Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P-2), synthesised by PIKfyve, regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex, leading to even a mild reduction in PtdIns(3,5)P-2 results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity, using YM-201636, significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly, YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II, an effect that was potentiated by inhibition of lysosomal proteases, suggesting that alterations in autophagy could be a contributing factor to neuronal cell death

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Dynamic control of neuroexocytosis by phosphoinositides in health and disease

Phosphoinositides are a group of phospholipids whose inositol headgroups can be phosphorylated at three distinct positions thereby generating seven different isotypes. The conversion between these lipid species depends on the activity of specific sets of phosphoinositide kinases and phosphatases whose targeting and activity is critical to establish the landscape of phosphoinositides on the cytosol-facing hemi-membrane of all organelles and plasmalemma. Phosphoinositides play pleiotropic roles ranging from signalling and membrane trafficking to modulation of ion channels and survival. In neurons and neurosecretory cells, whose main function is to communicate through the release of neurotransmitter, most of the work has focused on the role played by phosphatidylinositol (4,5) bisphosphate in controlling the mechanism underpinning neurotransmitter release through the fusion of secretory vesicles with the plasmalemma. Emerging evidence supports a multi-faceted regulation of neuroexocytosis by 3-phosphorylated phosphoinositides. In this review, we summarise the molecular mechanism by which these lipids control exocytosis and how minute changes in their metabolism can have devastating effects in the nervous system and lead to neurodegeneration. © 2010 Elsevier Ltd. All rights reserved

Phosphoinositides in neuroexocytosis and neuronal diseases

Phosphoinositides (PIs) are a family of phospholipids derived from phosphatidylinositol (PtdIns), whose location, synthesis, and degradation depend on specific PI kinases and phosphatases. PIs have emerged as fundamental regulators of secretory processes, such as neurotransmitter release, hormone secretion, and histamine release in allergic responses. In neurons and neuroendocrine cells, regulated secretion requires the calcium-dependent fusion of transmitter-containing vesicles with the plasma membrane. The role played by PIs in exocytosis is best exemplified by the Ca2+-dependent binding of vesicular Synaptotagmin1 to the plasma membrane PtdIns(4,5)P2, and the recently demonstrated role of PtdIns(4,5)P2 in the mobilization of secretory vesicles to the plasma membrane. New evidence has also recently emerged of an alternative PI pathway that can control exocytosis positively (via PtdIn3P) or negatively (via PtdIns(3,5)P2). However, the positive or negative effectors for these pathways remain to be established. Reducing PtdIns(3,5)P2 potentiates neuroexocytosis but leads to neuronal degeneration and has been linked to certain forms of Charcot-Marie- Tooth disease and amyotrophic lateral sclerosis. The goal of this review is to describe the role of PIs in neuroexocytosis and explore the current hypotheses linking these effects to human diseases

Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2α (PI3K-C2α) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2α is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2α function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2α enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2α mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2α is required for acquisition of fusion competence in neurosecretion

Processing of tf-LC3 in PC12 cells.

<p>(A) PC12/tfLC3 cells were treated for 24 h with 1 µM YM-201636 or DMSO, fixed and nuclei labelled using DAPI. The distribution and fluorescence of GFP and RFP were analysed by confocal microscopy. (B) The number of autophagosomes (determined by colabeling for GFP and RFP) was compared to the total number of RFP puncta, mean ± SEM, ***p<0.001 (n = 16–18 images from 2 independent experiments).</p

The effect of YM-201636 on endosomal and retrograde trafficking in hippocampal neurons.

<p>(A) Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 for 2 h then supplemented with 1 µg/ml CTB-Alexa555 for a further 2 h. Cells were fixed and immunolabelled for GM130. Representative 3D projections are shown. (B) The integrated intensity (per µm²) of CTB-Alexa555 in Golgi complex, as defined by GM130, the integrated intensity of GM130 and the size of the area analysed was measured and the percentage change between conditions determined. (C) The change in total CTB-Alexa555 integrated intensity within the cell body and the area (µm²) of the cell body was determined. (mean ± SEM, n = 3 independent experiments, 11–20 cells per experiment). Significances relative to DMSO *p<0.05, **p<0.01, ***p<0.001 (D) Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 for 3.5 h then supplemented with 25 µg/ml transferrin-Alexa555 (Tf-Alexa555) for a further 30 min. Cells were fixed and immunolabelled for LAMP1. Scale bar = 10 µm.</p

Effect of YM-201636 on WGA trafficking by immunocytochemistry.

<p>Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 for 4 h, then supplemented with 5 µg/ml WGA-Alexa555 for the final 5 min (A,B) or 30 min (C,D). Cells were fixed, immunolabelled for EEA1 and LAMP1, and imaged by confocal microscopy. (B,D) The intensity of WGA-Alexa555 per µm² in the cell body and the total area of the cell body were determined in the YM-201636–treated cells relative to DMSO. (E,F) Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 in the presence of 5 µg/ml WGA-Alexa555 for the full 4 h. Cells were fixed, immunolabelled for GM130 and imaged by confocal microscopy. (F) The amount of WGA-Alexa555 fluorescent intensity in the cell body/µm² of the YM-2016363 treated cells was determined relative to DMSO. The area of the cell bodies analysed was also determined (mean ± SEM, n = 3 independent experiments, 9–28 cells per experiment). Significances relative to DMSO **p<0.01. Scale bar = 10 µm.</p