Multi-omics analysis of human brain tissue and an animal model of Parkinson’s Disease Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program “International Max Planck Research School for Neurosciences” of the Georg-August University School of Science (GAUSS) submitted by Lucas A. Caldi Gomes Born in Londrina, Paraná, Brasil Göttingen, September, 2019 Thesis Committee Prof. Dr. Paul Lingor (Rechts der Isar Hospital of the Technical University Munich, Dept. of Neurology – Translational Neurodegeneration Laboratory) Prof. Dr. André Fischer (University Medical Center Göttingen, Dept. Epigenetics and Systems Medicine in Neurodegenerative Diseases, DZNE Göttingen) Prof. Dr. Silvio Rizzoli (University Medical Center Göttingen, Dept. of Neuro- and Sensory Physiology) Members of the Examination Board 1st Referee: Prof. Dr. Paul Lingor (Rechts der Isar Hospital of the Technical University Munich, Dept. of Neurology – Translational Neurodegeneration Laboratory) 2nd Referee: Prof. Dr. André Fischer (University Medical Center Göttingen, Dept. Epigenetics and Systems Medicine in Neurodegenerative Diseases, DZNE Göttingen) Further members of the Examination Board Prof. Dr. Silvio Rizzoli (University Medical Center Göttingen, Department of Neuro- and Sensory Physiology) Dr. Camin Dean (European Neuroscience Institute Göttingen, Trans-synaptic Signaling Laboratory) Dr. Jens Gruber (German Primate Center, Medical RNA Biology Laboratory) Dr. Sebastian Kügler (University Medical Center Göttingen, Dept. of Neurology – Viral vectors Laboratory) Date of oral examination: 11.10.2019 “I am driven by two main philosophies: know more today about the world than I knew yesterday and lessen the suffering of others. You'd be surprised how far that gets you.” ― Neil deGrasse Tyson Table of Contents 1. Introduction .......................................................................................................... 1 1.1. Neurodegenerative diseases in modern society ................................................................ 1 1.2. Parkinson’s Disease ............................................................................................................ 1 1.2.1. Parkinson’s Disease – history, epidemiology and etiology ........................................ 1 1.2.2. Pathophysiology and progression of PD .................................................................... 3 1.2.3. PD diagnosis and therapeutic alternatives ................................................................. 6 1.3. Gene expression regulation and miRNA biology ................................................................ 7 1.3.1. Overview on epigenetics and miRNAs ....................................................................... 8 1.3.2. miRNA biogenesis and gene silencing ........................................................................ 9 1.3.3. The role of miRNAs in neurodegeneration and PD .................................................. 11 1.4. Objectives of this doctoral thesis ..................................................................................... 13 2. Material and Methods ......................................................................................... 15 2.1. Materials .......................................................................................................................... 15 2.1.1. Human samples ........................................................................................................ 15 2.1.2. Reagents ................................................................................................................... 16 2.1.3. Primers and Kits........................................................................................................ 17 2.1.4. Buffers ...................................................................................................................... 20 2.1.5. Equipment ................................................................................................................ 21 2.1.6. Software ................................................................................................................... 22 2.1.7. Genes selected for MLPA and gene panel sequencing experiments ....................... 23 2.2. Methods ........................................................................................................................... 25 2.2.1. Human samples ........................................................................................................ 25 2.2.2. Animal samples ........................................................................................................ 26 2.2.3. Molecular biology techniques .................................................................................. 28 3 Results ................................................................................................................ 44 3.1 Assessment of genetic alterations in PD patients by MLPA and gene panel sequencing 44 3.2 Small RNA sequencing overview and small RNA profiles in PD / control midbrains ....... 46 3.3 Differential expression analyses of small RNA sequencing results reveal regulated and potential signature miRNAs for PD .............................................................................................. 47 3.4 Sample correlation analyses ............................................................................................ 49 3.5 Literature screening links differentially expressed miRNAs to important biological processes both in health and disease .......................................................................................... 51 3.6 Functional annotation with targets of differentially expressed miRNAs identify important enriched pathways in the context of PD ..................................................................... 52 3.7 RNA sequencing overview and transcriptomic raw data processing ............................... 54 3.8 Transcriptomic differential expression analyses point towards dopaminergic depletion and massive inflammation/immune response in midbrains of PD subjects ................................ 56 3.9 Differences in protein content in PD and control midbrain are portrayed by proteomics analysis ......................................................................................................................................... 59 3.10 Functional enrichment of proteomics data reveals regulation in PD-related pathways . 61 3.11 Step-wise integration of multi-omics dataset for pathway identification ....................... 64 3.11.1 Integration of Small RNA and Transcriptomic datasets ........................................... 64 3.11.2 Integration of Transcriptomic and Proteomic datasets ........................................... 67 3.12 Validation of small RNA sequencing results in human midbrain tissue by q-RT-PCR ...... 69 3.13 Validation of transcriptomics results in human midbrain tissue by q-RT-PCR ................. 71 3.14 Comparison of human RNA sequencing results with data from a PD mouse model ....... 73 3.14.1 Validation of PD-deregulated miRNAs in αSyn.A53T midbrains .............................. 73 3.14.2 Validation of PD deregulated mRNAs in αSyn.A53T midbrains ............................... 76 4 Discussion ........................................................................................................... 79 4.1 No major genetic alterations are found in the PD patient cohort ................................... 79 4.2 Small RNA sequencing results reveals differentially expressed miRNAs in PD including candidates with potential discriminative power.......................................................................... 80 4.3 Pathway enrichment analysis reveals important biological roles for the targets of differentially expressed miRNAs in the context of PD ................................................................. 83 4.4 Transcriptomic profiling documents dopaminergic depletion and indicates an important inflammatory reaction in PD midbrains ....................................................................................... 86 4.5 Proteomic profiling reveals enrichment in PD-related pathways matching the transcriptomic results .................................................................................................................. 92 4.6 Multi-omics integration identifies common pathways in matched datasets .................. 95 4.6.1 Integration of small RNA and transcriptomics ......................................................... 95 4.6.2 Integration of and transcriptomics and proteomics ................................................ 97 4.7. Validation of sequencing results in human midbrain tissue by q-RT-PCR ....................... 98 4.7.1. miRNA data validation .............................................................................................. 98 4.7.2. Transcriptomics data validation ............................................................................. 100 4.8. Validation of PD-deregulated miRNAs and transcripts in αSyn.A53T midbrains ........... 101 5. Concluding remarks ............................................................................................ 106 6. Summary ............................................................................................................ 108 7. References ......................................................................................................... 109 8.