Knock-in models related to Alzheimer’s disease: synaptic transmission, plaques and the role of microglia

Background: Microglia are active modulators of Alzheimer’s disease but their role in relation to amyloid plaques and synaptic changes due to rising amyloid beta is unclear. We add novel findings concerning these relationships and investigate which of our previously reported results from transgenic mice can be validated in knock-in mice, in which overexpression and other artefacts of transgenic technology are avoided. Methods: AppNL-F and AppNL-G-F knock-in mice expressing humanised amyloid beta with mutations in App that cause familial Alzheimer’s disease were compared to wild type mice throughout life. In vitro approaches were used to understand microglial alterations at the genetic and protein levels and synaptic function and plasticity in CA1 hippocampal neurones, each in relationship to both age and stage of amyloid beta pathology. The contribution of microglia to neuronal function was further investigated by ablating microglia with CSF1R inhibitor PLX5622. Results: Both App knock-in lines showed increased glutamate release probability prior to detection of plaques. Consistent with results in transgenic mice, this persisted throughout life in AppNL-F mice but was not evident in AppNL-G-F with sparse plaques. Unlike transgenic mice, loss of spontaneous excitatory activity only occurred at the latest stages, while no change could be detected in spontaneous inhibitory synaptic transmission or magnitude of long-term potentiation. Also, in contrast to transgenic mice, the microglial response in both App knock-in lines was delayed until a moderate plaque load developed. Surviving PLX5266-depleted microglia tended to be CD68-positive. Partial microglial ablation led to aged but not young wild type animals mimicking the increased glutamate release probability in App knock-ins and exacerbated the App knock-in phenotype. Complete ablation was less effective in altering synaptic function, while neither treatment altered plaque load. Conclusions: Increased glutamate release probability is similar across knock-in and transgenic mouse models of Alzheimer’s disease, likely reflecting acute physiological effects of soluble amyloid beta. Microglia respond later to increased amyloid beta levels by proliferating and upregulating Cd68 and Trem2. Partial depletion of microglia suggests that, in wild type mice, alteration of surviving phagocytic microglia, rather than microglial loss, drives age-dependent effects on glutamate release that become exacerbated in Alzheimer’s disease

Association between -T786C NOS3 polymorphism and resistant hypertension: a prospective cohort study

<p>Abstract</p> <p>Background</p> <p>It is estimated that 5% of the hypertensive patients are resistant to conventional antihypertensive therapy. Polymorphisms in the endothelial nitric oxide synthase (NOS3) gene have been associated with high blood pressure levels, but not with resistant hypertension. The aim of the present study was to investigate if the -786T>C and G894T (Glu298Asp) polymorphisms of the NOS3 gene were associated with resistant hypertension.</p> <p>Methods</p> <p>A prospective case-control observational study was performed. From a series of 950 consecutive patients followed up during 42 months, 48 patients with resistant hypertension were detected. 232 patients with controlled high blood pressure were also included.</p> <p>Results</p> <p>No differences were observed in the distribution of G894T (Glu298Asp) NOS3 genotypes between the resistant hypertension group and the controlled hypertension patients. However, genotype -786CC was more frequent in the group of patients with resistant hypertension (33.3%) than in the group of patients with controlled high blood pressure (17.7%) (p 0.03). Furthermore carriers of allele T (-786TC and -786TT) were more frequent in patients with controlled hypertension (82.3%) than those with resistant hypertension (66.7%) (Multivariate analysis; RR 2.09; 95% CI 1.03–4.24; p 0.004).</p> <p>Conclusion</p> <p>Our results indicate that genotype -786CC of the NOS3 gene increase the susceptibility to suffer resistant hypertension, which suggest that resistance to conventional therapy could be determined at the endothelial level.</p

Onset and Progression of Behavioral and Molecular Phenotypes in a Novel Congenic R6/2 Line Exhibiting Intergenerational CAG Repeat Stability

In the present study we report on the use of speed congenics to generate a C57BL/6J congenic line of HD-model R6/2 mice carrying 110 CAG repeats, which uniquely exhibits minimal intergenerational instability. We also report the first identification of the R6/2 transgene insertion site. The relatively stable line of 110 CAG R6/2 mice was characterized for the onset of behavioral impairments in motor, cognitive and psychiatric-related phenotypes as well as the progression of disease-related impairments from 4 to 10 weeks of age. 110Q mice exhibited many of the phenotypes commonly associated with the R6/2 model including reduced activity and impairments in rotarod performance. The onset of many of the phenotypes occurred around 6 weeks and was progressive across age. In addition, some phenotypes were observed in mice as early as 4 weeks of age. The present study also reports the onset and progression of changes in several molecular phenotypes in the novel R6/2 mice and the association of these changes with behavioral symptom onset and progression. Data from TR-FRET suggest an association of mutant protein state changes (soluble versus aggregated) in disease onset and progression

