Kinesin-1 regulates synaptic strength by mediating delivery, removal and redistribution of ampars

The cellular processes that govern neuronal function are highly complex and tightly regulated in order to perform the elaborate information processing achieved by the brain. This is particularly evident in the trafficking of membrane proteins to and from synapses, which can travel long distances away from the cell body. Regulation of neurotransmitter receptors such as the AMPA-type glutamate receptor (AMPAR), the major excitatory neurotransmitter receptor in the brain, is a crucial mechanism for the modulation of synaptic transmission. Yet, the mechanisms by which AMPARs are transported over long distances are still unclear. We have addressed this question through genetic, cell biological and electrophysiological analysis of the C. elegans AMPAR GLR-1. This dissertation describes the role of long-range transport of AMPARs in the regulation of synaptic strength and provides insights into the cellular mechanisms underlying learning and memory. The pair of interneurons AVA expresses GLR-1 and are part of a welldefined circuit regulating the forward and backward movement of C. elegans in response to sensory inputs. To determine the mechanism for GLR-1 delivery to a synapse, we monitored the real-time trafficking of a fluorescently tagged GLR-1 chimera in AVA. We show that UNC-116, the C. elegans homolog of the vertebrate kinesin-1 (KIF5), is responsible for mediating the rapid, bidirectional transport of GLR-1. This motor-driven transport of GLR-1 modifies synaptic strength by mediating the rapid delivery, removal and redistribution of synaptic AMPARs. In the absence of unc-116, we found that although homomeric GLR-1 AMPARs can still diffuse to and accumulate at proximal synapses, glutamategated currents are decreased due to lack of heteromeric GLR-1/GLR-2-containing AMPARs. Furthermore, we show that transient expression of UNC- 116 can rescue defective glutamatergic signaling in adult unc-116 mutants, demonstrating that motor-dependent transport is ongoing in the adult nervous system and is involved in the regulation of synaptic strength. These data have allowed us to establish a link between motor-dependent transport of AMPARs and the strength of synaptic transmission.

KINESIN-1 REGULATES SYNAPTIC STRENGTH BY MEDIATING DELIVERY, REMOVAL AND REDISTRIBUTION OF AMPARS by Dane Arthur Maxfield A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology The University of Utah May 2014 Copyright © Dane Arthur Maxfield 2014 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Dane Arthur Maxfield has been approved by the following supervisory committee members: Andres Villu Maricq Chair 1/6/2014 Date Approved Michael Vershinin Member 1/6/2014 Date Approved Markus Babst Member 1/6/2014 Date Approved Julie Hollien Member 1/6/2014 Date Approved Erik Jorgensen Member 1/6/2014 Date Approved and by Neil Vickers Chair of the Department of _______________________ Biology and by David B. Kieda, Dean of The Graduate School. ABSTRACT The cellular processes that govern neuronal function are highly complex and tightly regulated in order to perform the elaborate information processing achieved by the brain. This is particularly evident in the trafficking of membrane proteins to and from synapses, which can travel long distances away from the cell body. Regulation of neurotransmitter receptors such as the AMPA-type glutamate receptor (AMPAR), the major excitatory neurotransmitter receptor in the brain, is a crucial mechanism for the modulation of synaptic transmission. Yet, the mechanisms by which AMPARs are transported over long distances are still unclear. We have addressed this question through genetic, cell biological and electrophysiological analysis of the C. elegans AMPAR GLR-1. This dissertation describes the role of long-range transport of AMPARs in the regulation of synaptic strength and provides insights into the cellular mechanisms underlying learning and memory. The pair of interneurons AVA expresses GLR-1 and are part of a well-defined circuit regulating the forward and backward movement of C. elegans in response to sensory inputs. To determine the mechanism for GLR-1 delivery to a synapse, we monitored the real-time trafficking of a fluorescently tagged GLR-1 chimera in AVA. We show that UNC-116, the C. elegans homolog of the vertebrate kinesin-1 (KIF5), is responsible for mediating the rapid, bidirectional transport of GLR-1. This motor-driven transport of GLR-1 modifies synaptic strength by mediating the rapid delivery, removal and redistribution of synaptic AMPARs. In the absence of unc-116, we found that although homomeric GLR-1 AMPARs can still diffuse to and accumulate at proximal synapses, glutamate-gated currents are decreased due to lack of heteromeric GLR-1/GLR-2- containing AMPARs. Furthermore, we show that transient expression of UNC- 116 can rescue defective glutamatergic signaling in adult unc-116 mutants, demonstrating that motor-dependent transport is ongoing in the adult nervous system and is involved in the regulation of synaptic strength. These data have allowed us to establish a link between motor-dependent transport of AMPARs and the strength of synaptic transmission. iv To my parents, Karl and Joni, and my wife, Tabitha for their constant support and encouragement. TABLE OF CONTENTS ABSTRACT..............................................................................................................iii LIST OF FIGURES .............................................................................................. viii ACKNOWLEDGMENTS........................................................................................xi CHAPTERS 1. INTRODUCTION............................................................................................... 1 Overview ...................................................................................................... 2 Communication Between Neurons............................................................ 3 Vertebrate Ionotropic Glutamate Receptors............................................. 5 Glutamate Receptor Topology, Stoichiometry and Kinetics.....................6 Glutamate Receptor Localization in the Brain......................................... 12 AMPAR Localization and Auxiliary Proteins........................................... 13 AMPAR Trafficking at the Synapse..........................................................15 Models of Long-Range AMPAR Transport............................................. 18 Caenorhabditis elegans............................................................................ 22 Glutamatergic Signaling in C. elegans.....................................................24 GLR-1 Stability, Localization and Function at the Synapse...................27 Regulation of the Long-Range Transport of GLR-1................................30 Concluding Remarks................................................................................. 32 References................................................................................................. 35 2. KINESIN-1 REGULTES SYNAPTIC STRENGTH BY MEDIATING DELIVERY, REMOVAL AND REDISTRIBUTION OF AMPARS.................47 Summary.................................................................................................... 48 Introduction................................................................................................ 48 Results........................................................................................................49 Discussion.................................................................................................. 60 Experimental Procedures..........................................................................62 References................................................................................................. 63 Supplemental Information.........................................................................64 Supplemental Experimental Procedures..................................................80 Supplemental References.........................................................................85 3. SUMMARY AND CONCLUSIONS................................................................ 87 Introduction................................................................................................ 88 Transport of GLR-1 In Vivo.......................................................................89 GLR-1 Is Transported by UNC-116/KIF5.................................................90 Transport Events Deliver GLR-1 to Synapses........................................91 GLR-1 Is Redistributed Between Synapses............................................ 93 GLR-1 Delivery and Removal Are Reduced in unc-116 Mutants.......... 94 Surface Expression of GLR-1 Is Increased in unc-116 Mutants........... 96 Glutamate-gated Currents Are Decreased in unc-116 Mutants.............97 Trafficking of GLR-2 Is Critically Dependent on UNC-116/KIF5........... 98 Ongoing Role of Motor Transport in the Adult Nervous System........... 99 Transport of AMPARs Is Evolutionarily Conserved............................. 101 Regulation of GLR-1 Transport.............................................................. 102 Trafficking of Auxiliary Proteins.............................................................. 105 Molecular Motors and Synaptic Plasticity............................................. 106 Concluding Remarks............................................................................... 107 References...............................................................................................108 APPENDIX: CORNICHONS CONTROL ER EXPORT OF AMPARS TO REGULATE SYNAPTIC EXCITABILITY..........................................................112 vii LIST OF FIGURES Figure Page 1.1 The chemical synapse.................................................................................4 1.2 Glutamate receptor stoichiometry and structure.......................................8 1.3 Desensitization of ionotropic glutamate receptors..................................11 1.4 Overview of AMPAR transport................................................................. 19 1.5 The C. elegans locomotory control circuit............................................... 26 1.6 The GLR-1 signaling complex.................................................................. 31 2.1 GLR-1::GFP is transported in both an anterograde and a retrograde direction along the AVA processes..........................................................49 2.2 GLR-1 is preferentially delivered to synaptic puncta in the AVA processes................................................................................................... 51 2.3 GLR-1 is redistributed between synapses.............................................. 52 2.4 Bidirectional transport of GLR-1 is dependent on UNC-116/KIF5........ 54 2.5 Delivery and removal of synaptic GLR-1 is mediated by UNC-116/KIF5...........................................................................................55 2.6 Surface expression of GLR-1 is increased in unc-116 mutants.............57 2.7 Glutamate-gated currents are reduced in unc-116 mutants..................58 2.8 GLR-2 is decreased in unc-116 mutants................................................ 59 2.9 Transient expression of UNC-116/KIF5 in adult unc-116 mutants rescues GLR-1 transport and synaptic transmission............................. 61 2.S1 GLR-1::GFP is expressed in a punctate pattern along the length of the AVA processes................................................................................... 65 2.52 GLR-1 insertion events occur near existing synapses........................... 67 2.53 GLR-1 transport is dependent on ATP and microtubules...................... 70 2.54 GLR-1 transport is normal in klp-4 mutants and only minor defects were observed in unc-104 mutants.......................................................... 71 2.55 Synaptic localization and transport of the vertebrate GluA1 AMPAR subunit depends on UNC-116/KIF5.........................................................73 2.56 The distribution of GLR-1 in AVA is consistent with diffusion-mediated transport in unc-116 mutants....................................................................74 2.57 Local protein synthesis does not contribute to fast delivery of synaptic AMPARs.................................................................................................... 76 2.58 klp-4 is epistatic to unc-116 in the regulation of GLR-1 transport......... 77 2.59 Glutamate-gated currents are reduced in unc-116 mutants..................78 3.1 A hypothetical UNC-116 motor complex..................................................92 3.2 A model of long-range transport of GLR-1 by UNC-116/KIF5.............100 3.3 A model of GLR-1 transport in unc-116 mutants..................................103 A.1 Reversal frequency and glutamate-gated currents are increased in cni-1 mutants...........................................................................................115 A.2 Synaptic levels of GLR-1::GFP are increased in cni-1 mutants and decreased by overexpression of cornichon proteins............................ 116 A.3 The frequency of GLR-1 anterograde transport is increased in cni-1 mutants.................................................................................................... 117 A.4 CNI-1 is widely expressed in the nervous system where it colocalizes with GLR-1 in the ER.............................................................................. 118 A.5 Surface expressed CNI-1 colocalizes with synaptic GLR-1.................119 A.6 Overexpression of CNI-1 results in GLR-1::GFP accumulation in neuronal cell bodies and its subsequent degradation.......................... 120 A.7 Overexpressing CNI-1 or CNIH-2 modifies glutamate-gated current and AMPAR surface expression............................................................ 121 ix A.8 Cornichon proteins decrease GluA1-mediated current and synaptic GluA1 levels when coexpressed in C. elegans AVA neurons..............122 A.9 CNI-1 modifies neuron excitability by regulating the export of AMPARs from the ER............................................................................. 123 A.S1 C. elegans cni-1 encodes a member of the family of cornichon proteins.................................................................................................... 128 A.S2 cni-1 is not required for either the nose touch or osmotic avoidance response.................................................................................................. 130 A.S3 Kainate- and NMDA-gated currents are increased in cni-1 mutants.... 131 A.S4 GLR-1 surface expression is increased in cni-1 mutants.....................133 A.S5 CNI-1 is coexpressed with GLR-1 in the nervous system....................135 A.S6 CNI-1 is expressed on the cell surface..................................................136 A.S7 CNI-1 reduces glutamate-gated current and GluA1 surface expression................................................................................................ 137 x ACKNOWLEDGMENTS A number of people have had a profound effect on my graduate school experience. First and foremost, I'd like to express my gratitude to my advisor, Dr. A. Villu Maricq. Thank you for taking a chance on this mathematician and for all your support and encouragement throughout the years. You have helped me to realize my scientific potential and pushed me to succeed both personally and professionally. There are many members of the scientific community that I would like to acknowledge. Thank you to the members of my thesis committee: Dr. Markus Babst, Dr. Erik Jorgensen, Dr. Julie Holien and Dr. Michael Vershinin, for all of your guidance and enthusiasm throughout my graduate career. Additionally, I would like to thank Dr. James Keener and Dr. Paul Bressloff in the Mathematical Biology Program for initially bringing me to Utah and for their support and advice during my master's work. Finally, I'd like to extend a special thank you to Tony Cooke and Dr. Robin Battye for teaching me all aspects of microscopy and getting me into the "family." Life in the lab would have not been the same without my awesome lab mates in the Maricq and Jorgenson laboratories. I am grateful for all your help and encouragement. I would like to thank Jerry Mellem, Penny Brockie, Fred Horendli, Dave Madsen, Jann Gardner, Mike Jensen, Craig Walker, Rui Wang, Colin Thacker, Angy Kallarackal, Randi Rawson, Rob Hobson and Christian Fr0kj®r-Jensen. To my "work wife" and good friend, Amber Smith, thank you for your friendship and valuable advice throughout the years. No matter what the situation, everything from last minute text edits to sanity beverages, you have always been just a phone call away. Finally, I would like to thank my friends and family for their never-ending encouragement throughout the years, especially my wife, Tabitha. Tabitha, you are my source of inspiration and a driving force in my life. This journey would not have been possible without you and your support. xii CHAPTER 1 INTRODUCTION Overview Learning and memory are essential for all aspects of life. The ability to process information and learn in response to experience is due to continual changes in the efficacy of neuronal communication. It is thought that experiences can modify neuronal communication by strengthening some neuronal pathways within a circuit and weakening others (Hebb, 1949). Identifying how these connections are modified, where in the brain the modifications occur, and how this leads to changes in behavior, learning and memory is a major goal of neuroscience. The adult human brain contains over 100 billion neurons, which are interconnected with one another via trillions of specialized points of contact. These interconnected webs of neurons form networks of neural circuits that regulate the behavioral abilities and thought processes of an animal. The complex nervous system of humans and other vertebrates has hindered progress in understanding how the nervous system facilitates changes in the strength of neuronal communication at the circuit, cellular and molecular levels. To circumvent this, many studies have taken advantage of the simple nervous systems of model organisms such as the soil nematode Caenorhabditis elegans. Manipulation of this simple nervous system can be used to uncover the pathways regulating changes in the strength of synaptic communication. We sought to uncover the gene products and pathways that regulate synaptic strength in C. elegans by using genetic, molecular and electrophysiological techniques. We first characterize the pathway required for 2 trafficking of molecules necessary for neuronal communication, then identify the gene products that regulate trafficking, and lastly determine the mechanism by which trafficking regulates synaptic strength. This work offers new insights into the mechanisms underlying experience-dependent changes in neuronal communication and the promise of novel strategies for the treatment of mental health and neurological disorders. Communication Between Neurons Neurons send and receive information through specialized points of contact called synapses (Sudhof, 2004). At the synapse, a presynaptic neuron and a postsynaptic neuron are separated by a small gap called the synaptic cleft. Electrical activity in the presynaptic nerve terminal results in the rapid fusion of synaptic vesicles filled with small signaling molecules (neurotransmitters) with the plasma membrane. Consequently, the neurotransmitters contained in the synaptic vesicle are released into the synaptic cleft, diffuse across, bind to and activate the corresponding neurotransmitter receptors on the postsynaptic membrane (Figure 1.1). Binding of a neurotransmitter to its respective receptor initiates a signaling cascade that is specific to the type of neurotransmitter released (Sudhof, 2004). There are two distinct classes of neurotransmitter receptors in the membrane of postsynaptic cells - metabotropic and ionotropic (Kew and Kemp, 2005). Metabotropic receptors are G-protein-coupled receptors that signal on a slow timescale through diverse second messenger pathways in cells. 3 4 Action Potiential Figure 1.1. The chemical synapse. Depolarization of the presynaptic neuron by an action potential (lightning bolt) triggers synaptic vesicles filled with neurotransmitters to fuse to the presynaptic membrane, releasing neurotransmitters into the synaptic cleft. Neurotransmitters diffuse across the synaptic cleft and bind to the corresponding neurotransmitter receptors on the plasma membrane of the postsynaptic cell. Binding of neurotransmitter to its respective ionotropic neurotransmitter receptor gates open the ion pore, allowing charged ions (cations or anions) to pass through the postsynaptic membrane. Conversely, ionotropic receptors are composed of several protein subunits that combine together to form either homomeric or heteromeric channels that contain an ion pore. Upon binding of the ligand, the pore of the ionotropic receptor gates open, allowing ions to traverse the cell membrane. This influx of ions causes a rapid change in the membrane potential of the cell. Ion channels that are permeable to cations (Na+, K+, Ca2+) depolarize the cell membrane causing neuronal excitation, whereas channels permeable to anions (Cl-) hyperpolarize the cell and are inhibitory. Glutamate is an excitatory neurotransmitter that controls a broad range of neuronal functions by activating a diverse set of ionotropic and metabotropic glutamate receptors (Kew and Kemp, 2005; Nakanishi, 1994). Vertebrate Ionotropic Glutamate Receptors The neurotransmitter glutamate mediates the vast majority of fast excitatory neurotransmission in the brain (Brockie and Maricq, 2010). The importance of glutamatergic neurotransmission is illustrated by the wide range of neurological processes that glutamate influences and the variety of disorders that arise when it is disrupted. For example, glutamate receptors have been shown to have critical roles in development, learning and memory (reviewed in Chen and Tonegawa, 1997). In addition, the disruption of glutamatergic signaling has been implicated in a variety of neurological disorders including schizophrenia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and excitotoxicity associated with epileptic seizures and ischemic brain damage 5 (Meldrum, 1994; Mody, 1998; Montastruc et al., 1997; Nakajima et al., 2012; Ulas et al., 1994). Eighteen ionotropic glutamate receptor (iGluR) subunits, which co-assemble to form functional receptors with highly varying properties, have been identified in the rat. Comparing the sequences of various iGluR subunits shows up to 80% similarity and the conservation of intron and exon structures (Suchanek et al., 1995; Wenthold et al., 1992). These different iGluR subunits can be separated into two different classes based on reactivity to the pharmacological agonist W-methyl-D-aspartate (NMDA): the NMDA and non- NMDA classes (reviewed in Dingledine et al., 1999). The 11 subunits of the non- NMDA class can be further subdivided based on their sensitivity to a-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and those sensitive to kainate (KA) (reviewed in Dingledine et al., 1999). There is also an additional class of iGluRs, the delta class, which fail to respond to any known iGluR agonist yet show molecular identity to other iGluR subunits (Mayer, 2006; Watkins and Jane, 2006; Yamazaki et al., 1992; Zuo et al., 1997). Glutamate Receptor Topology, Stoichiometry and Kinetics Ionotropic glutamate receptors are integral membrane proteins composed of four large subunits, each around 900 residues, which form an ion channel. Detailed crystallographic descriptions of iGluRs along with functional and biochemical data show that iGluRs form as tetrameric channels (Armstrong and Gouaux, 2000; Armstrong et al., 1998; Dingledine et al., 1999; Laube et al., 6 1998; Rosenmund et al., 1998; Sobolevsky et al., 2009). A functional iGluR is formed when subunits of the same class co-assemble to create a ligand-gated ion channel, e.g., AMPA subunits assemble with other members of the AMPA subfamily (Ayalon and Stern-Bach, 2001; Laube et al., 1998; Mano and Teichberg, 1998; Rosenmund et al., 1998). These oligomeric combinations are formed in the endoplasmic reticulum (ER), possibly assembling as a dimer-of-dimers (Tichelaar et al., 2004). In addition to heteromeric receptors, some subunits are also capable of forming homomeric receptors. This diversity is further increased through posttranscriptional splice variants and RNA editing of iGluRs (Seeburg et al., 1998). These mechanisms result in a complex and diverse family of iGluRs that differ in kinetics, ion permeability and pharmacological specificity (detailed below); which are capable of regulating a wide range of neurological functions. Due to the high sequence homology between the different iGluR subunits, all iGluRs are thought to have the same structure and topology (Figure 1.2A). Individual receptor subunits have discrete domains; an extracellular N-terminal domain; two extracellular domains, S1 and S2, that form the ligand binding site; four hydrophobic transmembrane domains (three true transmembrane domains and one re-entrant loop); and an intracellular C-terminal domain. Each of these domains plays a different role in receptor function. The N-terminal domain regulates the association between subunits (Ayalon and Stern-Bach, 2001; Ayalon et al., 2005), the S1 and S2 domains regulate desensitization 7 8 A NH Extracellular Intracellular B Tetrameric Channel COOH Figure 1.2. Glutamate receptor stoichiometry and structure. A) Ionotropic glutamate receptor subunits contain three transmembrane domains (blue) and a re-entrant loop (red). The binding site for glutamate is formed by the S1 domain in the extra-cellular N-terminus and S2 domain in the extracellular loop between M3 and M4. B) The iGluR channel is formed by a tetrameric assembly of subunits with the re-entrant loop lining the pore of the receptor (top view). (Stern-Bach et al., 1994), and the C-terminal domain mediates receptor localization (Daw et al., 2000; Osten et al., 2000). Once iGluRs have been assembled and localized, receptors can transduce the appropriate signal in response to glutamate binding. In the absence of the presynaptic release of glutamate, the iGluR channel remains in the closed state. Opening, and thus activation of the channel, can be achieved by glutamate binding to each receptor subunit in the S1 and S2 ligand-binding domain. To facilitate ligand binding, the S1 and S2 domains adopt a clamshell­like formation with each domain forming half of the clamshell and the agonist-binding pocket located between them (reviewed in Dingledine et al., 1999). When glutamate binds in this pocket, the resulting conformational change moves the S1 and S2 domains closer together. How this conformational change leads to the pore opening is still not fully understood. The ion pore of the iGluR is formed by the amino acids of the M2 re­entrant loops of each of the four subunits (Figure 1.2B). The amino acids of the inner cavity of the pore determine the ion selectivity. All iGluRs are permeable to Na+ and K+, but Ca2+ permeability is highly regulated due to its role in intracellular signaling cascades. The Ca2+ permeability of iGluRs is largely dependent on the subunit composition of the channel and on a specific amino acid at the mouth of the pore, called the Q/R site. Both AMPA and kainate receptor subunits encode a glutamine (Q) at this site, which allows these subunits to have a high permeability to Ca2+. However, posttranscriptional RNA editing can modify this site to an arginine (R) residue, resulting in decreased Ca2+ permeability (Bass, 9 2002; Dingledine et al., 1999; Sommer et al., 1991). On the other hand, NMDA receptors (NMDAR) are Ca2+ permeable when activated. However, the activation of NMDARs requires both presynaptic neurotransmitter release and post-synaptic depolarization (Ho et al., 2011). Termination of synaptic signaling through iGluR channels is essential for proper synaptic signaling and can occur in one of two ways. The first is through agonist dissociation from the receptor. This allows the conformational change induced by agonist binding to be relaxed, thereby restoring the iGluR to the closed inactive state. Alternatively, the receptor can adopt an additional conformational state where the iGluR remains bound to glutamate yet the channel closes (Figure 1.3) (Jones and Westbrook, 1996). This is called a desensitized state and occurs during the continued presence of glutamate. Receptors can then recover from the desensitized state by releasing the agonist and again become competent for activation. Desensitization of receptors is an important process for maintaining the proper function and signaling of iGluRs. This step is of critical importance for the cessation of receptor signaling when glutamate clearance from the synaptic cleft is not achieved due to a high frequency of release events, multiple presynaptic inputs at a synapse, or spillover from nearby synapses (Jones and Westbrook, 1996; Otis et al., 1996; Stern- Bach et al., 1998; Trussell and Fischbach, 1989). Differential rates of desensitization and of receptor recovery from desensitization also play a role in the extent and duration of the postsynaptic response (Dingledine et al., 1999). 