Correction: DHA Supplemented in Peptamen Diet Offers No Advantage in Pathways to Amyloidosis: Is It Time to Evaluate Composite Lipid Diet?

[This corrects the article DOI: 10.1371/journal.pone.0024094.].

Detrimental effects of arachidonic acid and its metabolites in cellular and mouse models of Alzheimer's disease: structural insight.

Inflammation is believed to be integral to the pathogenesis of Alzheimer's disease (AD). Arachidonic acid (AA) is the most important omega-6 fatty acid and a mediator of inflammatory pathways. High-sensitivity enzyme linked immunosorbent assay shows that AA and its various metabolites; prostaglandins, thromboxanes, and leukotriene B4 resulted in significantly higher secretion of both Abeta40 and 42 peptides. A combination of identical number of alternate cis and trans double bonds either at positions Δ5 or 7Z,13 or 15E (such as PGE(2), PGF(2α), THXB2 and PGF(2α)EA) or at positions Δ6Z,8E,10E,14Z (such as LB4) built in the 3-dimensional structure of 20-carbon fatty acyl chains believed to be responsible for their detrimental action. CP 24,879 and sesamin, 2 inhibitors of the AA pathway suppressed the production of amyloid-beta (Aß) peptides. Immunoblotting experiments and use of SP-C99 transfected COS-7 cells suggested that AA and its metabolites-driven altered production of Aß is mediated through gamma-secretase cleavage of amyloid precursor protein (APP). An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings by showing higher levels of Abeta and amyloid plaques in the brains, when they were fed chow supplemented with 2% AA. Our work not only supports that AA and its metabolites are involved in the production of Aß and in the pathogenesis of AD but also contributes to clarify aspects of structure-activity relationship helpful for future nonsteroidal anti-inflammatory drugs (NSAIDs) research.

DHA supplemented in peptamen diet offers no advantage in pathways to amyloidosis: is it time to evaluate composite lipid diet?

Numerous reports have documented the beneficial effects of dietary docosahexaenoic acid (DHA) on beta-amyloid production and Alzheimer's disease (AD). However, none of these studies have examined and compared DHA, in combination with other dietary nutrients, for its effects on plaque pathogenesis. Potential interactions of DHA with other dietary nutrients and fatty acids are conventionally ignored. Here we investigated DHA with two dietary regimes; peptamen (pep+DHA) and low fat diet (low fat+DHA). Peptamen base liquid diet is a standard sole-source nutrition for patients with gastrointestinal dysfunction. Here we demonstrate that a robust AD transgenic mouse model shows an increased tendency to produce beta-amyloid peptides and amyloid plaques when fed a pep+DHA diet. The increase in beta-amyloid peptides was due to an elevated trend in the levels of beta-secretase amyloid precursor protein (APP) cleaving enzyme (BACE), the proteolytic C-terminal fragment beta of APP and reduced levels of insulin degrading enzyme that endoproteolyse beta-amyloid. On the contrary, TgCRND8 mice on low fat+DHA diet (based on an approximately 18% reduction of fat intake) ameliorate the production of abeta peptides and consequently amyloid plaques. Our work not only demonstrates that DHA when taken with peptamen may have a tendency to confer a detrimental affect on the amyloid plaque build up but also reinforces the importance of studying composite lipids or nutrients rather than single lipids or nutrients for their effects on pathways important to plaque development.

Structural insight into the differential effects of omega-3 and omega-6 fatty acids on the production of Abeta peptides and amyloid plaques.

Several studies have shown the protective effects of dietary enrichment of various lipids in several late-onset animal models of Alzheimer Disease (AD); however, none of the studies has determined which structure within a lipid determines its detrimental or beneficial effects on AD. High-sensitivity enzyme-linked immunosorbent assay (ELISA) shows that saturated fatty acids (SFAs), upstream omega-3 FAs, and arachidonic acid (AA) resulted in significantly higher secretion of both Aß 40 and 42 peptides compared with long chain downstream omega-3 and monounsaturated FAs (MUFA). Their distinct detrimental action is believed to be due to a structural template found in their fatty acyl chains that lack SFAs, upstream omega-3 FAs, and AA. Immunoblotting experiments and use of APP-C99-transfected COS-7 cells suggest that FA-driven altered production of Aß is mediated through γ-secretase cleavage of APP. An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein (APP); Swedish (K670N/M671L) and Indiana (V717F), corroborated in vitro findings by showing lower levels of Aß and amyloid plaques in the brain, when they were fed a low fat diet enriched in DHA. Our work contributes to the clarification of aspects of structure-activity relationships.

Oleic acid ameliorates amyloidosis in cellular and mouse models of Alzheimer's disease.

Several lines of evidence support protective as well as deleterious effects of oleic acid (OA) on Alzheimer's disease (AD) and other neurological disorders; however, the bases of these effects are unclear. Our investigation demonstrates that amyloid precursor protein (APP) 695 transfected Cos-7 cells supplemented with OA have reduced secreted amyloid-beta (Aß) levels. An early-onset AD transgenic mouse model expressing the double-mutant form of human APP, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings when they were fed a high-protein, low-fat (18% reduction), cholesterol-free diet enriched with OA. These mice exhibited an increase in Aß40/Aß42 ratio, reduced levels of beta-site APP cleaving enzyme (BACE) and reduced presenilin levels along with reduced amyloid plaques in the brain. The decrease in BACE levels was accompanied by increased levels of a non-amyloidogenic soluble form of APP (sAPPα). Furthermore, the low-fat/+OA diet resulted in an augmentation of insulin-degrading enzyme and insulin-like growth factor-II. These results suggest that OA supplementation and cholesterol intake restriction in a mouse model of AD reduce AD-type neuropathology.

Amelioration of cystic fibrosis intestinal mucous disease in mice by restoration of mCLCA3.

BACKGROUND & AIMS: Mice deficient of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) exhibit severe intestinal lesions, particularly mucous overproduction/secretion and accumulation, which is similar to meconium ileus in CF patients. Moreover, severity of the intestinal disease in CF mice is strongly influenced by genetic modifiers, and CFTR deficiency affects the expression of multiple secondary genes that may impact on the phenotype. The murine orthologue of human hCLCA1 (mCLCA3) is expressed by goblet cells and implicated in their normal function, particularly with mucus production/secretion that is exaggerated in CF; however, its influence on the CF intestinal disease, although suggested, remains unclear. METHODS: To investigate the role of mCLCA3 on the CF intestinal disease in mice, its expression in this tissue has been assessed, and a CF mouse line maintaining elevated mCLCA3 levels has been developed and comprehensively characterized. RESULTS: Expression of mCLCA3 is significantly reduced in CF mouse intestines, although the number of goblet cells is elevated, indicating marked reduction per cell. Importantly, correction of this deficiency results in amelioration of the mucous-based disease leading to a marked improvement of intestinal pathology and survival, although goblet cell hyperplasia and hypertrophy were augmented. This intestinal amelioration did not appear to be related to rectification of the CF electrophysiologic defect. CONCLUSIONS: mCLCA3 has a role in intestinal goblet cell function that includes modification of the mucous properties and/or secretion that are altered in CF. Thus, elevation of mCLCA3 (hCLCA1) levels could provide a means to reduce intestinal mucous-based lesions in CF and related diseases.

Excess of nicastrin in brain results in heterozygosity having no effect on endogenous APP processing and amyloid peptide levels in vivo.

Nicastrin is an integral member of PS-complexes that perform gamma-secretase cleavage of numerous type I membrane proteins including amyloid precursor protein that underlies Alzheimer's disease; thus, diminishing gamma-secretase activity by reducing levels of functional PS-complexes is suggested as a possible preventative/therapeutic avenue for the disease. One means of reducing PS-complex activity entails decreasing the levels of one or more of its components, such as nicastrin, which is fundamental to its assembly. Two previous studies detailing the effects of decreased nicastrin on gamma-secretase cleavage of APP in nicastrin heterozygous mouse fibroblast, which express relatively low levels of endogenous nicastrin compared to neurons, were contradictory. One report documented a 50% reduction in gamma-secretase cleavage of APP while the second showed markedly higher levels of this activity. Here we report that brains of heterozygous nicastrin mice show no difference in levels of APP gamma-secretase cleavage, APP C-terminal fragments or beta-amyloid peptides, compared to wild-type. This result is explained by the levels of nicastrin protein and functional presenilin complexes being similar between the heterozygous and wild-type brains, though nicastrin mRNA levels were diminished appropriately in the former. These in vivo results indicate that nicastrin mRNA and its immature protein are likely in overabundance in neurons and not limiting for assembly of PS-complexes, and that a 50% reduction of its mRNA or protein production would not affect APP processing, in contrast to fibroblast. Thus, partial reduction (maintaining a level above 50% of normal) of brain nicastrin would likely not be efficacious in reducing functional PS-complexes and gamma-secretase activity as a therapeutic strategy for Alzheimer's disease.

Cysteine based novel noncompetitive inhibitors of urease(s)--distinctive inhibition susceptibility of microbial and plant ureases.

Based on the catalysis mechanism of urease, a homologous series of 10 cysteine derivatives (CysDs) was designed and synthesized, and their inhibitory activities were evaluated for microbial ureases (Bacillus pasteurii, BPU, and Proteus mirabilis, PMU) and for a plant urease [jack bean (Cavavalia ensiformis), JBU]. As already described, thiol-compounds might inhibit urease activity by chelating the nickel atoms involved in the catalysis process. In contrast to cysteine, which has been reported to be a very weak urease inhibitor, we verified a potential inhibitory activity of these CysDs. The kinetic data demonstrate that thiol derivatives are more effective than the respective thioether derivatives. Besides, thiol-CysDs had a reduced activity in acidic pH (5.0). Lineweaver-Burk plots indicated that the nature of inhibition was of noncompetitive type for all 10 compounds, with the minimum Ki value of 2 microM for N,N-dimethyl L-cysteine. It is proposed that these classes of compounds are more potent inhibitors of the bacterial ureases, compared with the plant-originated urease. Since microbial urease is directly involved in the infection process of many pathological organisms, this work demonstrates that thiol-CysDs represent a class of new potential urease inhibitors.

Loss of nicastrin elicits an apoptotic phenotype in mouse embryos.

Nicastrin is a member of the high molecular weight presenilin complex that plays a central role in gamma-secretase cleavage of numerous type-1 membrane-associated proteins required for cell signaling, proliferation and lineage development. We have generated a nicastrin-null mouse line by disruption of exon 3. Similar to previously described nicastrin-null mice, these animals demonstrate severe growth retardation, mortality beginning at embryonic age 10.5 days, and marked developmental abnormalities indicative of a severe Notch phenotype. Preceding their mortality, 10.5-day-old nicastrin-null embryos were found to also exhibit specific apoptosis within regions showing profound deformities, particularly in the developing heart and brain. This result suggests that complete disruption of presenilin complexes elicits programmed cell death, in addition to a Notch phenotype, which may contribute to the developmental abnormalities and embryonic mortality of nicastrin-null mice and possibly neurodegeneration in Alzheimer's disease.

Expression of S100A8 correlates with inflammatory lung disease in congenic mice deficient of the cystic fibrosis transmembrane conductance regulator.

BACKGROUND: Lung disease in cystic fibrosis (CF) patients is dominated by chronic inflammation with an early and inappropriate influx of neutrophils causing airway destruction. Congenic C57BL/6 CF mice develop lung inflammatory disease similar to that of patients. In contrast, lungs of congenic BALB/c CF mice remain unaffected. The basis of the neutrophil influx to the airways of CF patients and C57BL/6 mice, and its precipitating factor(s) (spontaneous or infection induced) remains unclear. METHODS: The lungs of 20-day old congenic C57BL/6 (before any overt signs of inflammation) and BALB/c CF mouse lines maintained in sterile environments were investigated for distinctions in the neutrophil chemokines S100A8 and S100A9 by quantitative RT-PCR and RNA in situ hybridization, that were then correlated to neutrophil numbers. RESULTS: The lungs of C57BL/6 CF mice had spontaneous and significant elevation of both neutrophil chemokines S100A8 and S100A9 and a corresponding increase in neutrophils, in the absence of detectable pathogens. In contrast, BALB/c CF mouse lungs maintained under identical conditions, had similar elevations of S100A9 expression and resident neutrophil numbers, but diverged in having normal levels of S100A8. CONCLUSION: The results indicate early and spontaneous lung inflammation in CF mice, whose progression corresponds to increased expression of both S100A8 and S100A9, but not S100A9 alone. Moreover, since both C57BL/6 and BALB/c CF lungs were maintained under identical conditions and had similar elevations in S100A9 and neutrophils, the higher S100A8 expression in the former (or suppression in latter) is a result of secondary genetic influences rather than environment or differential infection.

Dissociated phenotypes in presenilin transgenic mice define functionally distinct gamma-secretases.

Gamma-secretase depends on presence of presenilins (PS), Nct, Aph-1, and PEN-2 within a core complex. This endoproteolytic activity cleaves within transmembrane domains of amyloid-beta precursor protein (APP) and Notch, and familial Alzheimer's disease (FAD) mutations in PS1 or PS2 genes shift APP cleavage from production of amyloid-beta (Abeta) 40 peptide to greater production of Abeta42. Although studies in PS1/PS2-deficient embryonic cells define overlapping activities for these proteins, in vivo complementation of PS1-deficient animals described here reveals an unexpected spectrum of activities dictated by PS1 and PS2 alleles. Unlike PS1 transgenes, wild-type PS2 transgenes expressed in the mouse CNS support little Abeta40 or Abeta42 production, and FAD PS2 alleles support robust production of only Abeta42. Although wild-type PS2 transgenes failed to rescue Notch-associated skeletal defects in PS1 hypomorphs, a "gained" competence in this regard was apparent for FAD alleles of PS2. The range of discrete and divergent processing activities in mice reconstituted with different PS genes and alleles argues against gamma-secretase being a single enzyme with intrinsically relaxed substrate and cleavage site specificities. Instead, our studies define functionally distinct gamma-secretase variants. We speculate that extrinsic components, in combination with core complexes, may tailor functional variants of this enzyme to their preferred substrates.

Generation and functional characterization of arylamine N-acetyltransferase Nat1/Nat2 double-knockout mice.

Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a variety of arylamine drugs and carcinogens and may play diametrically opposing roles in enhancing either the detoxification of these chemicals or their metabolic activation into DNA-binding electrophiles. To facilitate the study of these processes, we have generated a Nat1/Nat2 double-knockout mouse model by gene targeting in embryonic stem cells. Nat1/2(-/-) mice were born at the expected frequency and seemed normal and viable with no overt phenotype, indicating that these genes are not critical for development or physiological homeostasis. In wild-type mice, NAT1 and NAT2 transcripts were detectable by RT-PCR in all tissues assayed including liver, kidney, colon, brain, bladder, and spleen. NAT1 and NAT2 transcripts were completely undetectable in the Nat1/2(-/-) mice. The in vitro N-acetylation of p-aminosalicylate was detected at significant levels in liver and kidney cytosols from either wild-type inbred 'rapid acetylator' C57BL/6 mice or from outbred CD-1 mice possessing homozygous rapid, heterozygous, or homozygous 'slow acetylator' Nat2 genotypes. Activity was undetectable in cytosol preparations from Nat1/2(-/-) mice. Nat1/2(-/-) mice also displayed severely compromised in vivo pharmacokinetics of p-aminosalicylate (PAS) and sulfamethazine (SMZ), with a drastically increased plasma area under the curve for PAS and a complete absence of their acetylated metabolites (AcPAS or AcSMZ) from plasma, confirming the functional absence of these enzymes and impaired drug metabolism capacity. This knockout mouse model should be helpful in delineating the role that variation in acetylating enzymes plays in mediating interindividual differences in susceptibility to arylamine-induced chemical toxicity and/or carcinogenesis.

Detection of modifier loci influencing the lung phenotype of cystic fibrosis knockout mice.

The variable severity of lung disease associated with cystic fibrosis (CF) cannot be explained by the genotype of the cystic fibrosis transmembrane conductance regulator (CFTR) locus alone. Lung disease has been reported in a congenic CF mouse model of C57BL/6J genetic background (B6 CF), in the absence of detectable infection, but not in CF mice of mixed genetic background, nor in wild-type animals maintained in identical environments. In this report, studies are presented to show that the same CF mutation in mice of a BALB/c background (BALB CF) results in minimal lung disease. By 12 weeks of age B6 CF mice developed a lung disease consisting of mononuclear cell interstitial infiltrate and fibrosis, and BALB CF or littermate control mice developed minimal histopathology. Therefore, it is possible to identify the chromosomal locations of genes that can contribute to the susceptibility to lung disease in B6 CF mice compared with BALB CF mice by means of a quantitative trait loci (QTL) mapping strategy based on the variable histology of the (B6 x BALB) F2 CF mice. Significant linkage of the fibrotic lung phenotype was detected for a region on Chromosome (Chr) 6, defined by markers D6Mit194 to D6Mit201, and suggestive linkage was found for regions on Chr 1, 2, 10, and 17. Additional loci, suggestive of linkage, were also detected for the interstitial thickening phenotype. Most of these putative loci are specific to the sex of the animals. These results suggest that multiple genes can influence the severity of CF lung disease in mice.

Alleles at the Nicastrin locus modify presenilin 1- deficiency phenotype.

Presenilin 1 (PS1), presenilin 2, and nicastrin form high molecular weight complexes that are necessary for the endoproteolysis of several type 1 transmembrane proteins, including amyloid precursor protein (APP) and the Notch receptor, by apparently similar mechanisms. The cleavage of the Notch receptor at the "S3-site" releases a C-terminal cytoplasmic fragment (Notch intracellular domain) that acts as the intracellular transduction molecule for Notch activation. Missense mutations in the presenilins cause familial Alzheimer's disease by augmenting the "gamma-secretase" cleavage of APP and overproducing one of the proteolytic derivatives, the Abeta peptide. Null mutations in PS1 inhibit both gamma-secretase cleavage of APP and S3-site cleavage of the Notch receptor. Mice lacking PS1 function have defective Notch signaling and die perinatally with severe skeletal and brain deformities. We report here that a genetic modifier on mouse distal chromosome 1, coinciding with the locus containing Nicastrin, influences presenilin-mediated Notch S3-site cleavage and the resultant Notch phenotype without affecting presenilin-mediated APP gamma-site cleavage. Two missense substitutions of residues conserved among vertebrates have been identified in nicastrin. These results indicate that Notch S3-site cleavage and APP gamma-site cleavage are distinct presenilin-dependent processes and support a functional interaction between nicastrin and presenilins in vertebrates. The dissociation of Notch S3-site and APP gamma-site cleavage activities will facilitate development of gamma-secretase inhibitors for treatment of Alzheimer's disease.

Differential display analysis of presenilin 1-deficient mouse brains.

Missense mutations in presenilin 1 (PS1) gene are the most common cause of early onset familial Alzheimer's disease (FAD). AD pathogenic PS1 mutations result in elevated gamma-secretase cleavage of APP and diminished S3-site cleavage of Notch. We have previously described a PS1-hypomorphic mouse line that could survive postnatally with markedly reduced gamma-secretase cleavage of APP and S3-site cleavage of Notch, resulting in a Notch developmental phenotype similar to PS1-null mice. This model was exploited to identify genes whose expression is altered due to the loss of PS1. A global gene expression study by differential display was performed on whole brains of PS1-hypomorphic mice and their wild type siblings. In total, more than 16,000 bands corresponding to cDNAs were compared between the mutant and wild-type brains. This analysis identified 19 cDNAs showing significantly altered expression resulting from PS1 deficiency. Four of the identified cDNAs corresponded to genes that could be associated with AD or presenilin function. Hypoxia inducible factor 1a (Hif1a), NPRAP (delta-catenin) and cell division cycle 10 (CDC10) showed significantly reduced expression in the PS1-hypomorphic compared to wild-type brains, whereas expression of nucleoside diphosphate kinase sub-unit A (NDPK-A) was markedly elevated in the respective brains. Clarification of the possible role of these genes in AD and the basis for their differential expression induced by PS1-deficiency may provide insight into the disease, presenilin function and consequences of its loss, as well as possible deleterious effects of AD therapeutics aimed at inhibiting PS1.