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Modern humans, archaic humans and great apes are genetically closely related and share many behavioral and anatomical similarities. However, modern humans differ from the others by a fast development of complex culture and technologies, that rely on complex cognitive abilities. Cognition is directly linked to brain structure and neuronal function. In this thesis, I study neuronal differences between humans and chimpanzees and bonobos as well as morphological differences of “ancestralized” and “modernized” human neurons, that could possibly contribute to cognitive differences among the different hominid species. The comparison of human and ape induced pluripotent stem cell-derived neurons (iNeurons) revealed that the human neurons mature transcriptionally, morphologically and functionally slower than their ape counterparts. By injecting the mRNA of 16 genes that are relevant for neuronal function and carry amino acid substitutions on the modern human lineage, I could show that the 16 proteins are able to increase the total neurite length and suggest a potential slower development of neurons injected with the modern human variants. In a single gene approach, I investigated the effect of modern and ancestral human SSH2 variants on neurite outgrowth and found differences in neurite length and branching pattern, making SSH2 a promising candidate for being involved in human neuron-specific morphology. I showed that iNeurons can serve as a model system for evolutionary neurobiology. I gained insights into features of neurons that are unique to modern humans in comparison to their closest relatives, the great apes and archaic humans.:BIBLIOGRAPHISCHE DARSTELLUNG 2 TABLE OF CONTENTS 3 1. THESIS SUMMARY 6 COMPARISON OF HUMAN AND APE INDUCED NEURONS 7 MICROINJECTION AS A TOOL TO STUDY RECENT HUMAN HISTORY 8 MODELING THE EFFECT OF A SINGLE GENE BY USING TRANSFECTION OF PRIMARY NEURONS 9 CONCLUSION 10 2. ZUSAMMENFASSUNG 11 VERGLEICH VON NERVENZELLEN VON MENSCHEN UND MENSCHENAFFEN 12 MIKROINJEKTION ZUR UNTERSUCHUNG DER JÜNGSTEN MENSCHLICHEN VERGANGENHEIT 14 MODELLIERUNG DES EFFEKTS EINES EINZELNEN GENS AUF DAS NEURITEN WACHSTUM VON PRIMÄREN NEURONEN 15 FAZIT 16 3. INTRODUCTION 17 3.1 THE HOMINID FAMILY 17 3.2 THE HOMINID BRAIN 19 3.3 THE INEURON MODEL SYSTEM 23 3.4 MICROINJECTION OF INS: A TOOL TO STUDY ANCIENT AND MODERN HUMAN NEURONS 24 4. MATERIAL AND METHODS 27 4.1 METHODS 27 4.1.1 GENERATION OF RTTA/NGN2-POSITIVE PLURIPOTENT STEM CELL LINES 27 4.1.2 CULTURING OF PLURIPOTENT STEM CELL LINES 27 4.1.3 CRYOPRESERVATION OF PLURIPOTENT STEM CELLS 28 4.1.4 DIFFERENTIATION OF RTTA/NGN2-POSITIVE PLURIPOTENT STEM CELLS TO INEURONS 28 4.1.5 SINGLE CELL TRANSCRIPTOMIC ANALYSIS 29 4.1.5.1 SINGLE CELL RNA-SEQ DATA GENERATION 29 4.1.5.2 DATA PROCESSING 30 4.1.5.3 IDENTIFICATION OF NEURONAL CELLS AND DIFFERENTIALLY EXPRESSED GENES 30 4.1.5.4 GENE ONTOLOGY ENRICHMENT ANALYSIS 31 4.1.6 ELECTROPHYSIOLOGY 32 4.1.6.1 RECORDINGS 33 4.1.6.2 ANALYSIS 33 4.1.7 LIPOFECTION OF INEURONS 34 4.1.8 IMMUNOSTAINING OF INEURONS 34 4.1.8.1 PREPARATION OF PARAFORMALDEHYDE FIXATIVE 34 4.1.8.2 FIXATION OF GFP-LABELLED INEURONS 34 4.1.8.3 QUENCHING AND IMMUNOSTAINING OF GFP-LABELLED INEURONS 35 4.1.9 IMAGE ACQUISITION 35 4.1.10 IMAGE QUANTIFICATION 35 4.1.10.1 QUANTIFICATION OF NEURONAL MORPHOLOGY. 35 4.1.10.2 QUANTIFICATION OF TUJI SIGNAL. 36 4.1.11 ASSIGNMENT OF CELL IDENTITY. 