Doctor of Philosophy in Neuroscience (PhD)
Role of axon guidance genes in the maintenance of adult nervous system
Installment #4 for #GreatSupervisor wk: Dr. Tim O’Connor: He works w/ fruit flies. We rarely talk #science bec we work on such diff things. But we do talk #grants, #scholarships, #jobs, & other #academic woes. Tim calls me “just the right amount of shit disturber.” A month before I started #GradSchool, I started a new treatment that put me in a perpetual fog. Some days, I couldn’t say #GABA, one of the shortest words in #neuroscience. In a world where we pride ourselves in knowing everything (so unrealistic!), it was tough.
#Professors made snide comments. I had come in w/ #funding & it felt like I was just not living up to expectations. At all. Tim would say repeatedly: “You have nothing to prove. We know you’re smart. We just have to figure out how u can show it.” (He got me a #tutor.)
Despite all the crap that 1st yr, I continued to get #funding. Tim's #confidence made such a diff. It felt sincere. (*Some* well-meaning folks are glib: they say the right things, but it's not hard to detect a well-polished "we believe in u" script fr yrs of practice.)
“Ha! I knew it! We are going to go celebrate your scholarship.” He is the 1st & only prof to say that. Not YOU should go celebrate, but WE should go celebrate. (#AcademicTwitter sidebar: NONE of us got where we are alone) He told me his door is open if I needed a space to vent, #cry, rant, & foam at the mouth. I took him up on it once after some miserable encounters w/ an inappropriate #male #professor, crawled in there, & had a big ugly cry. He sat there quietly & handed me tissue after tissue. No glib, no hug, no awkwardness.
Just presence. It was exactly what I needed. In #academia, we have #advisors, #collaborators, #colleagues, & #bosses. What we often don't have are #allies. Find those allies. And appreciate them. #ProfLife #PhDchat #PhDlife #PhD #WomenInSTEM #WomenInMedicine #SayThankYou
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Next-generation sequencing has made genetic variant discovery routine clinical practice. To assess a variant’s pathogenicity classical clinical genetics methods, require familial transmission or the identification of the variant in multiple affected individuals. However, most identified variants are rare, often de novo, or present in few individuals. Therefore, classical genetics methods such as case-control or co-segregation studies cannot be applied. Computational methods can be used to predict the function of thousands of gene variants, but these methods are not reliable. Experimental assays to assess the relative function of variants can provide a path to interpretation of variant activity. Drosophila melanogaster offers a robust platform for variant functional testing at scale, but remains unvalidated for accurate assessment of the function of high numbers of variants in well-established assays.The first section of my thesis describes my role in a multi-model organism variant functionalization project characterizing 100 human PTEN variants. In flies, ubiquitous overexpression of PTEN-WT resulted in developmental delay. By comparing eclosion delay for overexpressed PTEN variants, we categorized variants as wildtype, gain of function or loss of function. This work demonstrates the utility of Drosophila as a powerful platform for high volume screening for the function of human gene variants from healthy and diseased individuals.However, clinically interpreting the functional consequence of the identified variation is challenging. Therefore, my third chapter aimed to establish a structured ‘well-established’ assay calibrated against known pathogenic and benign PTEN variants to convert variant function in an assay into a clinically-relevant interpretation in adherence to ClinGen Sequence Variant Interpretation (SVI) Working Group recommended guidelines. We screened ~100 human PTEN variants for suppression of PI3K/AKT signaling dependent cellular proliferation in Drosophila, a pathway that underlies the primary cause of PTEN’s contribution to cancer. The assay correctly assigned the function of known pathogenic and benign variants and exhibited a high correlation with human cell line functional data. We also showed that PTEN functionally replaces its Drosophila ortholog in developmental growth. Our work provides evidence that well- established assays, directly testing disease-relevant protein activity in Drosophila, can be used to generate reliable functional data appropriate for clinical variant interpretation.
