Endocytic Trafficking Routes of Wild Type and ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. ΔF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and ΔF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16°C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations ΔF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis

Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid beta 42 production by direct modulation of gamma-secretase activity

Chronic use of nonsteroidal anti-inflammatory drugs ( NSAIDs) is associated with a lower risk of developing Alzheimer's disease. Recent evidence indicates that some NSAIDs specifically inhibit secretion of the amyloidogenic Abeta42 peptide in cultured cells and mouse models of Alzheimer's disease. The reduction of Abeta42 peptides is not mediated by inhibition of cyclooxygenases ( COX) but the molecular mechanism underlying this novel activity of NSAIDs has not been further defined. We now demonstrate that NSAIDs efficiently reduce the intracellular pool of Abeta42 in cell-based studies and selectively decrease Abeta42 production in a cell-free assay of gamma-secretase activity. Moreover, we find that presenilin-1 (PS1) mutations, which affect gamma-secretase activity, differentially modulate the cellular Abeta42 response to NSAID treatment. Overexpression of the PS1-M146L mutation enhances the cellular drug response to Abeta42 lowering NSAIDs as compared with cells expressing wildtype PS1. In contrast, expression of the PS1-DeltaExon9 mutation strongly diminishes the Abeta42 response, showing that PS1 mutations can modulate the cellular drug response to NSAID treatment both positively and negatively. Enhancement of the NSAID drug response was also observed with overexpression of the APP V717F mutation but not with Swedish mutant APP, which affects beta-secretase cleavage. In sum, these results strongly suggest that NSAIDs represent a founding group of compounds that lower Abeta42 production by direct modulation of gamma-secretase activity or its substrate

NSAIDs and enantiomers of flurbiprofen target γ-secretase and lower Aβ42 in vivo

Epidemiologic studies demonstrate that long-term use of NSAIDs is associated with a reduced risk for the development of Alzheimer disease (AD). In this study, 20 commonly used NSAIDs, dapsone, and enantiomers of flurbiprofen were analyzed for their ability to lower the level of the 42-amino-acid form of amyloid β protein (Aβ42) in a human H4 cell line. Thirteen of the NSAIDs and the enantiomers of flurbiprofen were then tested in acute dosing studies in amyloid β protein precursor (APP) transgenic mice, and plasma and brain levels of Aβ and the drug were evaluated. These studies show that (a) eight FDA-approved NSAIDs lower Aβ42 in vivo, (b) the ability of an NSAID to lower Aβ42 levels in cell culture is highly predicative of its in vivo activity, (c) in vivo Aβ42 lowering in mice occurs at drug levels achievable in humans, and (d) there is a significant correlation between Aβ42 lowering and levels of ibuprofen. Importantly, flurbiprofen and its enantiomers selectively lower Aβ42 levels in broken cell γ-secretase assays, indicating that these compounds directly target the γ-secretase complex that generates Aβ from APP. Of the compounds tested, meclofenamic acid, racemic flurbiprofen, and the purified R and S enantiomers of flurbiprofen lowered Aβ42 levels to the greatest extent. Because R-flurbiprofen reduces Aβ42 levels by targeting γ-secretase and has reduced side effects related to inhibition of cyclooxygenase (COX), it is an excellent candidate for clinical testing as an Aβ42 lowering agent

Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens.

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens