Molecular Mechanisms of Alzheimer's Disease III.

This Special Issue of IJMS is the third in the series: Molecular Mechanisms of Alzheimer's Disease [...].

Simvastatin, Its Antimicrobial Activity and Its Prevention of Alzheimer's Disease.

Simvastatin, a blockbuster drug for treating hypercholesterolemia, has multifactorial benefits as an antimicrobial agent and plays a preventative role in reducing the incidence of Alzheimer's Disease (AD). Although most of the beneficial effects of simvastatin have been attributed to its ability to reduce cholesterol levels, recent scientific studies have suggested that its benefits are largely due to its pleiotropic effects in targeting other pathways, e.g., by inhibiting protein lipidation. There are certain pleiotropic effects that can be predicted from the inhibition of the mevalonate pathway; however, some of the effects of simvastatin in proteostasis lead to reduced levels of amyloid beta, the key contributor to AD. This review discusses the use of simvastatin as an antimicrobial agent and anti-AD drug.

Polyphasic Characterisation of Cedecea colo sp. nov., a New Enteric Bacterium Isolated from the Koala Hindgut.

The Cedecea genus is comprised of six rarely isolated species within the Enterobacteriaceae family. Representatives are Gram-negative motile bacilli, and are typically oxidase-negative, lipase-positive and resistant to colistin and cephalothin. In this study, a putative novel Cedecea species (designated strain ZA_0188T), isolated from the koala hindgut, was characterised using a polyphasic taxonomic approach. Maximum average nucleotide identity (ANI) and 16S ribosomal RNA (rRNA) similarity scores well below thresholds of species demarcation were reported, at 81.1% and 97.9%, respectively. Multilocus phylogenetic analysis indicated strain ZA_0188T was most similar to but divergent from recognised Cedecea species. The isolate's genomic G+C content was determined as 53.0 mol%, >1% lower than previously reported in Cedecea. Phenotypically, strain ZA_0188T was distinct from recognised Cedecea species such as colistin- and cephalothin-sensitive, lipase-, sorbitol-, sucrose-, and Voges-Proskauer-negative, and melibiose-, arabinose-, arginine-, and rhamnose-positive. In preliminary experiments, strain ZA_0188T exhibited cellulase activity and high-level tolerance to eucalyptus oil compared to other enteric species surveyed. Collectively, these findings suggest that strain ZA_0188T represents a novel enteric species, for which the name Cedecea colo is proposed.

Siccibacter turicensis from Kangaroo Scats: Possible Implication in Cellulose Digestion.

Microbiota in the kangaroo gut degrade cellulose, contributing to the kangaroo's energy and survival. In this preliminary study, to discover more about the gut microbes that contribute to the survival of kangaroos, cellulose-degrading bacteria were isolated from kangaroo scats by selection on solidified media containing carboxymethyl cellulose as the main carbon source. One frequently occurring aerobic bacterium was Siccibacter turicensis, a microbe previously isolated in fruit powder and from a patient with angular cheilitis. The whole genome sequence of the kangaroo isolate was obtained using the Illumina MiSeq platform. Its sequence shared 97.98% identity of the S. turicensis Type strain, and the ability of the Type strain to degrade cellulose was confirmed. Analysis of the genomic data focused on the cellulose operon. In addition to genes from the operon, we suggest that a gene following the operon may have an important role in regulating cellulose metabolism by signal transduction. This is the first report of S. turicensis found within microbiota of the animal gut. Because of its frequent presence in the kangaroo gut, we suggest that S. turicensis plays a role in cellulose digestion for kangaroos.

Abeta aggregation and possible implications in Alzheimer's disease pathogenesis.

Amyloid beta protein (Abeta) has been associated with Alzheimer's disease (AD) because it is a major component of the extracellular plaque found in AD brains. Increased Abeta levels correlate with the cognitive decline observed in AD. Sporadic AD cases are thought to be chiefly associated with lack of Abeta clearance from the brain, unlike familial AD which shows increased Abeta production. Abeta aggregation leading to deposition is an essential event in AD. However, the factors involved in Abeta aggregation and accumulation in sporadic AD have not been completely characterized. This review summarizes studies that have examined the factors that affect Abeta aggregation and toxicity. By necessity these are studies that are performed with recombinant-derived or chemically synthesized Abeta. The studies therefore are not done in animals but in cell culture, which includes neuronal cells, other mammalian cells and, in some cases, non-mammalian cells that also appear susceptible to Abeta toxicity. An understanding of Abeta oligomerization may lead to better strategies to prevent AD.