10 11 O- Ligand f f l Closed Figure 1.3. Desensitization of ionotropic glutamate receptors. The binding of ligand (blue) to a closed receptor causes a conformational change, opening the receptor (horizontal arrows) and allowing ions to flow through (vertical arrow). The receptor rapidly closes (horizontal arrows) in the presence of ligand, entering a desensitized state. Removal of the ligand returns the receptor to the closed state, allowing for activation upon ligand binding. Open Desensitized Glutamate Receptor Localization in the Brain Within the vertebrate central nervous system, iGluRs are expressed in almost all neurons and in some glial cells. Glutamate receptor expression is not static; rather, it varies in a cell-specific manner throughout development and in response to environmental factors. The level of expression of each iGluR subunit is determined at any particular time by a balance in the rates of gene transcription, mRNA translation, mRNA degradation, and protein degradation. Additional processes, such as receptor assembly in the ER and synaptic targeting, allow for further control over the levels of functional receptors in the cell. This is further complicated by the expression of several classes of iGluR subunits in most cells. Thus, the postsynaptic response is defined by the various iGluRs expressed within a particular cell and how they are arranged at individual synapses. In the brain, iGluRs are required for the expression of synaptic plasticity, the cellular model of learning and memory (reviewed in Peng et al., 2011). There are two major types of synaptic plasticity, known as long-term potentiation (LTP) and long-term depression (LTD) (reviewed in Song and Huganir, 2002). LTP results in the strengthening of glutamatergic signaling between neurons, whereas LTD results in the weakening of this signal. Perturbations in glutamatergic signaling prevent the induction of LTP and LTD, leading to defects in learning and memory (Henley and Wilkinson, 2013; Huganir and Nicoll, 2013). Over the last 25 years, a myriad of studies have focused on the cellular trafficking of the AMPA-type glutamate receptor (AMPAR) due to its central role in synaptic 12 13 plasticity. These studies have proved useful in elucidating many of the cell biological processes involved in synaptic function. AMPAR Localization and Auxiliary Proteins Proper AMPAR localization to a region of the synapse called the post­synaptic density (PSD) is crucial for efficient synaptic transmission. One of the major molecular mechanisms that controls the localization of AMPARs within the PSD is the interaction of the C-termini of AMPARs with PSD-95/DLG/ZO-1 (PDZ) containing intracellular scaffolding molecules. Several glutamate receptor-associated proteins containing PDZ domains have been identified, including PSD-95 family members GRIP1 and GRIP2, also known as ABP (AMPA receptor-binding protein) (Scannevin and Huganir, 2000; Tomita et al., 2001). These proteins have been shown to regulate the stability of AMPARs in the membrane through interactions of their respective PDZ domains (Daw et al., 2000; Osten et al., 2000). In the PSD, AMPARs are not uniformly distributed but are confined to subsynaptic domains and positioned near presynaptic release sites (Ehlers et al., 2007; Kerr and Blanpied, 2012; MacGillavry et al., 2013). Once properly localized to the synapse, AMPARs must then be expressed on the cell surface and able to open in response to glutamate. Recently, it was discovered that AMPARs associate with auxiliary proteins, which regulate their surface expression and function at the synapse. The first AMPAR auxiliary protein was identified from a spontaneous mouse mutation in a gene encoding a multiple transmembrane protein called stargazin (Letts et al., 1998). Recordings from cerebellar granule cells expressing the stargazin mutation revealed a significant loss of AMPA-mediated currents while showing normal NMDA-mediated currents (Chen et al., 2000), demonstrating that stargazin is critically required for surface expression of AMPARs. Stargazin and its closely related paralogs belong to a family of proteins that interact with AMPAR subunits termed transmembrane AMPA-receptor regulatory proteins (TARPs), which direct the proper expression and localization of AMPARs (Chen et al., 2000; Letts et al., 1998; Schnell et al., 2002; Tomita et al., 2003). In addition to regulating surface expression and localization, TARPs have also been shown to modify AMPAR function. AMPARs associated with TARPs show increased single-channel conductance, open probability and activation rates while having a reduced rate of desensitization and a slower deactivation time course (Priel et al., 2005; Tomita et al., 2005; Yamazaki et al., 2004). More recently, an additional class of proteins, called cornichons, have been implicated as AMPAR auxiliary proteins (Schwenk et al., 2009). Cornichon was originally identified in Drosophila as a protein required for the ER export of the EGF-like ligand Gurken (Bokel et al., 2006; Roth et al., 1995). However, affinity purification of AMPARs from rat brain followed by mass spectroscopy analysis identified the cornichon proteins CNIH-2 and CNIH-3 as AMPAR-interacting proteins. When co-expressed with AMPARs in heterologous systems, CNIH-2 and CNIH-3 modified AMPAR desensitization, deactivation and surface expression (Coombs et al., 2012; Kato et al., 2010; Schwenk et al., 2009; Shi et al., 2010). Recently, two studies have shown that cornichons function to regulate 14 the trafficking of AMPARs in neurons (Brockie et al., 2013; Herring et al., 2013); yet, it is still unclear if cornichon functions as an auxiliary protein at synapses in vivo. AMPAR Trafficking at the Synapse Changes in the number of AMPARs at the synapse tunes synaptic efficacy. Such modifications are dependent on the availability of receptor binding sites and on the equilibrium of receptor efflux and influx (Newpher and Ehlers, 2008; Shepherd and Huganir, 2007). Synaptic AMPARs are not stable, but rather are constantly diffusing in and out of the PSD and recycling between intracellular compartments and the postsynaptic membrane. This dynamic process controls the number of surface-expressed AMPARs and is of central importance for synaptic plasticity (Contractor and Heinemann, 2004; Malinow and Malenka, 2002; McGee and Bredt, 2003). The number of AMPARs at the synapse is estimated to be 50-100 by anatomical methods (Tanaka et al., 2005) and between 60-190 using physiological methods (Matsuzaki et al., 2001; Smith et al., 2003). Single molecule tracking and fluorescent recovery after photobleaching (FRAP) experiments revealed both mobile and immobile synaptic receptors numbering from 50-200 (Ashby et al., 2006; Tardin et al., 2003). The cell controls the number of surface expressed receptors through a dynamic balance of exocytosis, endocytosis and trapping of AMPARs in the PSD. AMPARs are first delivered to the surface via SNARE-dependent exocytosis (Lu et al., 2001). This process has been shown to be regulated in response to 15 synaptic activity (Yudowski et al., 2007). However, where these AMPAR exocytosis events take place is still a matter of debate. Some studies propose that AMPAR exocytosis occurs directly at the synapse (Gerges et al., 2006; Patterson et al., 2010), while others suggest exocytosis takes place in the dendrite near the synapse (Jaskolski et al., 2009; Makino and Malinow, 2009; Yudowski et al., 2007). Once AMPARs are on the plasma membrane, they can move in and out of the synapse and between neighboring synapses via lateral diffusion (Borgdorff and Choquet, 2002; Groc et al., 2004; Opazo et al., 2010; Tardin et al., 2003). At the synapse, AMPARs exist in two populations: mobile and immobile. AMPARs inside the PSD show less mobility than AMPARs in the extrasynaptic space (Tardin et al., 2003). In addition, the mobility of AMPARs in the membrane is altered in response to synaptic activity. Active synapses capture diffusing AMPARs and inactive synapses release AMPARs to diffuse away (Ehlers et al., 2007; Lu et al., 2010; Makino and Malinow, 2009; Patterson et al., 2010; Tardin et al., 2003). This diffusional trapping is dependent on the interaction of the cytoskeletal protein PSD-95 with stargazin-bound AMPARs as they diffuse on the plasma membrane (Bats et al., 2007). Surface-expressed AMPARs can be removed from the plasma membrane by endocytosis. Endocytic zones can be found in the lateral margins of excitatory synapses next to the PSD (Blanpied et al., 2002). Displacing endocytic zones disrupts AMPAR removal from the synapse and reduces the mobile pool of receptors at the surface (Petrini et al., 2009). After internalization, 16 AMPARs can be sorted into one of two pathways. Endocytosed AMPARs can be trafficked to early endosomes or to specialized recycling endosomes that allow for rapid reintroduction to the surface (Hanley, 2010). Alternatively, AMPARs can be sorted to late endosomes, which ultimately sends receptors to the lysosomes for degradation (Lee et al., 2004; Lu et al., 2007). The trafficking of AMPARs at the synapse is also critically dependent on their subunit composition (Lu et al., 2009). In the hippocampus, it has been shown that the short-tailed subunits of the heteromeric GluA2/GluA3 AMPARs continuously cycle in and out of the synapse in an activity-independent manner (Passafaro et al., 2001; Shi et al., 2001). This process, termed constitutive recycling, is hypothesized to preserve the total number of AMPARs at the synapse and to maintain synaptic strength in the face of protein turnover (Zhu et al., 2000). Thus, in the absence of neuronal activity, AMPARs can go into synapses without changing the magnitude of synaptic transmission, suggesting a one-to-one exchange of AMPARs between extrasynaptic and synaptic sites (Kakegawa et al., 2004; Shi et al., 2001). Conversely, AMPARs with long-tailed subunits, such as GluA1, GluA2L (the long splice isoform of GluA2) or GluA4, are added to synapses in an activity-dependent manner during LTP (Hayashi et al., 2000; Kolleker et al., 2003; Makino and Malinow, 2009; Zhu et al., 2000). Formally, synaptic strengthening involves activity-dependent addition of long­tailed receptors, whereas synaptic weakening occurs through endocytosis of long-or short-tailed receptors. 17 18 Models of Long-Range AMPAR Trafficking Neurons pose a unique problem for the long-range trafficking of proteins due to their elaborate, highly polarized structure. Membrane proteins must travel extremely long distances and may be inserted at the plasma membrane far from their final destination. Although much is known about the local dynamics of AMPARs at the level of the synapse (discussed above), it is still an open question as to how AMPARs are trafficked to the synapse in the first place (Figure 1.4). It is generally accepted that AMPARs are synthesized in the cell body or soma of a neuron. Then, AMPARs must be trafficked to the synapse where they function, although this trafficking pathway is still unresolved. Given the central importance of AMPARs in synaptic plasticity, it is imperative to understand the molecular mechanisms governing the long-range transport of AMPARs in neurons. However, relatively few studies have addressed this fundamental question. One model of how AMPARs are trafficked from the soma to the synapse is that AMPARs are inserted into the plasma membrane at the cell body and are then trafficked to synapses via lateral diffusion. This model is supported by work that used a membrane-impermeable, irreversible, photoreactive AMPAR agonist 6-azido-7-nitro-1,4-dihydroquinoxaline-2,3-dione (ANQX) (Chambers et al., 2004) to inactivate surface AMPARs. By monitoring the recovery of AMPA-mediated currents with electrophysiology after photoinactivation of surface AMPARs, Adesnik et al. showed that it took approximately 16 hours for AMPA-mediated currents to recover (Adesnik et al., 2005). These data suggest that most 19 Figure 1.4. Overview of AMPAR transport. A cartoon schematic showing the trafficking pathways of AMPARs. At synapses, AMPARs can move via lateral diffusion in the plasma membrane between neighboring synapses. A dynamic balance of endocytosis and exocytosis regulates the level of surface-expressed receptors at synapses. The mechanism of long-range AMPAR transport to the synapse is unresolved. long-range transport of AMPARs occurs by lateral diffusion in the membrane. Although the rapid recycling of AMPARs with internal stores does occur, it is not the major source of functional synaptic AMPARs (Adesnik et al., 2005). However, mathematical modeling studies of long-range AMPAR transport concluded that lateral diffusion alone would be insufficient for the delivery of AMPARs from the soma to distal dendrites (Earnshaw and Bressloff, 2008), suggesting there must be an additional active component to long-range AMPAR trafficking. One such active process that would allow for the rapid delivery of proteins over long distances is molecular-motor-mediated transport. The molecular motors kinesin and dynein are part of a large family of proteins that move along microtubules (MTs) (Goldstein and Yang, 2000). Microtubules have defined polarity with a plus end and a minus end. In general, the motor protein family dynein moves towards the minus end of MTs, whereas the kinesin family moves to the plus end (Goldstein and Yang, 2000). Because dendrites contain MTs of mixed polarities (Baas, 1999), both dynein and kinesin could mediate transport of cargo from the soma into dendrites. In fact, disruption of both kinesin and dynein function with monoclonal antibodies has been shown to reduce AMPAR-mediated currents in vitro (Kim and Lisman, 2001), suggesting a role for motor-mediated transport in the delivery of AMPARs to synapses. Furthermore, yeast-two- hybrid experiments have shown that the AMPAR-interacting protein, GRIP1, can bind to the kinesin-1 KIF5 (Setou et al., 2002). The interaction between GRIP1 and KIF5 steers the motor complex into dendrites and is predicted to be 20 involved in the transport of AMPARs (Setou et al., 2002). However, direct measurements of AMPAR localization and trafficking after the disruption of motor-driven transport are not yet available. Another model for the long-range transport of AMPARs to the synapse is that AMPARs are locally synthesized in the dendrite. Having AMPARs synthesized in proximity to the synapse provides a local pool of receptors, which can rapidly transport to nearby synapses via lateral diffusion. Protein synthesis was historically thought to occur exclusively in neuronal cell bodies. However, since the identification of poly-ribosomes in hippocampal dendrites (Steward and Levy, 1982), local translation of proteins in dendrites has become widely accepted. Studies of hippocampal slices in which the dendrites have been severed from the cell bodies were found to retain the ability to express LTP and LTD, indicating that local translation can mediate long-term modifications in synaptic strength (Huber et al., 2000; Kang and Schuman, 1996). In agreement with this, direct evaluation of AMPAR mRNA distribution (Grooms et al., 2006) and AMPAR local synthesis (Ju et al., 2004) have shown dendritic synthesis of AMPARs to be an effective regulator of the local abundance and composition of receptors. To date, the question of how AMPARs are transported over long distances to synapses is still without a definitive answer. Multiple studies using various techniques have lead to differing results on the relative roles of lateral diffusion, motor-mediated transport and local synthesis in long-range AMPAR transport. These competing models derive almost exclusively from in vitro studies in 21 cultured neurons and may not accurately reflect AMPAR transport in vivo. Therefore, in order to elucidate how AMPARs are transported to the synapse in vivo, we investigated the long-range AMPAR transport in the model organism Caenorhabditis elegans. Caenorhabditis elegans C. elegans is a free-living soil-dwelling nematode that has been used as a model organism for studies of the cellular and molecular mechanisms that regulate synaptic function for nearly 40 years (Brenner, 1974). As a relatively simple organism, the worm is comprised of approximately 1000 cells and its genome has been fully sequenced. C. elegans exists as a self-fertilizing hermaphrodite with a life cycle of approximately 4 days at 20°C and generates a typical brood size of 300 progeny. The ability of C. elegans to self propagate is a tremendous advantage for genetic studies as recessive homozygous mutations can be readily isolated and maintained. Hermaphroditic worms also have the ability to mate with male worms, aiding in genetic manipulation and also providing a simple way to achieve specific genetic backgrounds. The C. elegans nervous system has many attributes that make it a perfect choice for neurobiological studies. Relative to the nervous system of rat, mouse and Drosophila, the C. elegans nervous system is extremely small, consisting of only 302 neurons. In addition, the neuronal circuitry and synaptic connectivity of the hermaphroditic nervous system has been fully reconstructed from serial section electron microscopy (White et al., 1986). The C. elegans nervous system 22 consists of several identifiable head and tail ganglia that send out neuronal processes, which run longitudinally along the ventral nerve cord (VNC). Since the neuronal lineage is invariant among animals, this allows for reproducible identification of individual neurons. Importantly, C. elegans can survive elimination of genes that are highly detrimental to the function of the nervous system. Since the initial use of C. elegans as a model system by Sydney Brenner (1974), there have been tremendous advances in the techniques and methodology used to manipulate the worm for biological studies. Transgenic animals can be generated through injecting DNA directly into the gonad of the worm (Berkowitz et al., 2008; Evans, 2006). As the cuticle of the worm is transparent, fluorescently labeled proteins can easily be imaged in live animals (Boulin, 2006). Mutant alleles can be easily generated by screening through animals that have been exposed to chemical mutagens or x-rays (Jorgensen and Mango, 2002). RNA interference (RNAi) is an extremely powerful genetic tool used to knock down expression of specific gene products. In the worm, this can be achieved by feeding worms bacteria that expresses a vector containing double-stranded RNA (dsRNA) specific to a particular gene. Upon entering the cell, dsRNA is cleaved into small interfering RNAs (siRNAs) which then bind to the their mRNA targets, causing them to be degraded (Kamath et al., 2001; Mello and Conte, 2004). In addition, dsRNA can also be used for tissue specific knockdown of specific gene products. This is achieved by generating transgenic 23 animals that express dsRNA under a tissue specific promoter (Esposito et al., 2007). To effectively study the function of particular genes in the nervous system, methods that access and monitor the electrical responses of individual neurons are essential. The nervous system of C. elegans spans the length of the worm. The nerve ring, generally regarded as the brain of the worm, is a large bundle of many axons that form numerous synapses in the head of the animal. Electrophysiological procedures have been developed to achieve neuronal access in live immobilized worms where electrical activity can be monitored (Mellem et al., 2002). This is done by dissecting the cuticle along the anterior portion of the worm, allowing for electrical access to the head neurons. In this way, individual neurons can be tested for their responses to agonists as well as the effect of mutations on neuronal function (Brockie et al., 2001a; Mellem et al., 2002). Glutamatergic Signaling in C. elegans Similar to the vertebrate nervous system, C. elegans also uses glutamatergic signaling as a major component of synaptic communication. At least 10 putative iGluR subunits are expressed in C. elegans (Brockie et al., 2001b). As with vertebrate iGluRs, C. elegans iGluRs can be separated via pharmacology and sequence similarity into NMDA and non-NMDA classes of receptors. Members of the non-NMDA class (GLR-1 - GLR-8) include subunits most similar to the AMPA and kainate class of receptors, whereas NMR-1 and 24 NMR-2 make up the NMDA class of receptors (Brockie et al., 2001 b). The large number of iGluRs expressed in C. elegans suggests that the worm has the potential to express a wide variety of functional iGluRs. The expression of fusion proteins between iGluRs and the green fluorescent reporter GFP in transgenic worms has revealed a detailed map of the expression pattern for each subunit (Brockie et al., 2001b). All iGluR subunits in C. elegans are restricted to expression within the nervous system, with varying degrees of overlapping expression patterns. The kainate receptor subunits GLR- 3 and GLR-6 have the most limited expression pattern, appearing in only a single pair of interneurons called RIA. The non-NMDA subunits GLR-1, GLR-2, GLR-4 and GLR-5 and the NMDA subunits NMR-1 and NMR-2 are expressed in many of the command interneurons - AVA, AVB, AVD, AVE and PVC - a circuit that has been shown to control worm forward and backward movement (Figure 1.5) (Brockie et al., 2001a; Chalfie et al., 1985). The remaining two subunits, GLR-7 and GLR-8, are expressed in the neurons of the pharyngeal nervous system (Brockie et al., 2001b). This suggests that iGluR subunits with overlapping expression patterns might function as heteromeric receptors in these neurons. The first iGluR subunit to be characterized in C. elegans was the AMPAR subunit GLR-1. Mutations in glr-1 disrupt the backward movement in response to nose touch stimulation (Hart et al., 1995; Maricq et al., 1995), resulting from defective glutamatergic signaling between sensory neurons and the command interneurons expressing GLR-1 (Mellem et al., 2002). In addition, the GLR-2 subunit is expressed in many of the same neurons that express GLR-1, including 25 26 BACKWARD MOVEMENT FORWARD MOVEMENT Sensory Neurons Command Interneurons Motor Neurons Behavioral Output Figure 1.5. The C. elegans locomotory control circuit. The locomotory control circuit includes the sensory neurons ALM, AVM, ASH and PLM. These neurons receive sensory input from the external environment and relay this information to the GLR-1 expressing command interneurons AVA, AVB, AVD, AVE and PVC. The command interneurons process this information and activate VB or VA motor neurons to generate forward or backward movement, respectively. Chemical synapses are indicated by arrowheads and gap junctions by boxes. the command interneurons. Mutations in glr-1 and glr-2 have the same behavioral defect, but are less severe in glr-2 mutants (Mellem et al., 2002). Interestingly, glutamate-gated currents in the command interneuron AVA are completely absent in glr-1 mutants. Yet, a small rapid current is still present in glr-2 mutants, indicating that GLR-1 might form a homomeric receptor or a heteromeric receptor with other iGluR subunits (Mellem et al., 2002). Taken together, these data suggest that the vast majority of glutamatergic signaling in AVA is mediated by GLR-1/GLR-2 heteromeric AMPARs to facilitate backing in response to nose touch stimulation. In addition, this allows for the correlation between a known glutamate-dependent behavior and electrophysiology. GLR-1 Stability, Localization and Function at the Synapse Which molecules are required for the proper trafficking of GLR-1 from the cell body to synaptic sites? To address this question, GLR-1::GFP fusion proteins have been used to observe the subcellular localization of GLR-1 (Brockie et al., 2001b; Burbea et al., 2002; Mellem et al., 2002; Rongo et al., 1998). These functional GLR-1::GFP proteins have proven to be invaluable for studies of GLR-1 trafficking, stability and localization to the synapse. In the neuronal process of transgenic animals, GLR-1::GFP is distributed in a punctate pattern. These GLR-1::GFP puncta have been shown to align with presynaptic markers and are likely to represent postsynaptic sites (Burbea et al., 2002; Rongo et al., 1998). 27 Since the initial identification of GLR-1 (Hart et al., 1995; Maricq et al., 1995), many gene products that regulate its localization have been identified. The first such discovery was the gene encoding lin-10. LIN-10 is a PDZ-domain protein involved in GLR-1 localization (Rongo et al., 1998). Mutations in lin-10 change the distribution of GLR-1::GFP in the VNC, going from a punctate distribution in wild-type animals to a more uniform distribution in lin-10 mutants (Rongo et al., 1998). Similar to glr-1, lin-10 mutants are nose touch defective, further supporting the hypothesis that LIN-10 is involved in regulating GLR-1 localization and function (Rongo et al., 1998). During C. elegans development, the density of GLR-1::GFP puncta remains constant despite a 10-fold increase in the size of the worm. The gene unc-43, which encodes the calmodulin-dependent protein kinase II (CaMKII), has been shown to be an important regulator of this process (Rongo and Kaplan, 1999). In unc-43 loss-of-function mutants, the density of GLR-1::GFP puncta along the VNC is decreased and there is an observed increase of GLR-1::GFP in the cell body (Rongo and Kaplan, 1999). These findings suggest that UNC-43 functions during development to regulate trafficking of GLR-1 out of the cell body for the formation of new synapses (Rongo and Kaplan, 1999). Once the appropriate synaptic density has been established, UNC-43 might have an additional role at the synapse to maintain GLR-1 density (Rongo and Kaplan, 1999). After GLR-1 has been localized to the synapse, endocytosis and membrane recycling play an important role in regulating the levels of GLR-1 at 28 the plasma membrane. An important regulator of this process is the C. elegans gene unc-11, which encodes an orthologue of the AP180 clathrin adaptor protein. Mutations in unc-11 disrupt clathrin-mediated endocytosis (Nonet et al., 1999) and result in accumulations of GLR-1::GFP in the VNC (Burbea et al., 2002). GLR-1::GFP is also accumulated at synapses when ubiquitination of GLR-1 is blocked, suggesting that ubiquitinated GLR-1 receptors are endocytosed in an UNC-11-dependent manner (Burbea et al., 2002). In addition, GLR-1 can be recycled from the membrane via a clathrin-independent endocytosis pathway. In this pathway, the small GTPase RAB-10 and the PDZ-domain protein LIN-10 mediate the recycling of GLR-1 (Glodowski et al., 2007). GLR-1 trafficked through this pathway is thought to be mediated via the ubiquitin-conjugating enzyme uev-1 (Kramer et al., 2010). In uev-1 mutants, GLR-1 accumulates in RAB-10-containing endosomes and is predicted to have less surface-expressed GLR-1 based on behavioral data (Kramer et al., 2010). Once at the synapse, GLR-1 must be competent to gate open in response to ligand binding. For years, iGluRs were thought to be stand-alone molecules; however, work in vertebrate systems and C. elegans has shown that this is not the case. The first AMPAR auxiliary protein identified in C. elegans was the CUB-domain transmembrane protein SOL-1 (Zheng et al., 2004). SOL-1 is required for GLR-1-dependent glutamate-gated currents and for behaviors that are dependent on GLR-1-mediated synaptic transmission (Zheng et al., 2004). Formally, SOL-1 functions to regulate GLR-1-mediated currents by modulating receptor desensitization (Walker et al., 2006a; Zheng et al., 2006). However, 29 GLR-1 function is not solely regulated by SOL-1, but also by the stargazin-like TARP proteins STG-1 and STG-2 (Walker et al., 2006b; Wang et al., 2008). That is, to form a fully functional GLR-1 complex that conducts physiologically relevant currents in vivo, the complex must contain the minimum set of proteins SOL-1, GLR-1, GLR-2 and either STG-1 or STG-2 (Figure 1.6) (Mellem et al., 2002; Walker et al., 2006a, 2006b; Wang et al., 2008; Zheng et al., 2004, 2006). Additional regulators of GLR-1-mediated currents in vivo have also recently been identified. SOL-2 is a CUB-domain protein related to SOL-1 that associates with both GLR-1 and SOL-1, and modifies GLR-1 desensitization and pharmacology (Wang et al., 2012). Regulation of the Long-Range Transport of GLR-1 The underlying molecular mechanisms regulating the long-range transport of the AMPAR subunit GLR-1 have yet to be discovered. However, many gene products implicated in regulating the transport of GLR-1 from the neuronal cell body to the synapse have been identified. One such proposed regulator of GLR- 1 transport is the cyclin-dependent kinase CDK-5. Mutations in cdk-5 decrease the amount of GLR-1::GFP in neuronal processes, whereas over-expression of CDK-5 results in an accumulation of GLR-1::GFP (Juo et al., 2007). This suggests that CDK-5 functions as a positive regulator of anterograde GLR-1 transport. Additional studies looking for suppressors of GLR-1 accumulation in CDK-5-overexpressing animals led to the identification of the kinesin-like protein KLP-4 (Monteiro et al., 2012). Mutations in klp-4 decrease the amount of GLR-1 30 31 Figure 1.6. The GLR-1 signaling complex. GLR-1-mediated glutamate-gated currents in vivo require multiple auxiliary proteins. The vast majority of AMPARs in the AVA interneuron are GLR-1/GLR-2 heteromeric receptors. Channel opening requires the presence the TARP proteins STG-1 or STG-2. The CUB-domain proteins SOL-1 and SOL-2 modulate receptor desensitization and channel kinetics. at synapses in the VNC (Monteiro et al., 2012). Further genetic studies suggest that KLP-4 and CDK-5 function in the same pathway in the cell body to regulate the anterograde transport of GLR-1 to synapses (Monteiro et al., 2012). Concluding Remarks Throughout the nervous system, AMPARs mediate rapid excitatory signaling between neurons. The number of functional postsynaptic AMPARs at the synapse is involved in establishing the activity-induced changes in synaptic strength associated with learning and memory (Malinow and Malenka, 2002). Many studies have focused on how local processes such as exocytosis, endocytosis and lateral diffusion regulate the abundance of AMPARs in the postsynaptic membrane (Ashby et al., 2006; Borgdorff and Choquet, 2002; Kessels and Malinow, 2009; Passafaro et al., 2001; Shi et al., 2001; Yudowski et al., 2007); however, much less is known about the mechanisms involved in the trafficking of AMPARs from the cell body to the synapse (Bredt and Nicoll, 2003; Shepherd and Huganir, 2007; van der Sluijs and Hoogenraad, 2011). To address this fundamental question, we have undertaken a comprehensive study to understand the molecular mechanisms governing the long-range trafficking of the AMPA-type glutamate receptor. This work utilizes the powerful genetic tools and simple nervous system of the model organism C. elegans to understand how AMPARs are transported in vivo (Chapter 2). To accomplish this, we took advantage of the clear worm cuticle and genetically engineered fluorescently tagged proteins and looked at AMPAR trafficking in the 32 unipolar process of the interneuron AVA. By looking at fluorescently tagged GLR-1 in AVA, we unequivocally show that AMPARs are transported by the molecular motor kinesin-1, encoded by the gene unc-116. Motor-based movement is required for the delivery of AMPARs to synapses, which occurs when a molecular motor stops near an existing synapse. We further demonstrate that AMPARs are not destined for a single synapse, but rather that GLR-1 can be reutilized at distant synapses through molecular motor-mediated removal and redistribution (Chapter 2). To understand the role of kinesin-mediated transport of GLR-1 in AVA, we took advantage of multiple hypomorphic genetic mutations and RNAi knockdowns of unc-116 (Chapter 2). In these mutants, AMPAR transport was severely disrupted in both anterograde and retrograde, suggesting that a single molecular motor can mediate the bidirectional transport of AMPARs. Unexpectedly, mutations in unc-116 resulted in accumulations of surface-expressed GLR-1 at synapses. Despite these accumulations of GLR-1 in unc- 116 mutants, glutamate-gated currents were paradoxically decreased due to a preferential degradation of the GLR-2 AMPAR subunit in the absence of motor transport. Finally, we show that motor-mediated transport of GLR-1 in the adult nervous system plays a major role in maintaining synaptic transmission by providing a constant source of functional glutamate receptors. Altogether, this work provides the first in vivo genetic, electrophysiological and cell biological characterization of AMPAR transport by molecular motors. 33 Given the conservation of the molecular mechanisms of neurotransmission between invertebrates and vertebrates, this work will aid in further understanding the role of motor-dependent transport in more complex nervous systems. 34 35 References Adesnik, H., Nicoll, R.A., and England, P.M. (2005). Photoinactivation of native AMPA receptors reveals their real-time trafficking. Neuron 48, 977-985. Armstrong, N., Sun, Y., Chen, G.Q., and Gouaux, E. (1998). Structure of a glutamate-receptor ligand-binding core in complex with kainate. Nature 395, 913-917. Armstrong, N., and Gouaux, E. (2000). Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28, 165-181. Ashby, M.C., Maier, S.R., Nishimune, A., and Henley, J.M. (2006). Lateral diffusion drives constitutive exchange of AMPA receptors at dendritic spines and is regulated by spine morphology. J. Neurosci. Off. J. Soc. Neurosci. 26, 7046­7055. Ayalon, G., and Stern-Bach, Y. (2001). Functional assembly of AMPA and kainate receptors is mediated by several discrete protein-protein interactions. Neuron 31, 103-113. Ayalon, G., Segev, E., Elgavish, S., and Stern-Bach, Y. (2005). Two regions in the N-terminal domain of ionotropic glutamate receptor 3 form the subunit oligomerization interfaces that control subtype-specific receptor assembly. J. Biol. Chem. 280, 15053-15060. Baas, P.W. (1999). Microtubules and neuronal polarity: lessons from mitosis. Neuron 22, 23-31. Bass, B.L. (2002). RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817-846. Bats, C., Groc, L., and Choquet, D. (2007). The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking. Neuron 53, 719-734. Berkowitz, L.A., Knight, A.L., Caldwell, G.A., and Caldwell, K.A. (2008). Generation of stable transgenic C. elegans using microinjection. J. Vis. Exp. 18, e833. Blanpied, T.A., Scott, D.B., and Ehlers, M.D. (2002). Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines. Neuron 36, 435-449. Bokel, C., Dass, S., Wilsch-Brauninger, M., and Roth, S. (2006). Drosophila Cornichon acts as cargo receptor for ER export of the TGFalpha-like growth factor Gurken. Dev. Camb. Engl. 133, 459-470. 36 Borgdorff, A.J., and Choquet, D. (2002). Regulation of AMPA receptor lateral movements. Nature 417, 649-653. Boulin, T. (2006). Reporter gene fusions. WormBook. ed. The C. elegans Research Community. https://www.wormbook.org. Bredt, D.S., and Nicoll, R.A. (2003). AMPA receptor trafficking at excitatory synapses. Neuron 40, 361-379. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94. Brockie, P.J., Mellem, J.E., Hills, T., Madsen, D.M., and Maricq, A.V. (2001a). The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion. Neuron 31, 617-630. Brockie, P.J., Madsen, D.M., Zheng, Y., Mellem, J., and Maricq, A.V. (2001b). Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC- 42. J. Neurosci. Off. J. Soc. Neurosci. 21, 1510-1522. Brockie, P.J., and Maricq, A.V. (2010). In a pickle: is cornichon just relish or part of the main dish? Neuron 68, 1017-1019. Brockie, P.J., Jensen, M., Mellem, J.E., Jensen, E., Yamasaki, T., Wang, R., Maxfield, D., Thacker, C., Hoerndli, F., Dunn, P.J., et al. (2013). Cornichons control ER export of AMPA receptors to regulate synaptic excitability. Neuron 80, 129-142. Burbea, M., Dreier, L., Dittman, J.S., Grunwald, M.E., and Kaplan, J.M. (2002). Ubiquitin and AP180 regulate the abundance of GLR-1 glutamate receptors at postsynaptic elements in C. elegans. Neuron 35, 107-120. Chalfie, M., Sulston, J.E., White, J.G., Southgate, E., Thomson, J.N., and Brenner, S. (1985). The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci. Off. J. Soc. Neurosci. 5, 956-964. Chambers, J.J., Gouda, H., Young, D.M., Kuntz, I.D., and England, P.M. (2004). Photochemically knocking out glutamate receptors in vivo. J. Am. Chem. Soc. 126, 13886-13887. Chen, C., and Tonegawa, S. (1997). Molecular genetic analysis of synaptic plasticity, activity-dependent neural development, learning, and memory in the mammalian brain. Annu. Rev. Neurosci. 20, 157-184. Chen, L., Chetkovich, D.M., Petralia, R.S., Sweeney, N.T., Kawasaki, Y., Wenthold, R.J., Bredt, D.S., and Nicoll, R.A. (2000). Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936-943. 37 Contractor, A., and Heinemann, S.F. (2004). AMPA receptor cycling in the synapse. Sci. STKE Signal Transduct. Knowl. Environ. 2004, tr7. Coombs, I.D., Soto, D., Zonouzi, M., Renzi, M., Shelley, C., Farrant, M., and Cull- Candy, S.G. (2012). Cornichons modify channel properties of recombinant and glial AMPA receptors. J. Neurosci. Off. J. Soc. Neurosci. 32, 9796-9804. Daw, M.I., Chittajallu, R., Bortolotto, Z.A., Dev, K.K., Duprat, F., Henley, J.M., Collingridge, G.L., and Isaac, J.T. (2000). PDZ proteins interacting with C-terminal GluR2/3 are involved in a PKC-dependent regulation of AMPA receptors at hippocampal synapses. Neuron 28, 873-886. Dingledine, R., Borges, K., Bowie, D., and Traynelis, S.F. (1999). The glutamate receptor ion channels. Pharmacol. Rev. 51, 7-61. Earnshaw, B.A., and Bressloff, P.C. (2008). Modeling the role of lateral membrane diffusion in AMPA receptor trafficking along a spiny dendrite. J. Comput. Neurosci. 25, 366-389. Ehlers, M.D., Heine, M., Groc, L., Lee, M.-C., and Choquet, D. (2007). Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity. Neuron 54, 447-460. Esposito, G., Di Schiavi, E., Bergamasco, C., and Bazzicalupo, P. (2007). Efficient and cell specific knock-down of gene function in targeted C. elegans neurons. Gene 395, 170-176. Evans, T. (2006). Transformation and microinjection. WormBook. ed. The C. elegans Research Community. https://www.wormbook.org. Gerges, N.Z., Backos, D.S., Rupasinghe, C.N., Spaller, M.R., and Esteban, J.A. (2006). Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane. EMBO J. 25, 1623-1634. Glodowski, D.R., Chen, C.C.-H., Schaefer, H., Grant, B.D., and Rongo, C. (2007). RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent endocytosis pathway. Mol. Biol. Cell 18, 4387-4396. Goldstein, L.S., and Yang, Z. (2000). Microtubule-based transport systems in neurons: the roles of kinesins and dyneins. Annu. Rev. Neurosci. 23, 39-71. Groc, L., Heine, M., Cognet, L., Brickley, K., Stephenson, F.A., Lounis, B., and Choquet, D. (2004). Differential activity-dependent regulation of the lateral mobilities of Am PA and NMDA receptors. Nat. Neurosci. 7, 695-696. Grooms, S.Y., Noh, K.-M., Regis, R., Bassell, G.J., Bryan, M.K., Carroll, R.C., and Zukin, R.S. (2006). Activity bidirectionally regulates AMPA receptor mRNA 38 abundance in dendrites of hippocampal neurons. J. Neurosci. Off. J. Soc. Neurosci. 26, 8339-8351. Hanley, J.G. (2010). Endosomal sorting of AMPA receptors in hippocampal neurons. Biochem. Soc. Trans. 38, 460-465. Hart, A.C., Sims, S., and Kaplan, J.M. (1995). Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature 378, 82-85. Hayashi, Y., Shi, S.H., Esteban, J.A., Piccini, A., Poncer, J.C., and Malinow, R. (2000). Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287, 2262-2267. Hebb, D.O. (1949). The Organization of Behavior: A Neuropsychological Theory (New York; Wiley), ISBN 9780471367277. Henley, J.M., and Wilkinson, K.A. (2013). AMPA receptor trafficking and the mechanisms underlying synaptic plasticity and cognitive aging. Dialogues Clin. Neurosci. 15, 11-27. Herring, B.E., Shi, Y., Suh, Y.H., Zheng, C.-Y., Blankenship, S.M., Roche, K.W., and Nicoll, R.A. (2013). Cornichon proteins determine the subunit composition of synaptic AMPA receptors. Neuron 77, 1083-1096. Ho, V.M., Lee, J.-A., and Martin, K.C. (2011). The cell biology of synaptic plasticity. Science 334, 623-628. Huber, K.M., Kayser, M.S., and Bear, M.F. (2000). Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science 288, 1254-1257. Huganir, R.L., and Nicoll, R.A. (2013). AMPARs and synaptic plasticity: the last 25 years. Neuron 80, 704-717. Jaskolski, F., Mayo-Martin, B., Jane, D., and Henley, J.M. (2009). Dynamin-dependent membrane drift recruits AMPA receptors to dendritic spines. J. Biol. Chem. 284, 12491-12503. Jones, M.V., and Westbrook, G.L. (1996). The impact of receptor desensitization on fast synaptic transmission. Trends Neurosci. 19, 96-101. Jorgensen, E.M., and Mango, S.E. (2002). The art and design of genetic screens: caenorhabditis elegans. Nat. Rev. Genet. 3, 356-369. Ju, W., Morishita, W., Tsui, J., Gaietta, G., Deerinck, T.J., Adams, S.R., Garner, C.C., Tsien, R.Y., Ellisman, M.H., and Malenka, R.C. (2004). Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors. Nat. Neurosci. 7, 244-253. 39 Juo, P., Harbaugh, T., Garriga, G., and Kaplan, J.M. (2007). CDK-5 regulates the abundance of GLR-1 glutamate receptors in the ventral cord of Caenorhabditis elegans. Mol. Biol. Cell 18, 3883-3893. Kakegawa, W., Tsuzuki, K., Yoshida, Y., Kameyama, K., and Ozawa, S. (2004). Input- and subunit-specific AMPA receptor trafficking underlying long-term potentiation at hippocampal CA3 synapses. Eur. J. Neurosci. 20, 101-110. Kamath, R.S., Martinez-Campos, M., Zipperlen, P., Fraser, A.G., and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2, RESEARCH0002. Kang, H., and Schuman, E.M. (1996). A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 273, 1402-1406. Kato, A.S., Gill, M.B., Ho, M.T., Yu, H., Tu, Y., Siuda, E.R., Wang, H., Qian, Y - W., Nisenbaum, E.S., Tomita, S., et al. (2010). Hippocampal AMPA receptor gating controlled by both TARP and cornichon proteins. Neuron 68, 1082-1096. Kerr, J.M., and Blanpied, T.A. (2012). Subsynaptic AMPA receptor distribution is acutely regulated by actin-driven reorganization of the postsynaptic density. J. Neurosci. Off. J. Soc. Neurosci. 32, 658-673. Kessels, H.W., and Malinow, R. (2009). Synaptic AMPA receptor plasticity and behavior. Neuron 61, 340-350. Kew, J.N.C., and Kemp, J.A. (2005). Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology (Berl.) 179, 4-29. Kim, C.H., and Lisman, J.E. (2001). A labile component of AMPA receptor-mediated synaptic transmission is dependent on microtubule motors, actin, and N-ethylmaleimide-sensitive factor. J. Neurosci. Off. J. Soc. Neurosci. 21, 4188­4194. Kolleker, A., Zhu, J.J., Schupp, B.J., Qin, Y., Mack, V., Borchardt, T., Kohr, G., Malinow, R., Seeburg, P.H., and Osten, P. (2003). Glutamatergic plasticity by synaptic delivery of GluR-B(long)-containing AMPA receptors. Neuron 40, 1199­1212. Kramer, L.B., Shim, J., Previtera, M.L., Isack, N.R., Lee, M.-C., Firestein, B.L., and Rongo, C. (2010). UEV-1 is an ubiquitin-conjugating enzyme variant that regulates glutamate receptor trafficking in C. elegans neurons. PloS One 5, e14291. Laube, B., Kuhse, J., and Betz, H. (1998). Evidence for a tetrameric structure of recombinant NMDA receptors. J. Neurosci. Off. J. Soc. Neurosci. 18, 2954-2961. 40 Lee, S.H., Simonetta, A., and Sheng, M. (2004). Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons. Neuron 43, 221­236. Letts, V.A., Felix, R., Biddlecome, G.H., Arikkath, J., Mahaffey, C.L., Valenzuela, A., Bartlett, F.S., 2nd, Mori, Y., Campbell, K.P., and Frankel, W.N. (1998). The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit. Nat. Genet. 19, 340-347. Lu, J., Helton, T.D., Blanpied, T.A., Racz, B., Newpher, T.M., Weinberg, R.J., and Ehlers, M.D. (2007). Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer. Neuron 55, 874­889. Lu, W., Man, H., Ju, W., Trimble, W.S., MacDonald, J.F., and Wang, Y.T. (2001). Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 29, 243-254. Lu, W., Shi, Y., Jackson, A.C., Bjorgan, K., During, M.J., Sprengel, R., Seeburg, P.H., and Nicoll, R.A. (2009). Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach. Neuron 62, 254-268. Lu, W., Isozaki, K., Roche, K.W., and Nicoll, R.A. (2010). Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1. Proc. Natl. Acad. Sci. U.S.A. 107, 22266-22271. MacGillavry, H.D., Song, Y., Raghavachari, S., and Blanpied, T.A. (2013). Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615-622. Makino, H., and Malinow, R. (2009). AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis. Neuron 64, 381-390. Malinow, R., and Malenka, R.C. (2002). AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103-126. Mano, I., and Teichberg, V.I. (1998). A tetrameric subunit stoichiometry for a glutamate receptor-channel complex. Neuroreport 9, 327-331. Maricq, A.V., Peckol, E., Driscoll, M., and Bargmann, C.I. (1995). Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor. Nature 378, 78-81. Matsuzaki, M., Ellis-Davies, G.C., Nemoto, T., Miyashita, Y., Iino, M., and Kasai, H. (2001). Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086-1092. 41 Mayer, M.L. (2006). Glutamate receptors at atomic resolution. Nature 440, 456­462. McGee, A.W., and Bredt, D.S. (2003). Assembly and plasticity of the glutamatergic postsynaptic specialization. Curr. Opin. Neurobiol. 13, 111-118. Meldrum, B.S. (1994). The role of glutamate in epilepsy and other CNS disorders. Neurology 44, S14-23. Mellem, J.E., Brockie, P.J., Zheng, Y., Madsen, D.M., and Maricq, A.V. (2002). Decoding of polymodal sensory stimuli by postsynaptic glutamate receptors in C. elegans. Neuron 36, 933-944. Mello, C.C., and Conte, D., Jr (2004). Revealing the world of RNA interference. Nature 431, 338-342. Mody, I. (1998). Ion channels in epilepsy. Int. Rev. Neurobiol. 42, 199-226. Montastruc, J.L., Rascol, O., and Senard, J.M. (1997). Glutamate antagonists and Parkinson's disease: a review of clinical data. Neurosci. Biobehav. Rev. 21, 477-480. Monteiro, M.I., Ahlawat, S., Kowalski, J.R., Malkin, E., Koushika, S.P., and Juo, P. (2012). The kinesin-3 family motor KLP-4 regulates anterograde trafficking of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans. Mol. Biol. Cell 23, 3647-3662. Nakajima, K., Yin, X., Takei, Y., Seog, D.-H., Homma, N., and Hirokawa, N. (2012). Molecular motor KIF5A is essential for GABA(A) receptor transport, and KIF5A deletion causes epilepsy. Neuron 76, 945-961. Nakanishi, S. (1994). Metabotropic glutamate receptors: synaptic transmission, modulation, and plasticity. Neuron 13, 1031-1037. Newpher, T.M., and Ehlers, M.D. (2008). Glutamate receptor dynamics in dendritic microdomains. Neuron 58, 472-497. Nonet, M.L., Holgado, A.M., Brewer, F., Serpe, C.J., Norbeck, B.A., Holleran, J., Wei, L., Hartwieg, E., Jorgensen, E.M., and Alfonso, A. (1999). UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Mol. Biol. Cell 10, 2343-2360. Opazo, P., Labrecque, S., Tigaret, C.M., Frouin, A., Wiseman, P.W., De Koninck, P., and Choquet, D. (2010). CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin. Neuron 67, 239-252. Osten, P., Khatri, L., Perez, J.L., Kohr, G., Giese, G., Daly, C., Schulz, T.W., Wensky, A., Lee, L.M., and Ziff, E.B. (2000). Mutagenesis reveals a role for 42 ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor. Neuron 27, 313-325. Otis, T., Zhang, S., and Trussell, L.O. (1996). Direct measurement of AMPA receptor desensitization induced by glutamatergic synaptic transmission. J. Neurosci. Off. J. Soc. Neurosci. 16, 7496-7504. Passafaro, M., Piech, V., and Sheng, M. (2001). Subunit-specific temporal and spatial patterns of AMPA receptor exocytosis in hippocampal neurons. Nat. Neurosci. 4, 917-926. Patterson, M.A., Szatmari, E.M., and Yasuda, R. (2010). AMPA receptors are exocytosed in stimulated spines and adjacent dendrites in a Ras-ERK-dependent manner during long-term potentiation. Proc. Natl. Acad. Sci. U. S. A. 107, 15951­15956. Peng, S., Zhang, Y., Zhang, J., Wang, H., and Ren, B. (2011). Glutamate receptors and signal transduction in learning and memory. Mol. Biol. Rep. 38, 453-460. Petrini, E.M., Lu, J., Cognet, L., Lounis, B., Ehlers, M.D., and Choquet, D. (2009). Endocytic trafficking and recycling maintain a pool of mobile surface AMPA receptors required for synaptic potentiation. Neuron 63, 92-105. Priel, A., Kolleker, A., Ayalon, G., Gillor, M., Osten, P., and Stern-Bach, Y. (2005). Stargazin reduces desensitization and slows deactivation of the AMPA-type glutamate receptors. J. Neurosci. Off. J. Soc. Neurosci. 25, 2682-2686. Rongo, C., Whitfield, C.W., Rodal, A., Kim, S.K., and Kaplan, J.M. (1998). LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia. Cell 94, 751-759. Rongo, C., and Kaplan, J.M. (1999). CaMKII regulates the density of central glutamatergic synapses in vivo. Nature 402, 195-199. Rosenmund, C., Stern-Bach, Y., and Stevens, C.F. (1998). The tetrameric structure of a glutamate receptor channel. Science 280, 1596-1599. Roth, S., Neuman-Silberberg, F.S., Barcelo, G., and Schupbach, T. (1995). cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell 81, 967-978. Scannevin, R.H., and Huganir, R.L. (2000). Postsynaptic organization and regulation of excitatory synapses. Nat. Rev. Neurosci. 1, 133-141. 43 Schnell, E., Sizemore, M., Karimzadegan, S., Chen, L., Bredt, D.S., and Nicoll, R.A. (2002). Direct interactions between PSD-95 and stargazin control synaptic AMpA receptor number. Proc. Natl. Acad. Sci. U.S.A. 99, 13902-13907. Schwenk, J., Harmel, N., Zolles, G., Bildl, W., Kulik, A., Heimrich, B., Chisaka, O., Jonas, P., Schulte, U., Fakler, B., et al. (2009). Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors. Science 323, 1313­1319. Seeburg, P.H., Higuchi, M., and Sprengel, R. (1998). RNA editing of brain glutamate receptor channels: mechanism and physiology. Brain Res. Brain Res. Rev. 26, 217-229. Setou, M., Seog, D.-H., Tanaka, Y., Kanai, Y., Takei, Y., Kawagishi, M., and Hirokawa, N. (2002). Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites. Nature 417, 83-87. Shepherd, J.D., and Huganir, R.L. (2007). The cell biology of synaptic plasticity: AMpA receptor trafficking. Annu. Rev. Cell Dev. Biol. 23, 613-643. Shi, S., Hayashi, Y., Esteban, J.A., and Malinow, R. (2001). Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105, 331-343. Shi, Y., Suh, Y.H., Milstein, A.D., Isozaki, K., Schmid, S.M., Roche, K.W., and Nicoll, R.A. (2010). Functional comparison of the effects of TARPs and cornichons on AMPA receptor trafficking and gating. Proc. Natl. Acad. Sci. U.S.A. 107, 16315-16319. Smith, M.A., Ellis-Davies, G.C.R., and Magee, J.C. (2003). Mechanism of the distance-dependent scaling of Schaffer collateral synapses in rat CA1 pyramidal neurons. J. Physiol. 548, 245-258. Sobolevsky, A.I., Rosconi, M.P., and Gouaux, E. (2009). X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462, 745-756. Sommer, B., Kohler, M., Sprengel, R., and Seeburg, P.H. (1991). RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67, 11­19. Song, I., and Huganir, R.L. (2002). Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci. 25, 578-588. Stern-Bach, Y., Bettler, B., Hartley, M., Sheppard, P.O., O'Hara, P.J., and Heinemann, S.F. (1994). Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins. Neuron 13, 1345-1357. 44 Stern-Bach, Y., Russo, S., Neuman, M., and Rosenmund, C. (1998). A point mutation in the glutamate binding site blocks desensitization of AMPA receptors. Neuron 21, 907-918. Steward, O., and Levy, W.B. (1982). Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus. J. Neurosci. Off. J. Soc. Neurosci. 2, 284-291. Suchanek, B., Seeburg, P.H., and Sprengel, R. (1995). Gene structure of the murine N-methyl D-aspartate receptor subunit NR2C. J. Biol. Chem. 270, 41-44. Sudhof, T.C. (2004). The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509­547. Tanaka, J., Matsuzaki, M., Tarusawa, E., Momiyama, A., Molnar, E., Kasai, H., and Shigemoto, R. (2005). Number and density of AMPA receptors in single synapses in immature cerebellum. J. Neurosci. Off. J. Soc. Neurosci. 25, 799­807. Tardin, C., Cognet, L., Bats, C., Lounis, B., and Choquet, D. (2003). Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 22, 4656-4665. Tichelaar, W., Safferling, M., Keinanen, K., Stark, H., and Madden, D.R. (2004). The three-dimensional structure of an ionotropic glutamate receptor reveals a dimer-of-dimers assembly. J. Mol. Biol. 344, 435-442. Tomita, S., Nicoll, R.A., and Bredt, D.S. (2001). PDZ protein interactions regulating glutamate receptor function and plasticity. J. Cell Biol. 153, F19-24. Tomita, S., Chen, L., Kawasaki, Y., Petralia, R.S., Wenthold, R.J., Nicoll, R.A., and Bredt, D.S. (2003). Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins. J. Cell Biol. 161, 805-816. Tomita, S., Adesnik, H., Sekiguchi, M., Zhang, W., Wada, K., Howe, J.R., Nicoll, R.A., and Bredt, D.S. (2005). Stargazin modulates AMPA receptor gating and trafficking by distinct domains. Nature 435, 1052-1058. Trussell, L.O., and Fischbach, G.D. (1989). Glutamate receptor desensitization and its role in synaptic transmission. Neuron 3, 209-218. Ulas, J., Weihmuller, F.B., Brunner, L.C., Joyce, J.N., Marshall, J.F., and Cotman, C.W. (1994). Selective increase of NMDA-sensitive glutamate binding in the striatum of Parkinson's disease, Alzheimer's disease, and mixed Parkinson's disease/Alzheimer's disease patients: an autoradiographic study. J. Neurosci. Off. J. Soc. Neurosci. 14, 6317-6324. 45 Van der Sluijs, P., and Hoogenraad, C.C. (2011). New insights in endosomal dynamics and AMPA receptor trafficking. Semin. Cell Dev. Biol. 22, 499-505. Walker, C.S., Francis, M.M., Brockie, P.J., Madsen, D.M., Zheng, Y., and Maricq, A.V. (2006a). Conserved SOL-1 proteins regulate ionotropic glutamate receptor desensitization. Proc. Natl. Acad. Sci. U.S.A. 103, 10787-10792. Walker, C.S., Brockie, P.J., Madsen, D.M., Francis, M.M., Zheng, Y., Koduri, S., Mellem, J.E., Strutz-Seebohm, N., and Maricq, A.V. (2006b). Reconstitution of invertebrate glutamate receptor function depends on stargazin-like proteins. Proc. Natl. Acad. Sci. U.S.A. 103, 10781-10786. Wang, R., Walker, C.S., Brockie, P.J., Francis, M.M., Mellem, J.E., Madsen, D.M., and Maricq, A.V. (2008). Evolutionary conserved role for TARPs in the gating of glutamate receptors and tuning of synaptic function. Neuron 59, 997-1008. Wang, R., Mellem, J.E., Jensen, M., Brockie, P.J., Walker, C.S., Hoerndli, F.J., Hauth, L., Madsen, D.M., and Maricq, A.V. (2012). The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors. Neuron 75, 838-850. Watkins, J.C., and Jane, D.E. (2006). The glutamate story. Br. J. Pharmacol. 147 Suppl 1, S100-108. Wenthold, R.J., Yokotani, N., Doi, K., and Wada, K. (1992). Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain. J. Biol. Chem. 267, 501-507. White, J.G., Southgate, E., Thomson, J.N., and Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 314, 1-340. Yamazaki, M., Araki, K., Shibata, A., and Mishina, M. (1992). Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family. Biochem. Biophys. Res. Commun. 183, 886-892. Yamazaki, M., Ohno-Shosaku, T., Fukaya, M., Kano, M., Watanabe, M., and Sakimura, K. (2004). A novel action of stargazin as an enhancer of AMPA receptor activity. Neurosci. Res. 50, 369-374. Yudowski, G.A., Puthenveedu, M.A., Leonoudakis, D., Panicker, S., Thorn, K.S., Beattie, E.C., and von Zastrow, M. (2007). Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors. J. Neurosci. Off. J. Soc. Neurosci. 27, 11112-11121. 46 Zheng, Y., Mellem, J.E., Brockie, P.J., Madsen, D.M., and Maricq, A.V. (2004). SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans. Nature 427, 451-457. Zheng, Y., Brockie, P.J., Mellem, J.E., Madsen, D.M., Walker, C.S., Francis, M.M., and Maricq, A.V. (2006). SOL-1 is an auxiliary subunit that modulates the gating of GLR-1 glutamate receptors in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 103, 1100-1105. Zhu, J.J., Esteban, J.A., Hayashi, Y., and Malinow, R. (2000). Postnatal synaptic potentiation: delivery of GluR4-containing AMPA receptors by spontaneous activity. Nat. Neurosci. 3, 1098-1106. Zuo, J., De Jager, P.L., Takahashi, K.A., Jiang, W., Linden, D.J., and Heintz, N. (1997). Neurodegeneration in Lurcher mice caused by mutation in delta2 glutamate receptor gene. Nature 388, 769-773. CHAPTER 2 KINESIN-1 REGULATES SYNAPTIC STRENGTH BY MEDIATING DELIVERY, REMOVAL AND REDISTRIBUTION OF AMPARS Reprinted from Neuron, 80 (6), Horendli, F., Maxfield, D.A., Brockie, P.J., Mellem, J.E., Jensen, E., Wang, R., Madsen, D.M. and Maricq, A.V., Kinesin-1 Regulates Synaptic Strength by Mediating Delivery, Removal and Redistribution of AMPARs, 1421-1437, Copyright (2013) with permission from Elsevier. 