Potential candidate for neuropsychiatric disorders:

MLC1, C15orf53 and OXTR

Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)

durch den Fachbereich I – Psychobiologie der Universität Trier

vorgelegt von

Thorsten Manfred Kranz

Betreuer: Prof. Dr. Jobst Meyer Prof. Dr. Claude P. Muller

Trier, 2012

Der Nutzen von Information liegt eindeutig in der Auswahl,

nicht in der Fülle,

in ihrer Relevanz,

nicht im Übertragungstempo.

Die Kunst vernetzt zu denken

Frederic Vester (1925 – 2003), deutscher Biochemiker und Biokybernetiker

Table of Contents

Table of Contents

Table of Contents ...... I Index of Figures ...... VII Index of Tables ...... IX List of Abbreviations ...... XI

1 Introduction ...... 1 1.1 The short outline on the history of psychiatry from ancient times till present ...... 1 1.2 Main characteristics of schizophrenia, bipolar disorder, periodic catatonia (SCZD10) and autism ...... 4 1.3 Neurodevelopmental hypothesis of mental disorders ...... 5 1.4 Biopsychosocial factors trigger the onset of mental disorders: – environment interaction ...... 6 1.5 Chronic stress deregulates the hypothalamus-pituitary-adrenal gland (HPA) axis and triggers the onset of psychiatric disorders ...... 9 1.6 Mental disorders follow a complex inheritance mode but in rare cases they appear to accumulate in multiplex families ...... 13 1.7 Multiplex families segregating periodic catatonia (SCZD10, OMIM: #605419) reveal susceptibility loci on 15q and 22q ...... 14 1.8 MLC1 mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC) ...... 16 1.9 SCZD10 (periodic catatonia): an entity on its own in psychiatric classification? ..... 17 1.10 Candidate genes for SCZD10, BD and ASD: C15orf53 (Chr. 15q14), oxytocin receptor ( OXTR , Chr. 3p25) and MLC1 (Chr. 22q13.33)...... 17 1.11 Non-coding RNAs and their influence in pathophysiology: regulation of microRNAs (miRNAs) in psychiatric disorders ...... 27 1.12 Transgenic mouse models are a suitable approach to investigate the etiological mechanisms of mental disorders ...... 32 1.13 Research objective ...... 38

I Table of Contents

2 Material & Methods ...... 40 2.1 Psychiatric disorder sample collections ...... 40 2.2 Individual patient samples (non-related) ...... 40 2.2.1 Bipolar disorder (BD) patients ...... 40 2.2.2 Periodic catatonia (SCZD10) patients ...... 41 2.3 Parents-offspring trios and multiplex families (related) ...... 41 2.3.1 Parents-offspring trios with autism spectrum disorder (ASD) ...... 41 2.3.2 Multiplex families segregating SCZD10 ...... 42 2.4 Cell lines ...... 42 2.4.1 Cell lines used for expression analysis and knock down experiments of MLC1 ...... 42 2.4.2 Cell lines cDNA used for gene expression analysis of C15orf53 ...... 43 2.5 Bacterial strains & transformation of DNA ...... 44 2.6 Bacterial DNA vectors ...... 45 2.7 Human brain tissue ...... 45 2.8 Genomic murine DNA & murine embryonic stem cells (ES) ...... 45 2.9 Buffers, media and reagents ...... 46 2.9.1 Used buffers and media ...... 46 2.10 Reagents ...... 48 2.11 Oligonucleotides ...... 51 2.11.1 Oligonucleotides (“primers”) used for PCR reactions ...... 51 2.11.2 Oligonucleotides (“primers”) used for SNP detection ...... 54 2.11.3 Oligonucleotides (“primers”) used for sequencing reactions ...... 54 2.12 Precursor sequences of miRNAs and applied antibodies ...... 55 2.13 Commercial kits ...... 56 2.14 Equipment, bioinformatic tools & databases ...... 57 2.14.1 Equipment ...... 57 2.14.2 Bioinformatic tools & databases ...... 58 2.15 Methods ...... 59 2.15.1 Non-conditional murine Mlc1 knockout mouse procedure ...... 59 2.16 PCR reactions on gDNA of ES ...... 62 2.16.1 Estimation of ES gDNA amount by PCR on Serpina6 ...... 62 2.16.2 Detection of aberrant Mlc1 gDNA seque