QTL analysis of high thermotolerance with superior and downgraded parental yeast strains reveals new minor QTLs and converges on novel causative alleles involved in RNA processing.

Revealing QTLs with a minor effect in complex traits remains difficult. Initial strategies had limited success because of interference by major QTLs and epistasis. New strategies focused on eliminating major QTLs in subsequent mapping experiments. Since genetic analysis of superior segregants from natural diploid strains usually also reveals QTLs linked to the inferior parent, we have extended this strategy for minor QTL identification by eliminating QTLs in both parent strains and repeating the QTL mapping with pooled-segregant whole-genome sequence analysis. We first mapped multiple QTLs responsible for high thermotolerance in a natural yeast strain, MUCL28177, compared to the laboratory strain, BY4742. Using single and bulk reciprocal hemizygosity analysis we identified MKT1 and PRP42 as causative genes in QTLs linked to the superior and inferior parent, respectively. We subsequently downgraded both parents by replacing their superior allele with the inferior allele of the other parent. QTL mapping using pooled-segregant whole-genome sequence analysis with the segregants from the cross of the downgraded parents, revealed several new QTLs. We validated the two most-strongly linked new QTLs by identifying NCS2 and SMD2 as causative genes linked to the superior downgraded parent and we found an allele-specific epistatic interaction between PRP42 and SMD2. Interestingly, the related function of PRP42 and SMD2 suggests an important role for RNA processing in high thermotolerance and underscores the relevance of analyzing minor QTLs. Our results show that identification of minor QTLs involved in complex traits can be successfully accomplished by crossing parent strains that have both been downgraded for a single QTL. This novel approach has the advantage of maintaining all relevant genetic diversity as well as enough phenotypic difference between the parent strains for the trait-of-interest and thus maximizes the chances of successfully identifying additional minor QTLs that are relevant for the phenotypic difference between the original parents.

Combined treatment with a BACE inhibitor and anti-Aß antibody gantenerumab enhances amyloid reduction in APPLondon mice.

Therapeutic approaches for prevention or reduction of amyloidosis are currently a main objective in basic and clinical research on Alzheimer's disease. Among the agents explored in clinical trials are anti-Aß peptide antibodies and secretase inhibitors. Most anti-Aß antibodies are considered to act via inhibition of amyloidosis and enhanced clearance of existing amyloid, although secretase inhibitors reduce the de novo production of Aß. Limited information is currently available on the efficacy and potential advantages of combinatorial antiamyloid treatment. We performed a chronic study in APPLondon transgenic mice that received treatment with anti-Aß antibody gantenerumab and BACE inhibitor RO5508887, either as mono- or combination treatment. Treatment aimed to evaluate efficacy on amyloid progression, similar to preexisting amyloidosis as present in Alzheimer's disease patients. Mono-treatments with either compound caused a dose-dependent reduction of total brain Aß and amyloid burden. Combination treatment with both compounds significantly enhanced the antiamyloid effect. The observed combination effect was most pronounced for lowering of amyloid plaque load and plaque number, which suggests effective inhibition of de novo plaque formation. Moreover, significantly enhanced clearance of pre-existing amyloid plaques was observed when gantenerumab was coadministered with RO5508887. BACE inhibition led to a significant time- and dose-dependent decrease in CSF Aß, which was not observed for gantenerumab treatment. Our results demonstrate that combining these two antiamyloid agents enhances overall efficacy and suggests that combination treatments may be of clinical relevance.

Reduced spine density in specific regions of CA1 pyramidal neurons in two transgenic mouse models of Alzheimer's disease.

One major hallmark of Alzheimer's disease (AD) is the massive loss of synapses that occurs at an early clinical stage of the disease. In this study, we characterize alterations in spine density and the expression of synapse-associated immediate early gene Arc (activity-regulated cytoskeleton-associated protein) in the hippocampal CA1 regions of two different amyloid precursor protein (APP) transgenic mouse lines before plaque development and their connection to performance in hippocampus-dependent memory tests. The density of mushroom-type spines was reduced by 34% in the basal dendrites proximal to the soma of CA1 pyramidal neurons in 5.5-month-old Tg2576 mice, carrying the Swedish mutation, compared with wild-type littermates. A similar reduction of 42% was confirmed in the same region of 8-month-old APP/Lo mice, carrying the London mutation. In this strain, the reduction extended to the distal dendritic spines (28%), although no differences were found in apical dendrites in either transgenic mouse line. Both transgenic mice lines presented a significant increase in Arc protein expression in CA1 compared with controls, suggesting rather an overactivity and increased spine turnover that was supported by a significant decrease in number of somatostatin-immunopositive inhibitory interneurons in the stratum oriens of CA1. Behaviorally, the transgenic mice showed decrease freezing in the fear contextual conditioning test and impairment in spatial memory assessed by Morris water maze test. These data indicate that cognitive impairment in APP transgenic mice is correlated with impairment of synaptic connectivity in hippocampal CA1, probably attributable to loss of inhibitory interneurons and subsequent hyperactivity.

Why do microorganisms have aquaporins?

Aquaporins are channel proteins that enhance the permeability of cell membranes for water. They have been found in Bacteria, Archaea and Eukaryotes. However, their absence in many microorganisms suggests that aquaporins do not fulfill a broad role such as turgor regulation or osmoadaptation but, instead, fulfill a role that enables microorganisms to have specific lifestyles. The recent discovery that aquaporins enhance cellular tolerance against rapid freezing suggests that they have ecological relevance. We have identified several examples of large-scale freeze-thawing of microbes in nature and we also draw attention to alternative lifestyle-related functions for aquaporins, which will be a focus of future research.

