INVESTIGATING THE NEUROBIOLOGICAL ROLE OF TUBBY, A PROTEIN INVOLVED IN OBESITY by RYAN MUI A THESIS SUBMITTED IN CONFORMITY WITH THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE DEPARTMENT OF MOLECULAR GENETICS UNIVERSITY OF TORONTO SAMUEL LUNENFELD RESEARCH INSTITUTE © Copyright by Ryan Mui, 2010 ii INVESTIGATING THE NEUROBIOLOGICAL ROLE OF TUBBY, A PROTEIN INVOLVED IN OBESITY Ryan Mui Master of Science 2010 Department of Molecular Genetics University of Toronto Samuel Lunenfeld Research Institute ABSTRACT Tubby mice succumb to blindness, deafness, and obesity. Vision and auditory deficits are attributed to neurodegeneration and tubby-associated obesity has been postulated to result from neuronal deficits in brain regions controlling weight regulation. TUB has been implicated in Gαq signaling and 2 isoforms of TUB, found exclusively in the brain, may have opposing effects on transactivation. Toward this end, I developed several cell culture assays to interrogate TUB function and found that TUB directs neuronal outgrowth in an isoform-specific manner. One isoform directs stable and polar outgrowth while the other directs multiple process outgrowths and branching. These effects can occur via Gαq signaling and require nuclear localization. Furthermore, I have found that the serotonergic system of tubby mice displays morphological and innervation deficits. Since the serotonergic system is implicated in modulating moods and behaviours, including appetite, these deficits may result in the obesity and motivational issues observed in tubby mice. iii ACKNOWLEDGEMENTS First and foremost, I would like to gratefully express my gratitude to my supervisor, Dr. Sabine Cordes, whose expertise, patience, and enthusiastic support has considerably enriched my graduate experience at Mt. Sinai Hospital. The mentorship and training that Dr. Cordes has provided me throughout these years is truly invaluable. Furthermore, I would also like to thank my committee members, Dr. Gabrielle Boulianne and Dr. James Dennis for their constructive feedback and guidance throughout the various stages of my personal development and throughout the revisions of this thesis. Finally, I would like to thank all the members of the Cordes lab. Thank you for all the laughs and memories- they are more than enough to last a lifetime. iv TABLE OF CONTENTS ABSTRACT ……………………………………………………………………... ii ACKNOWLEDGEMENTS ………………………………………………………. iii TABLE OF CONTENTS …………………………………………………………. iv LIST OF FIGURES …………………………………………………………......... vi LIST OF TABLES ………………………………………………………………... vii LIST OF ABBREVIATIONS ……………………………………………………. viii 1.0 INTRODUCTION …………………………………………………………… 1 1.1 Intrinsic regulation of neuronal outgrowth …………………………… 1 1.2 Tubby, a putative transcription factor involved in Gαq signaling ……. 2 1.3 Neuronal deficits resulting in the obesity phenotype are not fully Understood ……………………………………………………………. 5 1.4 The tubby mutation might result from deficits in serotonin-signaling .. 8 1.5 The tubby and anorexia mouse mutation manifest opposite phenotypes .. 9 1.6 Anorexia-associated lethality can be suppressed by the TUB protein ….. 11 2.0 MATERIALS AND METHODS …………………………………………… 14 2.1 Generation and genotyping of anorexia mice…………………………… 14 2.2 Analysis of TUB isoform protein levels in anx/anx and anx/+ brain regions via Western blotting ……………………………………………. 14 2.3 Generation of TUB Constructs and accessory constructs ………………. 15 2.4 N2A cell culture and reagents ………………………………………… 17 2.5 Generation of stable cell lines ………………………………………… 17 2.6 Western blotting to verify protein levels ……………………………… 17 2.7 Morphological studies in N2A ………………………………………….. 18 2.8 Quantification detection criteria used to evaluate neuritic outgrowth in N2A …………………………………………………………………… 20 2.9 Generation of primary striatal and hippocampal cultures ………………. 21 2.10 Immunocytochemistry ………………………………………………….. 22 2.11 Morphological studies in primary striatal and hippocampal neurons ….. 22 2.12 Generation of tub/tub mice ……………………………………………... 23 2.13 Analysis of serotonergic neuronal innervation in the hippocampus ……. 24 3.0 RESULTS ……………………………………………………………………. 25 3.1 Relative protein levels of full length versus Δ5 TUB are increased in brain regions of C57 anx/anx mice……………………………………… 25 v 3.2 PIP2 binding sequesters TUB from the nucleus in N2A cells ………….. 26 3.3 N2A cells undergo minor differentiation after 48 hours of incubation …. 28 3.4 Constitutively activated TUB promotes isoform-specific neuritic outgrowth formation in N2A cells ……………………………………… 29 3.5 Cells expressing constitutively activated full length and Δ5 TUB do not secrete factors into the media that promote isoform-specific neuritic outgrowth ……………………………………………………………….. 