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THE UNIVERSITY OF OKLAHOMA HEALTH SCIENCES CENTER
GRADUATE COLLEGE
OF AUTISM AND WORMS:
NEUROLIGIN MUTANTS AND SYNAPTIC FUNCTION IN C. elegans
A DISSERTATION
SUBMITTED TO THE GRADUATE FACULTY
In partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
BY
JERROD WAYNE HUNTER
Oklahoma City, Oklahoma
COPYRIGHT
May 13, 2011
ACKNOWLEDGMENTS
I would like to express my gratitude to all those who helped me complete this dissertation. I would like to sincerely thank my mentor, Dr. Jim Rand, Ph.D.- the scientific rigor, writing and presentation skills I learned from him will benefit me for my entire career. He gave me the freedom to explore new ideas and approaches, and to take my research where the data led me.
I also would like to thank my lab colleagues Dr. Greg Mullen, Dr. Ellie Mathews, Kiely Grundahl, John McManus and Jessica Heatherly.
I greatly appreciate the guidance and support of my committee members: Dr. Michael Dresser, M.D. Ph.D., Dr. Eric Howard, Ph.D., Dr. Yuechueng Liu, Ph.D., Dr. John Ash, Ph.D. and Dr. David Sherry, Ph.D. They provided valuable direction on my project, and more importantly, on my Ph.D. training.
I am grateful to the faculties and staff in the Department of Cell Biology at OUHSC, and the Genetic Models of Disease Research Program at OMRF for their constant help and support.
My heartfelt gratitude is also extended to my family, Qingzhao and Jaden, whose support made this possible. I love you.
TABLE OF CONTENTS
INTRODUCTION AND LITERATURE REVIEW..
- 1 Neuroligin and neurexin structure and function:
- 2 Autism and Autism Spectrum Disorders (ASDs)
- 3 The Use of C. elegans in Neurobiology
- 4 Research objectives
1.1 Figure 7. Sequence alignments of the C. elegans NLG-1 and the hNLGN 4 proteins
1.1.1 Figure 8. nlg-1 transcripts, deletion mutations and reporter constructs
2 Cellular Expression and Sub-Cellular Localization of the NLG-1 Protein
2.1 A neuroligin transcriptional reporter is expressed in body-wall muscles and ~45 neurons
2.1.1 Figure 9. Structure of the C. elegans neuroligin protein (NLG-1)
2.1.2 Figure 10. Cellular expression: nlg-1 transcriptional reporter
2.1.3 Figure 11. Cellular expression: nlg-1 transcriptional reporter vs. ttx-3 transcriptional
2.1.4 Figure 12. The relative position of nlg-1 positive cells with respect to cells of ...
3 The NLG-1 protein is localized to synaptic regions
4 Behavioral Phenotypes of Neuroligin Deficient (nlg-1) Mutants of C. elegans
4.1 Development, locomotion and nervous system structure are superficially normal in nlg-1 mutants
4.2 nlg-1 mutants lack a thermal response
4.2.1 Figure 15. nlg-1 deletion mutants have thermotaxis defects
4.3 Spontaneous reversal frequency is reduced in nlg-1 mutants; this is a progressive phenotype
4.3.1 Figure 16. nlg-1 deletion mutants have spontaneous reversal deficits
4.3.2 Figure 17. Spontaneous reversals in larvae and adults: the nlg-1 spontaneous
4.4 nlg-1 mutants have specific chemosensory deficits
4.4.1 Figure 18. nlg-1 deletion mutants have octanol avoidance defects
4.5 nlg-1 mutants have altered processing of conflicting chemosensory cues
4.5.1 Figure 19. nlg-1 deletion mutants have Approach-Avoidance deficits
5 nlg-1 Mutants Have Elevated Oxidative Stress
5.1 Hypersensitivity to paraquat
5.2 An oxidative damage biomarker is elevated in nlg-1 mutants
5.3 nlg-1 mutants have reduced lifespan
5.4 Additional Stress Phenotypes of nlg-1 Mutants
5.5 nlg-1 mutants have normal sensitivity to thermal stress
5.6 Analysis of tissue aging in nlg-1 deficient mutants
5.6.3 Figure 22. nlg-1 mutants are hypersensitive to copper, but not cadmium
5.6.4 Figure 23. nlg-1 mutants show signs of premature aging
6 Are Nematode and Mammalian Neuroligins Functionally Equivalent?
6.1 Rescue of mutant behavioral phenotypes by transgenic expression of mammalian neuroligins
6.1.1 Figure 24. hNLGN4 rescues the nlg-1 spontaneous reversal phenotype
6.1.2 Figure 25. hNLGN4 and rNlgn1 rescue the nlg-1 thermotaxis defect
6.1.3 Figure 26. hNLGN4 and rNlgn1 rescue the nlg-1 Approach-Avoidance defect.
6.1.4 Figure 27. hNLGN4 and rNlgn1 rescue the nlg-1 octanol chemotaxis deficit
6.1.5 Figure 28. Relative rescue for mammalian neuroligin transgenic animals
7 Effects of ASD-related neuroligin mutations in C. elegans
7.1 Table 4 Amino acid substitutions in hNLGN4 and hNLGN3 associated with autism,
7.1.1 Figure 29. ASD associated mutations: effects on spontaneous reversal
7.1.2 Figure 30 ASD associated mutations: effects on thermotaxis behavior
7.1.3 Figure 31. ASD associated mutations: effects on Approach-Avoidance
7.1.4 Figure 32. ASD associated mutations: effects on octanol chemotaxis
7.1.5 Figure 33. Relative rescue of ASD-associated constructs
8 Analysis of Factors Required for C. elegans Neuroligin Localization and Function
8.1.1 Figure 34 Schematic diagram of the neuroligin (NLG-1) deletion constructs
8.1.2 Figure 35. Deletion analysis of NLG-1: spontaneous reversal
8.1.3 Figure 36. Deletion analysis of NLG-1: thermotaxis
8.1.4 Figure 37.Deletion analysis of NLG-1: Approach-Avoidance
8.2 Mutagenesis of putative PKC and Mek/Jnk binding sites
8.2.1 Figure 38 Diagram of a deletion eliminating two potential PKC sites
8.3 RNAi screen to identify putative localization factors
8.4 Mutagenesis and screen to identify putative localization mutants
1 Neuroligin Expression and Localization
2 Phenotypes of nlg-1 Null Mutants
2.1 Control of Spontaneous Reversals
2.2 Sensory behaviors and circuits
2.3 Integration of Multiple Sensory Inputs
3 nlg-1 Mutants and Oxidative Stress
3.1 Autism and Oxidative Stress
4 Analysis of Deletion Constructs, ASD Associated Mutations and Site Directed Mutations
APPENDIX I SUPLEMENTARY DATA.