This thesis describes two projects that examined protein function in neural plasticity and cancer development. The first project created assays that tested the pathogenicity of human variants identified in SMAD4 and BMPR1A, genes that are associated with juvenile polyposis syndrome. As exome sequencing becomes easier and more cost-effective, many human variants are being identified. However, for most variants, their impact on protein function and their ability to cause disease are unknown. Drosophila melanogaster offers an efficient system for testing human variant protein functionality in a panel of assays to screen through many variants. I have used simple overexpression assays in Drosophila to test human SMAD4 and BMPR1A variants. I developed two assays in which wildtype SMAD4, but not loss of function variants, caused either lethality or wing vein defects. I screened through seven human SMAD4 variants implicated in disease to assess their relative function and identified four that exhibit functional differences to wildtype. I also tested human BMPR1A but found that overexpression of this gene in Drosophila had no effect. I postulated this is due to a lack of ligand binding. Therefore, I created reagents for alternative methods to screen BMPR1A variant function. First, I generated mimetic mutations in the orthologous tkv gene. Second, I created a chimeric gene comprising the extracellular domain of Tkv and the intracellular domain of BMPR1A. I postulate that this chimera should bind Drosophila BMP ligands and activate canonical BMP signaling, allowing for assays of BMPR1A variants in the intracellular domain. These reagents and assays are important for experimentally determining ariant activity and for improving our understanding of structure/function relationships for SMAD4 and BMPR1A. Going forward, functionally testing large numbers of variants will inform personalized medicine approaches and improve computer models for projecting pathogenicity of human variants.In the second project, I created CalpA and CalpB double mutants to test whether a reduction of calpain activity could stimulate de novo neurite formation. Also, I overexpressed a proteolytic target of Calpain, Cortactin, and created a Calpain proteolysis resistant version of Cortactin. Surprisingly, I was unable to identify any phenotype in the nervous system.
Although autism spectrum disorders (ASDs) have long been known to have a strong heritability, the genetic basis of these disorders has remained largely elusive. Hundreds of genes have been linked to ASDs, but most of them only contribute a small increase in risk. In 2009, a genome-wide association study identified Semaphorin 5A (SEMA5A) as a novel autism susceptibility gene. Sema5A is a member of the Semaphorin family consisting of secreted and membrane-associated proteins characterized by the Sema domain. Although initially identified as axon guidance cues, Semaphorins have been found to play numerous key roles in the development and function of the nervous system. Here, I provide evidence that Sema5A, along with Sema5B, regulates dendritic morphology and excitatory synaptic elimination in hippocampal neurons. The overexpression of Sema5A/Sema5B negatively impacted dendrite complexity and reduced excitatory synapse density without affecting inhibitory synapses, in contrast the knockdown of Sema5A/Sema5B increased excitatory synapse density. I also investigated the relationship between Sema5A/Sema5B and activity-dependent plasticity including long-term potentiation (LTP) and long-term depression (LTD), which are cellular models of learning and memory. It was demonstrated that the overexpression of Sema5A/Sema5B attenuated the LTP-mediated increase of synapse density, whereas the knockdown of Sema5A/Sema5B blocked the LTD-mediated decrease of synapse density. Furthermore, soluble Sema5A treatment altered the surface expression of the AMPA receptor subunit GluA1 with total level of GluA1 unchanged. Finally, I examined the signaling mechanisms of Sema5A-mediated synapse elimination and plasticity. I found that in vitro Sema5A signalled through two members (Plexin A1 and Plexin A2) of the Plexin family, which are known as the neuronal receptors for the Semaphorin family. Moreover, TAG-1, a cell adhesion molecule also known as Contactin-2, was necessary for the function of Sema5A and Sema5B. Lastly I found that ALLN, an inhibitor of protease calpain, significantly rescued Sema5A-mediated synapse elimination, suggesting that calpain was downstream of Sema5A signaling in hippocampal neurons. Thus, my data revealed a new role for class 5 Semaphorins in synapse density and plasticity, and may therefore provide insights into the critical roles of Sema5A in the general mechanisms of circuit formation and the specific etiology of ASDs.
During prenatal and early postnatal development, the mammalian nervous system has theremarkable ability to build its intricate array of connections and circuitries with the help of avariety guidance cues. When the nervous system matures, it appears to lose its ability to rebuilddamaged connections following traumatic insults. This can be attributed in part to the expressionof inhibitory molecules that hinder axon regeneration and reconnection. The goal of this thesis isto identify novel compounds that can stimulate axon outgrowth in the unfavourable environmentof the adult central nervous system (CNS) by manipulating the axon outgrowth machinery in theneuronal growth cone. To isolate compounds of therapeutic potential, we first developed a novelhigh-throughput screening technology to rapidly identify candidate neurite outgrowth promotingmolecules from a bioactive marine sponge extract library. Using the high-throughput screeningtechnology, we identified a natural diketopiperazine DKP101516 that demonstrated robust axonoutgrowth promoting activity through the phosphotidyl-3-inositol kinase (PI3K) signallingpathway. Further in vivo studies revealed that while DKP101516 did not stimulate afferentregeneration, it markedly enhanced intraspinal axon sprouting following dorsal rhizotomy.Lastly, behavioural studies suggest that DKP10516 also promoted rapid and transient recovery inmechanosensation, concomitant to the sprouting of VGLUT1 positive mechanosensory afferents.Collectively, our data suggest that DKP101516 may be a promising therapeutic to stimulate axonrepair and functional recovery following injuries in the CNS.
