Rare A360T Mutation Alters GSK3ß(Ser9) Binding in the Cytosolic Loop of Presenilin 1, Influencing ß-Catenin Nuclear Localization and Pro-Death Gene Expression in Alzheimer's Disease Case.

Presenilin 1 (PS1) forms, via its large cytosolic loop, a trimeric complex with N-cadherin and ß-catenin, which is a key component of Wnt signaling. PS1 undergoes phosphorylation at 353 and 357 serines upon enhanced activity and elevated levels of the GSK3ß isoform. PS1 mutations surrounding these serines may alter the stability of the ß-catenin complex. Such mutations are found in some cases of familial early-onset Alzheimer's disease (fEOAD), but their functional impact remains obscure. One of such variants of PS1, the A360T substitution, is located close to GSK3ß-targeted serine residues. This variant was recently demonstrated in the French population, but more detail is needed to understand its biological effects. To assess the significance of this variant, we employed functional studies using a fibroblast cell line from an Alzheimer's disease case (a female proband) carrying the A360T mutation. Based on functional transcriptomic, cellular, and biochemical assays, we demonstrated atypically impaired ß-catenin/GSK3ß signaling in the A360T patient's fibroblasts. In detail, this was characterized by a decreased level of active cytosolic ß-catenin and bound by PS1, an increased level of nuclear ß-catenin, an increased level of inhibited GSK3ß phosphorylated on Ser9, and enhanced interaction of GSK3ß(Ser9) with PS1. Based on the transcriptomic profile of the A360T fibroblasts, we proposed a dysregulated transcriptional activity of ß-catenin, exemplified by increased expression of various cyclin-dependent kinases and cyclins, such as cyclin D1, potentially inducing neurons' cell cycle re-entry followed by apoptosis. The A360T cells did not exhibit significant amyloid pathology. Therefore, cell death in this PS1 cytosolic loop mutation may be attributed to impaired ß-catenin/GSK3ß signaling rather than amyloid deposition per se. We further estimated the biological and clinical relevance of the A360T variant by whole exome sequencing (WES). WES was performed on DNA from the blood of an A360T female proband, as well as an unrelated male patient carrying the A360T mutation and his mutation-free daughter (both unavailable for the derivation of the fibroblast cell lines). WES confirmed the highest-priority AD causality of the A360T variant in PS1 and also profiled the pathways and processes involved in the A360T case, highlighting the greatest importance of altered Wnt signaling.

Promising effects of xanthine oxidase inhibition by allopurinol on autonomic heart regulation estimated by heart rate variability (HRV) analysis in rats exposed to hypoxia and hyperoxia.

BACKGROUND: It has long been suggested that reactive oxygen species (ROS) play a role in oxygen sensing via peripheral chemoreceptors, which would imply their involvement in chemoreflex activation and autonomic regulation of heart rate. We hypothesize that antioxidant affect neurogenic cardiovascular regulation through activation of chemoreflex which results in increased control of sympathetic mechanism regulating heart rhythm. Activity of xanthine oxidase (XO), which is among the major endogenous sources of ROS in the rat has been shown to increase during hypoxia promote oxidative stress. However, the mechanism of how XO inhibition affects neurogenic regulation of heart rhythm is still unclear. AIM: The study aimed to evaluate effects of allopurinol-driven inhibition of XO on autonomic heart regulation in rats exposed to hypoxia followed by hyperoxia, using heart rate variability (HRV) analysis. MATERIAL AND METHODS: 16 conscious male Wistar rats (350 g): control-untreated (N = 8) and pretreated with Allopurinol-XO inhibitor (5 mg/kg, followed by 50 mg/kg), administered intraperitoneally (N = 8), were exposed to controlled hypobaric hypoxia (1h) in order to activate chemoreflex. The treatment was followed by 1h hyperoxia (chemoreflex suppression). Time-series of 1024 RR-intervals were extracted from 4kHz ECG recording for heart rate variability (HRV) analysis in order to calculate the following time-domain parameters: mean RR interval (RRi), SDNN (standard deviation of all normal NN intervals), rMSSD (square root of the mean of the squares of differences between adjacent NN intervals), frequency-domain parameters (FFT method): TSP (total spectral power) as well as low and high frequency band powers (LF and HF). At the end of experiment we used rat plasma to evaluate enzymatic activity of XO and markers of oxidative stress: protein carbonyl group and 8-isoprostane concentrations. Enzymatic activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measures in erythrocyte lysates. RESULTS: Allopurinol reduced oxidative stress which was the result of hypoxia/hyperoxia, as shown by decreased 8-isoprostane plasma concentration. XO inhibition did not markedly influence HRV parameters in standard normoxia. However, during hypoxia, as well as hyperoxia, allopurinol administration resulted in a significant increase of autonomic control upon the heart as shown by increased SDNN and TSP, with an increased vagal contribution (increased rMSSD and HF), whereas sympathovagal indexes (LF/HF, SDNN/rMSSD) remained unchanged. CONCLUSIONS: Observed regulatory effects of XO inhibition did not confirm preliminary hypothesis which suggested that an antioxidant such as allopurinol might activate chemoreflex resulting in augmented sympathetic discharge to the heart. The HRV regulatory profile of XO inhibition observed during hypoxia as well as post-hypoxic hyperoxia corresponds to reported reduced risk of sudden cardiovascular events. Therefore our data provide a new argument for therapeutical use of allopurinol in hypoxic conditions.