36 4.1.12 MICROINJECTION 36 4.13 TRANSFECTION OF PRIMARY NEURONS WITH SSH2 PLASMIDS 40 4.1.3.1 CELL CULTURE 40 4.1.3.2 TRASFECTION 40 4.2 MATERIALS 41 5. RESULTS 48 5.1 COMPARISON OF INDUCED NEURONS REVEALS A SLOWER STRUCTURAL AND FUNCTIONAL MATURATION IN HUMANS THAN IN APES 48 5.1.1 ABSTRACT 49 5.1.2 INTRODUCTION 49 5.1.3 RESULTS 51 5.1.3.1 MATURATION OF HUMAN AND APE INDUCED NEURONS IN VITRO 51 5.1.3.2 MORPHOLOGICAL HETEROGENEITY IN IN POPULATIONS 53 5.1.3.3 MORPHOLOGICAL MATURATION OF APE AND HUMAN INS 55 5.1.3.4 SCRNASEQ REVEALED THAT NGN2 INDUCES CORTICAL AND SENSORY NEURON FATES 56 5.1.3.5 NGN2 ALSO INDUCES CORTICAL SENSORY NEURON FATE 59 5.1.3.6 TRANSCRIPTIONAL MATURATION OF HUMAN AND CHIMPANZEE INS 60 5.1.3.7 INTRINSIC PASSIVE ELECTROPHYSIOLOGICAL PROPERTIES OF HUMAN AND APE INS 62 5.1.3.8 ACTIVE ELECTROPHYSIOLOGICAL PROPERTIES OF APE AND HUMAN INS 63 5.1.4 DISCUSSION 65 5.1.4.1 NGN2 INDUCES HETEROGENEOUS NEURONAL FATES 65 5.1.4.2 EVOLUTIONARY ASPECTS OF NEURONAL MATURATION 65 5.5. SUPPLEMENTARY INFORMATION 68 5.5.1 SUPPLEMENTARY FIGURES 68 5.5.2 SUPPLEMENTARY TABLES 81 5.2 MRNA MICROINJECTION AS A TOOL TO STUDY RECENT HUMAN BRAIN EVOLUTION 88 5.2.1 ABSTRACT 89 5.2.2 INTRODUCTION 89 5.2.3 RESULTS 91 5.2.3.1 NEURONAL GENES CARRYING AMINO ACID SUBSTITUTIONS BETWEEN MODERN AND ARCHAIC HUMANS 91 5.2.3.2 SCREEN OF DISTINCT TRANSCRIPTOME DATASETS FOR EXPRESSION ANALYSES OF THE 16 NEURONAL GENES 94 5.2.3.3 TRANSCRIPTIONAL ANALYSES OF THE 16 NEURONAL GENES IN INEURONS 97 5.2.3.4 MICROINJECTION OF THE 16 NEURONAL GENES INTO INEURONS 99 5.2.3.5 EFFECT OF SSH2 GENE VARIANTS ON HUMAN PRIMARY NEURONS 101 5.2.4. DISCUSSION 103 6. DISCUSSION 108 6.1 COMPARISON OF HUMAN AND APE INDUCED NEURONS 108 6.2 EXPERIMENTAL SYSTEMS TO MODEL RECENT HUMAN HISTORY 109 6.3 MRNA MICROINJECTION TO MODEL MULTIGENIC HUMAN TRAITS 110 6.4 MODELING THE EFFECT OF A SINGLE GENE BY USING TRANSFECTION OF PRIMARY NEURONS 111 6.5 CONCLUDING REMARKS 111 INDEX OF FIGURES 113 SUPPLEMENTARY FIGURES 113 INDEX OF TABLES 114 SUPPLEMENTARY TABLES 114 REFERENCES 115 SOFTWARE AND SCRIPTS 128 ACKNOWLEDGEMENTS 129 CURRICULUM VITAE 130 PUBLICATIONS 133 SELECTED TALKS 134 POSTER PRESENTATIONS 134 SELBSTÄNDIGKEITSERKLÄRUNG 135

<p>Parkinson’s disease (PD) is characterized by progressive cell death of the dopaminergic neurons of nigrostriatal pathway. Several causes have been implicated for PD via neurochemical research including mitochondrial dysfunction, oxidative stress, and protein misfolding, to list a few. The novel Catecholamine Regulated Protein 40 (CRP40) has certain dopaminergic and neuroprotective features that implicate its importance for PD research. Recent studies using post-mortem brain tissue of patients with PD found MOT-2/CRP40 depletion in the frontal cortex and substantia nigra. MOT-2/CRP40 reduction is also observed in striatal brain tissue samples from a hemi-lesioned preclinical animal model of PD. Most recently, work done at the University of Laval in collaboration McMaster University suggests that levels of CRP40 mRNA are in deficit in blood platelet samples from a primate model of PD.</p> <p>The studies presented in this thesis suggest that the CRP40 protein has a dual function with regards to PD. The full-length CRP40 binds dopamine and, upon injection at the striatum of 6-hydroxydopamine hemi-lesioned rats, alleviates behavioural symptoms for up to 7 days. On the other hand, a 7kDA fragment of CRP40 does not bind dopamine, but does confer the same alleviatory effect upon intra-striatal injection in 6-hydroxydopamine hemi-lesioned rats. Not only has a protein now been identified with novel potential as a therapeutic agent for PD, but also the approximate region of the CRP40 protein responsible for behavioural effects.</p> <p>The later studies of this thesis show that CRP40 is found dysregulated in platelets of PD patients and lymphocytes of SCZ patients. This evidence has revealed CRP40 as a novel PD biomarker, for which on going studies are now in place to explore the potential of CRP40 as a diagnostic for PD.</p> / Doctor of Philosophy (PhD)