Proper neuronal development and function requires precise sorting and delivery of variouselements from the soma to the synapse. Important mediators of intracellular transport events arethe actin-based class V myosin motors, which are involved in organelle transport in various celltypes. Two myosin V family members, myosin Va and Vb, are present in the brain, however, theidentity of cargoes transported by these motors is unknown. The objective of this thesis was toconduct molecular and cell biological studies to identify and characterize novel myosin Vcargoes in neurons.The first approach I used was to characterize the distribution of candidate protein cargoes afterblocking the function of endogenous myosin Va and Vb with dominant-negative (DN) versions.I found that in developing neurons, expression of DN myosin Vb, but not DN myosin Va,resulted in the accumulation in the soma of the AMPA-type glutamate receptor subunit, GluR1,and a reduction of its surface expression. I also found that myosin Vb-mediated trafficking ofGluR1 required an interaction with the GTPase Rab11. These results reveal a novel mechanismfor the transport of a specific glutamate receptor subunit mediated by myosin Vb and Rab11.As an alternative approach to identify myosin Va binding partners in the brain, we conducted ayeast-two hybrid screen of a rat brain cDNA library using the cargo binding domain of myosinVa. Among the proteins identified in our screen, I selected a protein of unknown functionpreviously identified as Rab-lysosomal-interacting protein like 2 (RILPL2) and further assessedits function. I found that RILPL2 expression in non-neuronal cells resulted in morphologicalchanges and activation of the Rho GTPase Rac1. In developing neurons, gain or loss of RILPL2function altered the density of dendritic spine protrusions and increased phosphorylation of theRac1 effector Pak. These findings uncover a novel role for the myosin Va-interacting protein,RILPL2, in regulating dendritic spine development, possibly through Rac signaling.Taken together, the work presented in this thesis provides novel insights into the function ofclass V myosins in neurons, and into the critical machinery involved in trafficking of AMPARsand dendritic spine morphogenesis.
Corticofugal axons projecting to the thalamus, brainstem and spinal cord must travel the same initial trajectory through the subcortical lateral and medial ganglionic eminences, and are therefore likely subject to the same sets of guidance cues. These cues direct the course of corticofugal axons bringing them to each intermediate target, until they reach the diencephalic-telencephalic boundary, where axons targeting the thalamus turn dorsally and brainstem and spinal cord targeting axons turn ventrally. Many of these guidance cues have been elucidated, yet there are still gaps in our understanding of the formation of the corticofugal projection. I found that Sema5B expression flanked the presumptive internal capsule during its formation, and was therefore ideally situated both spatially and temporally to act as an instructive cue for descending cortical axons. In Chapter 2, I show that Sema5B is not only capable of inhibiting cortical axons in vitro, but can cause misguidance of cortical axons in slice culture when placed ectopically over normally non-Sema5B expressing regions. In addition, I show that the loss of Sema5B from the neocortical VZ resulted in aberrant penetration of this normally avoided region. Therefore Sema5B is both necessary and sufficient to inhibit the corticofugal projection. Semaphorins and their plexin receptors are frequently proteolyzed to modulate the elicited responses in navigating growth cones. In Chapter 3 I show that Sema5B cleavage results in an inhibitory fragment that in heterologous cells can produce inhibitory gradients for cortical explants in collagen gel co-cultures, and collapse dissociated cortical neuronal growth cones, an effect that can be blocked with a function disrupting antibody to the cell adhesion molecule TAG-1. This thesis shows that Sema5B, a guidance cue with a hitherto unknown function, is responsible for a very important aspect of cortical development. My work leads to a final proposal that Sema5B is in fact a two-in-one protein with separable inhibitory and alternate complex functions, the implications of which are discussed thoroughly in Chapter 4.