48 Neuron Article Kinesin-1 Regulates Synaptic Strength by Mediating the Delivery, Removal, and Redistribution of AMPA Receptors Frederic J. Hoerndli,1,2 Dane A. Maxfield,1,2 Penelope J. Brockie,1 Jerry E. Mellem,1 EricaJensen,1 Rui Wang,1 David M. Madsen,1 and Andres V. Maricq1,* 1Department of Biology, Centerfor Cell and Genome Science, University of Utah, Salt Lake City, UT 84112, USA 2These authors contributed equally to this work *Correspondence: [e-mail] http://dx.doi.org/10.1016/j.neuron.2013.10.050 SUMMARY A primary determinant of the strength of neuro­transmission is the number of AMPA-type glutamate receptors (AMPARs) at synapses. However, we still lack a mechanistic understanding of how the number of synaptic AMPARs is regulated. Here, we show that UNC-116, the C. elegans homolog of vertebrate kine-sin- 1 heavy chain (KIF5), modifies synaptic strength by mediating the rapid delivery, removal, and redistribution of synaptic AMPARs. Furthermore, by studying the real-time transport of C. elegans AMPAR subunits in vivo, we demonstrate that although homomeric GLR-1 AMPARs can diffuse to and accumulate at synapses in unc-116 mutants, glutamate-gated currents are diminished because heteromeric GLR-1/GLR-2 receptors do not reach synapses in the absence of UNC-116/KIF5-mediated transport. Our data support a model in which ongoing motor-driven delivery and removal of AMPARs controls not only the number but also the composition of synaptic AMPARs, and thus the strength of synaptic transmission. INTRODUCTION The number of functional postsynaptic glutamate receptors is a major determinant of the strength of synaptic signaling. Thus, experience-dependent changes in the number of receptors contribute to fundamental network properties such as learning and memory (Jackson and Nicoll, 2011; Kerchner and Nicoll, 2008; Malinow and Malenka, 2002). Because most neurons have long processes, synapses are often far removed from the cell body, imparting a major challenge for the modulation and maintenance of synaptic machinery. Although we have consider­able insight into the local mechanisms that contribute to synaptic strength by regulating the recycling of a-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) between the postsynaptic membrane and endosomal compartments (Henley et al., 2011; Kennedy and Ehlers, 2011; Kessels and Malinow, 2009; Petrini et al., 2009; Rusakov et al., 2011; Shepherd and Huganir, 2007; Yudowski et al., 2006), we have far fewer mechanistic insights into the long-range transport of AMPARs and how transport impacts synaptic strength and plasticity. These questions are particularly timely, considering the strong association of transport defects with synaptopathies and neurodegenerative disorders such as Alzheimer's disease (Stokin and Goldstein, 2006). At least three different mechanisms have been proposed for the long-range delivery of AMPARs to synapses, including local synthesis(Ho etal., 2011; Ju etal., 2004), lateral diffusion (Ades-nik et al., 2005), and motor-dependent transport (Greger and Esteban, 2007; Kim and Lisman, 2001; Setou et al., 2002). How­ever, it has been difficult to establish the relative contributions of these various processes to synaptic function. These competing models derive almost exclusively from in vitro studies in cultured neuronal preparations, and thus might not accurately reflect the effects of the local cellular environment, signaling molecules, and the extracellular matrix, all of which can influence neuronal development and synaptic function. Therefore, we developed techniques that allowed us to directly observe the in vivo delivery of AMPARs to synapses in a specific neuron in C. elegans. Studying AMPAR delivery in C. elegans allows us to integrate in vivo cell biological and electrophysiological studies of synaptic function. C. elegans are transparent and have only 302 neurons, a subset ofwhich communicate bythe synaptic release of gluta­mate to mediate specific behaviors (de Bono and Maricq, 2005). Glutamate gates a variety of receptors, including the GLR-1 AMPAR signaling complex, which is expressed in interneurons that contribute to worm locomotion (de Bono and Maricq, 2005). Previous studies have identified the molecular compo­nents o fthe GLR-1 signaling complex (Brockie et al., 2001; Mel­lem et al., 2002; Walker et al., 2006; Wang et al., 2008, 2012; Zheng et al., 2004, 2006) and the mechanisms that regulate the localization and stability of synaptic GLR-1 (Burbea et al., 2002; Glodowskietal., 2007; Juoetal., 2007; Rongoand Kaplan, 1999; Rongo et al., 1998; Zhang et al., 2012). We now demonstrate that the microtubule-dependent motor, UNC-116/KIF5, and the associated kinesin light chain, KLC-2, mediatethetransportofGLR-1 tosynapses. Inaseriesofinvivo studies, we evaluated the relative contributions of motor trans­port, receptor diffusion, and local synthesis to the delivery of GLR-1 to synapses. We found that motor-mediated transport CrossMark Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 1421 49 Cell P R E S S Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs Anterograde movement | | Anterograde | Retrograde Figure 1. GLR-1::GFP Is Transported in Both an Anterograde and a Retrograde Direction along the AVA Processes (A) Confocal images of GLR-1::GFP puncta in the proximal AVA processes before (top) and after (bottom) photobleaching. Scale bar represents 2.5 mm. (B) Higher gain images o f the region shown in (A) at various tim e points after photobleaching. The arrowheads indicate anterograde (blue) and retrograde (red) movement. (C) Kymograph showing mobile and immobile GLR-1::GFP vesicles in the photobleached region shown in (A). (D) Measurement of the area (left) and average total fluorescence (right) o f immobile and mobile GLR-1::GFP. n > 100 immobile; n > 450 mobile; ***p < 0.001. (E) Quantification o f the velocity (left) and run length (right) o f mobile GLR-1::GFP vesicles. n > 450 vesicles. Error bars indicate SEM. See also Figure S1. is the predominant mechanism for delivery, removal, and redis­tribution of GLR-1. In unc-116 mutants, GLR-1 diffused out of the cell body to proximal synapses, where it reached higher than normal levels secondary to the loss of motor-driven removal of synaptic receptors. Despite the synaptic accumulation of GLR-1 in unc-116 mutants, glutamate-gated currents were severely diminished because the AMPAR signaling complex lacked GLR-1/GLR-2 heteromeric receptors. Defective AMPAR signaling in unc-116 mutants was rescued by transient expres­sion of UNC-116 in the adult nervous system, demonstrating that ongoing motor-dependent transport is required for the regulation of synaptic strength. RESULTS In Vivo Measurement of GLR-1 Transport In C. elegans, the two AVA interneurons are part of a well-defined circuit that regulates worm reversal behavior (Brockie et al., 2001). These neurons express the GLR-1 AMPAR subunit and each neuron extends a single process into the ventral cord that runs the length of the worm. We were able to specifically visualize these processes by using promoter sequences (Prig-3 and Pflp- 18) that limited transgene expression in the ventral cord to AVA (Figure S1A available online) (Feinberg et al., 2008; Wang et al., 2012). In transgenic worms that expressed a functional GFP-tagged variant of GLR-1 (GLR-1::GFP) in AVA, we observed discrete GFP puncta, which mark postsynaptic sites along the processes (Figure S1B) (Rongo et al., 1998). To address how GLR-1 receptors are delivered to these synapses, we used real-time streaming confocal microscopy to image receptor movement. Unless otherwise indicated, we imaged a region of the proximal AVA process of young adult worms (Figure S1A, boxed region). To better image receptor transport, we first reduced background fluorescence by photobleaching a region of interest that was approximately 45 mm in length (Figure 1A). Transport events were more apparent after photobleaching, and we noted no adverse effects of photobleaching on transport (Figures S1C and S1D). We then captured a series of confocal images that revealed numerous small, fluorescent GLR-1::GFP puncta that moved either anterogradely or retrogradely along the AVA processes. We refer to these as vesicles, given that their movement was consistent with known vesicular transport of transmembrane proteins (Figure 1B). The bidirectional vesicle transport, interrupted by occasional pauses or stops, is most apparent in kymographs generated from the full series of images (Figure 1C). The mobile vesicles were considerably smaller and dimmer than the large, immobile puncta (Figure 1D), which we refer to as synaptic puncta. Vesicles moved at approximately 1422 Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 50 Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs 1.6 mm/s in both anterograde and retrograde directions, with an average run length of approximately 6 mm (Figure 1E). Transport Vesicles Are Directed to Synapses Most of the transport vesicles had brief pauses in their move­ment that were of variable duration (Figure 2A). A more detailed analysis of the kymographs revealed that stop events (defined as pauses in movement lasting at least 1.2 s) were clustered near existing GLR-1 synaptic puncta (Figures 2B and 2C). We also observed vesicles that stopped moving for extended periods, at times lasting many minutes. In one kymograph, we observed a vesicle moving anterogradely (Figure 2A, blue arrow) and another vesicle moving retrogradely (Figure 2A, red arrow) and observed both vesicles stop at the same synapse (Figure 2A, green box). Twenty minutes later, a kymograph of the same re­gion revealed sustained recovery of GLR-1::GFP fluorescence at the synapse where the two vesicles stopped (Figure 2A, lower panel, filled arrowhead), suggesting thatthese stops might have been permanent delivery events. We also detected long-lived increases in fluorescence at additional synapses (Figure 2A, lower panel, open arrowheads), which we assume reflect stop­page events that occurred in the 20 min interval between kymographs. We next asked whether vesicles were delivered directly to the surface membrane or first to a subsynaptic compartment. To simultaneously measure GLR-1 transport and surface deliv­ery, we generated a transgenic strain that expressed a func­tional GLR-1 protein fused to both superecliptic pHluorin (SEP) and mCherry at the extracellular N-terminal domain (SEP:: mCherry::GLR-1) (Kennedy and Ehlers, 2011) (Figure 2D). The SEP variant of GFP is pH sensitive and not appreciably fluores­cent when localized to the relatively acidic environment of sub-cellular organelles in C. elegans (Dittman and Kaplan, 2006; Miesenbocketal.,1998;Wangetal., 2012).Thus, we rarelydetect intracellular transport in the green channel (Figure 2E). Further­more, photobleaching eliminates SEP fluorescence of surface GLR-1, but does not affect the SEP fluorophore on internalized receptors. Following photobleaching of both fluorophores, we acquired two-color streaming confocal movies to simultaneously monitorvesicle movement in the red channel and surface delivery of receptors thatwe detected by the appearance of a fluorescent signal in the green channel. Although we observed long-lived stops in vesicle movement, these were not immediately associ-atedwith GLR-1 surfacedelivery. However, wedidobserve inser­tion events occurring at variable intervals following vesicle stops (Figure 2E). This suggests that receptors were first delivered to a subsynaptic compartment rather than directly to the surface of the synapse and that stoppage and insertion are separable processes. Insertion events were observed most frequently at the same location as the synaptic puncta (Figures 2F and S2A). These data indicate that delivery of GLR-1 to synapses occurs in at least two steps. First, transport vesicles stop and deliver GLR-1 to a subsynaptic compartment in the region of a synapse. Second, after some delay, receptors are inserted into the synap­tic membrane. Interestingly, the insertion rate (Figure 2E) is a fraction ofthe stoppage rate (Figure 2C), even though the synap­tic delivery (Figure S2B) and transport parameters (data not shown) were unaltered by the location of the fluorophore tag. This result suggests that not all longer-duration stops (>1.2 s) are destined for eventual insertion at a particular synapse. GLR-1 ReceptorsAre Redistributed between Synapses To determine the fate of GLR-1 receptors at synapses, we fused a photoactivatable florophore (PAGFP) to GLR-1 and expressed the functional protein in the AVA neurons. Following photoac­tivation of GLR-1::PAGFP puncta, we occasionally observed vesicles leaving synaptic puncta and traveling in either an anter­ograde (Figure 3A) or a retrograde (Figure 3A, insert) direction. These observations raised the question ofwhetherGLR-1 recep­tors could be utilized at multiple synapses. We therefore evalu­ated the source of synaptic receptors using a photoconversion strategy to follow the fate of receptors from the cell body, and from proximal and distal synapses. We tagged GLR-1 with Den-dra2, a photoconvertible fluorophore that can be switched from green to red fluorescence using UV illumination (Gurskaya et al., 2006) and expressed the functional fusion protein in the AVA neurons. Four hours after photoconversion of GLR-1::Dendra2 in the AVA cell bodies, we found that approximately 25% of the fluorescent signal at distal synapses was red (Figure 3B). In contrast, we did not observe an appreciable red signal in the distal processes of sham-converted worms (Figure S3A). Next, we photoconverted both synaptic puncta and interpunc-tal GLR-1::Dendra2 fluorescence in the proximal region of the AVA processes. Fours hours after photoconversion, we moni­tored the appearance of red fluorescence at distal synapses and found that red signal originating in the proximal processes had been redistributed to distal puncta (Figure 3C). In separate experiments, we photoconverted only the synaptic puncta in the proximal processes, leaving the interpuncta regions uncon­verted, and again observed red signal at distal synapses 4 hr