Search for high-mass resonances decaying to dilepton final states in pp collisions at s√=7 TeV with the ATLAS detector

The ATLAS detector at the Large Hadron Collider is used to search for high-mass resonances decaying to an electron-positron pair or a muon-antimuon pair. The search is sensitive to heavy neutral Z′ gauge bosons, Randall-Sundrum gravitons, Z * bosons, techni-mesons, Kaluza-Klein Z/γ bosons, and bosons predicted by Torsion models. Results are presented based on an analysis of pp collisions at a center-of-mass energy of 7 TeV corresponding to an integrated luminosity of 4.9 fb−1 in the e + e − channel and 5.0 fb−1 in the μ + μ −channel. A Z ′ boson with Standard Model-like couplings is excluded at 95 % confidence level for masses below 2.22 TeV. A Randall-Sundrum graviton with coupling k/MPl=0.1 is excluded at 95 % confidence level for masses below 2.16 TeV. Limits on the other models are also presented, including Technicolor and Minimal Z′ Models

The formation of actin waves during regeneration after axonal lesion is enhanced by BDNF

During development, axons of neurons in the mammalian central nervous system lose their ability to regenerate. To study the regeneration process, axons of mouse hippocampal neurons were partially damaged by an UVA laser dissector system. The possibility to deliver very low average power to the sample reduced the collateral thermal damage and allowed studying axonal regeneration of mouse neurons during early days in vitro. Force spectroscopy measurements were performed during and after axon ablation with a bead attached to the axonal membrane and held in an optical trap. With this approach, we quantified the adhesion of the axon to the substrate and the viscoelastic properties of the membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term live imaging. Here we demonstrate that BDNF regulates neuronal adhesion and favors the formation of actin waves during regeneration after axonal lesion

Leishmania-Induced Inactivation of the Macrophage Transcription Factor AP-1 Is Mediated by the Parasite Metalloprotease GP63

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl β-Cyclodextrin (MβCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity

A Network-Based Multi-Target Computational Estimation Scheme for Anticoagulant Activities of Compounds

BACKGROUND: Traditional virtual screening method pays more attention on predicted binding affinity between drug molecule and target related to a certain disease instead of phenotypic data of drug molecule against disease system, as is often less effective on discovery of the drug which is used to treat many types of complex diseases. Virtual screening against a complex disease by general network estimation has become feasible with the development of network biology and system biology. More effective methods of computational estimation for the whole efficacy of a compound in a complex disease system are needed, given the distinct weightiness of the different target in a biological process and the standpoint that partial inhibition of several targets can be more efficient than the complete inhibition of a single target. METHODOLOGY: We developed a novel approach by integrating the affinity predictions from multi-target docking studies with biological network efficiency analysis to estimate the anticoagulant activities of compounds. From results of network efficiency calculation for human clotting cascade, factor Xa and thrombin were identified as the two most fragile enzymes, while the catalytic reaction mediated by complex IXa:VIIIa and the formation of the complex VIIIa:IXa were recognized as the two most fragile biological matter in the human clotting cascade system. Furthermore, the method which combined network efficiency with molecular docking scores was applied to estimate the anticoagulant activities of a serial of argatroban intermediates and eight natural products respectively. The better correlation (r = 0.671) between the experimental data and the decrease of the network deficiency suggests that the approach could be a promising computational systems biology tool to aid identification of anticoagulant activities of compounds in drug discovery. CONCLUSIONS: This article proposes a network-based multi-target computational estimation method for anticoagulant activities of compounds by combining network efficiency analysis with scoring function from molecular docking

Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

<p>Abstract</p> <p>Background</p> <p>Bacterial taxonomy and phylogeny based on <it>rrs </it>(16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of <it>rrs </it>sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their <it>rrs </it>phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, <it>Clostridium </it>represents a large genus of around 110 species of significant biotechnological and medical importance. Certain <it>Clostridium </it>strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods.</p> <p>Results</p> <p>Seven hundred sixty five <it>rrs </it>sequences (> 1200 nucleotides, nts) belonging to 110 <it>Clostridium </it>species were analyzed. On the basis of 404 <it>rrs </it>sequences belonging to 15 <it>Clostridium </it>species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) <it>in silico </it>restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 <it>Clostridium </it>sp. which are presently classified up to genus level, (ii) identification of 84 novel <it>Clostridium </it>spp. and (iii) potential reduction in the number of <it>Clostridium </it>species represented by small populations.</p> <p>Conclusions</p> <p>This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality rates.</p