Overexpression of GSK3βS9A Resulted in Tau Hyperphosphorylation and Morphology Reminiscent of Pretangle-Like Neurons in the Brain of PDGSK3β Transgenic Mice

It has been demonstrated that GSK3beta is involved in Alzheimer Disease (AD) pathogenesis. In order to understand the underlying mechanism, we have generated and characterized transgenic mice in which the constitutively active human GSK3beta (with S9A mutation) was overexpressed in the brain under the control of the platelet-derived growth factor (PDGF) B-chain promoter. Varying levels of human GSK3betaS9A transgene protein expression was observed in six of the seven founders generated. Line 3083, 3107, 3112 and 3125 displayed higher GSK3betaS9A protein expression levels. Immunostaining analysis demonstrated that transgene expression was observed mainly in cortex and hippocampus of transgenic brain. Expression of human GSK3beta transgene did not significantly change the brain total GSK3beta protein levels in any of the generated mouse lines, as comparing to age matched wild type mice. Although significant kinase activity was detected in human GSK3betaS9A transgene protein extracted from brains of all six expressing lines, significant increase in total GSK3betaS9A kinase activity was observed only in the offspring of line 3083 and 3107. By analyzing the offspring from several transgenic mouse lines, including lines other than 3083 and 3107, it was found that overexpressed constitutively active human GSK3betaS9A resulted in hyperphosphorylation of tau and morphology reminiscent of pretangle-like neurons in cortex and hippocampus.     

The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila

Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, we have expressed the human SCA8 noncoding RNA in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, we have identified mutations in four genes: staufen, muscle-blind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induce neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. Our demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in our SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease.     

Macrophages induce invasiveness of epithelial cancer cells via NF-kappa B and JNK

Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-alpha- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-alpha-dependent activation of JNK and NF-kappaB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-kappaB activity exhibited enhanced invasiveness. Inhibition of the NF-kappaB pathway by TNF-alpha neutralizing Abs, an NF-kappaB inhibitor, RNAi to RelA, or overexpression of IkappaB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-kappaB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-kappaB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. We conclude that TNF-alpha, via NF-kappaB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells.     

Trends of reproductive hormones in male rats during psychosocial stress: role of glucocorticoid metabolism in behavioral dominance

Stress in socially subordinate male rats, associated with aggressive attacks by dominant males, was studied in a group-housing context called the visible burrow system (VBS). It has been established that subordinate males have reduced serum testosterone (T) and higher corticosterone (CORT) relative to dominant and singly housed control males. The relationship of the decreased circulating T levels in subordinate males to changes in serum LH concentrations has not been evaluated previously. Since decreases in LH during stress may cause reductions in Leydig cell steroidogenic activity, the present study defined the temporal profiles of serum LH, T, and CORT in dominant and subordinate males on Days 4, 7, and 14 of a 14-day housing period in the VBS. The same parameters were followed in serum samples from single-housed control males. Leydig cells express glucocorticoid receptors and may also be targeted for direct inhibition of steroidogenesis by glucocorticoid. We hypothesize that Leydig cells are protected from inhibition by CORT at basal concentrations through oxidative inactivation of glucocorticoid by 11beta-hydroxysteroid dehydrogenase (11betaHSD). However, Leydig cell steroidogenesis is inhibited when 11betaHSD metabolizing capacity is exceeded. Therefore, 11betaHSD enzyme activity levels were measured in Leydig cells of VBS-housed males at the same time points. Significant increases in LH and T relative to control were observed in the dominant animals on Day 4, which were associated with the overt establishment of behavioral dominance as evidenced by victorious agonistic encounters. Serum LH and T were lower in subordinate males on Day 7, but T alone was lower on Day 14, suggesting that lowered LH secretion in subordinates may gradually be reversed by declines in androgen-negative feedback. Serum CORT levels were higher in subordinate males compared to control at all three time points. In contrast, oxidative 11betaHSD activity in Leydig cells of dominant males was higher relative to control and unchanged in subordinates. These results suggest the following: 1) failure of Leydig cells of subordinate males to compensate for increased glucocorticoid action during stress, by increasing 11betaHSD oxidative activity, potentiates stress-mediated reductions in T secretion; and 2) an inhibition of the reproductive axis in subordinate males at the level of the pituitary.     

