Molecular and functional analyses of mutant Idh1 in murine neural stem and progenitor cells Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Miriam Knühmann aus Kamp-Lintfort Düsseldorf, April 2019 aus dem Institut für Neuropathologie der Heinrich-Heine-Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. med. Guido Reifenberger Koreferent: Prof. Dr. Andreas Weber Tag der mündlichen Prüfung: 05.Juli 2019 • FOR MY FAMILY • “IT DOESN’T MATTER IF YOU FALL DOWN AS LONG AS YOU PICK SOMETHING UP FROM THE FLOOR WHEN YOU GET UP.” EFRAIM RACKER I. CONTENTS I. CONTENTS I. Contents ....................................................................................................................... 1 II. Abbreviations .............................................................................................................. 5 III. SI units .......................................................................................................................10 IV. Zusammenfassung ...................................................................................................11 V. Summary ....................................................................................................................12 1.0 Introduction ..............................................................................................................13 1.1 Classification of diffuse gliomas and glioblastomas ................................................13 1.2 Implications of wildtype and mutant IDH .................................................................15 1.3 2-HG in health and disease ....................................................................................18 1.4 ROS and oxidative stress-responsive signaling ......................................................19 1.4.1 The oxidative stress response by NF-țB, NRF2, HSF1 and AP-1 ...................21 1.4.2 The oxidative stress response by MAPKs .......................................................22 1.4.3 The integrated stress response .......................................................................23 1.5 Cytoprotective signaling and programmed cell death .............................................24 1.6 Cell cycle regulation ...............................................................................................26 1.7 The conditional CreERtam/loxP mouse model .........................................................27 1.8 4-OHT and its effects on the cell ............................................................................29 1.9 Neural stem cells as a model for the characterization of IDH1 mutation .................30 1.10 Aims .....................................................................................................................32 2.0 Materials and methods ............................................................................................33 2.1 Materials ................................................................................................................33 2.1.1 Chemicals and kits ..........................................................................................33 2.1.2 Materials and technical devices .......................................................................36 2.1.3 Software ..........................................................................................................38 2.1.4 Solutions and buffer ........................................................................................39 2.1.5 Primers ............................................................................................................41 2.1.6 Antibodies .......................................................................................................43 1 I. CONTENTS 2.1.7 Inhibitors .........................................................................................................44 2.2 Methods .................................................................................................................46 2.2.1 Cell culture ......................................................................................................46 2.2.1.1 Conditional knock-in mouse model ...........................................................46 2.2.1.2 Isolation and culturing of murine NSC/NPCs ............................................47 2.2.2 Genotyping ......................................................................................................48 2.2.2.1 Extraction of genomic DNA from mice tails ...............................................48 2.2.2.2 Genotyping PCR reaction .........................................................................48 2.2.3 Quantitative real-time PCR ..............................................................................49 2.2.3.1 RNA extraction with Trizol® ......................................................................49 2.2.3.2 cDNA synthesis ........................................................................................49 2.2.3.3 qRT-PCR ..................................................................................................50 2.2.4 Transcriptome profiling ....................................................................................50 2.2.4.1 RNA extraction using the QIAGEN RNeasy Mini Kit .................................50 2.2.4.2 RNA sequencing analyses ........................................................................51 2.2.4.3 Microarray analyses .................................................................................51 2.2.5 Western Blotting ..............................................................................................52 2.2.5.1 Cell lysis ...................................................................................................52 2.2.5.2 BCA assay for protein quantification .........................................................52 2.2.5.3 SDS-PAGE ...............................................................................................52 2.2.5.4 Transfer of proteins onto nitrocellulose membranes .................................53 2.2.5.5 Immunostaining of blotted proteins ...........................................................53 2.2.6 Metabolome profiling .......................................................................................54 2.2.7 Flow cytometric analyses ................................................................................55 2.2.7.1 FITC Annexin V / 7-AAD apoptosis staining..............................................55 2.2.7.2 Cell cycle analysis using BrdU incorporation ............................................56 2.2.7.3 ROS detection with the DHE/DCF assay ..................................................57 2.2.8 Viability assay (MTT assay) .............................................................................57 2.2.9 Kinase inhibitor screening ...............................................................................57 2.2.10 Caspase-Glo® 3/7 assay...............................................................................59 2 I. CONTENTS 2.2.11 Proteome profiling .........................................................................................59 3.0 Results ......................................................................................................................61 3.1 Validation of the conditional knock-in mouse model ...............................................61 3.1.1 Expression of recombinant DNA decreases over time after 4-OHT treatment .62 3.1.2 Concentrations of the oncometabolite 2-HG decrease over time after 4-OHT treatment ..................................................................................................................63 3.2 The effect of 4-OHT on NSC/NPCs ........................................................................64 3.2.1 4-OHT mediates ER-independent cytotoxic effects on NSC/NPCs ..................64 3.2.2 4-OHT induces autophagic cell death in NSC/NPCs .......................................66 3.2.3 4-OHT induces apoptotic cell death in NSC/NPCs ..........................................68 3.2.4 4-OHT treatment affects Jak2/Stat3 signaling in NSC/NPCs ...........................71 3.2.5 4-OHT-mediated cytotoxicity on NSC/NPCs is attenuated by Jak2 inhibition and amplified by IKK inhibition ........................................................................................74 3.2.6 4-OHT-treated NSC/NPCs arrest in G0/G1- and in G2/M-phase .....................78 3.3 Characterization of Idh1-mutant NSC/NPCs using a conditional knock-in model ....80 3.3.1 Mutant Idh1 induces Hmox1 expression in NSC/NPCs ...................................80 3.3.2 Idh1-mutant NSC/NPCs show increased 2-HG and decreased glycolate metabolite levels ......................................................................................................83 3.4 Validation of Idh1 mutation-mediated effects in 2-HG-treated Idh1wt/wt Cre-/- NSC/NPCs ...................................................................................................................87 3.4.1 NSC/NPCs are highly sensitive to 2-HG treatment ..........................................87 3.4.2 2-HG treatment results in stress-responsive gene expression in NSC/NPCs ...88 3.4.3 2-HG causes elevated ROS levels and induces an antioxidative response in NSC/NPCs ...............................................................................................................89