LM, Lahiri DK: Targeting APP metabolism for the treatment of Alzheimer's disease. Drug Dev Res. 2002, 56: 211-227. 10.1002/ddr.10077.View ArticleGoogle Scholar
- Selkoe DJ, Schenk D: Alzheimer's disease: Molecular understanding predicts amyloid-based therapeutics. Ann Rev Pharmacol Toxicol. 2003, 43: 545-584. 10.1146/annurev.pharmtox.43.100901.140248.View ArticleGoogle Scholar
- Citron M: Strategies for disease modification in Alzheimer's disease. Nat Rev Neurosci. 2004, 5: 677-685. 10.1038/nrn1495.View ArticlePubMedGoogle Scholar
- Hunt CE, Turner AJ: Cell biology, regulation and inhibition of β-secretase (BACE-1). FEBS J. 2009, 276: 1845-1859. 10.1111/j.1742-4658.2009.06929.x.View ArticlePubMedGoogle Scholar
- Fukumoto H, Cheung BS, Hyman BT, Irizarry MC: β-secretase protein and activity are increased in the neocortex in Alzheimer disease. Arch Neurol. 2002, 59: 1381-1389. 10.1001/archneur.59.9.1381.View ArticlePubMedGoogle Scholar
- Holsinger RMD, McLean CA, Beyreuther K, Masters CL, Evin G: Increased expression of the amyloid precursor β-secretase in Alzheimer's disease. Ann Neurol. 2002, 51: 783-786. 10.1002/ana.10208.View ArticlePubMedGoogle Scholar
- Li R, Lindholm K, Yang L-B, Yue X, Citron M, Yan R, Beach T, Sue L, Sabbagh M, Cai H, Wong P, Price D, Shen Y: Amyloid β peptide load is correlated with increased β-secretase activity in sporadic Alzheimer's disease patients. Proc Natl Acad Sci USA. 2004, 101: 3632-3637. 10.1073/pnas.0205689101.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhao J, Fu Y, Yasvoina M, Shao P, Hitt B, O'Connor T, Logan S, Maus E, Citron M, Berry R, Binder L, Vassar R: β-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: Implications for Alzheimer's disease pathogenesis. J Neurosci. 2007, 27: 3639-3649. 10.1523/JNEUROSCI.4396-06.2007.View ArticlePubMedGoogle Scholar
- He W, Zhong Z, Lindholm K, Berning L, Lee W, Lemere C, Staufenbiel M, Li R, Shen Y: Deletion of tumor necrosis factor death receptor inhibits amyloid β generation and prevents learning and memory deficits in Alzheimer's mice. J Cell Biol. 2007, 178: 829-841. 10.1083/jcb.200705042.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamamoto M, Kiyota T, Horiba M, Buescher JL, Walsh SM, Gendelman HE, Ikezu T: Interferon-γ and tumor necrosis factor-α regulate amyloid-β plaque deposition and β-secretase expression in swedish mutant APP transgenic mice. Am J Pathol. 2007, 170: 680-692. 10.2353/ajpath.2007.060378.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun X, He G, Qing H, Zhou W, Dobie F, Cai F, Staufenbiel M, Huang LE, Song W: Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression. Proc Natl Acad Sci USA. 2006, 103: 18727-18732. 10.1073/pnas.0606298103.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang X, Zhou K, Wang R, Cui J, Lipton SA, Liao F-F, Xu H, Zhang Y-W: Hypoxia-inducible factor 1α (HIF-1α)-mediated hypoxia increases BACE1 expression and β-amyloid generation. J Biol Chem. 2007, 282: 10873-10880. 10.1074/jbc.M608856200.View ArticlePubMedGoogle Scholar