A new method to measure cellular toxicity of non-fibrillar and fibrillar Alzheimer's Abeta using yeast.

The 42 amino acid amyloid-beta (Abeta) can exist in multiple physical states including oligomers and fibrils. This study shows that fibril formation is hastened by the biological buffers required to support the growth of mammalian cells, but is prevented if Abeta is maintained in water. Here we describe a method to produce Abeta in oligomeric form and the comparison of stable fibrillar and non-fibrillar forms in cell toxicity studies in water, achieved through the use of yeast. We show that extracellular, non-fibrillar Abeta causes a dose dependent loss of cell viability while fibrillar Abeta has low toxicity.

The three-dimensional structure of the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae and other fungi, the enzymes dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) are encoded by a polycistronic gene that is translated into a single polypeptide having all three functions. These enzymatic functions are essential to both prokaryotes and lower eukaryotes, and catalyse sequential reactions in folate biosynthesis. Deletion or disruption of either function leads to cell death. These enzymes are absent from mammals and thus make ideal antimicrobial targets. DHPS is currently the target of antifolate therapy for a number of infectious diseases, and its activity is inhibited by sulfonamides and sulfones. These drugs are typically used as part of a synergistic cocktail with the 2,4-diaminopyrimidines that inhibit dihydrofolate reductase. A gene encoding the S.cerevisiae HPPK and DHPS enzymes has been cloned and expressed in Escherichia coli. A complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue has been crystallized, and its structure solved by molecular replacement and refined to 2.3A resolution. The polypeptide consists of two structural domains, each of which closely resembles its respective monofunctional bacterial HPPK and DHPS counterpart. The mode of ligand binding is similar to that observed in the bacterial enzymes. The association between the domains within the polypeptide as well as the quaternary association of the polypeptide via its constituent DHPS domains provide insight into the assembly of the trifunctional enzyme in S.cerevisiae and probably other fungal species.

Isolation of the Pneumocystis carinii dihydrofolate synthase gene and functional complementation in Saccharomyces cerevisiae.

The Pneumocystis carinii gene encoding the enzyme dihydrofolate synthase (DHFS), which is involved in the essential biosynthesis of folates, was isolated from clones of the Pneumocystis genome project, and sequenced. The deduced P. carinii DHFS protein shares 38% and 35% identity with DHFS of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. P. carinii DHFS expressed from a plasmid functionally complemented a S. cerevisiae mutant with no DHFS. Comparison of available DHFSs with highly similar folylpolyglutamate synthases allowed the identification of potential signatures responsible for the specificities of these two classes of enzymes. The results open the way to experimentally analyse the structure and function of P. carinii mono-functional enzyme DHFS, to investigate a possible role of DHFS in the resistance to antifolates of P. jirovecii, the species infecting specifically humans, and to develop a new class of antifolates.

Growth inhibition of Candida species and Aspergillus fumigatus by statins.

Statins are a class of drugs widely used for lowering high cholesterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the synthesis of cholesterol. We studied the effects of two major statins, simvastatin and atorvastatin, on five Candida species and Aspergillus fumigatus. The statins strongly inhibited the growth of all species, except Candida krusei. Supplementation of Candida albicans and A. fumigatus with ergosterol or cholesterol in aerobic culture led to substantial recovery from the inhibition by statins, suggesting specificity of statins for the mevalonate synthesis pathway. Our findings suggest that the statins could have utility as antifungal agents and that fungal colonization could be affected in those on statin therapy.

Direct integrin alphavbeta6-ERK binding: implications for tumour growth.

Blockade of the mitogen-activated protein (MAP) kinase pathway suppresses growth of colon cancer in vivo. Here we demonstrate a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule, integrin alphavbeta6, in colon cancer cells. Down-regulation of beta6 integrin subunit expression inhibits tumour growth in vivo and MAP kinase activity in response to serum stimulation. In alphavbeta6-expressing cells ERK2 is bound only to the beta6 subunit. The increase in cytosolic MAP kinase activity upon epidermal growth factor stimulation is all accounted for by beta6-bound ERK. Deletion of the ERK2 binding site on the beta6 cytoplasmic domain inhibits tumour growth and leads to an association between ERK and the beta5 subunit. The physical interaction between integrin alphavbeta6 and ERK2 defines a novel paradigm of integrin-mediated signalling and provides a therapeutic target for cancer treatment.

Analysis of Pneumocystis jirovecii DHPS alleles implicated in sulfamethoxazole resistance using an Escherichia coli model system.

Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP). Drug treatment failure has been associated epidemiologically with point mutations in the gene for dihydropteroate synthase which is part of a gene that encodes three covalently linked enzymes involved in folic acid synthesis (FAS). The evaluation of whether mutations found in P. jirovecii FAS lead to sulfa drug resistance is hampered by the lack of a culture system for P. jirovecii as well as the failure of P. jirovecii FAS to complement in a heterologous system. Therefore, we chose to model the P. jirovecii mutations in the Saccharomyces cerevisiae FAS protein (encoded by FOL1) via its expression in Escherichia coli. An optimized drug diffusion assay was used to evaluate the FAS mutants against 15 sulfa drugs. It was established that the single amino acid substitution, P599S, in the (DHPS) domain of FAS led to sulfa drug resistance, whereas the T597A substitution led to increased sensitivity. The presence of both mutations (T597A and P599S) was cooperative and led to increased sulfa drug resistance. Analysis of a novel double mutant, (T597V P599S) was found to have significantly higher sulfa drug resistance than the T597A P599S mutant. These data suggest that further amino acid substitutions may lead to the evolution of higher sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine and sulfathiazole) were identified that had higher inhibitory potential than sulfamethoxazole, which is currently the preferred treatment for PCP.

Quorum protection, growth and survival.

For the growth of a cell culture, one inoculates not with one cell but with a quorum of cells. This most often a requirement, not just a convenience, and most of us take this for granted without question. Here this observation is re-examined to understand why a quorum may be required to grow cells. The importance of quorums may be widespread in the aspects of microbiology they affect. It is very likely that quorums are connected with and have a large impact on the determination of Minimal Inhibitory Concentrations. It is also possible that low cell density may adversely affect cell survival, however, this is an area where even less is known. The need for a quorum might affect other aspects of microbial cell culture, cell isolation and cell preservation. Effects also extend to mammalian cell culture. Here I seek to review studies that have been documented and speculate on how the information might be utilized in the future.

Exogenous folates stimulate growth and budding of Candida glabrata.

Folate, vitamin B9, is well recognized as being essential for cell growth. The utilization of folate is common to all cells, but the source of it may be quite different. For example, mammalian cells depend on exogenous uptake of folates, while plants and microbes can synthesize them. There has been little consideration of uptake of folate in microbial cells, and studies on the effects of folates in mammalian cells, where conditions are restricted. This study shows that exogenous folates (folic acid or folinic acid), causes Candida glabrata cells suspended in water alone to undergo two cycles of cell division and to form multiple buds. The effect was limited to cells in the stationary phase and more profound in quiescent cells. These data indicate a novel response of yeast to folates that may increase the utility of yeast as a model to study folate transport and signaling.

Yeast Model of Amyloid-ß and Tau Aggregation in Alzheimer's Disease.

The amyloid-ß peptide (Aß) and the phosphorylated protein tau have been widely implicated in Alzheimer's disease and are the focus of most research. Both agents have been extensively studied in mammalian cell culture and in animal studies, but new research is focusing on yeast models. Yeast are eukaryotes, just like us, and are amenable to effects and expression of Aß and tau and appear able to 'report' with considerable relevance on the effects of these biomolecules. The use of yeast enables powerful new approaches to understanding how to overcome the effects of Aß and tau, and such advances could lead to new therapies to prevent the progression of Alzheimer's disease.

Novel endoperoxide antimalarials: synthesis, heme binding, and antimalarial activity.

We report the synthesis of a series of novel epoxy endoperoxide compounds that can be prepared in high yields in one to three steps from simple starting materials. Some of these compounds inhibit the growth of Plasmodium falciparum in vitro. Structure-activity studies indicate that an endoperoxide ring bisubstituted with saturated cyclic moieties is the pharmacophore. To study the molecular basis of the action of these novel antimalarial compounds, we examined their ability to interact with oxidized and reduced forms of heme. Some of the compounds interact with oxidized heme in a fashion similar to chloroquine and other 4-aminoquinolines, while some of the compounds interact with reduced heme. However, the level of antimalarial potency is not well correlated with these activities, suggesting that some of the endoperoxides may exert their antimalarial activities by a novel mechanism of action.

