I N T R A C E L L U L A R M E C H A N I S M S U N D E R L Y I N G G R O W T H C O N E C O L L A P S E by K E N N E T H C H I - W A N T O B . S c , University of British Columbia, 2001 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Neuroscience) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A August, 2007 © Kenneth Chi-Wan To, 2007 ABSTRACT During the course of development, expression of attractive and inhibitory guidance cues play a pivotal role in the pathfinding decisions of a growing neuron. In addition, injury-induced recapitulation of their expression, particularly inhibitory cues, likely influences the course of axonal regeneration, thus providing a rationale for the intense focus in this area o f research. Though significant progress has been made, it remains poorly described what signaling cascades, and in what combination, are involved during inhibitory-cue induced growth cone collapse. Therefore, to further understand why neurons are repelled or inhibited by certain cues, the aim of this thesis is to identify the underlying intracellular mechanisms regulating growth cone collapse induced by inhibitory cues. Us ing a novel anti-invasive compound called Motuporamine C (MotC), I have characterized in chapter 2 its effects as,a regulator of neuronal outgrowth. I found that MotC was a robust stimulator of growth cone collapse leading to a cessation of neurite growth. This was partially mediated through R h o - R O C K signaling, a pathway involved in regulating actin dynamics. Based on this partial response, I hypothesized that other signal transduction pathways were involved. I addressed this in chapter 3 by identifying calcium-activated calpain, a protease well-characterized in playing a role in adhesion regulation, was also activated during MotC-induced growth cone collapse. Furthermore, I show that concurrent inhibition of both R h o - R O C K and calpain pathways are necessary for maximum attenuation of the MotC-mediated collapse response. Since these results were identified using an organic molecule not endogenously expressed in vertebrate organisms, I hypothesized in chapter 4 that similar pathways would be activated in response to a physiological in vivo guidance cue. Using the inhibitory cue i i Semaphorin 5B (Sema5B), I found in addition to the activation of calpain, the phosphatase calcineurin was also involved in mediating Sema5B-induced growth cone collapse. Moreover, it is the combination of calpain- and calcineurin-mediated pathways that is required for evoking maximal growth cone collapse and that cross-talk between these two effector molecules occurs. These results are of particular interest since previously it was proposed by Gomez and Zheng (2006) that calpain and calcineurin signaling cascades were parallel pathways. Taken together, my findings show that different inhibitory cues activate multiple intracellular pathways that appear to impinge on different aspects of the intracellular machinery regulating motility. The combinatorial activation of these pathways is necessary for mediating maximal growth cone collapsing effects. Moreover, the elucidation of common signaling cascades between inhibitory cues to induce growth cone collapse may eventually provide novel targets for the development of new therapeutic strategies to promote functional recovery following neuronal injury. i i i T A B L E OF CONTENTS Abstract i i Table of Contents iv List o f Figures v i Acknowledgements v i i i 1. Introduction 1 1.1 Cytoskeletal dynamics mediating growth cone collapse 2 1.2 Molecular mechanisms mediating growth cone collapse 13 1.3 Extracellular cues governing growth cone motility 25 1.4 Summary 30 1.5 Research Objectives 32 1.6 References 33 2. The anti-invasive compound Motuporamine C is a robust stimulator o f neuronal growth cone collapse 44 2.1 Introduction. ' 44 2.2 Material and Methods 46 2.3 Results 50 2.4 Discussion 74 2.5 References 78 3. Motuporamine C activates multiple concurrent pathways to stimulate growth cone collapse 83 3.1 Introduction 83 3.2 Material and Methods 86 3.3 Results 89 3.4 Discussion 107 3.5 References 113 4. Combined activation of calpain and calcineurin during ligand-induced growth cone collapse 116 4.1 Introduction 116 4.2 Material and Methods 119 4.3 Results '. 124 4.4 Discussion 145 4.5 References 152 5. General Discussion 155 5.1 Utilization of M o t C as a research tool to identify pathways implicated in growth cone collapse 155 iv 5.2 Inhibitory cues evoke a combination of multiple signaling pathways to induce growth cone collapse 160 5.3 Application of pathways identified in MotC-mediated growth cone collapse using a physiological in vivo guidance cue, Sema5B 162 5.4 Sema5B activates both calcineurin- and calpain-mediated pathways during growth cone collapse 165 5.5 Combined activation of multiple downstream targets - a model for growth cone collapse 168 5.6 Potential mechanisms utilized by cues influencing motility to regulate adhesion in a calpain-independent manner 180 5.7 Involvement of microtubules in M o t C - and Sema5B-mediated growth cone collapse 182 5.8 Conclusion 185 5.9 References 186 v LIST OF FIGURES Figure 1.1 Chicken dorsal root ganglion growth cone stained for F-actin and microtubules.... 3 Figure 1.2 Schematic of the clutch model for directed neurite outgrowth 11 Figure 1.3 Schematic highlighting signal transduction cascades regulated by different amplitude fluxes in [Ca 2 + ]i 18 Figure 1.4 Signaling cascades implicated in Rho GTPase activity 21 Figure 2.1 Inhibitory effects of M o t C on E8 D R G explants 51 Figure 2.2 Growth cone morphology in response to M o t C 54 Figure 2.3 Removal of M o t C allows D R G explants to recover 57 Figure 2.4 Timelapse videomicroscopy confirms collapsing effect and reversibility o f M o t C 59 Figure 2.5 Inhibitory effect of M o t C is independent of growth factor used to culture D R G explants 63 Figure 2.6 Application of M o t C activates the Rho pathway 65 Figure 2.7 Morphological changes to the growth cone in response to M o t C and the R O C K inhibitor, Y27632 68 Figure 2.8 Outgrowth inhibition by M o t C requires the downstream Rho effector, R O C K 70 Figure 3.1 M o t C addition causes a high-amplitude rise in [Ca ]j in chick D R G growth cones 90 Figure 3.2 Absence of extracellular calcium inhibits MotC-induced growth cone collapse 93 Figure 3.3 Inhibition of C a 2 + influx via C o 2 + blocks MotC-induced growth cone collapse 95 Figure 3.4 Calpain is activated following M o t C application and inhibition of calpain attenuates M o t C effectiveness 98 Figure 3.5 Inhibition of calcineurin does not attenuate MotC-mediated collapse 101 vi Figure 3.6 Inhibition of both calpain and R O C K attenuates the collapsing effect of M o t C in an additive fashion 104 Figure 3.7 Proposed model of multiple pathways modulated by M o t C to induce growth cone collapse I l l Figure 4.1 Addit ion of Sema5B induces a significant rise in [Ca 2 + ]j in chick D R G growth cones 125 Figure 4.2 Addition of netrin-1 induces a mid-amplitude rise in [Ca 2 +]j 128 Figure 4.3 Sema5B-induced growth cone collapse is markedly attenuated in the presence of the external Ca 2 +-chelator, E G T A 130 Figure 4.4 C o 2 + , a broad spectrum blocker of high voltage-activated C a 2 + channels, attenuates Sema5B-induced growth cone collapse 133 Figure 4.5 Inhibition of calpain activity significantly reduces the Sema5B-mediated collapse response 135 Figure 4.6 Inhibition of calcineurin with C s A or D M significantly reduces the Sema5B-mediated collapse response 138 Figure 4.7 Inhibition o f both calpain and calcineurin reduces growth cone response to Sema5B in an additive fashion. 