The rat bone marrow micronucleus test: Statistical considerations on historical negative control data.

Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo test. As the updated guideline gives high importance to adequate statistical assessment of historical negative control data to estimate validity of experiments and judge results, the present study evaluated statistical methodologies for handling of historical negative control data sets, and comes forward with respective proposals and reference data. Therefore, the working group "Statistics" within the German-speaking "Gesellschaft für Umwelt-Mutationsforschung e.V." (GUM) compiled a data set of 891 negative control rats from valid OECD 474-studies of four laboratories. Based on these data, Analysis-of-Variance (ANOVA) identified "laboratory" and "strain", but not "gender" as relevant stratification parameters, and argued for approximately normally distributed micronucleus frequencies in polychromatic erythrocytes per animal. This assumption provided the basis for further specifying one-sided parametric tolerance intervals for determination of corresponding upper historical negative control limits. Finally, the stability of such limits was investigated as a function of the number of experiments performed, using a simulation-based statistical strategy.

The APP intracellular domain (AICD) potentiates ER stress-induced apoptosis.

Here we employed human SHEP neuroblastoma cells either stably or inducibly expressing the amyloid precursor protein (APP) intracellular domain (AICD) to investigate its ability to modulate stress-induced cell death. Analysis of effector caspase activation revealed that AICD overexpression was specifically associated with an increased sensitivity to apoptosis induced by the 2 endoplasmic reticulum (ER) stressors thapsigargin and tunicamycin, but not by staurosporine (STS). Basal and ER stress-induced expression of Bip/Grp78 and C/EBP-homologous protein/GADD153 were not altered by AICD implying that AICD potentiated cell death downstream or independent of the conserved unfolded protein response (UPR). Interestingly, quantitative polymerase chain reaction analysis and reporter gene assays revealed that AICD significantly downregulated messenger RNA levels of the Alzheimer's disease susceptibility gene ApoJ/clusterin, indicating transcriptional repression. Knockdown of ApoJ/clusterin mimicked the effect of AICD on ER stress-induced apoptosis, but had no discernible effect on staurosporine-induced cell death. Our data suggest that altered levels of AICD may abolish the prosurvival function of ApoJ/clusterin and increase the susceptibility of neurons to ER stress-mediated cell death, a pathway that may contribute to the pathogenesis of Alzheimer's disease.

Liposome-incorporated DHA increases neuronal survival by enhancing non-amyloidogenic APP processing.

The fluidity of neuronal membranes plays a pivotal role in brain aging and neurodegeneration. In this study, we investigated the role of the omega-3 fatty acid docosahexaenoic acid (DHA) in modulation of membrane fluidity, APP processing, and protection from cytotoxic stress. To this end, we applied unilamellar transfer liposomes, which provided protection from oxidation and effective incorporation of DHA into cell membranes. Liposomes transferring docosanoic acid (DA), the completely saturated form of DHA, to the cell cultures served as controls. In HEK-APP cells, DHA significantly increased membrane fluidity and non-amyloidogenic processing of APP, leading to enhanced secretion of sAPPα. This enhanced secretion of sAPPα was associated with substantial protection against apoptosis induced by ER Ca(2+) store depletion. sAPPα-containing supernatants obtained from HEK-APP cells exerted similar protective effects as DHA in neuronal PC12 cells and HEK293 control cells. Correlating to further increased sAPPα levels, supernatants obtained from DHA-treated HEK-APP cells enhanced protection, whereas supernatants obtained from DHA-treated HEK293 control cells did not inhibit apoptosis, likely due to the low expression of endogenous APP and negligible sAPPα secretion in these cells. Further experiments with the small molecule inhibitors LY294002 and SP600125 indicated that sAPPα-induced cytoprotection relied on activation of the anti-apoptotic PI3K/Akt pathway and inhibition of the stress-triggered JNK signaling pathway in PC12 cells. Our data suggest that liposomal DHA is able to restore or maintain physiological membrane properties, which are required for neuroprotective sAPPα secretion and autocrine modulation of neuronal survival.

sAPPalpha antagonizes dendritic degeneration and neuron death triggered by proteasomal stress.

Impaired proteasomal function is a major hallmark in the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD). Here we investigated the biological properties of the secreted cleavage product of APP (sAPPalpha) in antagonizing stress signalling, dendritic degeneration and neuronal cell death induced by the proteasome inhibitor epoxomicin. Analysis of executioner caspase activation demonstrated that sAPPalpha was able to protect PC12 cells from apoptosis triggered by epoxomicin, as well as by genotoxic stress (UV irradiation). This anti-apoptotic effect of sAPPalpha was associated with inhibition of the stress-triggered c-Jun N-terminal kinase (JNK)-signalling pathway. The anti-apoptotic effect of sAPPalpha could also be confirmed in organotypic slice cultures of Thy1-GFP mouse hippocampi. Confocal time-lapse imaging of CA1 pyramidal neurons revealed that preincubation with sAPPalpha preserves the structural integrity of neurons after epoxomicin treatment. Taken together, our data demonstrate that sAPPalpha is neuroprotective under conditions of proteasomal stress.