Investigation Into the Roles of Ndel1 in the Postnatal Hippocampus

University of Calgary PRISM: University of Calgary's Digital Repository

Graduate Studies The Vault: Electronic Theses and Dissertations

2017 Investigation into the Roles of Ndel1 in the Postnatal Hippocampus

Kiroski, Ivana

Kiroski, I. (2017). Investigation into the Roles of Ndel1 in the Postnatal Hippocampus (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26651 http://hdl.handle.net/11023/3871 master thesis

University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY

Investigation into the Roles of Ndel1 in the Postnatal Hippocampus

Ivana Kiroski

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN NEUROSCIENCE

CALGARY, ALBERTA

MAY, 2017

Abstract Ndel1 is a microtubule-associated protein (MAP) widely recognized as an integrator of the cytoskeleton. In the developing brain, Ndel1 impacts neurogenesis, nucleokinesis and neuronal migration through interactions with its binding partners, Lis1 and Dynein. Ndel1 knockout mice are embryonic lethal; therefore, the Nguyen lab created conditional knockout mice for Ndel1 (Ndel1 CKO) in CA1 hippocampal excitatory neurons 1 month post birth, to investigate its roles in the postnatal brain. It has been shown that Ndel1 CKO animals exhibit microtubule (MT) fragmentation in CA1 pyramidal neurons associated with synaptic and dendritic pathology, as well as spatial memory impairment. Additionally, Reelin, a secreted glycoprotein involved in the MTs regulation, synaptic plasticity and cell positioning, is reduced in the hippocampus of Ndle1 CKO mice. Interestingly, a single injection of Reelin reduces the MT fragmentation in CA1 pyramidal neurons as well as the synaptic/dendritic pathology. My thesis investigates the significance of Ndel1 and Reelin in postnatal hippocampal integrity and how it impacts spatial learning and memory. My work shows that cellular/anatomical defects do not lead to cell death in the hippocampus of mutant mice. Additionally, CA1 pyramidal neurons become dispersed postnatally, which is decreased with Reelin supplementation. I found an upregulation of Lis1 in Ndel1 CKO hippocampi. As overexpression of Lis1 in the brief presence of nocodazole (MT destabilization agent) generates fragments of MTs disconnected from the centrosomes (Smith et al, Nat Cell Biol 2000), I reasoned that the MTs fragmentation in CA1 pyramidal neurons of Ndel1 CKO mice involves increased Lis1 dosage. Finally, I found that a single injection of Reelin improves the spatial memory of Ndel1 CKO mice during the Morris Water Task. My work provides insight into the postnatal roles of Ndel1 and Reelin in CA1 integrity and how that ultimately impacts spatial learning and memory in Ndel1 CKO mice.

ii Table of Contents

Abstract...... ii Table of Contents...... iii List of Tables...... vi List of Figures...... vii

CHAPTER 1: INTRODUCTION...... 1 1.1 Identification and origin of Ndel1...... 1 1.2 Ndel1 gene expression and regulation...... 1 1.3 Ndel1 protein...... 5 1.4 Embryonic lethality of Ndel1...... 5 1.5 Diverse cellular roles of Ndel1...... 5 1.5.1 Cytoskeletal protein...... 5 1.5.1.1 Ndel1 as a binding partner for Lis1...... 7 1.5.1.2 Cytoplasmic Dynein is a retrograde MT transport complex...... 9 1.5.1.3 Ndel1/Lis1 regulate cytoplasmic Dynein...... 9 1.5.1.5 Functions of the Ndel1/Lis1/Dynein complex...... 11 1.5.1.6 Ndel1 as a regulator of polarized cargo...... 11