A nuclear SH3 domain-binding protein that colocalizes with mRNA splicing factors and intermediate filament-containing perinuclear networks

A protein (SNP70) has been isolated that binds to the Src homology domain 3 of p47(phox), p85alpha, and c-src. Cloning and sequencing of the polypeptide revealed it to be a 70-kDa protein that has a number of potential domains, including Src homology 3 binding motifs and several nuclear localization signals. Immunofluorescence using anti-peptide antibodies revealed SNP70 to be primarily concentrated in the nucleus but excluded from nucleoli, in interphase cells. However, it was distributed throughout the cytoplasm in dividing cells. Extraction and subfractionation experiments indicated that SNP70 did not bind directly to DNA but did bind to poly(G)-rich oligonucleotides and was resistant to extraction with non-ionic detergents but was solubilized by treatment with RNase, high salt, or ammonium sulfate. Double-immunofluorescence experiments showed that SNP70 co-localized with two pre-mRNA splicing factors SC35 and U2B" within the nucleus. A population of SNP70 was found outside the nucleus, and double-immunofluorescence and immunoelectron microscopy demonstrated that it associated with vimentin-containing intermediate filaments, particularly those surrounding the nucleus. The data suggest that SNP70 associates with nuclear or perinuclear filaments and may play a role in the regulation of pre-mRNA processing.     

Prenatal stress decreases Bdnf expression and increases methylation of Bdnf exon IV in rats

There is ample evidence that exposure to stress during gestation increases the risk of the offspring to develop mood disorders. Brain-derived neurotrophic factor (Bdnf) plays a critical role during neuronal development and is therefore a prime candidate to modulate neuronal signaling in adult offspring of rat dams that were stressed during gestation. In the current study, we tested the hypothesis that alterations in Bdnf expression in prenatally stressed (PNS) offspring are mediated by changes in DNA methylation in exons IV and VI of the Bdnf gene. We observed decreased Bdnf expression in the amygdala and hippocampus of prenatally stressed rats both at weaning and in adulthood. This decrease in Bdnf expression was accompanied by increased DNA methylation in Bdnf exon IV in the amygdala and hippocampus, suggesting that PNS-induced reduction in Bdnf expression may, at least in part, be mediated by increased DNA methylation of Bdnf exon IV. Expression of DNA methyltransferases (Dnmt) 1 and 3a was increased in PNS rats in the amygdala and hippocampus. Our data suggest that PNS induces decreases in Bdnf expression that may at least in part be mediated by increased DNA methylation of Bdnf exon IV.     

Molecular pathways differentiate hepatitis C virus (HCV) recurrence from acute cellular rejection in HCV liver recipients

Acute cellular rejection (ACR) and hepatitis C virus (HCV) recurrence (HCVrec) are common complications after liver transplantation (LT) in HCV patients, who share common clinical and histological features, making a differential diagnosis difficult. Fifty-three liver allograft samples from unique HCV LT recipients were studied using microarrays, including a training set (n = 32) and a validation set (n = 19). Two no-HCV-ACR samples from LT recipients were also included. Probe set intensity values were obtained using the robust multiarray average method (RMA) method. Analysis of variance identified statistically differentially expressed genes (P ≤ 0.005). The limma package was used to fit the mixed-effects models using a restricted maximum likelihood procedure. The last absolute shrinkage and selection operator (LASSO) model was fit with HCVrec versus ACR as the dependent variable predicted. N-fold cross-validation was performed to provide an unbiased estimate of generalization error. A total of 179 probe sets were differentially expressed among groups, with 71 exclusive genes between HCVrec and HCV-ACR. No differences were found within ACR group (HCV-ACR vs. no-HCV-ACR). Supervised clustering analysis displayed two clearly independent groups, and no-HCV-ACR clustered within HCV-ACR. HCVrec-related genes were associated with a cytotoxic T-cell profile, and HCV-ACR-related genes were associated with the inflammatory response. The best-fitting LASSO model classifier accuracy, including 15 genes, has an accuracy of 100% in the training set. N-fold cross-validation accuracy was 78.1%, and sensitivity, specificity and positive and negative predictive values were 50.0%, 90.9%, 71.4% and 80.0%, respectively. Arginase type II (ARG2), ethylmalonic encephalopathy 1 (ETHE1), transmembrane protein 176A (TMEM176A) and TMEM176B genes were significantly confirmed in the validation set. A molecular signature capable of distinguishing HCVrec and ACR in HCV LT recipients was identified and validated.     

Reprogramming virus nanoparticles to bind metal ions upon activation with heat

We have reprogrammed the stimulus-responsive conformational change property of a virus nanoparticle (VNP) to enable the surface exposure of metal binding motifs upon activation with heat. The VNP is based on the widely investigated adeno-associated virus (AAV). An intrinsic bioactive functionality of AAV was genetically replaced with a hexahistidine (His) tag. The peptide domain with the inserted His tag is normally inaccessible. Upon external stimulation with heat, the VNP undergoes a conformational change, resulting in externalization of His tag-containing domains and the conferred ability to bind metal. We show that beyond this newfound functionality of the capsid, the VNPs maintain many of the wild-type capsid properties. Our work lays the groundwork for developing stimulus-responsive VNPs that can be used as "smart" building blocks for the creation of higher order structures.