- Tamagno E, Guglielmotto M, Aragno M, Borghi R, Autelli R, Giliberto L, Muraca G, Danni O, Zhu X, Smith MA, Perry G, Jo D-G, Mattson MP, Tabaton M: Oxidative stress activates a positive feedback between the γ- and β-secretase cleavages of the β-amyloid precursor protein. J Neurochem. 2008, 104: 683-695.PubMed CentralPubMedGoogle Scholar
- O'Connor T, Sadleir KR, Maus E, Velliquette RE, Zhao J, Cole SL, Eimer WA, Hitt B, Bembinster LA, Lammich S, Lichtenthaler SF, Hébert SS, De Strooper B, Haass C, Bennett DA, Vassar R: Phosphorylation of the translation initiation factor eIF2α increases BACE1 levels and promotes amyloidogenesis. Neuron. 2008, 60: 988-1009. 10.1016/j.neuron.2008.10.047.PubMed CentralView ArticlePubMedGoogle Scholar
- Costantini C, Scrable H, Puglielli : An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β-peptide generation. EMBO J. 2006, 25: 1997-2006. 10.1038/sj.emboj.7601062.PubMed CentralView ArticlePubMedGoogle Scholar
- Costantini C, Ko MH, Jonas MC, Puglielli L: A reversible form of lysine acetylation in the ER and Golgi lumen controls the molecular stabilization of BACE1. Biochem J. 2007, 407: 383-395. 10.1042/BJ20070040.PubMed CentralView ArticlePubMedGoogle Scholar
- Jonas MC, Costantini C, Puglielli L: PCSK9 is required for the disposal of non-acetylated intermediates of the nascent membrane protein BACE1. EMBO Reports. 2008, 9: 916-922. 10.1038/embor.2008.132.PubMed CentralView ArticlePubMedGoogle Scholar
- Sambamurti K, Sevlever D, Koothan T, Refolo LM, Pinnix I, Gandhi S, Onstead L, Younkin L, Prada CM, Yager D, Ohyagi Y, Eckman CB, Rosenberry TL, Younkin SG: Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid β-protein. J Biol Chem. 1999, 274: 26810-26814. 10.1074/jbc.274.38.26810.View ArticlePubMedGoogle Scholar
- Tun H, Marlow L, Pinnix I, Kinsey R, Sambamurti K: Lipid rafts play an important role in Aβ biogenesis by regulating the β-secretase pathway. J Mol Neurosci. 2002, 19: 31-35. 10.1007/s12031-002-0007-5.View ArticlePubMedGoogle Scholar
- Cordy JM, Hussain I, Dingwall C, Hooper NM, Turner AJ: Exclusively targeting β-secretase to lipid rafts by GPI-anchor addition up-regulates β-site processing of the amyloid precurso protein. Proc Natl Acad Sci USA. 2003, 100: 11735-11740. 10.1073/pnas.1635130100.PubMed CentralView ArticlePubMedGoogle Scholar
- Phiel CJ, Wilson CA, Lee VM-Y, Klein PS: GSK3α regulates production of Alzheimer's disease amyloid-β peptide. Nature. 2003, 423: 435-439. 10.1038/nature01640.View ArticlePubMedGoogle Scholar
- Desire L, Bourdin J, Loiseau N, Pellion H, Picard V, De Oliviera C, Bachelot F, Leblond B, Taverne T, Beausoleil E, Lacombe S, Drouin D, Schweighoffer F: RAC1 inhibition targets amyloid precursor protein processing by γ-secretase and decreases Aβ production in vitro and in vivo. J Biol Chem. 2005, 280: 37516-37525. 10.1074/jbc.M507913200.View ArticlePubMedGoogle Scholar
