Durham E-Theses INVESTIGATION OF HOW ENDOPLASMIC RETICULUM STRESS CAUSES INSULIN RESISTANCE AND NEUROINFLAMMATION BROWN, MAX,ADAM How to cite: BROWN, MAX,ADAM (2015) INVESTIGATION OF HOW ENDOPLASMIC RETICULUM STRESS CAUSES INSULIN RESISTANCE AND NEUROINFLAMMATION , Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/11438/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 INVESTIGATION OF HOW ENDOPLASMIC RETICULUM STRESS CAUSES INSULIN RESISTANCE AND NEUROINFLAMMATION Volume I Max Adam Brown This thesis is submitted as part of the requirements for the award of Degree of Doctor of Philosophy School of Biological and Biomedical Sciences Durham University July 2015 ABSTRACT Endoplasmic reticulum (ER) stress is caused by the accumulation of mis/unfolded proteins in the ER. ER stress signalling pathways termed the unfolded protein response are employed to alleviate ER stress through increasing the folding capacity and decreasing the folding demand of the ER as well as removing mis/unfolded proteins. However, ER stress signalling pathways induce diverse cellular changes beyond changes to the ER. This study aims to further investigate some of these ER stress-mediated events. ER stress can cause activation of JNK. Prolonged ER stress-mediated JNK activation is reported to promote apoptosis whilst both acute and long-lasting JNK activation is proposed to cause insulin resistance. To begin with it is reported in this thesis that acute ER stress-induced JNK activation, which is dependent on IRE1α and TRAF2, promotes survival. In contrast to other studies, this thesis provides evidence that acute ER stress- mediated JNK activation does not inhibit insulin signalling during ER stress in several cell lines. However, prolonged ER stress, in four different cell lines, does inhibit insulin signalling in a JNK independent manner. This study argues that ER-stress-induced insulin resistance during prolonged ER stress involves inhibition of trafficking of newly synthesised insulin receptors through the secretory pathway to the plasma membrane. Finally ER stress can activate inflammatory signalling pathways other than JNK and thus ER stress may promote inflammation. Neuroinflammation and ER stress are reported in Parkinson’s disease (PD) yet a link between them has so far not been investigated. Using a cellular model of PD, it is reported in this thesis that ER stress has the potential to activate neuroinflammation in PD. TABLE OF CONTENTS 1 Introduction .................................................................................................................... 1 1.1 The secretory pathway ............................................................................................. 1 1.1.1 The endoplasmic reticulum .............................................................................. 2 1.1.2 The Golgi apparatus ......................................................................................... 3 1.2 Endoplasmic reticulum stress .................................................................................. 6 1.2.1 The unfolded protein response ......................................................................... 6 1.3 Inflammation ......................................................................................................... 11 1.3.1 MAPK Signalling pathways activating AP-1 ................................................ 12 1.3.2 Macrophage activation ................................................................................... 16 1.4 The UPR and inflammation ................................................................................... 18 1.4.1 The UPR and JNK .......................................................................................... 19 1.4.2 The UPR and p38 ........................................................................................... 21 1.4.3 The UPR and NF-B ...................................................................................... 23 1.4.4 The UPR, cytokine production and macrophage activation........................... 24 1.5 The UPR and diabetes ........................................................................................... 25 1.5.1 Diabetes .......................................................................................................... 25 1.5.2 ER stress in obesity and insulin resistance ..................................................... 28 1.5.3 How ER stress causes insulin resistance ........................................................ 31 1.6 The UPR and Parkinson’s disease ......................................................................... 37 1.6.1 Parkinson’s disease ........................................................................................ 37 1.6.2 Genetic factors ............................................................................................... 37 1.6.3 Parkinson’s disease mimetic drugs and the UPR ........................................... 43 1.6.4 Oxidative stress, ER stress and mitochondrial stress ..................................... 45 1.6.5 Inflammatory signalling in PD ....................................................................... 46 1.6.6 Insulin signalling in PD .................................................................................. 51 i 1.7 Aims ...................................................................................................................... 53 2 Materials and methods ................................................................................................. 55 2.1 Materials................................................................................................................ 55 2.1.1 Oligodeoxynucleotides .................................................................................. 55 2.1.2 Antibodies ...................................................................................................... 58 2.1.3 Cell lines ........................................................................................................ 60 2.1.4 Cell culture reagents ...................................................................................... 61 2.1.5 Reagents ......................................................................................................... 63 2.1.6 Special consumables ...................................................................................... 66 2.1.7 Commercially available kits .......................................................................... 67 2.1.8 Solutions for protein work ............................................................................. 68 2.1.9 Solutions for DNA work ................................................................................ 70 2.1.10 Solutions for RNA work ................................................................................ 70 2.1.11 E. coli strains ................................................................................................. 71 2.1.12 Plasmids ......................................................................................................... 71 2.2 Methods ................................................................................................................. 72 2.2.1 Mammalian cell culture ................................................................................. 72 2.2.2 Molecular Biology ......................................................................................... 81 2.2.3 Microscopy .................................................................................................... 94 2.2.4 Statistical Analysis ......................................................................................... 95 3 Early JNK activation by the ER stress sensor IRE1 inhibits cell death early in the ER stress response ..................................................................................................................... 97 3.1 Rationale ............................................................................................................... 97 3.2 ER stress transiently activates JNK before XBP1 splicing reaches maximal levels 97 3.3 Early transient JNK activation in ER stressed cells inhibits cell death .............. 106 3.4 Discussion ........................................................................................................... 108 4 Acute endoplasmic reticulum stress separates JNK and TRB3 activation from insulin resistance ............................................................................................................................ 111 ii 4.1 Rationale .............................................................................................................. 111 4.2 ER stress for up to ~8 h does not inhibit insulin-stimulated AKT activation ..... 112 4.3 ER stress does not inhibit insulin-dependent