32 3.6 Unactivated TUB is nuclear upon activation by substance P through the neurokinin-1 receptor and directs isoform-specific neuritic outgrowth … 32 3.7 TUB can exist in cytoplasmic vesicle-like entities ……………………... 36 3.8 Nuclear localization of TUB is essential for isoform-specific neuritic outgrowth in N2A cells …………………………………………………. 37 3.9 Constitutively activated full length and Δ5 TUB direct isoform-specific neuritic outgrowth in primary striatal neuronal cultures ………………... 40 3.10 Constitutively activated full length and Δ5 TUB direct isoform-specific neuritic outgrowth in primary hippocampal neuronal cultures …………. 43 3.11 Summary of TUB-effects on neuritic outgrowth in different cell lines … 45 3.12 Serotonergic neuronal innervation and morphology is affected in the hippocampus of tub/tub mice …………………………………………… 46 4.0 DISCUSSION ………………………………………………………………... 50 4.1 Relative levels of full length versus Δ5 TUB are increased in the hippocampus of anx/anx mice …………………………………………... 50 4.2 TUB directs isoform-specific neuritic outgrowth through Gαq signaling and translocation to the nucleus ………………………………………… 50 4.3 Constitutively activated TUB directs isoform-specific neuritic outgrowth in primary striatal and hippocampal neurons ……………….. 55 4.4 Analyzing TUB function and regulation using cell culture-based assays . 58 4.5 The regulation of Tubby splicing on neuritic outgrowth ………………... 59 4.6 Aberrant morphology and innervation of serotonergic neurons in the hippocampus of tub/tub mice …………………………………………… 60 5.0 CONCLUSIONS …………………………………………………………….. 62 6.0 APPENDIX …………………………………………………………………... 63 6.1 The mood stabilizers lithium and valproic acid promote process formation and elongation in N2A cells and enhance the isoform-specific cell shape changes of constitutively activated TUB ……………………. 63 7.0 REFERENCES ………………………………………………………………. 67 vi LIST OF FIGURES Figure 1: TUB is implicated in Gaq signaling as a possible transcription regulator ……………………………………………………………….. 4 Figure 2: Isoform levels of TUB in the brain ……………………………………. 12 Figure 3: Immunoblotting for TUB-isoforms in anx/anx mice and anx/+ mouse brain regions …………………………………………………………… 25 Figure 4: Stable expression of TUB-RFP isoforms (unactivated and constitutively activated) in N2A cells …………………………………. 27 Figure 5: Percentage of neurite-bearing cells in stable cell lines ………………... 28 Figure 6: Isoform-specific cell shape changes occur in constitutively activated TUB-expressing N2A cells ……………………………………………. 31 Figure 7: TUB isoform-specific neuritic outgrowth is not the result of a cellular secreted factor ………………………………………………………… 32 Figure 8: TUB can be activated by substance P through the neurokinin-1 receptor to direct isoform-specific cell shape changes ………………. 35 Figure 9: The TUB protein can exist in cytosolic vesicles in N2A cells ……….. 36 Figure 10: The K39KKR to LAAA mutation in the nuclear localization signal attenuates nuclear accumulation and TUB-isoform specific neuritic outgrowth …………………………………………………………….. 39 Figure 11: Constitutively activated TUB directs isoform-specific cell shape changes in primary striatal neurons ………………………………….. 42 Figure 12: Constitutively activated TUB directs isoform-specific cell shape changes in primary hippocampal neurons ……………………………. 45 Figure 13: Mice homozygous for the tubby mutation display aberrant serotonergic innervation in the hippocampus ………………………... 49 Figure 14: Relative levels of full length TUB and Δ5 TUB could be important for regulating neuronal outgrowth ……………………………………….. 59 Figure 15: The mood stabilizers lithium and valproic acid promote process formation and elongation in N2A cells and enhance the isoform- specific cell shape changes of constitutively activated TUB ………… 63 vii LIST OF TABLES Table 1: Hypothalamic dysregulation in tub/tub mice ………………………… 8 Table 2: Analysis of process outgrowth (mean percentage average ± standard error) and degree of branching (average number of processes ± standard error) in stable cell lines ……………………………………. 29 Table 3: Analysis of process outgrowth (mean percentage average ± standard error) and degree of branching (average number of processes ± standard error) before and after activation of TUB via substance P and the neurokinin-1 receptor ………………………………………... 34 Table 4: Analysis of process outgrowth (mean percentage average ± standard error) and degree of branching (average number of processes ± standard error) when nuclear accumulation of TUB is attenuated …… 37 LIST OF APPENDICES Appendix: The mood stabilizers lithium and valproic acid promote process formation and elongation in N2A cells and enhance the isoform- specific cell shape changes of constitutively activated TUB …………. 63 viii LIST OF ABBREVIATIONS 5-HT serotonin 5-HTT serotonin transporter AgRP agouti-related protein α-MSH α-melanocyte stimulating hormone