141 2+ Figure 4.8 Mode l of the signal transduction cascades by which increases in [Ca ]; via Sema5B-mediated growth cone collapse act 150 Figure 5.1 Schematic summarizing signal transduction cascades associated with different amplitude-rises in [Ca 2 + ]i 169 Figure 5.2 Schematic summarizing the intracellular pathways implicated in Sema5B-mediated growth cone collapse 171 Figure 5.3 Schematic outlining the downstream targets implicated in M o t C -mediated growth cone collapse 173 Figure 5.4 Schematic diagram showing the signaling cascades involved in M A G -mediated growth cone collapse 176 Figure 5.5 Schematic outlining the pathways regulated in netrin-1 -mediated neurite outgrowth 178 Figure 5.6 The collapsing effect of Sema5B, but not M o t C , is significantly attenuated in the presence of the G S K - 3 (3 inhibitor, SB216763 183 vn A C K N O W L E D G E M E N T S I thank my supervisor, Dr. T i m O'Connor, for his support and guidance over the years. T im has always allowed me the freedom to pursue my research interests and has made himself available whenever I sought advice. I am also thankful for Tim's patience in allowing me to pursue my non-academic interests. A s a side note, I am continuously amazed by Tim's knowledge of the wine industry and have learnt a great deal. I am thankful to Dr. John Church for providing invaluable advice on experimental design, technical support, and manuscript proof-editing. A s I can appreciate the hectic schedule of a principal investigator, I am grateful for the time John spent personally training me on the use of ratiometric imaging equipment. I thank Dr. Ca l Roskelley for all of his advice over the years. Ca l has provided invaluable feedback during experimental design and manuscript preparation. I also thank my committee members Dr. Matter Ramer and Dr. Alaa El-Husseini for their valuable input and guidance. I thank my peers, Dr. Aruna Somasiri, M r . Arthur Legg, M s . Robyn Lett, Mrs . Marcia Graves, Dr. Wenyan Wang, and Dr. A n a Le Meur for their support and advice during my graduate studies. These individuals have truly impacted my academic and personal endeavours in the most positive of ways. Finally, I am thankful to my family for the unequivocal support they have provided me over the years. M y most important acknowledgement is to my partner, M s . Loretta Wong, for her unconditional support and unquestionable patience. She has allowed me to pursue my goals and ambitions without hesitation, and as such I am. truly indebted. v i i i 1. INTRODUCTION A fundamental question in neuroscience is how the nervous system forms its elaborate architecture. Specifically, what are the precise morphological steps required for the development o f the functional circuitry underlying the nervous system? Neurons must first migrate to specific locations, extend axons and dendrites towards their proper targets, and then form functional synapses. Following development, many o f these complex processes are recapitulated during learning, memory, and neuronal regeneration. Due to the importance of these morphological steps during development, gaining a greater understanding of the underlying mechanisms governing these processes remains intensely studied. The growth cone, a specialized structure at the motile tip of an extending neurite, was first described by Santiago Ramon y Cajal in 1890 as a "battering ram" that traverses through obstacles in the environment en route to its final connective target (Cajal, 1890). Since Ramon y Cajal's landmark observations over a century ago, growth cone behaviour has been examined in detail. Stereotyped growth cone behaviours include pause, collapse, retract, and turning. These distinct actions reflect the growth cone's ability to encounter and decode cues in the extracellular environment both temporally and spatially, and then modulate appropriate intracellular mechanisms in response to these cues (Henley et al., 2004; Mattson et al., 1988; McCobb et al., 1988; Wong et al., 2002). Though fundamental during development and following neuronal injury, the complex mechanisms associated with growth cone motility remain poorly resolved. Moreover, the studies that have examined the signaling cascades involved tend to focus on individual 1 pathways. Therefore, the aim of this thesis is to identify the underlying mechanisms, and their combinatorial activities, that govern growth cone motility. Specifically, I w i l l address what signal transduction pathways are involved in inhibitory cue-induced growth cone collapse. In chapter 2,1 w i l l characterize a novel anti-invasive compound termed Motuporamine C (MotC) as a robust stimulator of growth cone collapse and provide evidence of a potential signal transduction pathway utilized by M o t C . Based on the partial involvement of this putative pathway in MotC-mediated growth cone collapse, I w i l l show in Chapter 3 the involvement of a second pathway that acts in a combinatorial fashion to mediate the collapsing effect of MotC . In Chapter 4,1 w i l l examine whether the putative pathways identified by M o t C apply to a physiological in vivo guidance cue, Semaphorin 5B (Sema5B). B y gaining a greater understanding of the intracellular pathways controlling outgrowth inhibition, the development of new therapeutic strategies to promote regeneration following neuronal injury may be expedited. 1.1 Cytoskeletal dynamics mediating growth cone motility The growth cone is a dynamic structure at the tip of the neurite responsible for determining directional outgrowth. It does this by decoding informational cues from the extracellular environment and translating this into appropriate signal transduction pathways that ultimately impinge on the growth cone's cytoskeleton to regulate motility (Bentley and O'Connor, 1994; Tanaka and Sabry, 1995). The cytoskeleton is composed of microtubules, actin microfilaments, and intermediate filaments. The growth cone can be divided into three regions; the peripheral, transient, and central domains (Fig. 1.1) 2 Figure 1.1. Chicken dorsal root ganglion growth cone stained for F-actin (red) and microtubules (green). (A) Normal motile growth cone differentiated regionally by peripheral (P-), transitional (T-), and central (C-) domains. Hol low arrowhead highlights filopodia and hollow arrow indicates lamellipodia. (B) Collapsed growth cone highlighting the dramatic loss of structure, in particular, in the P-domain. 3 4 (Bridgman and Dailey, 1989; Forscher and Smith, 1988; Smith, 1988). The peripheral (P-) domain contains the bulk of filamentous actin (Dent and Gertler, 2003; Forscher and Smith, 1988). This actin is localized within finger-like projections termed filopodia and veil-like structures termed lamellipodia (Dent and Gertler, 2003; Meyer and Feldman, 2002). The central (C-) domain is rich in microtubules and contains organelles and vesicles (Gordon-Weeks, 2004). Lastly, the transitional (T-) domain serves as an interface between the P-domain and C-domain (Forscher and Smith, 1988; Gordon-Weeks, 2004). In the case of growth cone collapse, these regions become much less defined, with the greatest contraction occurring in the P-domain (Fig. 1.1). While it has been established that both microtubule and actin components play a major role in growth cone motility (Bentley and O'Connor, 1994; Smith, 1988), intermediate filaments are only found within neurites and therefore not considered to play a major role in outgrowth (Phillips et al., 1983; Shaw et al., 1985). Ac t in dynamics A n actin filament (F-actin) consists of a double helical polymer composed of globular actin subunits (G-actin) (Dent and Gertler, 2003). They are polar in nature, with net growth and addition taking place at the plus (or barbed) end and disassembly occurring at the minus (or pointed) end (Pollard and Borisy, 2003). The barbed end is oriented towards the distal membrane, while the pointed end is directed inwards (Pollard and Borisy, 2003). While monomers can associate and dissociate from both the barbed and pointed ends in vitro, addition of G-actin on the barbed end and removal of 5 monomers on the pointed end is kinetically favoured (Pollard and Borisy, 2003). Retrograde transport of F-actin towards the central domain of growth cones occurs in both filopodia and lamellipodia and is a myosin-driven process (Dent and K a l i l , 2001; Diefenbach et al., 2002; L i n et al., 1996; L i n and Forscher, 1993), although the exact type(s) of myosin isoform(s) involved remains unclear. The observation that actin is localized to the leading edge of the growth