The caspase-derived C-terminal fragment of betaAPP induces caspase-independent toxicity and triggers selective increase of Abeta42 in mammalian cells

During its physiopathological maturation, the beta-amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Abeta40 and Abeta42, two highly aggregable betaAPP-derived fragments generated by combined cleavages by beta- and gamma-secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids betaAPP C-terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31-toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31-stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine-induced caspase 3-like activity and pro-caspase-3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly-(ADP-ribose)-polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Abeta42 but not Abeta40 production by HEK293 cells expressing wild-type betaAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic betaAPP maturation pathway by increasing selectively Abeta42 species in wild type-betaAPP-expressing human cells.     

Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor

The micrographic phytopathogen Botrytis cinerea causes gray mold diseases in a large number of dicotyledonous crop plants and ornamentals. Colonization of host tissue is accompanied by rapid killing of plant cells ahead of the growing hyphen, probably caused by secretion of nonspecific phytotoxins, e.g., the sesquiterpene botrydial. Although all pathogenic strains tested so far had been shown to secrete botrydial and although the toxin causes comparable necrotic lesions as infection by the fungus, the role of botrydial in the infection process has not been elucidated so far. Here, we describe the functional characterization of bcbot1, encoding a P450 monooxygenase and provide evidence that it is involved in the botrydial pathway, i.e., it represents the first botrydial biosynthetic gene identified. We show that bcbot1 is expressed in planta and that expression in vitro and in planta is controlled by an alpha-subunit of a heterotrimeric GTP-binding protein, BCG1. Deletion of bcbot1 in three standard strains of B. cinerea shows that the effect on virulence (on several host plants) is strain-dependent; only deletion in one of the strains (T4) led to reduced virulence.     

The ERRalpha orphan nuclear receptor controls morphogenetic movements during zebrafish gastrulation

Gastrulation is a process involving cellular commitment and movements whereby the three fundamental germ layers are established in vertebrates embryos. Estrogen Receptor-Related (ERR) alpha is a nuclear receptor displaying high sequence identity to the Estrogen Receptors (ERs). However, ERRalpha is unable to bind and to be regulated by estrogens or any natural ligand to date. Whereas recent studies have suggested roles for ERRalpha in bone and adipose tissue metabolism in the mouse, little is known about its roles during embryonic development. In zebrafish embryos, ERRalpha is expressed from the beginning of gastrulation at the margin of the blastoderm that represents the presumptive mesendoderm. Using loss of function (morpholinos or a dominant-negative version of the protein) and gain of function (mRNA injection) strategies, we show here that ERRalpha is involved in epiboly and convergent-extension (CE) processes in the zebrafish. Altogether, these results propose ERRalpha as a new regulator of morphogenetic movement during gastrulation, independently of cell fate determination.     

G protein-coupled receptor endocytosis in ADP-ribosylation factor 6-depleted cells

The internalization of G protein-coupled receptors is regulated by several important proteins that act in concert to finely control this complex cellular process. Here, we have applied the RNA interference approach to demonstrate that ADP-ribosylation factor 6 (ARF6) is essential for the endocytosis of a broad variety of receptors. Reduction of endogenous expression of ARF6 in HEK 293 cells resulted in a correlated inhibition of the beta(2) -adrenergic receptor internalization previously characterized as being sequestered via the clathrin-coated vesicle pathway. Furthermore, other receptors internalizing via this endocytic route, namely the angiotensin type 1 receptor and the vasopressin type 2 receptor, were also impaired in their ability to be sequestered when levels of endogenous ARF6 in cells were reduced. Interestingly, endocytosis of the endothelin type B receptor, characterized as being internalized via the caveolae pathway, was also markedly inhibited in ARF6-depleted cells. In contrast, internalization of the vasoactive intestinal peptide receptor was unaffected by reduced levels of ARF6. Finally, internalization of the acetylcholine-muscarinic type 2 receptor via the non-clathrin-coated vesicle pathway was also inhibited in ARF6-depleted cells. Taken together, our results demonstrate that ARF6 proteins play an essential role in the internalization process of most G protein-coupled receptors regardless of the endocytic route being utilized. However, this phenomenon is not general. In some cases, another ARF isoform or other proteins may be essential to regulate the endocytic process.     

mTOR/p70S6k signalling alteration by Abeta exposure as well as in APP-PS1 transgenic models and in patients with Alzheimer's disease

In Alzheimer's disease, neuropathological hallmarks include the accumulation of beta-amyloid peptides (Abeta) in senile plaques, phosphorylated tau in neurofibrillary tangles and neuronal death. Abeta is the major aetiological agent according to the amyloid cascade hypothesis. Translational control includes phosphorylation of the kinases mammalian target of rapamycin (mTOR) and p70S6k which modulate cell growth, proliferation and autophagy. It is mainly part of an anti-apoptotic cellular signalling. In this study, we analysed modifications of mTOR/p70S6k signalling in cellular and transgenic models of Alzheimer's disease, as well as in lymphocytes of patients and control individuals. Abeta 1-42 produced a rapid and persistent down-regulation of mTOR/p70S6k phosphorylation in murine neuroblastoma cells associated with caspase 3 activation. Using western blottings, we found that phosphorylated forms of mTOR and p70S6k are decreased in the cortex but not in the cerebellum (devoid of plaques) of double APP/PS1 transgenic mice compared with control mice. These results were confirmed by immunohistochemical methods. Finally, the expression of phosphorylated p70S6k was significantly reduced in lymphocytes of Alzheimer's patients, and levels of phosphorylated p70S6k were statistically correlated with Mini Mental Status Examination (MMSE) scores. Taken together, these findings demonstrate that the mainly anti-apoptotic mTOR/p70S6k signalling is altered in cellular and transgenic models of Alzheimer's disease and in peripheral cells of patients, and could contribute to the pathogenesis of the disease.     