Effects of sub-chronic donepezil on brain Abeta and cognition in a mouse model of Alzheimer's disease.

RATIONALE: Acetylcholinesterase inhibitors (AChEIs) are approved to treat the symptoms of mild to moderate Alzheimer's disease by restoring acetylcholine levels at synapses where the neurotransmitter has been depleted due to neurodegeneration. This assumption is challenged by more recent clinical studies suggesting the potential for disease-modifying effects of AChEIs as well as in vitro studies showing neuroprotective effects. However, few preclinical studies have assessed whether the improvement of cognitive symptoms may be mediated by reductions in Abeta or Tau pathology. OBJECTIVES: The objective of the present study was to determine whether short-duration treatment with donepezil could improve spatial learning and memory in transgenic mice overexpressing mutant human amyloid precursor protein (hAPP) and presenilin 1 (PS1) (Dewachter et al., J Neurosci 20(17):6452-6458, 2000) after amyloid pathology has fully developed, consistent with early stages of Alzheimer'sdisease in humans. In parallel, the effect of donepezil treatment on brain amyloid, Tau, and glial endpoints was measured. RESULTS: This study showed a significant improvement in reference memory in hAPP/PS1 mice along with dose-dependent reductions in brain amyloid-ß (Aß). CONCLUSION: These results suggest that the observed cognitive improvement produced by donepezil in Alzheimer's disease may be due, at least in part, to reduction of brain Aß.

Pathological Hallmarks, Clinical Parallels, and Value for Drug Testing in Alzheimer's Disease of the APP[V717I] London Transgenic Mouse Model.

The APP[V717I] London (APP-Ld) mouse model recapitulates important pathological and clinical hallmarks of Alzheimer's disease (AD) and is therefore a valuable paradigm for evaluating therapeutic candidates. Historically, both the parenchymal and vascular amyloid deposits, and more recently, truncated and pyroglutamate-modified Abeta(3(pE)-42) species, are perceived as important hallmarks of AD-pathology. Late stage symptoms are preceded by robust deficits in orientation and memory that correlate in time with Abeta oligomerization and GSK3ß-mediated phosphorylation of endogenous murine Tau, all markers that have gained considerable interest during the last decade. Clinical parallels with AD patients and the value of the APP-Ld transgenic mouse model for preclinical in vivo testing of candidate drugs are discussed.

Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26.

Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation.

Water channels are important for osmotic adjustments of yeast cells at low temperature.

The importance of aquaporin expression in water permeability in Saccharomyces cerevisiae was assessed by measuring the osmotic water permeability coefficient (P(f)) and the activation energies (E(a)) from both hypo- and hypertonic experiments performed with whole protoplasts from four strains differing in aquaporin level of expression: parental, double-deleted and overexpressing AQY1 or AQY2. Double-deleted (lower P(f)) and AQY1-overexpressing strains (higher P(f)) presented linear Arrhenius plots with E(a) consistent with fluxes mainly through the lipids [16.3 kcal mol(-1) (68.2 kJ mol(-1))] and with a strong contribution of channels [9.6 kcal mol(-1) (40.2 kJ mol(-1))], respectively. The Arrhenius plots for the parental (swelling experiments) and overexpressing AQY2 strains (swelling and shrinking experiments) were not linear, presenting a break point with a change in slope around 23 degrees C. The E(a) values for these strains, calculated for temperatures ranging from 7 to 23 degrees C, were lower [9.5 kcal mol(-1) (39.7 kJ mol(-1))] than the values obtained from 23 to 38 degrees C [17 kcal mol(-1) (71.1 kJ mol(-1))]. This behaviour indicates that only in the lower temperature range did the water fluxes occur predominantly via the water channels. The permeabilities for each strain relative to the deletion strain show that an increase in permeability due to the presence of aquaporins was more relevant at low temperatures. Following our results, we propose that water channels play an important role for osmotic adjustment of yeast cells at low temperature.

Heterologous aquaporin (AQY2-1) expression strongly enhances freeze tolerance of Schizosaccharomyces pombe.

Aquaporin membrane proteins enable the transport of water across membranes in various organisms. In yeast their expression has been shown to correlate strongly with freeze tolerance. When we analyzed the freeze tolerance of Schizosaccharomyces pombe, an organism whose genome sequence has revealed no genes encoding a bona fide water channel, we found very low intrinsic freeze tolerance compared to other yeast species with aquaporin-encoding genes. Deletion of Spac977.17, which encodes a putative glycerol facilitator, resulted in no significant differences in freeze tolerance with its corresponding wild-type strain in all growth conditions tested. However, when we expressed the Saccharomyces cerevisiae aquaporin-encoding gene AQY2-1 in S. pombe cells, we found that the relatively low freeze tolerance of S. pombe could be significantly enhanced. Therefore, (i) the absence of a bona fide water channel in S. pombe might provide in part an explanation for its overall low freeze tolerance compared to other yeast species, and (ii) aquaporin overexpression might be a tool to improve cryopreservation of many other cell types as well, as has recently been shown for mouse oocytes and fish embryos.

Aquaporin expression and freeze tolerance in Candida albicans.

Aquaporins are members of the major intrinsic protein superfamily of integral membrane proteins which enable the transport of water, glycerol, and other solutes across membranes in various organisms. In microorganisms, the physiological role of aquaporins is not yet defined. We found a clear correlation between expression of the Candida albicans aquaporin-encoding gene AQY1 and freeze tolerance. A connection with the function for the aquaporin in the natural environment of C. albicans is, however, not obvious.

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions.

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.

Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains.

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.

Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs.

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.