cone highlighted its importance in motility (Bray and Chapman, 1985; Forscher and Smith, 1988; Yamada et al., 1970; Yamada et al., 1971). In the P-domain of the growth cone, the bulk of filamentous (F-) actin forms two types of arrays. The first is a polarized bundled array of F-actin that composes the core of filopodia. Filopodia play an essential role in sampling the environment for guidance cue information and transduce these signals into directionality and outgrowth (Bentley and Toroian-Raymond, 1986; Bray and Chapman, 1985; Chien et al., 1993; O'Connor and Bentley, 1993). The second array that F-actin can adopt is a meshwork array that forms lamellipodia. These veil-like structures are thought to be important for growth cone protrusion and play a significant role in substrate adhesion during growth cone motility (Conklin et al., 2005; Jay, 2000). It has been shown that lamellipodial extension between filopodia encountering a positive cue leads to turning (Bentley and Toroian-Raymond, 1986; Kleitman and Johnson, 1989; O'Connor et al., 1990). Previously, it was proposed that actin served as a physical barrier to prevent microtubules from invading the P-domain of the growth cone (Forscher and Smith, 1988), although it has since been shown that microtubules can and do invade the periphery (Gordon-Weeks, 1991; Tanaka and Kirschner, 1995), indicating a much more active role for microtubules in motility. 6 Microtubule dynamics Microtubules are an integral component of the cytoskeletal machinery important for motility. They are present along the neurite shaft as well as the growth cone (Fig. 1.1). Within the neurite, microtubules are bundled, but within the C-domain of the growth cone, they tend to be de-fasiculated (Tanaka and Sabry, 1995; Yamada et al., 1971). A s with actin, microtubules have a polarized distribution in axons, with the fast-growing (plus) end directed towards the growth cone periphery, while the slow-growing (minus) end is oriented proximally towards the cell body (Dent et al., 1999; Dent and K a l i l , 2001). Microtubules are hollow cylindrical filaments composed of 13 protofilaments, comprised of a and P tubulin subunits (Gordon-Weeks, 2004). Microtubule populations can be classified as either stable, characterized by acetylation of tubulin as a post-translational modification (Kozminski et al., 1993), or dynamic, characterized by tyrosination of tubulin post-translationally (Lai et al., 1994). Stable microtubules are typically localized along the neurite shaft and in the C-domain of the growth cone (L im et al., 1989). Dynamic microtubules exist not only in the C-domain, but can also extend into the actin-rich P-domain, and at times the proximal region of the filopodium (Bush et al., 1996; Gordon-Weeks, 1991; Sabry et al., 1991; Zhou et a l , 2002). Mitchison and Kirschner (1984a; 1984b) postulated that this dynamic instability afforded microtubules the ability to probe the actin network, allowing microtubules to be "captured" by the actin network in response to a morphogenetic signal and thus subsequently stabilized against catastrophe. Applied to the context of the motile growth cone, dynamic instability permits microtubules to investigate the actin network in the peripheral domain, and specifically 7 within the filopodia, for changes following an encounter with a guidance cue, upon which capture and stabilization can occur (Dent et al., 1999; Gordon-Weeks, 1991). This was first demonstrated using motile Xenopus spinal growth cones. B y fluorescently visualizing microtubule direction within growth cones sharply turning at borders between laminin-permissive and collagen IV-non-permissive substrates, these experiments showed re-orientation of microtubules along the direction of turn (Tanaka and Kirschner, 1995). Adhesive Focal Contacts The cytoskeleton is intimately linked to the extracellular environment through the binding of cell adhesion molecules at sites termed focal contacts (Letourneau and Shattuck, 1989). The components which make up focal contacts include integrins, kinases, phosphatases, and the actin-associated proteins talin and vinculin (Gomez et a l , 1996; Letourneau and Shattuck, 1989; Schmidt et al., 1995). Integrins serve as the migration-promoting receptors of the growth cone by acting as the primary link between the extracellular matrix and the actin cytoskeleton and have been shown to activate motility-related signaling cascades (Jay, 2000; Ridley et a l , 2003). One kinase family associated with adhesion regulation, Src tyrosine kinases, has been shown to regulate tyrosine phosphorylation at the tips of growth cone filopodia (Robles et al., 2005). B y inhibiting Src kinase activity in the growth cone, Robles et al. (2005) demonstrated phospho-tyrosine signaling and downstream targets such as the focal contact marker vinculin were down-regulated, suggesting an uncoupling of the actin 8 cytoskeleton from adhesion receptors and thus resulting in diminished growth cone motility. Furthermore, previous studies have shown that guidance cue receptors such as Plexin-A2 and P l ex in -B l can regulate Src kinase activity to regulate motility (Basile et al., 2005; Sasaki et al., 2002). These results highlight the importance of signaling pathways governing focal contacts, and presumably the tractional force required, for neurite outgrowth. It is also important to note differences in adhesion requirements for non-neuronal versus neuronal cells. A t the leading edge of non-neuronal cells, adhesion is necessary to provide the tractional forces necessary for forward movement, whereas at the trailing edge, de-adhesion is important to allow the cell's rear to retract towards the direction of movement (Ridley et al., 2003). It is thus the balance between these two processes which promotes forward migration. In neurons, no trailing edge is present and thus retraction in this context is not observed (Dent and Gertler, 2003; K a l i l and Dent, 2005). One exception may be the exploratory nature of filopodia as they sample the environment, although it remains unknown what the dynamic relationship between adhesion and de-adhesion is in this case. The molecular clutch model for directed growth cone motility To account for the interdependency of the cytoskeletal and adhesive elements governing the motile growth cone, the molecular clutch model was proposed to describe the force-coupling required for productive forward movement (Mitchison and Kirschner, 1988). Based on this model, an extending lamellipodium that encounters an extracellular ligand that binds a cell surface receptor on that lamellipodium w i l l couple this complex to 9 the actin cytoskeleton, and thus engaging the "clutch" (Lin et al., 1996; L i n and Forscher, 1995; Sheetz et al., 1998). In turn, actomyosin contraction of actin filaments w i l l not be able to pull the actin network back towards the proximal end of the growth cone. A s a result, lamellipodial protrusion occurs by net actin assembly at the distal barbed end of the actin filament that is fixed with respect to the substrate (Mitchison and Kirschner, 1988). Providing protrusion is occurring as a result of an encounter with a chemoattractant cue in the environment, engorgement of the growth cone in this protrusive direction occurs (Goldberg and Burmeister, 1986). This involves microtubule capture in this protrusive region, and subsequently the transport of organelles and vesicles (Dent et al., 1999; Gordon-Weeks, 1991). Following engorgement, consolidation occurs as the proximal region of the growth cone assumes a cylindrical shape and organelle transport becomes bidirectional (Fig. 1.2). In addition, when this "clutch" is disengaged, retrograde flow resumes to direct proximal movement of F-actin (Forscher and Smith, 1988; Mitchison and Kirschner, 1988). When the rate of retrograde flow is faster than actin assembly, retraction of the lamellipodium occurs. The balance between actin assembly, the coupling of the growth cone cytoskeleton with cell adhesion molecules, and retrograde flow thus serves as a fundamental mechanism of motility. i Cytoskeletal dynamics implicated in growth cone collapse Growth cone collapse represents an extreme motile event in which the cytoskeletal machinery contributing to motility appears to break down. Though not well defined, it appears collapse involves the retraction and/or depolymerization of both the 10 Figure 1.2. Schematic of the clutch model for directed neurite outgrowth. Adapted from Dent and Gertler (2003), Jay (2000), and Mitchison and Kirschner (1988). (A) Filopodial protrusion towards a chemoattractive cue in the context of the clutch hypothesis. Protrusion is dependent on the assembly of actin at the filopodial tip and a clutch mechanism to prevent actomyosin contraction on F-actin. (B ,C ,D) Directed neurite outgrowth towards a chemoattractive cue as defined by the stages o f (B) protrusion, (C) engorgement, and (D) consolidation. 11 • Vesicle :4- F-actin meshwork Microtubule Chemoattractive cue Bundled F-actin Region of interest 12 actin and microtubule network from the growth cone periphery (Pollard and Borisy, 2003; Gordon-Weeks, 1991). This in turn results in filopodial and lamellipodial retraction. Presumably, this event also requires detachment from the extracellular surface to facilitate retraction, ultimately producing a collapsed phenotype characterized by a "club-like" appearance at the neurite tip (Fig. L I B ) . 1.2 Molecular mechanisms mediating growth cone motility While the mechanisms underlying cytoskeletal rearrangement are relatively well defined, the identification of the signal transduction pathways regulating the protrusive and retractive properties of the cytoskeleton remains poorly described, and at times, contradictory. Previous studies from the Poo lab have identified c A M P as an important second messenger regulating the turning behaviour of growth cones (Henley and Poo, 2004; Song et al., 1998; Song et al., 1997). C a 2 + has also been shown to act as a key second messenger in modulating growth cone motility. In this case, the downstream effectors of Ca are only beginning to emerge, with some of the identified targets being the phosphatase calcineurin (Lautermilch and Spitzer, 2000; Wen et al., 2004), the kinases Ca 2 +/CaM-dependent protein kinase II (Wen et al., 2004) and protein kinase C (Jin et al., 2005; Sivasankaran et al., 2004), and the protease calpain (Robles et al., 2003). Furthermore, examination of the Rho family of small GTPases, the most widely studied of which are Rho, Rac, and Cel l division cycle 42 (Cdc42), has proven their importance in regulation of motility, albeit at times contradictory (Jin and Strittmatter, 1997; L i u and Strittmatter, 2001; Luo, 2000). With Rho GTPases as the exception, identification of 13 these downstream effectors has been achieved primarily through direct manipulation of C a 2 + or c A M P levels within the growth cone (Lautermilch and Spitzer, 2000; Robles et al., 2003; Wen et al., 2004). Therefore, it remains unclear whether these signaling cascades are also regulated by extracellular cues involved in regulating growth cone motility. In addition, even less understood are what intracellular pathways modulate microtubule re-organization in the motile growth cone. Cyc l ic nucleotides Cyclic nucleotides have been shown to play important roles as second messengers. In the case of c A M P , it is converted from A T P via adenylyl cyclase, while c G M P is converted from G T P via guanylyl cyclase. Important studies from the Pop laboratory have identified the importance of cyclic nucleotides in regulating growth cone motility. Using a growth cone turning assay, Song et al. (1997) showed that the normally chemoattractive brain-derived neurotrophic factor (BDNF) could be switched to a chemorepellant by reducing intracellular c A M P levels. Follow-up studies demonstrated Sema3A-mediated growth cone repulsion could be switched to attraction by raising intracellular c G M P levels (Song et al., 1998). Based on these observations, it was proposed that the ratio of specific nucleotides could modulate the turning response of the motile growth cone (Song et al., 1998). To date, cues have been divided into two classes based on responsiveness to specific cyclic nucleotides. Cues sensitive to c A M P levels include nerve growth factor (NGF) , B D N F , netrin, and myelin-associated glycoprotein ( M A G ) , while cues sensitive to c G M P include sema3A, neurotrophin-3, and the 14 chemokine SDF-1 (Song et al., 1998; Song et al., 1997; Song and Poo, 1999; Xiang et al., 2002). Furthermore, evidence indicates that these increases in cyclic nucleotide levels result in a positive feedback loop (Bolsover, 2005; M i n g et al., 2001). For instance, increases in [Ca ]j result in a raised c A M P x G M P ratio, which in turn increases the open probability of cAMP-sensitive C a 2 + channels/stores (Ming et al., 2001). Thus, the inter-relationship between cyclic nucleotides and C a 2 + further underscores the complexity of the molecular mechanisms regulating growth cone motility. Ca l c ium 2+ * Ca is another second messenger that plays a central role in transducing extracellular guidance information to outgrowth and directional motility. Regulation of the intracellular free C a 2 + concentration ([Ca 2 +]j) in the growth cone is precisely maintained by C a 2 + influx through plasma membrane C a 2 + channels, C a 2 + release from internal stores, and C a 2 + extrusion through plasma membrane Ca 2 + -ATPases and plasmalemmal N a + - C a 2 + exchangers (Bolsover, 2005; Henley and Poo, 2004; Henley et al., 2004; Hong et al., 2000). Based on this regulation, the C a 2 + set-point hypothesis has been proposed, which states that an optimal range of [Ca 2 +]j is required for normal growth cone motility and neurite outgrowth, while C a 2 + fluctuations above or below this optimal concentration results in outgrowth inhibition (Kater and M i l l s , 1991). This hypothesis has been substantiated in numerous neuronal cell types (al-Mohanna et al., 1992; Gu et al., 1994; Henley and Poo, 2004; Lankford and Letourneau, 1991). Furthermore, the spatial presentation of an extracellular stimulus the growth cone 15 encounters can dictate its motile behaviour. For example, in the case o f extracellular bath application of dopamine or serotonin, a global high amplitude rise in [Ca ]j was observed, leading to growth cone collapse and inhibition of outgrowth (Mattson et al., 1988; McCobb et al., 1988). On the contrary, when an extracellular gradient o f glutamate or acetylcholine was presented, a moderate rise in [Ca 2 +]j was found, inducing an attractive growth cone steering response (Zheng et al., 1994; Zheng et al., 1996). These experiments not only support the C a 2 + set-point hypothesis, but also substantiate the idea that global changes in [Ca 2 +]j regulate neurite outgrowth, while asymmetric local changes in [Ca 2 +]j influence growth cone steering. Therefore, based on how an extracellular cue is spatially presented to a growth cone, one can begin to address questions specifically concerning outgrowth versus steering. Calcineurin, protein kinases, and calpain Recently, efforts have been made to identify the downstream effector molecules regulated by C a 2 + signaling. The most extensively studied Ca 2 + -binding protein is calmodulin (CaM) (Bolsover, 2005). Upon binding, C a 2 + / C a M interacts with several targets, including Ca 2 +/CaM-dependent protein kinases (CaMKs) . Studies on the role of C a M K I I , a kinase localized to the actin cytoskeleton and shown to regulate neurite extension, indicates it plays a central role in Ca 2 +-dependent chemoattraction in Xenopus growth cones (Wen et al., 2004; Zheng et al., 1994). B y modestly elevating local [Ca 2 +]j via photolysis of caged C a 2 + , it was shown that C a M K I I specifically promotes attractive growth cone turning and that this response is mediated via regulation of the Rho family 16 of GTPases (Jin et al., 2005; Wen et al., 2004). Another kinase, protein kinase C (PKC) , has also been implicated in growth cone motility. It has been shown to be activated following increased [Ca 2 + ] i stimulated by the chemoattractant, B D N F (Jin et al., 2005). Subsequently, P K C was found to inhibit Rho, while promoting Rac and cdc42 activity (Jin et al., 2005). Furthermore, pathways responsible for the repulsive behaviour in motility arising from low- and high-amplitude [Ca 2 + ] f increases have also begun to be teased out (Lautermilch and Spitzer, 2000; Robles et al., 2003; Wen et a l , 2004). B y inducing a low-amplitude rise in [Ca 2 +]j via photolysis of caged C a 2 + in Xenopus growth cones, Wen et al. (2004) demonstrated the Ca 2 +/CaM-dependent phosphatase calcineurin is required for repulsive turning. Examination of potential substrates of calcineurin under these conditions led to the identification that both protein phosphatase 1 (PP1) and the growth and plasticity associated protein 43 (GAP-43) may be involved (Lautermilch and Spitzer, 2000; Wen et al., 2004). In the case of induction of large C a 2 + transients, the Ca 2 +-sensitive protease calpain has been shown to be activated and that calpain is necessary for growth cone repulsion in this case (Robles et al., 2003). Taken together, this has led to a recent revision of the Ca 2 +-set point hypothesis; repulsion/inhibition via low-amplitude rises in [Ca ]; requires calcineurin activity, attraction/outgrowth via mid-amplitude rises in [Ca 2 +]j require C a M K I I or P K C , and repulsion/inhibition via high-amplitude rises in [Ca ]j require calpain (Gomez and Zheng, 2006) (Fig. 1.3). However, since most of these studies directly modulated C a 2 + levels within the growth cone, it remains unknown whether this model holds true for most extracellular cues that regulate motility. Specifically, it is not known whether inhibitory cues that induce growth cone collapse through changes in [Ca ]j do in fact activate calcineurin- or calpain-mediated 17 Figure 1.3. Schematic highlighting signal transduction cascades regulated by different amplitude fluxes in [Ca 2 +]j . Low- , mid-, and high-amplitude increases in [Ca 2 +]j are represented by small, medium, and large [Ca 2 + ]i schematic balloons, respectively. Varying C a 2 + signals are proposed to regulate distinct downstream pathways. Red schematic balloons indicate proteins typically associated with induction of growth cone collapse, while blue schematic balloons indicate proteins usually associated with promoting outgrowth. Adapted from Gomez and Zheng (2006). 18 Growth cone collapse Outgrowth Growth cone collapse Adapted from Gomez and Zheng (2006) 19 pathways. Moreover, since previous studies have shown that calcineurin is a proteolytic target of calpain (Shioda et al., 2006), it is unclear whether these pathways are mutually exclusive of one another during growth cone collapse. R h o G T P a s e s a n d t h e i r d o w n s t r e a m targets The small GTPases of the Rho family represent a major class of downstream second messenger effectors in growth cone motility. Critical regulators of the actin cytoskeleton, the most widely studied have been Rho, Rac, and Cdc42. These GTPases exist either in an inactive, GDP-bound state, or an active, GTP-bound state. Three protein classes regulate the nucleotide-binding state of this family: guanine nucleotide exchange factors (GEFs), which facilitate the exchange of G D P for G T P , GTPase activating proteins (GAPs) , which increase the GTPase activity of this subfamily and thereby inactivate them, and guanine dissociation inhibitors (GDIs), which regulate both the G D P / G T P cycle and the membrane association/dissociation cycle (Luo, 2002). In neurons, Rho activation has been shown to mediate neurite retraction and growth cone collapse (Jalink et al., 1994; Katoh et al., 1998; Wahl et al., 2000). It is of interest to note that in non-neuronal cells, Rho activation differs slightly in that it results in stress fiber and adhesion complex formation; processes necessary for generating contractile forces to pull the cell forward during migration (Ridley, 2001). In the case of Rac and Cdc42 activation, these GTPases have, for the most part, been shown to promote neurite outgrowth, with Cdc42 thought to be involved in filopodial protrusion and Rac mediating lamellipodial formation (Brown et al., 2000; Kozma et al., 1997; Lamoureux et al., 1997; 20 Figure 1.4. Signaling cascades implicated in Rho GTPase activity in growth cone motility. Red schematic balloons indicate proteins typically associated with promoting growth cone collapse, blue schematic balloons show proteins usually associated with promoting outgrowth, while green schematic balloons have been shown to play roles in both outgrowth and inhibitory responses. It should be noted these signaling cascades do not represent all potential downstream targets of Rho GTPases associated with growth cone motility. Abbreviations: M R L C , myosin regulatory light chain; M R L C - P h , myosin regulatory light chain phosphatase. 21 22 Luo et al., 1994). However, exceptions to the commonly ascribed functions of these three Rho GTPases have been identified, such as Semaphorin 3 A signaling to induce outgrowth inhibition as described below (Jin and Strittmatter, 1997). A s a growth cone encounters extracellular guidance cues, a series of signaling cascades are triggered by the ligand-receptor complex. In many cases, these complexes initiate signal transduction pathways that eventually target Rho GTPases, which in turn regulate cytoskeletal dynamics (Fig. 1.4) (Gallo and Letourneau, 2003; Giniger, 2002; Luo, 2000). In the case of Rac and Cdc42, p21-activated kinase ( P A K ) is a common effector, and NGF-induced neurite outgrowth in P C 12 cells is inhibited by dominant-negative forms of either GTPase (Daniels et al., 1998). Furthermore, P A K signaling has been shown to activate LIM-domain-containing protein kinase (LIM-kinase), which in turn phosphorylates and inhibits the actin depolymerizing factor, cofilin, leading to protrusion (Arber et al., 1998; Yang et al., 1998). In the case o f Rho, the best-studied effector is the serine/threonine Rho-kinase ( R O C K ) . Rho-mediated R O C K activation has been shown in numerous neuronal cell types to promote growth cone collapse and neurite retraction (Bito et al., 2000; Hirose et al., 1998; Loudon et al., 2006; Niederost et al., 2002). Effectors downstream of R O C K include the regulatory light chain o f myosin II ( M R L C ) and the regulatory subunit of myosin light chain phosphatase ( M R L C - P h ) , both of which in turn lead to increased phosphorylation of myosin light chain and ultimately myosin Il-mediated retraction (Gallo et al., 2002; Loudon et al., 2006; Mulder et al., 2004; Schmidt et al., 2002). Adding to the complexity of these signal transduction pathways, R O C K can also activate LIM-kinase (Maekawa et al., 1999; Ohashi et a l , 2000). A s LIM-kinase represents a common target to both P A K and R O C K , it remains unresolved 23 how the seemingly disparate effects of Rac/cdc42 and Rho are mediated through convergent effector molecules. It should also be noted that in addition to R O C K , the formin-related protein, mammalian diaphanous (mDia), has been implicated in Rho-mediated signaling to regulate both actin assembly and microtubule stabilization and thus serves as another potential target involved in growth cone motility (Eisenmann et al., 2007; Watanabe et al., 1999; Watanabe et al., 1997). Glycogen synthase kinase-3P While significant progress has been made in identifying the molecular mechanisms regulating the actin cytoskeleton, few studies have examined the intracellular cascades governing microtubule dynamics during growth cone collapse. Most studies to date have teased out pathways implicated in microtubule re-organization after growth cone exposure to the repellent cue, lysophosphatidic acid ( L P A ) . Upon binding to an endothelial differentiating gene (EDG) receptor family member, it has been demonstrated that in addition to inducing actin filament contraction via R h o - R O C K signaling, microtubule re-organization occurs through activation of the serine/threonine kinase, glycogen synthase kinase-3p (GSK-3P) (Sayas et al., 2002a; Sayas et al., 2002b). Subsequently, GSK-3P activates the microtubule binding protein, Tau, leading to microtubule instability and neurite retraction (Sayas et al., 2002a; Sayas et al., 1999). Furthermore, a recent study by Zhou et al. (2004) has shown that NGF-induced axon growth is regulated via inactivation of GSK-3P. This inactivation in turn led to an accumulation of the microtubule binding protein, adenomatous polyposis coli (APC) , at 24 the growing plus ends of microtubules, suggesting A P C plays a role in microtubule stabilization (Zhou et al., 2004). Future studies w i l l be required to examine whether and how microtubule organization is modulated by other guidance cues. 1.3 Extracellular cues regulating growth cone motility The activation of the intracellular mechanisms regulating growth cone motility is initiated by the extracellular cues the growth cone encounters. A s an axon navigates through the extracellular environment, either during development or following neural injury in the peripheral nervous system, the motile growth cone must integrate guidance information from extracellular cues and translate this information into an appropriate physiological response. Cues can be classified into 3 categories: positive guidance cues (e.g. - netrins), negative guidance cues (e.g. - semaphorins), and myelin-associated inhibitors (e.g. - M A G ) . The growth cone response to these cues depends on the spatial context in which they are presented. In the case where a gradient is applied, the growth cone wi l l turn towards attractive cues and away from negative cues, whereas i f the cue is presented globally to the growth cone, outgrowth wi l l be promoted with a positive cue and collapse/retraction with a negative cue (Gomez and Zheng, 2006; Henley and Poo, 2004; McFarlane, 2000). While the behavioural outcome of the motile tip reflects the spatial context of the signal, the downstream signaling cascades initiated are likely the same (Gomez and Zheng, 2006; Henley and Poo, 2004). However, how these pathways are regulated by specific cues to induce particular motile responses remains largely undefined. Previous studies have tended to focus on the involvement of individual 25 pathways modulated in response to a specific guidance cue. This most likely does not reflect the underlying mechanisms governing motility since it can be assumed that more than one pathway is activated during a given motile response. It is therefore of interest to elucidate which molecular mechanisms are regulated by extracellular cues that direct growth cone motility, and specifically collapse, and to examine whether combinatorial activation of multiple pathways is required. Semaphorins - A prototypical family of repellent guidance cues One of the most widely-studied guidance cue families, the semaphorins are secreted and membrane-associated glycoproteins that can be grouped into eight classes based on structure and amino acid sequence similarity. Class 1 and 2 are found in invertebrates, classes 3-7 are in vertebrates, and class 8 is encoded by viruses (Raper, 2000). A l l semaphorins contain a stereotypical -500 amino acid Semaphorin domain (Kolodkin, 1998). Classes 2, 3, 4, and 7 contain a single immunoglobulin-like domain near the C-terminus, whereas class 5 semaphorins have a domain containing seven thrombospondin type 1 and type 1-like repeats (Adams et al., 1996; Raper, 2000). Furthermore, semaphorin 1, 4, 5, 6, and 7 are membrane-associated, while class 2, 3, and 8 are secreted (Raper, 2000). Though typically thought of as playing an inhibitory role in axonal guidance, it is now clear these molecules can be bifunctional, mediating repulsion and attraction in different neurons. Plexins are the predominant family of semaphorin receptors and are categorized as isoforms A through D . It is thought plexins are autoinhibitory by nature, and that 26 semaphorin binding permits plexin activation (Takahashi and Strittmatter, 2001). Interestingly, semaphorin class 3 requires the transmembrane protein neuropilin as a co-receptor to signal through Plexin A (Antipenko et al., 2003). To date, the signal transduction pathways modulating this receptor-ligand interaction in growth cone motility remain poorly described. In the case of the inhibitory cue semaphorin 4D (Sema4D), upon binding Plexin B l , activated Rac is recruited to this receptor complex through a GTPase-binding domain in Plexin (Vikis et al., 2002; V i k i s et a l , 2000). Since active Rac normally promotes actin polymerization through Pak, it is thought that in this case Rac is sequestered, thus down-regulating Pak activity. Previous studies examining the prototypical inhibitory semaphorin 3 A (Sema3A) indicates that upon binding to Plexin A l and its co-receptor neuropilin-1, Rac co-localizes with this receptor complex (Fournier et al., 2000). In addition, a dominant-negative form of Rac inhibits Sema3A-induced growth cone collapse and outgrowth inhibition (Jin and Strittmatter, 1997). Examination of downstream targets of this signaling pathway have shown that Sema3A inhibits cofilin activity via LEVI-kinase activation to induce growth cone collapse (Aizawa et al., 2001). Although the involvement of Rac in Sema3A signaling implicates this GTPase as a repressor of outgrowth and motility, it appears to be more the exception rather than the rule. Moreover, relatively few studies have examined C a 2 + signaling in response to semaphorins. This is likely due to the fact that the prototypical Sema3 A has been shown to mediate its repulsive effects in a Ca 2 +-independent manner (Shim et al., 2005; Wen et al., 2004). Therefore, further studies are required to identify whether other semaphorins do in fact mediate their motile effects on growth cones through C a 2 + . 27 Netrins - A prototypical family of attractive guidance cues Netrins are laminin-like molecules that bind the receptors Deleted in Colon Cancer (DCC) and U N C 5 to mediate distinct motile behaviours. When netrin binds to D C C , growth cone attraction occurs (Keino-Masu et al., 1996; Nikolopoulos and Giancotti, 2005), whereas binding to U N C 5 , either as homodimers or heterodimers with D C C , elicits repulsive effects (Hong et al., 1999; Nikolopoulos and Giancotti, 2005). In the case of growth cone attraction, a mid-amplitude increase in [Ca 2 +]j has been shown to result from an influx of C a 2 + through voltage-dependent C a 2 + channels ( V D C C s ) and transient receptor potential (TRP) channels, as wel l as C a 2 + release from internal stores (Hong et al., 2000; Wang and Poo, 2005). Examination of the downstream effectors of netrin signaling identified an increase in Rac activity (Jin et al., 2005). To confirm that this Rac activity was indeed dependent on C a 2 + , chelation of intracellular C a 2 + via B A P T A - A M abolished this effect (Jin et al., 2005). Whether the regulation of [Ca 2 +]j and the downstream effects on Rho GTPases is required to mediate the motile effects of other extracellular cues remains undefined. Myelin-associated glycoprotein - A myelin-associated inhibitor M A G is an extensively studied, myelin-associated inhibitory protein belonging to the immunoglobulin superfamily. Normally found in both peripheral and C N S myelin, it is involved in the formation and maintenance of myelin sheaths (Fruttiger et al., 1995; Marcus et al., 2002). However, following injury to the C N S , the inhibitory nature of 28 M A G contributes to the non-permissive environment hindering axonal regeneration (Sandvig et al., 2004). M A G binds with high affinity to the Nogo receptor (NgR), but requires p 7 5 N T R as a co-receptor to stimulate inhibition of outgrowth and growth cone collapse (Wong et al., 2002). More recently, additional co-receptors of M A G signaling have been identified ( M i et al., 2004; Shao et al., 2005). Examination of downstream second messengers of M A G signaling has revealed C a 2 + and c A M P to play central roles in MAG-mediated inhibition. It has been shown that M A G induces a low-amplitude rise in [Ca 2 +]j via C a 2 + release from internal stores (Hong et al., 2000). Furthermore, Song et al. (1998) demonstrated that MAG-mediated growth cone repulsion can be converted to attraction by elevating c A M P levels in Xenopus spinal neurons. Henley et al. (2004) sought to determine the underlying relationship between C a 2 + - and cAMP-dependent signaling following M A G addition. In this study, it was found that upon increased c A M P signaling, which converts MAG-mediated repulsion to attraction, basal levels of [Ca 2 +]j increased to a mid-amplitude response (Henley et al., 2004), providing further support for the C a 2 + set-point hypothesis. Finally, it has also been demonstrated that upon M A G / N g R / p 7 5 N T R association, Rho is activated via p 7 5 N T R (Vinson et al., 2001; Wong et al., 2002; Yamashita et a l , 1999), while Rac and Cdc42 are concomitantly inactivated (Niederost et al., 2002). Therefore, similar to netrin-1, it appears that a common resulting mechanism of guidance cue stimulation is an amplitude rise in [Ca 2 + ] , followed by the downstream activation of intracellular pathways involved in regulating cytoskeletal dynamics. Sti l l , it remains unknown how broad this commonality is and specifically whether other negative cues induce similar signal transduction cascades to mediate outgrowth inhibition. 29 Motuporamine C - Utilization of an anti-invasive compound to study growth cone collapse Motuporamines are a novel family of alkaloids isolated from the marine sponge, Xestospongia exigua (Williams et al., 1998). B y utilizing an anti-invasive screen, Roskelley et al. (2001) found that Motuporamine C (MotC) inhibits the migration of a number of cancer cell lines. Furthermore, McHardy et al. (2004) showed M o t C increases stress fiber formation and that this is due to an upregulation of Rho activity. However, little is known about the mechanisms of M o t C function including what target M o t C directly modulates and whether the target is intracellular or at the cell surface. Nevertheless, based on the results in non-neuronal cultures, and given the inhibitory role Rho plays in neuronal growth cone motility, M o t C may serve as a useful research tool in elucidating the intracellular mechanisms underlying outgrowth inhibition in neurons. 1.4 Summary Recent progress has begun to identify the signal transduction cascades involved in regulating outgrowth inhibition. However, how these pathways are modulated by extracellular cues remains unclear. Presently, it is unknown whether these pathways may crosstalk and/or act in concert with one another to mediate the motile response stimulated by a specific cue. This information would be of significant interest as it w i l l identify potential key targets to develop more potent therapeutic strategies to stimulate neurite regeneration. Therefore, the aim of this thesis is to examine the signalling cascades that 30 underlie growth cone collapse and whether multiple pathways in combination are required. In chapter 2,1 characterize the anti-invasive compound M o t C as a robust collapsing cue of growth cones and provide evidence that M o t C signals, at least partially, through the R h o - R O C K pathway. Based on my experiments in which inhibition of the R h o - R O C K pathway only partially attenuates the growth cone collapsing effects of M o t C , I hypothesize in chapter 3 that other signaling cascades act in concert with R h o - R O C K . Indeed, I identify Ca 2 +-mediated calpain activation as a second pathway regulated by MotC during collapse. Since M o t C is an organic molecule not found in vertebrate organisms, I next examine whether similar intracellular pathways are modulated by a physiological in vivo guidance cue. Using the inhibitory cue Sema5B in my growth cone collapse assay, combined with the robust C a 2 + response stimulated by M o t C during outgrowth inhibition, I hypothesize in chapter 4 that C a 2 + and its downstream targets are involved in Sema5B-mediated collapse. I find that C a 2 + and the downstream targets, calpain and calcineurin, are involved. Interestingly, I also find in this case that the combined activation of calpain and calcineurin is necessary to promote complete collapse due to Sema5B signalling. Taken together, these studies show that inhibitory cues stimulate a complex network of downstream signal transduction pathways to induce growth cone collapse. I also show that it is indeed the activation o f these pathways in particular combinations that contributes to the complete collapsing effects of particular cues. 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For instance, the Rho GTPase family of small molecules plays a pivotal role in regulating cytoskeletal dynamics in both neuronal and non-neuronal cell types (Dickson, 2001; Ridley, 2001; Meyer and Feldman, 2002). Rho activation has been shown to induce contraction in yeast and fibroblasts (Ridley and Hal l , 1992). Similarly, activation in neuronal populations induces growth cone collapse and retraction (Suidan et al., 1992; Jalink et al., 1994). While such similarities exist, the downstream signaling pathways associated with these molecules have yet to be clearly defined (Luo et al., 1997; Dickson, 2001). Even more poorly understood is whether the mechanisms identified in migrating cells directly translate to similar machinery being 1 A version o f this chapter has been published. To, K . C . , Loh , K . T . , Roskelley, C D . , Andersen, R . J . and O'Connor, T .P. (2006) Neuroscience 139:1263-1274. 44 evoked during neurite outgrowth. Recently, a novel family of alkaloids, the motuporamines, was isolated from the Papua New Guinea marine sponge, Xestospongia exigua (Williams et al., 1998). In a previous study, these motuporamines, particularly isoform C, were found to inhibit cell migration of various human cancer cell lines (Roskelley et al., 2001). Furthermore, it was found that motuporamines attenuate membrane ruffling at the leading edge of lamellae and increase stress fiber formation (Roskelley et al., 2001; McHardy et al., 2004). Since growth cone and non-neuronal cell motility likely require similar cellular rearrangements, I hypothesized that motuporamines could affect growth cone motility in an inhibitory manner, thus allowing the exploitation of this compound as a novel means to study the intracellular pathways that are utilized by neurons for outgrowth. To investigate this, I utilized motuporamine C (MotC) in an effort to identify which molecular pathways are important for growth cone motility. M y results show that MotC acts in an inhibitory, dose-dependent manner. It was found that growth cone collapse occurs within minutes of M o t C application, followed by neurite retraction in the continued presence of this compound. These effects are reversible as growth cone , motility recovers after M o t C removal. Furthermore, I demonstrate that M o t C inhibits * motility through an upregulation of the Rho-Rho kinase pathway. I have thus shown that this organic molecule, originally identified for its anti-motility effects on cells, is a robust inhibitor of neurite outgrowth. M o t C may therefore be utilized as a tool to elucidate key intracellular pathways associated with neurite outgrowth inhibition and collapse. 45 2.2 Materials and Methods Motuporamine C collapse assays. 12-well culture dishes were treated overnight at 37°C with 1 ug/ml laminin (Chemicon) and 100 ug/ml poly-L-lysine (Sigma). Explants of embryonic day 8 (E8) chick D R G s and sympathetic neurons were subsequently cultured in these wells with 50 ng/ml N G F (Invitrogen)- or 50 ng/ml NT-3 (Chemicon)-supplemented D M E M (Sigma) overnight at 37°C. Following 24 hour incubation, M o t C (dissolved in water) was added to cultures for a further 24 hours (48 hour total). A n equivalent volume in micro litres of water was added to control cultures. Washout studies were performed by washing out M o t C for 24 hours with pre-warmed NGF-supplemented D M E M media after 24 hour exposure to this compound. Time-lapse videomicroscopy. Coverslips were coated with 1 (J.g/ml laminin (Chemicon) and 100 ug/ml poly-L-lysine (Sigma) overnight at 37 °C. E8 D R G explants were cultured on these coverslips containing D M E M supplemented with 20 m M H E P E S and 14 m M N a C l overnight at 37 °C. Cultures were then placed on an inverted N i k o n D I A P H O T 200 fluorescence microscope, with time-lapse imaging captured using a Princeton Instruments MicroMax C C D camera (Kodak chip K A F 1400) and MetaView Imaging System 3.6 (Universal Imaging Corporation). Cultures were filmed for 1 hour in the absence of M o t C , followed by 1 hour in its presence. For time-lapse of washout, M o t C was washed out with D M E M supplemented with 2 0 m M H E P E S and 14 m M N a C l and filmed for another 3 hours. Immunocytochemistry. Cultures were fixed with 3.7% formaldehyde in P E M (0.1 M PIPES, 2 m M E G T A , 1 m M M g S 0 4 ) for 10 min, and then washed with P B S containing 46 0.1% Triton-X and 0.1% B S A (3 x 1 minute, followed by 3 x 10 minutes). Rabbit-anti-neurofilament (Sigma) primary antibody (1:500) was applied overnight (4°C) and then washed again in P B S containing Triton X-100 and B S A as described above. Cells were then incubated at room temperature in 1:500 Cy3-conjugated goat-anti-rabbit (Jackson Laboratories) secondary antibody. Growth cone assay. D R G explants were cultured for 24 hours as described above, followed by 1 hour exposure to MotC . Explants were subsequently washed with P B S (2 x 1 minute), then fixed with 3.7% formaldehyde solution in P B S for 10 minutes at room temperature. After fixation, cultures were washed in P B S ( 2 x 1 minute), once for 5 minutes in P B S containing 0.1 % Triton X-100, followed by P B S ( 2 x 1 minute) once more. For actin staining, explants were then stained with 2 units of A lexa Fluor 488 phalloidin (Molecular Probes) diluted into 200 pi P B S containing 1% B S A for 30 minutes. For microtubule staining, explants were fixed using a microtubule stabilizing fixative (Dent et al., 1999). This fixative was 4% paraformaldehyde / 0.25% glutaraldehyde containing 0.1% Triton X-100, l O u M taxol, and 1.3uM phalloidin in P H E M buffer. Following a 15 minute fixation, neurons were washed in P B S and then incubated in mouse-anti-acetylated tubulin (Sigma) and rat-anti-tyrosinated tubulin antibodies for 1 hour at room temperature and then washed again in P B S containing Triton X-100 and B S A as described above. Cells were then incubated at room temperature in 1:500 Cy3-conjugated goat-anti-mouse (Jackson Laboratories) and 1:500 Alexa 488 goat-anti-rat secondary antibodies. Rho-GTP Affinity Precipitation and Immunoblot. D R G explants were dissected from E8 chicks. Using the Rho activation assay kit (Upstate), pull-down of Rho-GTP was 47 performed as described. Briefly, experimental lysates (1 mg/ml) were treated with M o t C for 1-3 hours at 37°C before the addition of Rhotekin Rho-binding domain agarose beads for 45 minutes at 4°C. The slurry of beads were then boiled for 5 minutes with 2x Laemmli reducing buffer and 1 M dithiothreitol, and then loaded into a 15% polyacrylamide gel at which point S D S - P A G E was performed using a 1:333 anti-Rho primary antibody (Upstate) and 1:1000 goat-anti-rabbit H R P conjugated IgG secondary antibody (Jackson Laboratories). A n ECL-based detection protocol was used for protein identification. Equal protein loading levels were determined using a colorimetric protein assay kit (BioRad) and a Western blot detecting total levels of Rho protein in lysates. These pull-downs were done in triplicate. ROCK Inhibition assay. D R G explants were cultured for 24 hours as described above. Following overnight incubation, the R O C K inhibitor Y27632 (Calbiochem) was added 1 hour prior to M o t C addition, and both compounds incubated with these cultures for another 24 hours (48 hour total). Immunocytochemistry or phalloidin stain was then performed as described above. C3 Exoenzyme Loading. E8 chick D R G explants were dissociated and triturated with C3 exoenzyme as previously described (Jin and Strittmatter, 1997). Briefly, explants were isolated and then re-suspended in 25 mM T r i s -HCl , 150 mM N a C l , 5 mM MgCL; , 1 mM dithiothreitol, and 5-20 pg/ml C3 exoenzyme from Clostridium botulinum (Calbiochem). This suspension was then passed 50 times through an Eppendorf P200 pipette tip and then plated in 20 volumes of D M E M media supplemented with 50 ng/ml N G F (Invitrogen). Cultured neurons were allowed to grow for 24 hours, followed by application of 5 u M M o t C for 1 hour in the case of the experimental condition, at which 48 point neurons were fixed and stained with Alexa Fluor 633 phalloidin (Molecular Probes) as described above. Quantification. Slides were viewed using a Nikon D I A P H O T 200 inverted fluorescence microscope, with images of neurites and growth cones captured using a cooled C C D camera (Princeton Instruments) and MetaView imaging software (Universal Imaging Corporation). Neurite length was measured by recording radially 1 in every -50 neuites per explant. Each experiment was done in triplicate with 15 explants per condition. Microsoft Excel was used to calculate the standard error of mean and standard deviation for each experimental condition. Statistical significance of differences between conditions was determined by A N O V A and Student's t-test using SPSS statistical software version 11.0 for Windows. For quantification of growth cone collapse, growth cones were scored as collapsed according to Fan et al. (1991). Specifically, growth cones lacking lamellipodia and extending 4 or less filopodia were scored as collapsed. 49 2.3 Results MotC inhibits neurite outgrowth and stimulates growth cone collapse To examine the effects of M o t C on neurite outgrowth, I cultured embryonic day 8 chick dorsal root ganglion (DRG) explants overnight in culture media containing N G F and subsequently added varying concentrations of M o t C . Following a 24 hour exposure to this compound, I found that as M o t C concentrations were increased, neurite outgrowth decreased significantly in a dose-dependent manner (Fig. 2.1). Initial baseline concentrations of M o t C were determined from previous studies carried out by Roskelley et al. (2001) on non-neuronal cultures. A t the lower concentrations, neurite outgrowth of D R G ' s appeared to halt. A t moderate to high amounts, neurite retraction likely occurred since neurite length following 24 hour MotC addition (48 hour total) was shorter than that at 24 hours initial growth (data not shown). A stepwise increase in concentration from 5 u M to 6 u M resulted in the most significant decrease in outgrowth. A t concentrations higher than 7 u M M o t C , I found cultured D R G s could not adhere to the tissue culture substrate following exposure to this compound. I observed that embryonic day 8 dissociated D R G cultures and sympathetic neurons responded in a similar inhibitory manner to M o t C (data not shown). Observations of bleb-like structures at neurite endings following addition of MotC suggested that M o t C may cause growth cone collapse. Growth cone collapse usually involves the loss of the actin cytoskeleton integrity in the filopodia and lamellipodia (Jin and Strittmatter, 1997). I examined whether M o t C was specifically causing growth cone 50 Figure 2.1. Inhibitory effects of M o t C on E8 D R G explants. (A) Control explant cultured for 48 hours in culture medium supplemented with N G F . (B-D) Explants cultured for 24 hours in culture medium supplemented with N G F , followed by a further 24 hours in D M E M supplemented with N G F and either 4, 5, or 6 u M M o t C , respectively. (E) Quantification of the effects of M o t C on D R G neurite outgrowth. The percentage o f neurites longer than a given length was plotted versus neurite length. For each explant of each condition, a measurement o f neurite length was taken every ~50 neurites radially along the explant. n=50 neurite length measurements from 15 D R G s per experimental condition, with experiments done in triplicate. Scale bar represents 100 um. 51 X A o 'C 120.0% 100.0% 80.0% 60.0% 40.0% 20.0% 0.0% - • - 0|jM MotC 3 M M MotC 4pM MotC - *— 5pM MotC - B - 6pM MotC - • - 7uM MotC r£> r f c rrb r ib r fa n