Cytotoxicity of dihydropteroate in Saccharomyces cerevisiae.

Microbes and plants synthesise folate using a unique biosynthetic pathway that is absent in animals. The end product, tetrahydrofolate, is utilised by all forms of life. In this study, an intermediate in this synthesis, dihydropteroic acid, was found to be toxic to Saccharomyces cerevisiae. Further tests were performed on mutants deficient in folate synthesis. One mutant specifically lacked dihydropteroate synthase and the second lacked dihydrofolate synthase. Dihydropteroic acid itself appeared to be toxic since both of these mutants were also inhibited. These results suggest novel ways in which antifolate therapy may be developed.

Over-production of dihydrofolate reductase leads to sulfa-dihydropteroate resistance in yeast.

Dihydropteroate synthase (DHPS) can metabolise sulfa drugs into sulfa-dihydropteroate (sulfa-DHP), which inhibits cell growth through competition with dihydrofolate (DHF), possibly indicating dihydrofolate reductase (DHFR) as the target of sulfa-DHP. The effect of over-production of DHFR on sulfa-DHP resistance was examined in Saccharomyces cerevisiae using a strain that requires DHF for growth. This strain was transformed with a plasmid which encodes over-production of DHFR in the presence of CuSO4. Over-production led to resistance to sulfa-DHP suggesting that sulfa-DHP targets DHFR. Spontaneous mutants hyper-resistant to sulfa-DHP did not show any changes within DHFR.

Pretreatment of chemically-synthesized Aß42 affects its biological activity in yeast.

The tendency of amyloid ß (Aß42) peptide to misfold and aggregate into insoluble amyloid fibrils in Alzheimer's disease (AD) has been well documented. Accumulation of Aß42 fibrils has been correlated with abnormal apoptosis and unscheduled cell division which can also trigger the death of neuronal cells, while oligomers can also exhibit similar activities. While investigations using chemically-synthesized Aß42 peptide have become common practice, there appear to be differences in outcomes from different preparations. In order to resolve this inconsistency, we report 2 separate methods of preparing chemically-synthesized Aß42 and we examined their effects in yeast. Hexafluoroisopropanol pretreatment caused toxicity while, ammonium hydroxide treated Aß42 induced cell proliferation in both C. glabrata and S. cerevisiae. The hexafluoroisopropanol prepared Aß42 had greater tendency to form amyloid on yeast cells as determined by thioflavin T staining followed by flow cytometry and microscopy. Both quiescent and non-quiescent cells were analyzed by these methods of peptide preparation. Non-quiescent cells were susceptible to the toxicity of Aß42 compared with quiescent cells (p < 0.005). These data explain the discrepancy in the previous publications about the effects of chemically-synthesized Aß42 on yeast cells. The effect of Aß42 on yeast cells was independent of the size of the peptide aggregates. However, the Aß42 pretreatment determined whether the molecular conformation of peptide resulted in proliferation or toxicity in yeast based assays.

Application of yeast to study the tau and amyloid-ß abnormalities of Alzheimer's disease.

The major molecules associated with Alzheimer's disease, the phosphorylated protein tau and the 42 amino acid peptide, amyloid-ß (Aß), have recently been analyzed in yeast. These yeast studies have provided major new insights into the effects of tau and Aß and, at the same time, offered new approaches to rapidly search for chemicals that may be involved in prevention of Alzheimer's disease. The following review summarizes the role of yeast and its contribution in Alzheimer's disease research, and highlights important studies that have been conducted in this model organism.

Lipid constituents of the edible mushroom, Pleurotus giganteus demonstrate anti-Candida activity.

Different solvent extracts of Pleurotus giganteus fruiting bodies were tested for antifungal activities against Candida species responsible for human infections. The lipids extracted from the ethyl acetate fraction significantly inhibited the growth of all the Candida species tested. Analysis by GC/MS revealed lipid components such as fatty acids, fatty acid methyl esters, ergosterol, and ergosterol derivatives. The sample with high amounts of fatty acid methyl esters was the most effective antifungal agent. The samples were not cytotoxic to a mammalian cell line, mouse embryonic fibroblasts BALB/c 3T3 clone A31. To our knowledge, this is the first report of antifungal activity of the lipid components of Pleurotus giganteus against Candida species.