Donepezil impairs memory in healthy older subjects: behavioural, EEG and simultaneous EEG/fMRI biomarkers

Rising life expectancies coupled with an increasing awareness of age-related cognitive decline have led to the unwarranted use of psychopharmaceuticals, including acetylcholinesterase inhibitors (AChEIs), by significant numbers of healthy older individuals. This trend has developed despite very limited data regarding the effectiveness of such drugs on non-clinical groups and recent work indicates that AChEIs can have negative cognitive effects in healthy populations. For the first time, we use a combination of EEG and simultaneous EEG/fMRI to examine the effects of a commonly prescribed AChEI (donepezil) on cognition in healthy older participants. The short- and long-term impact of donepezil was assessed using two double-blind, placebo-controlled trials. In both cases, we utilised cognitive (paired associates learning (CPAL)) and electrophysiological measures (resting EEG power) that have demonstrated high-sensitivity to age-related cognitive decline. Experiment 1 tested the effects of 5 mg/per day dosage on cognitive and EEG markers at 6-hour, 2-week and 4-week follow-ups. In experiment 2, the same markers were further scrutinised using simultaneous EEG/fMRI after a single 5 mg dose. Experiment 1 found significant negative effects of donepezil on CPAL and resting Alpha and Beta band power. Experiment 2 replicated these results and found additional drug-related increases in the Delta band. EEG/fMRI analyses revealed that these oscillatory differences were associated with activity differences in the left hippocampus (Delta), right frontal-parietal network (Alpha), and default-mode network (Beta). We demonstrate the utility of simple cognitive and EEG measures in evaluating drug responses after acute and chronic donepezil administration. The presentation of previously established markers of age-related cognitive decline indicates that AChEIs can impair cognitive function in healthy older individuals. To our knowledge this is the first study to identify the precise neuroanatomical origins of EEG drug markers using simultaneous EEG/fMRI. The results of this study may be useful for evaluating novel drugs for cognitive enhancement.     

The homeobox gene Msx in development and transdifferentiation of jellyfish striated muscle

Bilaterian Msx homeobox genes are generally expressed in areas of cell proliferation and in association with multipotent progenitor cells. Likewise, jellyfish Msx is expressed in progenitor cells of the developing entocodon, a cell layer giving rise to the striated and smooth muscles of the medusa. However, in contrast to the bilaterian homologs, Msx gene expression is maintained at high levels in the differentiated striated muscle of the medusa in vivo and in vitro. This tissue exhibits reprogramming competence. Upon induction, the Msx gene is immediately switched off in the isolated striated muscle undergoing transdifferentiation, to be upregulated again in the emerging smooth muscle cells which, in a stem cell like manner, undergo quantal cell divisions producing two cell types, a proliferating smooth muscle cell and a differentiating nerve cell. This study indicates that the Msx protein may be a key component of the reprogramming machinery responsible for the extraordinary transdifferentation and regeneration potential of striated muscle in the hydrozoan jellyfish.     

Role of Escherichia coli molecular chaperones in the protection of bacterial cells against irreversible aggregation induced by heat shock

All living organisms respond to environmental stresses, such as heat or ethanol by increasing the synthesis of a specific group of proteins termed heat shock proteins (Hsps) or stress proteins. Major Hsps are molecular chaperones and proteases. Molecular chaperones facilitate the proper folding of polypeptides, protect other proteins from inactivation, and reactivate aggregated proteins. Heat shock proteases eliminate proteins irreversibly damaged by stress. This review describes the role of heat shock proteins of the model bacterial cell, E. coli in the protection of other proteins against aggregation and in the mechanism of removal of protein aggregates from the cell. This mechanism remains unclear and it is believed to involve substrate renaturation and proteolysis by molecular chaperones and heat shock proteases. Recently, many studies have been focused on the disaggregation and reactivation of proteins by a bi-chaperone system consisting of DnaK/DnaJ/GrpE and ClpB, an ATPase from the AAA superfamily of proteins.     

Glycoprotein 96-activated dendritic cells induce a CD8-biased T cell response

Heat shock proteins (Hsps) are able to induce protective immune responses against pathogens and tumors after injection into immunocompetent hosts. The activation of components of the adaptive immune system, including cytotoxic T lymphocytes specific for pathogen- or tumor-derived peptides, is crucial for the establishment of immunoprotection. Hsps acquire these peptides during intracellular protein degradation and when released during necrotic cell death, facilitate their uptake and Minor Histocompatibility Complex (MHC)-restricted representation by professional antigen-presenting cells (APCs). In addition, the interaction of Hsps with APCs, including the Endoplasmatic Reticulum (ER)-resident chaperone glycoprotein 96 (Gp96), induces the maturation of these cells by Toll-like receptor (TLR)-mediated signaling events. We now provide evidence that in contrast to lipopolysaccharides (LPS)-mediated dendritic cell (DC) maturation, the interaction of Gp96 with DCs leads to the preferential expansion of antigen-specific CD8-positive T cells in vitro and in vivo. This CD8 preference induced by mouse and human DCs did not correlate with enhanced levels of interleukin-12 secretion. Thus, despite the fact that both LPS and Gp96 activate DCs in a TLR4-dependent manner, the experiments of this study clearly demonstrate qualitative differences in the outcome of this maturation process, which preferentially favors the expansion of CD8-positive T cells.     

Regulation of intracellular phosphatidylinositol-4-phosphate by the Sac1 lipid phosphatase

Phosphatidylinositol 4-phosphate (PtdIns(4)P) regulates diverse cellular processes, such as actin cytoskeletal organization, Golgi trafficking and vacuolar biogenesis. Synthesis and turnover of PtdIns(4)P is mediated by a set of specific lipid kinases and phosphatases. Here we show that the polyphosphoinositide phosphatase Sac1p has a central role in compartment-specific regulation of PtdIns(4)P. We have found that sac1Delta mutants show pleiotropic, synthetically lethal interactions with mutations in genes required for vacuolar protein sorting (Vps). Disruption of the SAC1 gene also caused a defect in the late endocytic pathway. These trafficking phenotypes correlated with a dramatic accumulation of PtdIns(4)P at vacuolar membranes. In addition, sac1 mutants displayed elevated endoplasmic reticulum PtdIns(4)P. The accumulation of PtdIns(4)P at the endoplasmic reticulum and vacuole and the endocytic defect could be compensated by mutations in the PtdIns 4-kinase Stt4p. Our results indicate that elimination of Sac1p causes accumulation of a Stt4p-specific PtdIns(4)P pool at internal membranes which impairs late endocytic and vacuolar trafficking. We conclude that Sac1p functions in confining PtdIns(4)P-dependent processes to specific intracellular membranes.     

Glycosaminoglycan destabilization of DNA-chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA-chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA-chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from approximately 10 to approximately 1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

Molecular discrimination of type-I over type-II methionyl aminopeptidases

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.     

Age-dependent suppression of SERCA2a mRNA in pediatric atrial myocardium

Differential expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) has been shown in heart failure and atrial arrhythmias. We investigated the influence of volume overload and age on their expression in pediatric atrial myocardium. Right atrial specimens from 18 children with volume overloaded right atrium (VO) and 12 patients without overload were studied. Each group was further divided into patients less than and older than 12 months of age. Only in the younger patients SERCA2a was significantly reduced in the VO group. In younger patients PLB mRNA level tended to be lower in VO. The PLB:SERCA protein ratio was significantly reduced in the VO group. Age itself did not influence the SERCA2a and PLB expression, if the hemodynamic overload was not taken into account. This study is the first to show a combined influence of volume overload and age on atrial SERCA2a expression.