- Flajolet M, He G, Lin A, Nairn AC, Greengard P: Regulation of Alzheimer's disease amyloid-β formation by casein kinase I. Proc Natl Acad Sci USA. 2007, 104: 4159-4164. 10.1073/pnas.0611236104.PubMed CentralView ArticlePubMedGoogle Scholar
- Thathiah A, Spittaels K, Hoffmann M, Staes M, Cohen A, Horré K, Vanbrabant M, Coun F, Baelkelandt V, Delacourte A, Fischer DF, Pollet D, De Strooper B, Merchiers P: The orphan G protein-coupled receptor 3 modulates amyloid-β peptide generation in neurons. Science. 2009, 323: 946-951. 10.1126/science.1160649.View ArticlePubMedGoogle Scholar
- Nunan J, Shearman MS, Chechler F, Cappai R, Evin G, Beyreuther K, Masters CL, Small DH: The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from γ-secretase. Eur J Biochem. 2001, 268: 5329-5336. 10.1046/j.0014-2956.2001.02465.x.View ArticlePubMedGoogle Scholar
- Espeseth AS, Xu M, Huang Q, Coburn CA, Jones KLG, Ferrer M, Zuck PD, Strulovici B, Price EA, Wu G, Wolfe AL, Lineberger JE, Sardana M, Tugusheva A, Pietrak BL, Crouthamel M-C, Lai M-T, Dodson EC, Bazzo R, Shi X-P, Simon AJ, Li Y, Hazuda DJ: Compounds that bind APP and inhibit Aβ processing in vitro suggest a novel approach to Alzheimer disease therapeutics. J Biol Chem. 2005, 280: 17792-17797. 10.1074/jbc.M414331200.View ArticlePubMedGoogle Scholar
- Kukar TL, Ladd TB, Bann MA, Fraering PC, Narlawar R, Maharvi GM, Healy B, Chapman R, Welzel AT, Price RW, Moore B, Rangachari V, Cusack B, Eriksen J, Jansen-West K, Verbeeck C, Yager D, Eckman C, Ye W, Sagi S, Cottrell BA, Torpey J, Rosenberry TL, Fauq A, Wolfe MS, Schmidt B, Walsh DM, Koo EH, Golde TE: Substrate-targeting γ-secretase modulators. Nature. 2008, 453: 925-930. 10.1038/nature07055.PubMed CentralView ArticlePubMedGoogle Scholar
- Pastorino L, Sun A, Lu P-J, Zhou XZ, Balastik M, Finn G, Wulf G, Lim J, Li S-H, Li X, Xia M, Nicholson LK, Lu KP: The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-β production. Nature. 2006, 440: 528-534. 10.1038/nature04543.View ArticlePubMedGoogle Scholar
- Tamboli IY, Prager K, Barth E, Heneka M, Sandhoff K, Walter J: Inhibition of glycosphingolipid biosynthesis reduces secretion of the β-amyloid precursor protein and amyloid β-peptide. J Biol Chem. 2005, 280: 28110-28117. 10.1074/jbc.M414525200.View ArticlePubMedGoogle Scholar
- He W, Lu Y, Qahwash I, Hu X-U, Chang A, Yan R: Reticulon family members modulate BACE1 activity and amyloid-β peptide generation. Nat Med. 2004, 10: 959-965. 10.1038/nm1088.View ArticlePubMedGoogle Scholar
- Murayama KS, Kametani F, Saito S, Kume H, Akiyama H, Araki W: Reticulons RTN3 and RTN4-B/C interact with BACE1 and inhibit its ability to produce amyloid β-protein. Eur J Neurosci. 2006, 24: 1237-1244. 10.1111/j.1460-9568.2006.05005.x.View ArticlePubMedGoogle Scholar
- Majercak J, Ray WJ, Espeseth A, Simon A, Shi X-P, Wolffe C, Getty K, Marine S, Stec E, Ferrer M, Strulovici B, Bartz S, Gates A, Xu M, Huang Q, Ma L, Shughrue P, Burchard J, Colussi D, Pietrak B, Kahana J, Beher D, Rosahl T, Shearman M, Hazuda D, Sachs AB, Koblan KS, Seabrook GR, Stone DJ: LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer's disease. Proc Natl Acad Sci USA. 2006, 103: 17967-17972. 10.1073/pnas.0605461103.PubMed CentralView ArticlePubMedGoogle Scholar