1.5.2 Ndel1 as a signal transducing adaptor protein...... 12 1.5.2.1 PDE4/cAMP/PKA cascade...... 12 1.5.2.2 AKT/GSK3 signaling...... 13 1.5.2.3 GTPase regulator...... 13 1.5.3 Ndel1 in cycling cells...... 14 1.5.3.1 Ndel1 in centrosomal maturation and separation...... 14 1.5.3.2 Ndel1 inhibits primary cilia assembly...... 15 1.5.3.3 Ndel1 in mitotic spindle regulation...... 15 1.6 Neurodevelopmental functions of Ndel1...... 16 1.6.1 Ndel1 in neurogenesis...... 16 1.6.2 Ndel1 in neurite outgrowth...... 17 1.6.3 Ndel1 in neuronal migration...... 17 1.7 Reelin and its function during neurodevelopment...... 19 1.7.1 Reelin in corticogenesis...... 21 1.8 Postnatal roles of Ndel1 in the brain...... 22 1.8.1 Ndel1 in neuronal and cell positioning integrity...... 22 1.8.2 Ndel1 in neurofilament assembly...... 22 1.8.3 Ndel1 in maintenance of dendritic arborization...... 22 1.9 Reelin and its function in the adult brain...... 23 1.9.1 Modulates dendritic spine density...... 23 1.9.2 Long-term potentiation and long-term depression...... 24 1.9.3 Cooperates with Ndel1 to maintain postnatal CA1 integrity...... 25 1.10 Ndel1 and its implication in mental disorders...... 25 1.11 Lissencephaly...... 25 1.12 Schizophrenia...... 26 1.12.1 Ndel1 and its interacting partner, DISC1...... 26

iii 1.11 Ndel1 CKO mouse model...... 27 1.12 Statement of thesis goals...... 28

CHAPTER 2: MATERIALS AND METHODS...... 32 2.1 Ndel1 conditional knockout (CKO) mouse model...... 32 2.2 Genotyping...... 32 2.3 Antibodies...... 32 2.4 Cell death analysis...... 32 2.5 Golgi staining...... 33 2.6 Neuron reconstruction using IMARIS...... 33 2.7 Western blotting...... 33 2.8 Stereotaxic Reelin injection...... 33 2.9 Cell dispersion quantification...... 34 2.10 Morris water task...... 34 2.11 24/7 video-electroencephalogram (EEG)...... 35

CHAPTER 3: NDEL1 AND REELIN ARE IMPORTANT FOR POSTNATAL CA1 INTEGRITY IN NDEL1 CKO MICE...... 37 3.1 Characterization of the anatomy of CA1 pyramidal neurons in Ndel1 CKO mice in the presence and absence of Reelin...... 37 3.1.1 No cell death in the hippocampus of Ndel1 CKO mice...... 37 3.1.2 No dendritic arborization deficit in the CA2 and CA3 hippocampal regions...... 37 3.1.3 CA1 cell dispersion in Ndel1 CKO mice...... 39 3.2 Characterization of the CA1 dendritic microtubule fragmentation of Ndel1 CKO mice in the presence and absence of Reelin...... 39 3.2.1 Upregulation of Ndel1 binding partners, Lis1 and Dynein...... 45 3.2.2 Upregulation of acetylated α-Tubulin in 10-week-old Ndel1 CKO mice...... 45 3.3 Investigation into the effects of Reelin supplementation on spatial learning and memory of Ndel1 CKO mice...... 45 3.3.1 Upregulation of Reelin receptors in 7-week-old Ndel1 CKO mice...... 50 3.3.2 Reelin supplementation improves spatial learning and memory in Ndel1 CKO mice...... 50 3.3.3 Reelin supplementation increases the lifespan of Ndel1 CKO mice...... 59 3.4 Discussion of results...... 59 3.4.1 Lifespan too short for cell death to occur?...... 59 3.4.2 Postnatal CA1 cell dispersion in Ndel1 CKO mice...... 61 3.4.3 Overexpression of Lis1 underlies MTs fragmentation I Ndel1 CKO?...... 61 3.4.4 Increased acetylation in 10-week-old Ndel1 CKO mice...too little too late?...... 62 3.4.5 7-weeks is an ideal age for Reelin injection in Ndel1 CKO mice...... 62 3.4.6 Spatial memory in Ndel1 CKO mice...... 62 3.4.7 Limits of the Ndel1 CKO mouse model...... 63 3.4.8 Summary of results...... 63

iv CHAPTER 4: GENERAL DISCUSSION...... 64 4.1 Ndel1 and Reelin in learning and memory...... 64 4.2 Do Ndel1 and Reelin interact or work independently?...... 64 4.3 What causes the short lifespan in Ndel2 CKO mice?...... 65 4.3 Ndel1 in epileptogenesis...... 65 4.4 Additional behavioral phenotypes in Ndel1 CKO mice...... 65 4.5 Future directions...... 66

REFERENCES: ...... 67

APPENDIX: FIGURES...... 96 List of Appendix Figures……...... 96

APPENDIX: SEIZURE ACTIVITY IN NDEL1 CKO MICE...... 101

APPENDIX: