TECHNISCHE UNIVERSITÄT MÜNCHEN FAKULTÄT WISSENSCHAFTSZENTRUM WEIHENSTEPHAN FÜR ERNÄHRUNG, LANDNUTZUNG UND UMWELT LEHRSTUHL FÜR ENTWICKLUNGSGENETIK From Novel Disease Genes to New Mouse Models - A Complementary Approach Caroline Alexandra Biagosch Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. Martin Hrab ĕ de Angelis Prüfer der Dissertation: 1. Priv.-Doz. Dr. Thomas Floss 2. Prof. Angelika Schniecke, Ph.D. Die Dissertation wurde am 10.10.2016 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 08.03.2017 angenommen. To my family. TABLE OF CONTENTS ABSTRACT ......................................................................................................................... 9 English .............................................................................................................................................. 9 German ........................................................................................................................................... 11 I. IDENTIFICATION OF A NEW DISEASE-ASSOCIATED GENE – FBXL4 .... 13 I.1. INTRODUCTION .............................................................................................................. 13 I.1.1. Genetic disease and mitochondriopathies .............................................................................13 I.1.2. Development of genetic disorders ..........................................................................................13 I.1.3. Mitochondria and oxidative phosphorylation .......................................................................14 I.1.4. Mitochondriopathies ...............................................................................................................15 I.1.5. Gene discovery approaches ....................................................................................................15 I.1.6. Functional validation of candidate genes ..............................................................................16 I.2. RESULTS ............................................................................................................................ 18 I.2.1. Identification of FBXL4 as a candidate gene for early-onset mitochondrial encephalopathy ........................................................................................................................................18 I.2.2. Molecular genetic analysis of FBXL4 ....................................................................................19 I.2.3. Clinical and biochemical features ..........................................................................................20 I.2.4. Validation of FBXL4 via cellular rescue experiments .........................................................21 I.2.5. Mutations in FBXL4 are related to a reduction of mitochondrial proteins .......................23 I.3. DISCUSSION ..................................................................................................................... 24 I.3.1. FBXL4 dysfunction causes a clinically and biochemically distinct phenotype ..................24 I.3.2. FBXL4 dysfunction causes a mitochondrial disorder ..........................................................25 I.3.3. Outlook: Relevance for diagnosis and protein function .......................................................27 II. GENERATION OF A MOUSE MODEL ( WDR45 KO) RESEMBLING THE HUMAN DISEASE BPAN ............................................................................................... 29 II.1. INTRODUCTION .............................................................................................................. 29 II.1.1. Generation of a knockout mouse model ................................................................................29 II.1.1.1. The use of mouse models and genetic engineering ......................................................................... 29 II.1.1.2. TALEN............................................................................................................................................... 30 II.1.1.3. CRISPR/Cas9 .................................................................................................................................... 31 5 TABLE OF CONTENTS II.1.1.4. The German Mouse Clinic and the establishment of a mouse model ........................................... 33 II.1.2. The human disease BPAN and the corresponding Wdr45 knockout ................................. 34 II.1.2.1. Neurodegeneration with brain iron accumulation (NBIA) ........................................................... 34 II.1.2.2. Beta-propeller protein-associated neurodegeneration (BPAN) .................................................... 35 II.1.2.3. Genetics of BPAN and the disease-associated gene WDR45 .......................................................... 36 II.1.2.4. The brain-specific Wdr45 knockout mouse model ......................................................................... 36 II.1.2.5. WDR45 and its function in autophagy ............................................................................................ 38 II.2. RESULTS ........................................................................................................................... 41 II.2.1. Clinical presentation in human BPAN patients ................................................................... 41 II.2.2. Generation of a Wdr45 knockout mouse via TALENs ........................................................ 43 II.2.3. Systemic Wdr45 KO is viable in mice .................................................................................... 45 II.2.4. Analysis of preliminary Wdr45 KO mice (F2) shows neurobehavioral deficits ................ 47 II.2.5. Screen of the disease progression Wdr45 cohort (F3) .......................................................... 50 II.2.5.1. Neurologic deterioration in Wdr45 KO mice .................................................................................. 50 II.2.5.2. Pathologic brain abnormalities in Wdr45 KO mice ....................................................................... 54 II.2.6. Secondary screen of aged Wdr45 KO GMC cohort (F4) ..................................................... 58 II.3. DISCUSSION ..................................................................................................................... 59 II.3.1. Of mice and men: the Wdr45 KO mouse as a model for BPAN ......................................... 59 II.3.2. The BPAN mouse model helps investigate disorders of autophagy ................................... 62 II.3.3. From the BPAN mouse model to therapeutic strategies ..................................................... 65 III. THERAPEUTIC STRATEGY FOR GLUTARIC ACIDURIA TYPE 1 .......... 67 III.1. INTRODUCTION .......................................................................................................... 67 III.1.1. The lysine degradation pathway ............................................................................................ 67 III.1.2. Glutaric aciduria type 1 - GCDH .......................................................................................... 69 III.1.3. 2-aminoadipic 2-oxoadipic aciduria – DHTKD1 ................................................................. 71 III.1.4. Glutaric aciduria type 3 – C7ORF10 .................................................................................... 72 III.1.5. DHTKD1 inhibition as a therapy for glutaric aciduria type 1 ........................................... 72 III.2. RESULTS ........................................................................................................................ 73 III.2.1. Generation of a Dhtkd1 KO mouse via TALENs ................................................................. 73 III.2.2. Clinical and biochemical characterization of the Dhtkd1 KO mouse ................................ 75 III.2.3. Generation of a Gcdh/Dhtkd1-double KO mouse................................................................. 77 III.2.4. Biochemical characterization of the double KO mouse ...................................................... 78 6 TABLE OF CONTENTS III.2.5. High-lysine diet challenges knockout lines ............................................................................79 III.3. DISCUSSION .................................................................................................................. 82 III.3.1. DHTKD1 inhibition is not a promising treatment strategy for GA-I .................................82 III.3.2. Murine double KO experiments question the current theory of lysine degradation ........82 III.3.3. Future investigations of the lysine degradation pathway towards GA-I therapy .............83 IV. CONCLUSION AND OUTLOOK ........................................................................ 85 V. MATERIAL AND METHODS ............................................................................. 88 V.1. Material ............................................................................................................................... 88 V.1.1. Chemicals and reagents ..........................................................................................................88 V.1.2. Enzymes and proteins