- Andersen OM, Reiche J, Schmidt V, Gotthardt M, Spoelgen R, Behlke J, von Arnim CAF, Breiderhoff T, Jansen P, Wu X, Bales KR, Cappai R, Masters CL, Gliemann J, Mufson EJ, Hyman BT, Paul SM, Nykjaer A, Willnow TE: Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein. Proc Natl Acad Sci USA. 2005, 102: 13461-13466. 10.1073/pnas.0503689102.PubMed CentralView ArticlePubMedGoogle Scholar
- Sakurai T, Kaneko K, Okuno M, Wada K, Kashiyama T, Shimizu H, Akagi T, Hasikawa T, Nukina N: Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing. J Cell Biol. 2008, 183: 339-352. 10.1083/jcb.200804075.PubMed CentralView ArticlePubMedGoogle Scholar
- Fahrenholz F: Alpha-secretase as a therapeutic target. Curr Alzheimer Res. 2007, 4: 412-417. 10.2174/156720507781788837.View ArticlePubMedGoogle Scholar
- Allinson TMJ, Parkin ET, Turner AJ, Hooper NM: ADAMs family members as amyloid precursor protein α-secretases. J Neurosci Res. 2003, 74: 342-352. 10.1002/jnr.10737.View ArticlePubMedGoogle Scholar
- Goodman AB, Pardee AB: Evidence for defective retinoid transport and function in late onset Alzheimer's disease. Proc Natl Acad Sci USA. 2003, 100: 2901-2905. 10.1073/pnas.0437937100.PubMed CentralView ArticlePubMedGoogle Scholar
- Corcoran JPT, So PL, Maden M: Disruption of the retinoid signalling pathway causes a deposition of amyloid-β in the adult rat brain. Eur J Neurosci. 2004, 20: 896-902. 10.1111/j.1460-9568.2004.03563.x.View ArticlePubMedGoogle Scholar
- Nitsch RM, Deng M, Tennis M, Schoenfeld D, Growdon JH: The selective muscarinic M1 agonist AF102B decreases levels of total Aβ in cerebraospinal fluid of patients with Alzheimer's disease. Ann Neurol. 2000, 48: 913-918. 10.1002/1531-8249(200012)48:6<913::AID-ANA12>3.0.CO;2-S.View ArticlePubMedGoogle Scholar
- Caccamo A, Oddo S, Billings LM, Green KN, Martinez-Coria H, Fisher A, LaFerla FM: M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006, 49: 671-682. 10.1016/j.neuron.2006.01.020.View ArticlePubMedGoogle Scholar
- Kojro E, Postina R, Buro C, Meiringer C, Gehrig-Burger K, Fahrenholz F: The neuropeptide PACAP promotes α-secretase pathway for processing Alzheimer amyloid precursor protein. FASEB J. 2006Google Scholar
- Zhu G, Wang D, Lin Y-H, McMahon T, Koo EH, Messing RO: Protein kinase C ε suppresses Aβ production and promotes activation of α-secretase. Biochem Biophys Res Comm. 2001, 285: 997-1006. 10.1006/bbrc.2001.5273.View ArticlePubMedGoogle Scholar
- Etcheberrigaray R, Tan M, Dewachter I, Kuipéri C, Van der Auwera I, Wera S, Qiao L, Bank B, Nelson TJ, Kozikowski AP, Van Leuven F, Alkon DL: Therapeutic effects of PKC activators in Alzheimer's disease transgenic mice. Proc Natl Acad Sci USA. 2004, 101: 11141-11146. 10.1073/pnas.0403921101.PubMed CentralView ArticlePubMedGoogle Scholar
