PhD PROGRAM IN TRANSLATIONAL AND MOLECULAR MEDICINE DIMET Functional analysis of m-AAA homo- and heterocomplexes: the role of mitochondrial protein quality control system in spinocerebellar neurodegeneration Coordinator: Prof. Andrea Biondi Tutor: Dr. Valeria Tiranti Cotutor: Dr. Franco Taroni Dr. Stefania Magri Matr. No. 725279 XXIV CYCLE ACADEMIC YEAR 2010-2011 1 2 3 4 Table of Contents Chapter 1 ........................................................... 13 General introduction ......................................................... 13 Mitochondrial dysfunction and neurodegenerative diseases ................................................................................ 13 Mitochondrial proteases and protein processing .............. 16 Mitochondrial proteases and quality control .................... 18 Matrix .............................................................. 20 Inner membrane ............................................... 20 Intermembrane space ........................................ 21 Outer membrane............................................... 21 Mitochondrial proteases and dynamics ........................... 22 OPA1 ............................................................... 23 PINK ................................................................ 25 AAA proteases ........................................................... 26 AAA+ superfamily .............................................. 26 AAA metalloprotease ......................................... 26 AAA metalloprotease domains ............................. 29 Structure-function relationship (operational model) ...................................................................... 32 Role of m-AAA complex ............................................... 35 In yeast saccharomyces cerevisiae ...................... 35 5 In humans ....................................................... 38 AFG3L2...................................................................... 39 Spinocerebellar ataxia ....................................... 40 SCA28 ............................................................. 41 Paraplegin .................................................................. 44 Hereditary spastic paraparesis and SPG7 ............. 44 Mouse models ............................................................ 45 Scope of the thesis ........................................................... 48 References ........................................................................ 49 Chapter 2 ........................................................... 64 Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28 ................................... 64 Abstract ..................................................................... 65 Introduction ............................................................... 66 Results ...................................................................... 67 Missense mutations affect functional domains of AFG3L2 ............................................................ 67 AFG3L2 substitutions impair respiration in yeast ... 68 AFG3L2 substitutions impair cytochrome c oxidase activity in yeast ................................................ 70 The mutations alter the proteolytic activity of AFG3L2 ............................................................ 71 Structural modeling of AFG3L2 mutants ............... 72 Protein expression studies in cells from affected individuals ........................................................ 73 Expression of AFG3L2 and paraplegin in the nervous system ............................................................ 73 Discussion .................................................................. 74 6 Reference .................................................................. 80 Figures ...................................................................... 83 Online Methods ........................................................... 95 Affected individuals, DNA samples and cell lines. ... 95 Mutation analysis. ............................................. 96 AFG3L2 and paraplegin yeast expression plasmids.96 Yeast strains and growth conditions. .................... 96 Assay of cytochrome c oxidase activity. ............... 97 Antibodies. ....................................................... 97 Protein immunoblot analysis. .............................. 98 Co-immunoprecipitation of AFG3L2 and paraplegin. ...................................................................... 99 Homology modeling. .......................................... 99 Confocal immunofluorescence analysis of human nervous tissue. ................................................. 99 In situ hybridization on mouse cerebellum. ......... 100 URLs. ............................................................ 100 Supplementary Information ........................................ 102 Genetic studies ............................................... 102 RNA analysis .................................................. 102 In silico analysis of the identified AFG3L2 mutations .................................................................... 102 Mutation screening .......................................... 103 Structural modeling of AFG3L2 mutations........... 104 Expression studies .......................................... 106 AFG3L2 and paraplegin yeast expression plasmids .................................................................... 106 Cytochrome c oxidase analysis in yeast.............. 108 7 Generation of antibodies against AFG3L2 and paraplegin ...................................................... 109 SK-N-SH cell culture and confocal immunofluorescence analysis ........................... 109 Immunoblot analysis of patients‘ cells ................ 110 Blue native gel electrophoresis.......................... 110 Supplementary Table 1. AFG3L2 polymorphic variants observed in this study ......................... 111 Supplementary Table 2. Oligonucleotide primers used for PCR amplification, DNA sequencing, and DHPLC analysis of AFG3L2 exons ...................... 112 DNA sequence analysis .................................... 112 Supplementary Table 3. Saccharomyces cerevisiae strains used in this study ................................. 113 Supplementary References ....................................... 129 Co-immunoprecipitation of human mitochondrial proteases AFG3L2 and paraplegin heterologously expressed in yeast cells ................................................................................ 131 Introduction ............................................................ 132 Materials .................................................................. 132 Time Taken ............................................................. 133 Procedure ................................................................ 133 Troubleshooting ....................................................... 136 Critical Steps ........................................................... 136 Keywords ................................................................ 136 Preparation of yeast mitochondria and in vitro assay of respiratory chain complex activities ............................... 138 Introduction ............................................................. 139 Materials .................................................................. 139 Time Taken .............................................................. 140 Procedure ................................................................ 141 Cell culture ..................................................... 141 8 Preparation of mitochondria by differential centrifugations ................................................ 141 Sample preparation ......................................... 142 Ubiquinol:cytochrome c oxidoreductase (Complex III) activity assay ............................................ 142 Cytochrome c oxidase (Complex IV) activity assay .................................................................... 143 ATP synthase (Complex V) activity assay ........... 144 Critical Steps ............................................................ 145 References ............................................................... 145 Keywords ................................................................. 145 Chapter 3 ......................................................... 147 Spinocerebellar ataxia type 28: identification and functional analysis of novel AFG3L2 mutations ............................... 147 Abstract ................................................................... 148 Introduction ............................................................. 149 Results .................................................................... 151 Mutation analysis ............................................ 151 Respiratory competence of AFG3L2 variants in yeast model ............................................................ 153 Proteolytic activity of AFG3L2 variants ............... 155 Evaluation of mitochondrial amount ................... 156 AFG3L2 mutations assemble with paraplegin ...... 157 Discussion ................................................................ 159 Materials and methods .............................................. 164 Patients and DNA samples ................................ 164 Mutation analysis ............................................ 164 9 AFG3L2 and paraplegin yeast expression plasmids .................................................................... 165 Yeast strains and growth conditions