Functional role of the cysteine 451 thiol group in the M4 helix of the gamma subunit of Torpedo californica acetylcholine receptor.

Previous chemical modification studies of the acetylcholine receptor [Yee, A.S., Corey, D.E., & McNamee, M.G. (1986) Biochemistry 25, 2110-2119] were extended by using fluorescent N-pyrenylmaleimide to alkylate purified Torpedo californica nicotinic acetylcholine receptor (AChR). Peptide sequencing of the tryptic fragments of the labeled AChR gamma subunit identified cysteines 416, 420, and 451 as the modified residues. The functional role of Cys-451 in the M4 transmembrane domain of the AChR gamma subunit was further investigated by studying the functional consequences of the site-specific mutation of this cysteine to either serine or tryptophan by using AChR mRNAs injected into Xenopus laevis oocytes. Both mutants displayed about 50% reduction in the normalized channel activity of the receptor measured as the ACh-induced conductance per femtomole of surface alpha-bungarotoxin binding sites. However, the mutations did not change other AChR functional properties such as agonist binding ability, the slow phase of desensitization, and blockade by competitive and noncompetitive antagonists. The significant reduction in AChR ion channel activity associated with the above point mutations, especially the simple change of the -SH group on Cys-451 to the -OH group, suggests that this thiol group in the M4 helix of gamma subunit may play an important role in AChR ion channel function. Previous site-directed mutations of the Cys-416 and -420 residues showed a decreased response when both of these residues were changed to phenylalanine, but not when they were changed to serine [Pradier, L., Yee, A.S., & McNamee, M.G. (1989) Biochemistry 28, 6562-6571].(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrocharcoal-Based Chronosequences Reveal Shifting Dominance of Conifer Boreal Forests Under Changing Fire Regime

Balsam fir (Abies balsamea) and black spruce (Picea mariana) forests are the main conifer forest types in the North American boreal zone. The coexistence of the two species as well as their respective canopy dominance in distinct stands raises questions about the long-term evolution from one forest type to the other in relation to environmental factors including climate and stand disturbance. We tested the hypothesis that repetitive fire events promote the succession of balsam fir forest to black spruce forest and vice versa. Postfire chronosequences of one black spruce (BSP) and one balsam fir (BFI) sites were reconstructed based on the botanical composition and ¹⁴C-dated soil macrocharcoals. The results support the hypothesis of a successional dynamics. The BSP site has been affected by fires for the last 7600 years, whereas the BFI site, after having been impacted by several fires during the first half of the Holocene, evolved in a fire-free environment for the last 4400 years. Periods of fire activity facilitated the dominance of black spruce forests. The cessation of fires around 4400 cal. years BP on BFI site marks the beginning of the transition from black spruce to balsam fir stands. This succession is a long process, due to the ability of black spruce to regenerate by layering in the absence of fire. The resulting balsam fir stands are ancient and precarious ecosystems, since fire generally leads to the return of black spruce. The increase in balsam fir to the detriment of black spruce in boreal forests is a response to a decrease in fire frequency.     

“As du Coeur” study: a randomized controlled trial on quality of life impact and cost effectiveness of a physical activity program in patients with cardiovascular disease

Background

Physical activity programs (PAP) in patients with cardiovascular disease require evidence of cost-utility. To assess improvement in health-related quality of life (QoL) and reduction of health care consumption of patients following PAP, a randomized trial was used.

Methods

Patients from a health insurance company who had experienced coronary artery disease or moderate heart failure were invited to participate (N?=?1891). Positive responders (N?=?50) were randomly assigned to a progressively autonomous physical activity (PAPA) program or to a standard supervised physical activity (SPA) program. The SPA group had two supervised sessions per week over 5?months. PAPA group had one session per week and support to aid habit formation (written tips, exercise program, phone call). To measure health-related quality of life EQ-5D utility score were used, before intervention, 6?months (T6) and 1 year later. Health care costs were provided from reimbursement databases.

Results

Mobility, usual activities and discomfort improved significantly in both group (T6). One year later, EQ-5D utility score was improved in the PAPA group only. Total health care consumption in the intervention group decreased, from a mean of 4097 euros per year before intervention to 2877 euros per year after (p?=?0.05), compared to a health care consumption of 4087 euros and 4180 euros per year, in the total population of patients (N?=?1891) from the health insurance company. The incremental cost effectiveness ratio was 10,928 euros per QALYs.

Conclusion

A physical activity program is cost-effective in providing a better quality of life and reducing health care consumption in cardiovascular patients.

Trial registration

ISRCTN77313697, retrospectively registered on 20 November 2015.

     

Building of an immunosensor: how can the composition and structure of the thiol attachment layer affect the immunosensor efficiency?

Immunosensors, based on the immobilization of a model rabbit antibody on mixed self-assembled monolayers and Protein A as a linking agent on gold transducers, were elaborated and characterized at each step by modulated polarization-infrared spectroscopy (PM-IRRAS) and occasionally by atomic force microscopy (AFM) and quartz crystal microbalance (QCM). By testing two different mixed SAMs comprising 11-mercaptoundecanoic acid (MUA), together with either decanethiol (C9CH3) or mercaptohexanol (C6OH), the role of the chemical composition and structure of the antibody attachment layer upon the sensor performance was demonstrated.