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NOVEL INSIGHTS INTO THE ROLE OF THE UNC-6/NETRIN RECEPTOR UNC-5 IN REGULATING GROWTH CONE POLARITY AND PROTRUSION
Mahadik, Snehal Sudhakar
Mahadik, Snehal Sudhakar
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Abstract
The central nervous system (CNS) is a complex network of connected neurons. In the developing nervous system, axons must reach their target for the formation of functional neuronal connections. During embryonic and postnatal development of a nervous system, neuronal precursor cells must migrate to their final destinations, and axons must navigate to the correct target to establish normal connectivity. Failure of axons to properly reach their target leads to incomplete development of a brain, resulting in several neurodevelopmental disorders such as mental retardation and autism, horizontal gaze palsy with progressive scoliosis (HGPPS) and schizophrenia (GUAN AND RAO 2003; NUGENT et al. 2012). Proper functioning of the nervous system depends on the development of proper neuronal connections, which in turn depends on the proper guidance of neurites from their neuronal cells of origin to their eventual synaptic targets. The outgrowth of developing axons is guided by growth cones, dynamic actin-based structures that sense and respond to extracellular guidance cues that drive the forward motion of the axon(TESSIER-LAVIGNE AND GOODMAN 1996). The growth are the dynamic structures which are present at the growing tip of an axon and made up of dynamic filopodial and lamellipodial protrusions, which are involved in axon outgrowth and guidance (GOLDBERG AND BURMEISTER 1986). The lamellipodium is an actin-based, membrane sheet-like structure at the leading edge of the growth cone consisting of a meshwork of actin filaments, which provide the forward motive force of the developing neurite(GALLO AND LETOURNEAU 2004). Filopodia, are the dominate structures of a growth cone that emanate from the growth cone lamellipodial body, consist of bundled filamentous actin (F-actin). Filopodia are thought to be involved in the sensation of directional cues and subsequent changes in directional locomotion as it contains receptors for various guidance cues. Netrins were the first family of guidance cues to be found in both invertebrate and vertebrate nervous systems. In C. elegans, a genetic screen for mutants that affects axon guidance identified UNC-6 which is involved in controlling both dorsally and ventrally guided projecting axons (MERZ AND CULOTTI 2000). UNC-6 is a laminin-related protein secreted by ventral cells in C. elegans and by floor-plate cells in vertebrates (HEDGECOCK et al. 1990; ISHII et al. 1992). UNC-6/Netrin is a bi-functional guidance cue which mediates its functions through two classes of single-pass transmembrane receptors, UNC-40 ad UNC-5. Much work has been done to identify role of UNC-6/ Netrins and its receptors in regulating end point axon guidance by studying the developed nervous system in vivo and by studying developing growth cones in vitro. However, very little is known about how UNC-6/ Netrin regulates in vivo growth cone morphology and dynamics to regulate proper navigation o axons to their targets. The work here attempts to do that by utilizing the free-living nematode Caenorhabditis elegans. C. elegans is a useful system to study axon pathfinding and growth cone development in vivo due to its simple, well-characterized nervous system, transparency and fully sequenced genome. In this work we focus on D-type GABAergic neurons 13 VD and 6 DD, which are motor neurons required for locomotion of C. elegans. VD/DD motor neurons resides in the ventral nerve cord their axons first migrate anteriorly before turning 90 degrees and forming commissures until reaching the dorsal nerve cord, at which point they spread both anteriorly and posteriorly and establish their synaptic targets. Previous work from lab using VD growth cone analysis display a polarity/protrusion model of growth cone repulsion from UNC-6/Netrin, UNC-6 first polarizes the growth cone of VD neuron via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. Through the UNC-40/DCC receptor, UNC-6 stimulates protrusion dorsally, and through UNC-5 inhibits protrusion ventrally and laterally, resulting in net dorsal growth (GUJAR et al. 2018). The work presented here is focused on how UNC-6/Netrin utilizes the receptor UNC-5 with the help of other signaling molecules and mediate inhibition of growth cone protrusions. In chapter III, we identify the role of MAX-1 in inhibiting growth cone protrusion by possibly affecting the trafficking or stability UNC-5. We show that max-1 acts independently of other pathways that regulate growth cone protrusion, including unc-40/DCC, unc-6/Netrin, unc-33/CRMP, and unc-34/Enabled. Our findings involving MAX-1 in the growth cone are in the contrast with endpoint axon guidance analysis which display synergistic interactions between max-1, unc-5, unc-6, unc-40, suggesting that we are still missing a link that translates how morphological changes in the growth cone affects axon guidance. In chapter IV, we show how regulation of vesicle exocytosis is exclusively used by receptor UNC-5 to inhibit growth cone protrusions. We identify tom-1 genetically interacts downstream of receptor unc-5 to inhibit the lateral and ventral protrusions in dorsally migrating VD growth cone. We also uncover the isoform specific role of TOM-1 in inhibiting UNC-5 signaling, where the short isoforms perform the canonical function inhibition and long isoform is pro-protrusive in nature. Further extension of this work is explained in chapter V, where we have shown that the TOM-1mediates inhibition of growth cone protrusions by inhibition AEX-3, which has pro-protrusive role in vesicle exocytosis. In chapter VI, we show that the long and short isoform of UNC-5 differentially regulates VD growth cone morphology. The long isoform of UNC-5 displays a conserved canonical role of inhibition of protrusions, but the short isoform has a pro-protrusive role. Lastly in chapter VII, we identify role of SRC-1 tyrosine kinase in mediation UNC-5 signaling by possible activation of UNC-5 through phosphorylation. In summary, the results contained here provide novel insights into the mechanisms by which receptor UNC-5 of UNC-6/Netrin utilizes the novel genes in repulsion, growth cone inhibition and regulates several aspects of growth cone protrusion during the process of axon guidance. The studies performed here also emphasis on the benefits of analyzing growth cones during outgrowth in addition to post-developmental end point analysis of axon guidance.
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Date
2022-12-31
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University of Kansas
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Keywords
Developmental biology, Cellular biology, Molecular biology, C.elegans, Growth cones, Guidance cues, Neurodevelopment, UNC-5, UNC-6