Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Novel Insights into the Pathophysiology of Peripherin-2 Mutations in Rod and Cone Photoreceptors Sybille Böhm aus Schäßburg, Rumänien 2018 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Martin Biel betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, den 12.10.2018 _______________________ (Sybille Böhm) Dissertation eingereicht am 12.10.2018 1. Gutachter: Prof. Dr. Martin Biel 2. Gutachter: PD Dr. Stylianos Michalakis Mündliche Prüfung am 18.12.2018 Table of contents 3 Table of contents 1 Preface .........................................................................................................................7 2 Introduction .................................................................................................................8 2.1 Anatomy of the retina .................................................................................................... 8 2.2 Anatomy of photoreceptors .......................................................................................... 9 2.3 Inherited retinal diseases ............................................................................................ 10 2.3.1 Retinitis pigmentosa ....................................................................................................... 10 2.4 Peripherin-2 .................................................................................................................. 11 2.5 Rom-1 ............................................................................................................................ 12 2.6 Mutations in PRPH2 and ROM1 .................................................................................. 12 2.7 Adeno-associated viruses ........................................................................................... 13 3 Aim of the study ........................................................................................................ 15 4 Materials and methods.............................................................................................. 16 3.1 Animals ......................................................................................................................... 16 3.2 Chemicals, solutions and buffers............................................................................... 16 3.3 Molecular biology ......................................................................................................... 16 3.3.1 Plasmids ......................................................................................................................... 16 3.3.2 Polymerase chain reaction (PCR) .................................................................................. 17 3.3.3 Precipitation of DNA fragments ...................................................................................... 17 3.3.4 Restriction analysis......................................................................................................... 18 3.3.5 Agarose gel electrophoresis and DNA fragment isolation.............................................. 18 3.3.6 Dephosphorylation and ligation ...................................................................................... 18 3.3.7 Transformation ............................................................................................................... 18 3.3.8 Inoculation of bacterial cells and isolation of plasmid DNA ............................................ 19 3.3.9 RNA isolation and cDNA synthesis ................................................................................ 20 3.3.10 Quantitative real-time PCR (qPCR) ................................................................................ 20 3.3.11 In silico prediction of splicing .......................................................................................... 20 3.3.12 Genomic DNA isolation .................................................................................................. 20 Table of contents 4 3.3.13 Construction and cloning of the PRPH2 minigene ......................................................... 21 3.3.14 Side-directed mutagenesis ............................................................................................. 22 3.4 Cell culture .................................................................................................................... 22 3.4.1 Cultivation of mammalian cell lines ................................................................................ 22 3.4.2 Transfection .................................................................................................................... 22 3.5 Protein biochemistry ................................................................................................... 23 3.5.1 Isolation and quantification of proteins ........................................................................... 23 3.5.2 Membrane preparations ................................................................................................. 23 3.5.3 Co-immunoprecipitation (co-IP) ...................................................................................... 24 3.5.4 SDS-PAGE ..................................................................................................................... 24 3.5.5 Western blotting .............................................................................................................. 25 3.5.6 Native protease cleavage assay .................................................................................... 26 3.5.7 Peptide competition assay ............................................................................................. 26 3.6 Sucrose density gradient centrifugation ................................................................... 27 3.7 Production of rAAVs .................................................................................................... 27 3.7.1 Transfection and harvest ................................................................................................ 27 3.7.2 Iodixanol gradient centrifugation .................................................................................... 28 3.7.3 Anion exchange chromatography ................................................................................... 29 3.7.4 Concentration of rAAVs .................................................................................................. 29 3.7.5 rAAV titer determination ................................................................................................. 30 3.8 Subretinal injection ...................................................................................................... 31 3.9 Immunohistochemistry ................................................................................................ 31 3.10 Confocal microscopy ................................................................................................... 32 3.11 Fluorescence-activated cell sorting (FACS) .............................................................. 32 3.12 Isolation of photoreceptor outer segments ............................................................... 33 3.13 Förster resonance energy transfer (FRET) ................................................................ 33 3.14 Statistics ....................................................................................................................... 35 4 Results ....................................................................................................................... 36 4.1 rAAV PRPH2 minigenes confer efficient protein expression in rods and cones .. 36 4.2 In silico mRNA splice analysis of exon 2-specific peripherin-2 point mutations .. 37 Table of contents 5 4.3 Comparative splice analysis of WT and mutant peripherin-2 minigenes in rods and cones ...................................................................................................................... 38 4.4 Quantitative analysis of native peripherin-2 splice isoforms .................................. 41 4.5 Peripherin-2 isoform encoded by exon 1 is mislocalized in photoreceptors ........ 43 4.6 Protein expression and localization of perMT in rods and cones ............................ 45 4.7 Splicing and protein expression of a rod-dominant perMT in cones and a cone- dominant perMT in rods ................................................................................................ 49 4.8 Impact of the disease-linked perMT on homo- and heteromeric protein-protein interactions ................................................................................................................... 50 4.9 P210 and C214 are crucial for the proper folding of the distal D2 loop ................. 50 4.10 Quantification of perWT, perMT, and Rom-1 protein-protein interactions in HEK293T cells using FRET .......................................................................................................... 52 4.11 Subunit assembly of homo- and heteromeric perWT, perMT, and Rom-1 complexes . ........................................................................................................................................ 54 4.12 Rod OS targeting of homo- and heteromeric perWT, perMT, and Rom-1 complexes .. .......................................................................................................................................