- Kojro E, Gimpl G, Lammich S, März W, Fahrenholz F: Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the α-secretase ADAM10. Proc Natl Acad Sci USA. 2001, 98: 5815-5820. 10.1073/pnas.081612998.PubMed CentralView ArticlePubMedGoogle Scholar
- Riddell DR, Zhou H, Comery TA, Kouranova E, Lo CF, Warwick HK, Ring RH, Kirksey Y, Aschmies S, Xu J, Kubek K, Hirst WD, Gonzales C, Chen Y, Murphy E, Leonard S, Vasylyev D, Oganesian A, Martone RL, Pangalos MN, Reinhart PH, Jacobsen JS: The LXR agonist LO901317 selectively lowers hippocampal Aβ42 and improves memory in the Tg2576 mouse model of Alzheimer's disease. Mol Cell Neurosci. 2007, 34: 621-628. 10.1016/j.mcn.2007.01.011.View ArticlePubMedGoogle Scholar
- Nalivaeva NN, Fisk LR, Belyaev ND, Turner AJ: Amyloid-degrading enzymes as therapeutics targets in Alzheimer's disease. Curr Alz Res. 2008, 5: 212-224. 10.2174/156720508783954785.View ArticleGoogle Scholar
- Dean R, Zlokovic BV: Role of the blood-brain barrier in the pathogenesis of Alzheimer's disease. Curr Alzheimer Res. 2007, 4: 191-197. 10.2174/156720507780362245.View ArticleGoogle Scholar
- Weller RO, Djuanda E, Yow H-Y, Carare RO: Lymphatic drainage of the brain and the pathophysiology of neurological disease. Acta Neurologica. 2009, 117: 1-14.Google Scholar
- Hickman SE, Allison EK, El Khoury J: Microglial dysfunction and defective β-amyloid clearance pathways in aging Alzheimer's disease mice. J Neurosci. 2008, 28: 8354-8360. 10.1523/JNEUROSCI.0616-08.2008.PubMed CentralView ArticlePubMedGoogle Scholar
- Mandrekar S, Jiang Q, Lee CYD, Koenigsknecht-Talboo J, Holtzman DM, Landreth GE: Microglia mediate the clearance of soluble Aβ through fluid phase macropinosytosis. J Neurosci. 2009, 29: 4252-4264. 10.1523/JNEUROSCI.5572-08.2009.PubMed CentralView ArticlePubMedGoogle Scholar
- Saito T, Iwata N, Tsubuki S, Takaki Y, Takano J, Huang S-M, Suemoto T, Higuchi M, Saido TC: Somatostatin regulates brain amyloid β peptide Aβ42 through modulation of proteolytic degradation. Nat Med. 2005, 11: 434-439. 10.1038/nm1206.View ArticlePubMedGoogle Scholar
- Jiang Q, Lee CYD, Mandrekar S, Wilkinson B, Cramer P, Zelcer N, Mann K, Lamb B, Willson TM, Collins JL, Richardson JC, Smith JD, Comery TA, Riddell D, Holtzman DM, Tontonoz P, Landreth GE: ApoE promotes the proteolytic degradation of Aβ. Neuron. 2008, 58: 681-693. 10.1016/j.neuron.2008.04.010.PubMed CentralView ArticlePubMedGoogle Scholar
- Marambaud P, Zhao H, Davies P: Resveratrol promotes clearance of Alzheimer's disease amyloid-β peptides. J Biol Chem. 2005, 280: 37377-37382. 10.1074/jbc.M508246200.View ArticlePubMedGoogle Scholar
- Serneels L, Biervliet JV, Craesserts K, Dejaegere T, Horré K, Houtvin TV, Esselmann H, Paul S, Schäfer MK, Berezovska O, Hyman BT, Sprangers B, Sciot R, Moons L, Jucker M, Yang Z, May PC, Karran E, Wiltfang J, D'Hooge R, De Strooper B: γ-Secretase heterogeneity in the Aph1 subunit: Relevance for Alzheimer's disease. Science. 2009, 324: 639-642. 10.1126/science.1171176.PubMed CentralView ArticlePubMedGoogle Scholar
- Yohrling GJ, Felsenstein KM, Conway KA, Zupa-Fernandez KA, Brenneman DE, Arnold HM: A comparative analysis of brain and plasma Aβ levels in eight common non-transgenic mouse strains: Validation of a specific immunoassay for total rodent Aβ. Curr Alzheimer Res. 2007, 4: 297-303. 10.2174/156720507781077269.View ArticlePubMedGoogle Scholar
- Grimme SJ, Westfall BA, Wiedman JM, Taron CH, Orlean P: The essential Smp3 protein is required for addition of the side-branching fourth mannose during the assembly of yeast glycosylphosphatidylinositols. J Biol Chem. 2001, 276: 27731-27739. 10.1074/jbc.M101986200.View ArticlePubMedGoogle Scholar
- Taron BW, Colussi PA, Wiedman JM, Orlean P, Taron CH: Human Smp3p adds a fourth mannose to yeast and human glycophosphatidylinositol precursors in vivo. J Biol Chem. 2004, 279: 36083-36092. 10.1074/jbc.M405081200.View ArticlePubMedGoogle Scholar
- Vetrivel KS, Meckler X, Chen Y, Nguyen PD, Seidah NG, Vassar R, Wong PC, Fukata M, Kounnas MZ, Thinakaran G: Alzheimer disease Aβ production in the absence of N-palmitoylation-dependent targeting of BACE1 to lipid rafts. J Biol Chem. 2009, 284: 3793-3803. 10.1074/jbc.M808920200.PubMed CentralView ArticlePubMedGoogle Scholar
- Stone TW: Kynurenines in the CNS: from endogenous obscurity to therapeutic importance. Prog Neurobiol. 2001, 64: 185-218. 10.1016/S0301-0082(00)00032-0.View ArticlePubMedGoogle Scholar
- Guillemin GJ, Brew BJ, Noonan CE, Takikawa O, Cullen KM: Indolemine 2,3 dioxygenase and quinolinic acid immunoreactivity in Alzheimer's disease hippocampus. Neuropath App Neurobiol. 2005, 31: 395-404. 10.1111/j.1365-2990.2005.00655.x.View ArticleGoogle Scholar
- Guillemin GJ, Brew BJ, Noonan CE, Knight TG, Smythe GA, Cullen KM: Mass spectrometric detection of quinolinic acid in microdissected Alzheimer's disease plaques. Int Congress Ser. 2007, 1304: 404-408. 10.1016/j.ics.2007.07.012.View ArticleGoogle Scholar
- Behan WMH, McDonald M, Darlington LG, Stone TW: Oxidative stress as a mechanism for quinolinic acid-induced hippocampal damage: protection by melatonin and diprenyl. British J Pharmacol. 1999, 128: 1754-1760. 10.1038/sj.bjp.0702940.View ArticleGoogle Scholar
- Borghi R, Patriarca S, Traverso N, Piccini L, Storace D, Garuti A, Cirmena G, Odetti P, Tabaton M: The increased activity of BACE1 correlates with oxidative stress in Alzheimer's disease. Neurobiol Aging. 2007, 28: 1009-1014. 10.1016/j.neurobiolaging.2006.05.004.View ArticlePubMedGoogle Scholar
- Kao S-C, Krichevsky KM, Kosik KS, Tsai L-H: BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem. 2004, 279: 1942-1949. 10.1074/jbc.M309219200.View ArticlePubMedGoogle Scholar
- Tamagno E, Bardini P, Obbili A, Vitali A, Borghi R, Zaccheo D, Pronzato MA, Danni O, Smith MA, Perry G, Tabaton M: Oxidative stress increases expression and activity of BACE in NT2 neurons. Neurobiol Dis. 2002, 10: 279-288. 10.1006/nbdi.2002.0515.View ArticlePubMedGoogle Scholar
- Tamagno E, Parola M, Bardini P, Piccini A, Borghi R, Guglielmotto M, Santoro G, Davit A, Danni O, Smith MA, Perry G, Tabaton M: β-Site APP cleaving enzyme up-regulation induced by 4-hydroxynonenal is mediated by stress-activated protein kinase pathways. J Neurochem. 2005, 92: 628-636. 10.1111/j.1471-4159.2004.02895.x.View ArticlePubMedGoogle Scholar
- Tong Y, Zhou W, Fung V, Christensen MA, Qing H, Sun X, Song W: Oxidative stress potentiates BACE1 gene expression and Aβ generation. J Neur Trans. 2005, 122: 455-469. 10.1007/s00702-004-0255-3.View ArticleGoogle Scholar
- Töpper R, Gehrmann J, Banati R, Schwarz M, Block F, Noth J, Kreutzberg DW: Rapid appearance of β-amyloid precursor protein immunoreactivity in glial cells following excitotoxic brain injury. Acta Neuropathol. 1995, 89: 23-28. 10.1007/BF00294255.View ArticlePubMedGoogle Scholar
- Hamazaki J, Iemura S-I, Natsume T, Yashiroda H, Tanaka K, Murata S: A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J. 2006, 25: 4524-4536. 10.1038/sj.emboj.7601338.PubMed CentralView ArticlePubMedGoogle Scholar
- Yao T, Song L, Xu W, DeMartino GN, Florens L, Swanson SK, Washburn MP, Conaway RC, Conaway JW, Cohen RE: Proteasome recruitment and activation of the Uch37 deubiquinating enzyme by Adrm1. Nat Cell Biol. 2006, 8: 994-1002. 10.1038/ncb1460.View ArticlePubMedGoogle Scholar
- Espeseth AS, Huang Q, Gates A, Xu M, Yu Y, Simon AJ, Shi X-P, Zhang X, Hodor P, Stone DJ, Burchard J, Cavet G, Bartz S, Linsley P, Ray WJ, Hazuda D: A genome-wide analysis of ubiquitin ligases in APP processing identifies a novel regulator of BACE1 mRNA levels. Mol Cell Neurosci. 2006, 33: 227-235. 10.1016/j.mcn.2006.07.003.View ArticlePubMedGoogle Scholar
- Upadhya SC, Hegde AN: Role of the ubiquitin proteasome system in Alzheimer's disease. BMC Biochem. 2007, 8: S12-10.1186/1471-2091-8-S1-S12. [http://www.biomedcentral.com/1471-2091/8/S1/S12]PubMed CentralView ArticlePubMedGoogle Scholar
- Meredith JE, Thompson LA, Toyn JH, Marcin L, Barten DM, Marcinkeviciene J, Kopcho L, Kim Y, Lin A, Guss V, Burton C, Iben L, Polson C, Cantone J, Ford M, Drexler D, Fiedler T, Lentz KA, Grace JE, Kolb J, Corsa J, Pierdomenico M, Jones K, Olson RE, Macor JE, Albright CF: P-glycoprotein efflux and other factors limit brain amyloid β reduction by β-site amyloid precursor protein-cleaving enzyme 1 inhibitors in mice. J Pharm Exp Ther. 2008, 326: 502-513. 10.1124/jpet.108.138974.View ArticleGoogle Scholar
- Zambrowicz BP, Sands AT: Knockouts model the 100 best-selling drugs - will they model the next 100?. Nat Rev. 2003, 2: 38-51.Google Scholar
- Lanz TA, Schachter JB: Demonstration of a common artifact in immunosorbent assays of brain extracts: Development of a solid-phase extraction protocol to enable measurement of amyloid-β from wild-type rodent brain. J Neurosci Meth. 2006, 157: 71-81. 10.1016/j.jneumeth.2006.03.023.View ArticleGoogle Scholar
- Zhang J-H, Chung TDY, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screening. 1999, 4: 67-73. 10.1177/108705719900400206.View ArticleGoogle Scholar
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