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Axon guidance cues are extracellular signals that direct the growth and steering of neuronal growth cones. Both attractive and repulsive cues are required to guide developing axons to their targets. Nonetheless, after axons have reached their targets and established functional circuits, many neurons continue to express these developmental cues. The expression of these genes in the adult indicates that there are likely additional roles for guidance cues beyond the initial phase of neuronal process outgrowth, growth cone navigation, and target innervation. The central goal of my work is to determine the functions of these cues in the mature nervous system. I hypothesize that axon guidance genes expressed in the adult nervous system have functional roles in the maintenance of neural circuits. Work in the past few decades has led to the discovery of numerous axon guidance genes and identified their functions during development. This study is the first to perform an RNA-mediated interference (RNAi) screen for axon guidance genes that have functional roles in the mature nervous system. Axon guidance genes expressed in the adult Drosophila melanogaster nervous system were identified using bio-informatics tools. In Drosophila, more than 96% of embryonic cues continue to be expressed in the adult. The axon guidance genes were knocked down in adult neurons using RNAi via spatial and temporal control of GAL4-UAS system. I have identified 15 axon guidance genes that are essential for survival and normal behavior (climbing, mobility, activity/rest cycle) in adult Drosophila. The results suggests that axon guidance genes are functional in the adult nervous system and may be involved in the maintenance of neural circuits underlying these phenotypes. Further studies on circuit morphology are required to understand better how axon guidance genes contribute to the maintenance of neuronal structure in adult brains.
The nervous system of an organism is exceedingly complex and yet highly specific in the connections that it makes. The mechanisms by which a functional nervous system is developed are therefore of great importance and interest. Neurons extend processes over long distances and through various environments in order to form connections with their appropriate targets. The neurites must sense their environment and make decisions based on guidance cues as to which direction to grow. The growth cone of the developing neurite is a dynamic, actin-rich domain at the leading edge and is the site for integration of various guidance cues and, therefore, of this decision making. The molecular mechanisms underlying outgrowth, in particular consolidation of developing axons – the process in which the actin network of the proximal region of the growth cone collapses creating a new segment of axon, remain unclear. Our lab previously uncovered a role for the actin-associated protein cortactin as an enhancer of membrane protrusions in cultured neurons and for calpain as an inhibitor of this process in consolidated regions. However, the physiological roles for cortactin and calpain in axon outgrowth and cell migration have remained elusive as others have observed both similar and opposite effects of these proteins. These discrepancies are likely due to the variability associated with in vitro studies. Therefore, I set out to elucidate the function of these molecules in developing axons in vivo within the model organism Drosophila melanogaster. Using two subsets of neurons within the central nervous system, I observed that the overexpression of cortactin combined with the inhibition of calpain increased the elongation of axons as well as the incidence of misguidance. Therefore, it appears that in vivo, cortactin acts as an enhancer of membrane protrusions and elongation and is actively inhibited by calpain. In addition, these two proteins appear to influence guidance, though through what mechanism remains to be investigated. Knowledge of the molecular pathways involved in axon outgrowth and guidance is key to the understanding of not only development, but also plasticity and repair within the nervous system.
The centrally projecting sensory axons of the dorsal root ganglia follow a well established pattern that is conserved across many species and offers a robust model for the study of axonal guidance. When primary sensory axons leave the sensory ganglia and project to the embryonic spinal cord, they do not immediately extend into the spinal cord dorsal horn, but bifurcate and travel long the rostrocaudal axis of the animal to form the dorsal funiculus and Lissaur’s tract. At a later stage they extend collateral fibres that enter the dorsal horn and target to specific laminae according to their sensory modality. The factors that prevent immediate entry into the dorsal horn or regulate the timing of sensory collateral formation and specificity of lamina innervation have not been clearly identified. Our lab previously showed that Semaphorin5B (Sema5B), a member of the semaphorin family of guidance molecules, is dynamically expressed in the embryonic spinal cord and correlates with these sensory axon targeting events. Using in vitro assays, I show systematically that Sema5B inhibits growth of both nerve growth factor-responsive and neurotrophin-3-responsive dorsal root ganglion neurites and that this inhibitory effect on the former is mediated in part through the cell adhesion molecule TAG-1. Using the technique of RNA interference, I show in vivo that a reduction-of-function of Sema5B in the spinal cord leads to cutaneous axons not only projecting prematurely into, but to erroneous targets within the dorsal horn of the spinal cord, while proprioceptive axons continued to pathfind correctly. Together, these results suggest that Sema5B acts as a repulsive barrier for centrally projecting primary sensory axons that first reach the spinal cord, and once collaterals form, Sema5B exerts a differential function on different types of sensory fibres to regulate their pathfinding. This is the first study to identify the specific cue that regulates sensory neuron entry and guidance into the spinal cord dorsal horn grey matter.