Up-regulation of ferritin ubiquitination in skeletal muscle of transgenic rats bearing the G93A hmSOD1 gene mutation.

In the present study we measured the levels of protein carbolnyls and the H and L subunits of ferritin in three hind limb muscles, [Extensor digitorum longus, Tibialis anterior and Soleus] of transgenic rats bearing the G93A hmSOD1 gene and of their non-transgenic littermates. All of the muscles from the transgenic animals showed significantly higher protein carbonyl levels, compared to the respective muscles from control non-transgenic animals. In two muscles (Tibialis anterior and Soleus) from transgenic rats, both L and H subunits of ferritin were upregulated. Moreover, we observed that the electrophoretic mobility of both ferritin subunits was retarded which indicates their post-translational modification. Ferritin immunoprecipitation experiments show an increased ubiquitination of both H and L ferritin in all muscles from the transgenic animals. Our data show for the first time that ferritin ubiquitination could be responsible for oxidative stress in muscles of rats bearing the G93A hmSOD1, consequently ferritin is not able to control the labile iron pool.

Reactions of *NO2 with chromium(III) complexes with histamine and pyridoxamine ligands studied by the stopped-flow technique.

This study demonstrated the direct formation of the nitrogen dioxide (*NO2) radical during the decomposition of 3-morpholinosydnonimine (SIN-1) in biological buffer 4-morpholinoethanosulfone acid solution. Consequently, at approximately pH 4, SIN-1 can be used successfully as a source of *NO2. This conclusion is drawn from a comparison of the reactions of cis-[Cr(C2O4)(L- L)(OH2)2]+, where L-L denotes pyridoxamine (Hpm) or histamine (hm), with the gaseous *NO2 radical obtained by two methods: from SIN-1 and from a simple redox reaction. These reactions were investigated using the stopped-flow technique. The measurements were carried out at temperatures ranging from 5 to 25 degrees C over a pH range from 6.52 to 9.11 for cis-[Cr(C2O4)(Hpm) (OH2)2]+ and from 6.03 to 8.15 for cis-[Cr(C2O4)(hm)(OH2)2] +. We also determined the thermodynamic activation parameter (E(a)) and the uptake mechanism for each of the coordination compounds studied.

Pivotal participation of nitrogen dioxide in L-arginine induced acute necrotizing pancreatitis: protective role of superoxide scavenger 4-OH-TEMPO.

For the first time, a direct sensitive method of *NO(2) detection and measurement in biological material has been established. It is based on the interaction of this radical with the coordination compound of Cr(III) with aminodeoxysugar as biosensor. Our new method makes it possible to precisely assess *NO(2) level in experimental acute necrotizing pancreatitis induced by L-arginine, where oxidative and nitrosative stresses are supposed to play a key role in the pathomechanism of the disease. As much as 20 nmol of *NO(2)/mg protein was detected which correlated with severe deterioration of pancreatic acinar cell ultrastructure. Protective effect of superoxide radical scavenger 4-OH-TEMPO expressed as *NO(2) level decrease confirmed by preserved acinar cell ultrastructure and decreased pancreatic amylase release to blood serum is demonstrated. This study reveals a possible pathomechanism of L-arginine induced acute pancreatitis.

4-OH-TEMPO prevents the morphological alteration of rat thymocytes primed to apoptosis by oxidative stress inducer ButOOH.

Thymocytes exposed to the pro-oxidant tert-butyl-hydroperoxide (ButOOH) display a number of dramatic changes in morphology similar to those observed in the case of dexamethasone-treated cells. Both reagents induce nuclear chromatin peripheral aggregation below the nuclear membrane. Some nuclei themselves break up producing two or more fragments. ButOOH-treated cells are morphologically characterised by cell shrinkage, extensive surface blebbing and, finally, fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed with or without nuclear fragments. An increased level of lipid hydroxyperoxides was detected after exposure of thymocytes to ButOOH. Both oxidative stress markers and morphological damage to cells were prevented by the antioxidant 4-OH-TEMPO.