Novel in vivo experimental viability assays with high sensitivity and throughput capacity using a bdelloid rotifer.

Rotifers have been used in biological research as well-characterized models of aging. Their multi-organ characters and their sensitivity for chemicals and environmental changes make them useful as in vivo toxicological and lifespan models. Our aim was to create a bdelloid rotifer model to use in high-throughput viability and non-invasive assays. In order to identify our species Philodina acuticornis odiosa (PA), 18S rDNA-based phylogenetic analysis was carried out and their species-specific morphological markers identified. To execute the rotifer-based experiments, we developed an oil-covered water-drop methodology adapted from human in vitro fertilization techniques. This enables toxicological observations of individual one-housed rotifers in a closed and controllable micro-environment for up to several weeks. Hydrogen peroxide (H2O2) and sodium azide (NaN3) exposures were used as well-understood toxins. The toxicity and survival lifespan (TSL), the bright light disturbance (BLD) the mastax contraction frequency (MCF) and the cellular reduction capacity (CRC), indices were recorded. These newly developed assays were used to test the effects of lethal and sublethal doses of the toxins. The results showed the expected dose-dependent decrease in indices. These four different assays can either be used independently or as an integrated system for studying rotifers. These new indices render the PA invertebrate rotifer model a quantitative system for measuring viability, toxicity and lifespan (with TSL), systemic reaction capacity (with BLD), organic functionality (with MCF) and reductive capability of rotifers (with CRC), in vivo. This novel multi-level system is a reliable, sensitive and replicable screening tool with potential application in pharmaceutical science.

Adiponectin Receptors Are Less Sensitive to Stress in a Transgenic Mouse Model of Alzheimer's Disease.

Background: Adiponectin and leptin are implicated in the initiation and pathomechanism of Alzheimer's disease (AD). The serum concentrations of these adipokines has been extensively studied in AD, however little is known about their receptors in this disease. Objective: We developed a novel approach to examine whether the receptors of adiponectin (AdipoR1 and -R2) and/or leptin (LepR) can contribute to AD pathomechanism. To achieve this, we investigated the effect of both genetic and environmental factors associated with AD on the expression of these receptors. Method: We used C57BL/6J (WT) and APP(swe)/Presen(e9d)1 (AD) mice. Both strains were exposed to restraint stress (RS) daily for 6h over different time periods. Then, we measured the mRNA expression of AdipoR1, AdipoR2 and LepR and the level of AdipoR1 and AdipoR2 proteins in the hippocampal and prefrontal cortical areas of each mouse. Results: We detected brain region specific transcriptomic changes of adiponectin receptors induced by APP and PS1 transgenes. Both acute and chronic RS caused significant elevations in AdipoR1 mRNA expression in the hippocampus of WT mice. In the prefrontal cortex, the mRNA expression of AdipoR1 followed a biphasic course. In AD mice, RS did not promote any changes in the expression of AdipoR1 mRNA and AdipoR1 protein levels. AdipoR2 mRNA in AD animals, however, showed a significant increase in the prefrontal cortex during RS. Regarding AdipoR1 and AdipoR2 mRNA and protein expression, relevant changes could be measured during stress exposure in both brain areas. Furthermore, stress exposed groups exhibited little change in LepR mRNA expression. Conclusion: Our findings indicate that carrying the transgenes associated with AD induces modification in the expression of both adiponectin receptors. In the case of a normal genetic background, these receptors also appear to be sensitive to environmental factors, while in a genetically determined AD model less response to stress stimuli could be observed. The results suggest that modification of adipokine receptors could also be considered in the therapeutic approach to AD.

Relevance of defensin ß-2 and α defensins (HNP1-3) in Alzheimer's disease.

The DEFB4 gene copy numbers were investigated in 206 AD patients and in 250 controls. The levels of the human defensin ß-2 (hBD2) and α-defensins (HNP 1-3) in the sera and in the cerebrospinal fluid (CSF) of the patients and the controls were determined. Higher copy numbers of the DEFB4 gene was observed in AD patients as compared with the controls. The levels of hBD-2 and HNP 1-3 were significantly elevated in the sera and in the CSF of the AD patients These data suggest that both defensin ß-2 and α-defensins have potential role in the development of AD.

Proteomic analysis of cerebrospinal fluid in Alzheimer's disease: wanted dead or alive.

Clinical diagnosis of Alzheimer's disease (AD) relying on symptomatic features has a low specificity, emphasizing the importance of the pragmatic use of neurochemical biomarkers. The most advanced and reliable markers are amyloid-ß (Aß42), total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) with relatively high levels of sensitivity, specificity, and diagnostic accuracy. Recent advances within the field of proteomics offer the potential to search for novel biomarkers in CSF by using modern methods, such as microarrays. The purpose of this study was to identify pathognostic proteins in CSF obtained from patients whose clinical AD diagnosis was confirmed by the "core" biomarkers. CSF samples were obtained from 25 AD patients and 25 control individuals. The levels of Aß42, t-tau, and p-tau were measured by ELISA. In the microarray experiments, ultrasensitive slides representing of 653 antigens were used. Apolipoprotein E genotyping was also determined. A decrease of seven CSF proteins in AD were found, four of them (POLG, MGMT, parkin, and ApoD) have a protective function against neuronal death, while the remaining three proteins (PAR-4, granzyme B, Cdk5) trigger multiple pathways facilitating neuronal cell death. Since these proteins from CSF samples could not be identified by western blot, their decreased levels in AD patients were not verified. Our results provide new information of pathognostic importance of POLG and granzyme B in AD. Although the function of MGMT, parkin, ApoD, PAR-4, and Cdk5 was previously known in AD, the findings presented here provide novel evidence of the significance of CSF analysis in the mapping of the AD pathomechanism.