Analyzing the Role of CyFIP2 in the Mouse Brain Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms Universität Bonn Vorgelegt von Ö Ilkin Deniz zer aus Trabzon, Türkei Bonn, 2020 Angefertigt mit Genehmigung der Mathematischen-Naturwissenschaftlichen-Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. 1. Gutachter: Prof. Dr. rer. nat. Walter Witke 2. Gutachter: Prof. Dr. rer. nat. Frank Bradke Tag der Promotion: 05.06.2020 Erscheinungsjahr: 2020 To my parents, for making everything possible. Summary An important regulator of neuron outgrowth, growth cone motility and dendritic spine formation is the actin nucleator ARP2/3, a protein complex that functions in forming branched actin networks. ARP2/3 complex activity is regulated by the class I nucleation promoting factor WAVE regulatory complex (WRC). In the brain, a crucial component of the pentameric WRC is the Cytoplasmic Fragile-X Mental Retardation Protein (FMRP) Interacting Protein 2 (CyFIP2) that is highly expressed in neurons. Knockout of the Cyfip2 gene in mice leads to perinatal lethality. When Cyfip2 is conditionally knocked out exclusively in the central nervous system (CNS), the mice fully recapitulate the complete knockout phenotype, thus suggesting a specific role for CyFIP2 in the CNS. Here we present our studies on a viable Cyfip2 conditional knockout mouse model, in which the deletion of the gene occurs at around postnatal day 18 (P18) mainly in hippocampal and cortical glutamatergic neurons. Upon complete loss of CyFIP2, we observed an increase of the small, dynamic actin filaments with no detectable changes in the stable actin pool. The analysis of excitatory synaptic transmission using patch-clamp recordings as well as Schaffer collateral stimulation recordings showed decreased excitatory synaptic transmission and defects in pre-synaptic vesicle exocytosis in the hippocampus. Using electron microscopy, we also showed reduced pre-synaptic bouton area accompanied by decreased neurotransmitter vesicle density in both Schaffer collaterals and mossy fibers, while post-synaptic densities did not appear significantly altered. In aged mice lacking CyFIP2, we observed increased dendritic spine number possibly in an attempt to compensate for the defects observed in the pre-synapse. These spines were mostly immature, phenocopying the Fragile-X syndrome mouse model. Lastly, a combination of behavioral tests showed a mild anxiety-like phenotype in Cyfip2 forebrain knockout mice with no defects in working and short-term memory according to Y-maze and novel object recognition experiments, respectively. Our electrophysiological, morphological and biochemical results together suggest a role of CyFIP2 in the structural maintenance of pre-synaptic terminals and dendritic spine maturation in the adult mouse brain that does not appear to be relevant for neurodegenerative processes, but rather for intellectual disability. iv Table of Contents List of Figures ............................................................................................................. x List of Tables ........................................................................................................... xiii Abbreviations ........................................................................................................... xiv 1. INTRODUCTION ................................................................................................... 1 1.1 The actin cytoskeleton ......................................................................................... 2 1.1.1. Actin binding proteins ................................................................................... 3 1.1.2. Actin nucleation factors ................................................................................ 4 1.1.3. The WAVE regulatory complex .................................................................... 6 1.1.4. The CyFIP1 isoform ..................................................................................... 7 1.1.5. The CyFIP2 isoform ..................................................................................... 9 1.1.5.1. CyFIP2 is expressed predominantly in brain........................................ 10 1.1.5.2. Alzheimer’s-like phenotype of Cyfip2+/- mice ....................................... 10 1.2. The nervous system .......................................................................................... 11 1.2.1. The synapse ............................................................................................... 12 1.2.2. Synaptic transmission ................................................................................ 13 1.2.3. Actin in the synapse ................................................................................... 16 1.2.3.1. Actin in synaptogenesis ....................................................................... 16 1.2.3.2. Actin in pre-synaptic boutons ............................................................... 17 1.2.3.3. Actin in dendritic spines ....................................................................... 18 1.3. Dementia .......................................................................................................... 19 1.3.1. Alzheimer’s disease ................................................................................... 20 1.3.1.1. APP processing ................................................................................... 21 1.3.1.2. Mouse models for Alzheimer’s disease ............................................... 22 1.3.2. Behavioral paradigms ................................................................................. 23 1.4. The Cyfip2flx/flx;Camk2a-Cre mouse model ....................................................... 25 1.5. Aim of the thesis ............................................................................................... 26 v 2. RESULTS ............................................................................................................ 27 2.1. CyFIP2 expression in the mouse brain ............................................................. 28 2.1.1. Glutamatergic neurons express CyFIP2 ..................................................... 28 2.1.2. Glial cells are devoid of CyFIP2 ................................................................. 30 2.2. Analysis of Cyfip2 conditional knockout mice ................................................... 31 2.2.1. CyFIP2 has a long half-life in vivo .............................................................. 32 2.2.2. WRC components are co-regulated with CyFIP2 ....................................... 33 2.2.3. CyFIP2 loss leads to an increase of small actin filaments .......................... 34 2.3. Synaptic analysis of Cyfip2 conditional knockout mice ..................................... 36 2.3.1. CyFIP2 loss leads to defects in synaptic transmission ............................... 36 2.3.2. Cyfip2 conditional knockout mice have decreased pre-synaptic vesicle exocytosis ............................................................................................................ 38 2.3.3. Loss of CyFIP2 leads to pre-synaptic terminal shrinkage in the hippocampus ............................................................................................................................. 41 2.3.4. CyFIP2 loss leads to altered levels of certain pre-synaptic proteins upon aging .................................................................................................................... 43 2.4. Evaluation of an Alzheimer’s-like phenotype in Cyfip2 conditional knockout mice ................................................................................................................................. 44 2.4.1. Cyfip2 conditional knockout mice show some molecular signatures of Alzheimer’s disease ............................................................................................. 45 2.4.2. CyFIP2 is involved in mRNA translation regulation .................................... 48 2.5. Morphological analysis of Cyfip2 conditional knockout mice reveals a phenotype similar to Fragile-X Syndrome .................................................................................. 49 2.5.1. Cyfip2 conditional knockout mice have increased number of spines .......... 49 2.5.2. CyFIP2 loss leads to decreased mature/immature spine ratios.................. 51 2.6. Behavioral analysis of Cyfip2 conditional knockout mice .................................. 54 2.6.1. Cyfip2 conditional knockout mice show decreased exploratory behavior ... 54 2.6.2. Cyfip2 conditional knockout mice have unaltered working memory ........... 57 2.6.3. CyFIP2 loss does not affect object recognition memory ............................. 59 vi 3. DISCUSSION ...................................................................................................... 61 3.1. CyFIP2 is expressed in glutamatergic and cholinergic neurons but not in glia cells.......................................................................................................................... 62 3.2. CyFIP2 is crucial for WRC integrity and regulation of dynamic actin filaments . 63 3.3. Cyfip2 conditional knockout mice show molecular and morphological signs of neurodegeneration with no memory defects ............................................................ 66 3.4. CyFIP2 loss leads to an FXS-like phenotype in the post-synapse .................... 68 3.5.