Involvement of neuronal nitric oxide synthase (NOS-I) PDZ ... Involvement of neuronal nitric oxide...

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  • Involvement of neuronal nitric oxide synthase (NOS-I) PDZ

    interactions in neuropsychiatric disorders

    Der Einfluss von PDZ Interaktionen der neuronalen Stickstoffmonoxidsynthase (NOS-I) auf

    neuropsychiatrische Störungen

    Doctoral thesis for a doctoral degree

    at the Graduate School of Life Sciences,

    Julius-Maximilians-Universität Würzburg,

    Section Neuroscience

    submitted by

    Esin Candemir

    from Istanbul, Turkey

    Würzburg 2017

  • Involvement of neuronal nitric oxide synthase (NOS-I) PDZ interactions in neuropsychiatric disorders

    Der Einfluss von PDZ Interaktionen der neuronalen Stickstoffmonoxidsynthase (NOS-I) auf

    neuropsychiatrische Störungen

    Doctoral thesis for a doctoral degree

    at the Graduate School of Life Sciences,

    Julius-Maximilians-Universität Würzburg,

    Section Neuroscience

    submitted by

    Esin Candemir

    from Istanbul, Turkey

    Würzburg 2017

  • Submitted on: ………………………………………………………..……..

    Office stamp

    Members of the Promotionskomitee:

    Chairperson: Prof. Dr. Michael Sendtner

    Primary Supervisor: Prof. Dr. Andreas Reif

    Supervisor (Second): Prof. Dr. Klaus-Peter Lesch

    Supervisor (Third): Prof. Dr. Erhard Wischmeyer

    Supervisor (Fourth): …………………………………………………….

    Date of Public Defence: …………………………………………….…………

    Date of Receipt of Certificates: …………………………………………….

  • Table of Contents

    Table of Contents Summary ........................................................................................................................................................ i

    Zusammenfassung ........................................................................................................................................ iii

    1. Introduction ........................................................................................................................................... 1

    1.1. Neuropsychiatric disorders ........................................................................................................... 1 1.1.1. Gene-environment interactions in neuropsychiatric disorders .................................................. 2 1.1.2. Synapse and dendrite maintenance ........................................................................................... 3 1.2. Nitric oxide (NO) system .............................................................................................................. 3

    1.2.1. NOS1 gene ............................................................................................................................. 4 1.2.2. Neuronal nitric oxide synthase (NOS-I) protein .................................................................... 4 1.2.3. NOS-I adaptor protein (NOS1AP) ......................................................................................... 7

    1.3. Research approaches ..................................................................................................................... 9 1.3.1. Behavioral testing in rodents ............................................................................................... 10

    2. Materials and Methods ........................................................................................................................ 17

    2.1. Molecular Methods ..................................................................................................................... 17 2.1.1. Plasmid cloning .................................................................................................................... 17 2.1.2. Recombinant AAV production and titration ........................................................................ 19 2.1.3. In vitro experiments ............................................................................................................. 21 2.1.4. Immunofluorescence and Microscopy ................................................................................. 23

    2.2. Behavioral Experiments .............................................................................................................. 24 2.2.1. Animals and ethics ............................................................................................................... 24 2.2.2. Viral vector delivery ............................................................................................................ 24 2.2.3. Chronic mild stress paradigm .............................................................................................. 25 2.2.4. Elevated zero maze (EZM) .................................................................................................. 26 2.2.5. Light-dark box (LDB) .......................................................................................................... 26 2.2.6. Open field (OF) .................................................................................................................... 27 2.2.7. Novel object exploration (OE) and recognition (NOR) ....................................................... 27 2.2.8. Social interaction and social novelty recognition ................................................................ 28 2.2.9. Prepulse inhibition (PPI) of the acoustic startle response (ASR) ........................................ 29 2.2.14. Forced Swim Test (FST) ...................................................................................................... 32 2.2.15. Nest building behavior and Sucrose Preference .................................................................. 33 2.2.16. Delay fear conditioning ....................................................................................................... 33 2.2.17. Perfusion of animals ............................................................................................................ 34 2.2.18. Statistics ............................................................................................................................... 35

    3. Results ................................................................................................................................................. 36

    3.1. In vitro results ............................................................................................................................. 36

  • Table of Contents

    3.1.1. Viruses and expression of virally encoded constructs ......................................................... 36 3.1.2. Interaction of viral constructs with endogenous NOS-I ....................................................... 36 3.1.3. Regulation of dendritic development and spinogenesis ....................................................... 39

    3.2. Behavioral consequences of disrupted NOS-I PDZ interactions ................................................ 45 3.2.1. Body weight of mice ............................................................................................................ 45 3.2.2. Locomotor Activity .............................................................................................................. 46 3.2.3. Anxiety-Related Behaviors .................................................................................................. 47 3.2.4. Novel object recognition ...................................................................................................... 49 3.2.5. Depressive-like behaviors .................................................................................................... 49 3.2.6. Spatial working memory ...................................................................................................... 52 3.2.7. Spatial reference memory .................................................................................................... 54 3.2.8. Sensorimotor gating deficits ................................................................................................ 55 3.2.9. Social interaction and social novelty recognition ................................................................ 57 3.2.10. Delay fear conditioning ....................................................................................................... 58

    4. Discussion ........................................................................................................................................... 60

    4.1. Interaction of virally expressed proteins with endogenous proteins ........................................... 60 4.2. Disruption of NOS-I PDZ interactions alters neurite growth ..................................................... 62 4.3. Behavioral consequences of disrupted NOS-I PDZ interactions ................................................ 64

    4.3.1. NOS-I interaction partners have different effects on locomotion ........................................ 65 4.3.2. Anxiety-related behaviors upon disrupted NOS-I PDZ interactions ................................... 66 4.3.3. Disruption of NOS-I PDZ interaction does not induce depressive-like behavior ................ 67 4.3.4. Sensorimotor gating remain substantially intact upon disruption of NOS-I PDZ interaction . ……………………………………………………………………………………………..68 4.3.5. Social interaction is dependent on NOS-I PDZ interactions ................................................ 68 4.3.6. Learning and memory is mildly i