after photoconversion (Figure 3D), but no red signal in sham-converted worms (Figure S3B). These data indicate that the red fluorescence, which appeared at distal synapses, was derived from receptors at proximal synapses rather than recep­tors that were photoconverted while in transit. To further evaluate the redistribution of receptors, we photo­activated GLR-1::PAGFP inthedistal halfoftheAVAprocesses. Fours hours after photoactivation, we observed GLR-1::PAGFP fluorescence at puncta in the proximal process (Figure 3E), but no signal in sham photoactivated controls (Figure S3C). Together, these data indicate that most synaptic GLR-1 recep­tors are delivered from the cell body to synapses, but receptors at a given synapse can be redistributed to other synapses. UNC-116/KIF5 Mediates GLR-1 Transport The speed and processivity of vesicle transport strongly sug­gested an energy-dependent, motor-driven process. In support of this hypothesis, we did not observe vesicle movement following treatment with Na-azide, a potent inhibitor of mito­chondrial respiration and ATP production (Bowler et al., 2006), or nocodazole, an inhibitor of microtubule polymerization (Fig­ure S3D), suggesting that microtubule-dependent motors drive the movement of GLR-1 vesicles. There are 21 known kinesin-like motors encoded by the C. elegans genome (Siddiqui, 2002), but only a few have been studied in detail, including OSM-3, UNC-104/KIF1, KLP-4, and UNC-116/KIF5. Of these, Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 1423 51 Cell P R E S S Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs Figure 2. GLR-1 Is Preferentially Delivered to Synaptic Puncta in the AVA Processes (A) Single-plane confocal images before and after photobleaching GLR-1::GFP in th e AVA processes with the corresponding kymograph showing anterograde (blue arrow) and retrograde (red arrow) delivery events to a synaptic puncta (green box). A second kymograph (bottom), taken 20 min after the first, shows the stable delivery event from the first kymograph (filled arrowhead), as well as additional delivery events during the interval between the tw o kymographs (open arrowheads). (B) Confocal image o f synaptic GLR-1::GFP puncta in AVA (top) and the corresponding linescan of fluorescence intensity (bottom). Green diamonds mark the peak fluorescence o f synaptic puncta and red d ots mark the relative positions of GLR-1::GFP vesicle s tops from a 5 min movie. (C) Quantification of GLR-1::GFP vesicle s tops in synaptic and extrasynaptic regions. n = 7. (legend continued on next page) 1424 Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 52 Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs Figure 3. GLR-1 Is Redistributed between Synapses (A) Photoactivation o f GLR-1::PAGFP expressed in AVA before and after UV photoactivation (blue, dashed box) w ith the corresponding kymograph. Arrowheads show anterograde (blue arrowheads) and retrograde (inset, red arrowheads) departure o f a GLR-1 vesicle from converted synaptic puncta. (B-E) All confocal images were taken before, immediately after, or 4 hr after photoconversion or photoactivation. (B) Schematic o f GLR-1::Dendra2 photo­conversion in the cell body (top). Images of G LR-1::Dendra2 in the cell body and in the distal processes (tail). Red arrowheads highlight synapses that received photoconverted GLR-1::Dendra2 from the cell body. (Cand D) Images o f GLR-1::Dendra2 after photoconversion o f total fluorescence (C) or puncta fluorescence only (D) in the proximal processes (only the red signal is shown). Red arrows indicate the appearance of photoconverted GLR-1::Dendra2 at d istal synapses (red and green signal shown). (E) Images o f GLR-1::PAGFP before and a fter photoactivation. Red arrows indicate the appearance o f photoactivated GLR-1::PAGFPat proximal synapses. Scale bars represent 5 mm. See also Figure S3. only UNC-104/KIF1 (kinesin-3), KLP-4 (a protein related to UNC- To determine the identity of the molecular motor(s) that trans- 104), and UNC-116/KIF5 are known to be expressed in the AVA port GLR-1 receptors, we measured the in vivo transport of GLR-interneurons (http://www.wormbase.org) (Siddiqui, 2002). 1::GFP vesicles in candidate motor protein mutants and found (D) Cartoon schematic o f the double-tagged SEP::mCherry::GLR-1 in transport vesicles and on the cell surface. (E) Simultaneous tw o -c o lo r confocal imaging of SEP::mCherry::GLR-1. Single-plane confocal images taken before and after photobleaching (top) and the corresponding kymograph (bottom) showing GLR-1 transport (mCherry signal) and GLR-1 insertion into the membrane (SEP signal). (F) Quantification o f GLR-1 insertion events in synaptic and extrasynaptic regions. n = 10. *p < 0.05, ***p < 0.001. Scale bars represent 5 mm. Error bars indicate SEM. See also Figure S2. Cell P R E S S Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 1425 53 Cel Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs that both anterograde and retrograde GLR-1 transport were dramatically disrupted in unc-116 loss-of-function mutants (Fig­ures 4A-4D). In contrast, we observed normal velocity and run length in klp-4(ok3537) null mutants and only mild disruption of transport in unc-104(e1265) null mutants (Figures S4Aand S4B). Interestingly, we observed an unc-116 allelic series with respect to GLR-1::GFP transport. Thus, the severity of the de­fects in GLR-1 transport speed, run length, and the number of transport events progressively increased from unc-116(e2310) to unc-116(rh24) (Figures 4A-4D; TableS1).The defective GLR- 1 transport observed in these unc-116 partial loss-of-function mutants suggested thattransport might beeliminated byacom-plete loss of UNC-116/KIF5 function. Unfortunately, we were un­able to measure GLR-1 transport in null mutants because the unc-116 nullallele islethal (Byrd etal., 2001). Wetherefore gener­ated transgenic worms that expressed a dominant-negative (DN) variant of UNC-116 (E160A) that is predicted to trap the protein in a rigor state (Klumpp et al., 2003). The rapid movement of GLR- 1::GFP vesicles was eliminated in worms that expressed UNC- 116(E160A) solely in AVA (Figures 4A-4D). We also failed to observe vesicle movement in transgenic worms where UNC- 116 was knocked down specifically in AVA using double­stranded RNAi (unc-116(RNAi); Figures4A-4D). We could rescue the defective transport of GLR-1::GFP in unc-116(wy270) mutants by specifically expressing a wild-type unc-116 trans­gene in AVA (Figures 4A-4D), indicating a cell-autonomous role for UNC-116-mediated transport of GLR-1. Kinesin-1 motors are tetrameric proteins composed of two heavy chains (UNC-116) and two light chains. In C. elegans, the genes encoding the kinesin light chains {klc-1 and klc-2) are broadly expressed (Sakamoto et al., 2005) (http://www. wormbase.org). Light chains regulate the binding of cargo to the motor and are involved in the recruitment of the motor to microtubuletracks (Hirokawa etal., 2010). Todeterminewhether KLC-1 or KLC-2 light chains regulate GLR-1 transport, we exam­ined GLR-1::GFP movement in light chain mutants. Transport was severely disrupted in klc-2, but not klc-1 mutants (Figures 4E and 4F), indicating that GLR-1 transport is dependent on a specific isoform of kinesin-1. To determine the subcellular distribution of UNC-116, we coexpressed fluorescently labeled UNC-116::mCherry with GLR-1::GFP in the AVA neurons. Although UNC-116 was detected throughout the processes, we noted that it appeared to accumulate at synapses (Figure 4G). We also simultaneously measured the movement of GLR-1::mCherry and UNC- 116::GFP using two-color streaming confocal movies. As pre­dicted for kinesin-driven transport of GLR-1, we found that the two signalscolocalized inasubsetoftransportevents, including retrograde movement toward the cell body (Figure 4H). Kinesin-1 motors direct movement toward the plus-end of mi­crotubules, which are typically oriented plus-end out, i.e., toward the distal ends of axonal processes (Stepanova et al., 2003). Because we observed bidirectional movement of GLR-1::GFP, we asked whether microtubules in AVA were of mixed polarity, similar to what has been observed in Drosophila dendrites (Stone et al., 2008; Zheng et al., 2008). Examination of microtubule growth dynamics in transgenicworms that expressed the micro-tubuleend- binding protein EBP-2(Stepanova et al., 2003; Zheng etal.,2008)fusedtoGFPrevealed both plus-end-outand minus-end- out microtubules, consistent with bidirectional transport by UNC-116/KIF5 (Figures S4C and S4D). Additionally, we did not find that microtubule orientation or dynamics were disrupted in unc-116 mutants (Figures S4C and S4D), indicating thatthe dis­rupted transport of GLR-1 in unc-116 mutants was not an indi­rect effect of altered microtubules. GLR-1 Removal from Synapses Is Reduced in unc-116 Mutants Although GLR-1 transport was severely disrupted in unc-116 mutants, we still observed accumulations of GLR-1::GFP in the proximal AVA processes (Figure 5A). Surprisingly, the average fluorescence intensity of synaptic puncta was considerably increased in unc-116 mutants compared to wild-type (Figures 5A and 5B) and with the same allelic dependence we observed for GLR-1 transport (Figure 4). The increased fluorescence in unc-116 mutants was not secondaryto possible presynapticde-fects, as we found that the intensity of synaptic GLR-1::GFP puncta was rescued by the selective expression of UNC-116 in AVA (Figures 5A and 5B). TodeterminewhetherC. elegans kinesin-1 could alsomediate the transport of vertebrate AMPARs, we expressed the verte­brate AMPAR subunit GluA1 fused to GFP in the AVA neurons. GluA1::GFP is functional and localized to puncta in the neural processes (Figure S5) (Brockie et al., 2013). Similar to what we observed for GLR-1, transport of GluA1::GFP was significantly impaired and the receptor accumulated at synaptic puncta in unc-116 mutants compared to wild-type worms (Figure S5). We reasoned that the accumulations of GLR-1 in unc-116 mutants might be secondary to defective removal of synaptic receptors. To test this hypothesis, we photoconverted GLR- 1::Dendra 2 at single synapses (Figure 5C). Following photocon­version, the red fluorescence decreased in wild-type worms with approximately 25% remaining 4 hr after conversion (Figure 5D). In contrast, decaywassignificantly reduced in unc-116 mutants, with the slowest decayobserved in unc-116(RNAi). These results indicate that the removal of synaptic receptors is dependent on UNC-116/KIF5, consistentwiththe observed increase in synap­tic GLR-1::GFP in unc-116 mutants. UNC-116/KIF5 Is Required for the Delivery of Synaptic GLR-1 In contrast to the increased GLR-1::GFP fluorescence in the proximal processes of unc-116 mutants, fluorescence intensity indistal regionsoftheAVAprocesseswasdecreased compared to that in wild-type worms (Figures 5E, 5F, and S6A). This finding, along with our analysis of vesicle stoppage and insertion (Figure 2), suggests that UNC-116/KIF5 is also required for the normal delivery of GLR-1 to synapses. Because diffusion depends on the square root of time, it is inefficient over long dis­tances. Although the young adult worms we study are approxi­mately 96 hr old, the number of synaptic GLR-1 receptors at more distal synapses was reduced in the absence of UNC- 116/KIF5-mediated transport (Figure 5E). Assuming that the diffusion constant for receptors in the cell membrane is approx­imately 0.1 mm2/s (Earnshaw and Bressloff, 2008), we estimate that 96 hr is sufficient time for GLR-1 receptors to diffuse to 1426 Neuron 80,1421-1437, December 18, 2013 ©2013 Elsevier Inc. 54 Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs Cel Figure 4. Bidirectional Transport of GLR-1 Is Dependent on UNC-116/KIF5 (A) Schematic o f UNC-116/KIF5 (top). A rrows and black bar indicate the location o f the m utation for each allele (Table S1). Red and yellow boxes represent the motor domain and coiled coil domains, respectively. Confocal images (middle) and kymographs (bottom) o f GLR-1 ::GFP in the AVA processes. Scale bar represents 2.5 mm. (B-D) Quantification o f anterograde (blue) and retrograde (red) vesicle velocity (B), run length (C), and the frequency o f transport events (D). n = 10 worms; ‘‘0 '' indicates no measurable mobile vesicles. (E) Confocal images (top) and kymographs (bottom) o f GLR-1::GFP in the AVA processes in wild-type (WT), klc-2(km11), and klc-1(ok2809). Scale bar represents 5 mm. (F) Quantification of the frequency o f transport events. Empty triangle in klc-2 represents anterograde = 0.22 events/min and retrograde = 0.17 e vents/min. n = 5. (G) Images o f GLR-1::GFP and UNC-116::mCherry in the AVA processes o f a transgenic worm. Scale bar represents 2 mm. (H) Kymograph showing retrograde comovement o f GLR-1::mCherry and UNC-116::GFP in AVA. The breaks in fluorescence are secondary to limited streaming capacity. Scale bar represents 2.5 mm. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate SEM. See also Figures S 3 -S 5 . Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 1427 55 Cell P R E S S Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs Figure 5. Delivery and Removal of Synaptic GLR-1 Is Mediated by UNC-116/KIF5 (A) Confocal images of GLR-1::GFP puncta in the proximal region o f the AVA processes in various transgenic worms. (B) Quantification of GLR-1::GFP synaptic puncta fluorescence normalized to WT. For all genotypes, n > 10 worms. (C) Confocal images o f G LR-1::Dendra2 synaptic puncta (red signal only) before and after photoconversion. Scale bar represents 1 mm. (D) Quantification o f th e red signal remaining 4 hr after photoconversion. n = 15 puncta per genotype. (E and F) Images (E) and q uantification (F) o f synaptic GLR-1::GFP puncta in AVA normalized to the proximal region of WT. n = 10 worms. Scale bar represents5 mm. (G) Cartoon schematic o f the distal photobleach experiment. (H) GLR-1::GFP images before, immediately after, and 4 hr after photobleaching in the regions indicated in (G). Scale bar represents 5 mm. (I) Linescans of GLR-1::GFP fluorescence intensity in the distal half of AVA before and after photobleaching. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate SEM. See also Figures S 4 -S 8 . 1428 Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 56 Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs proximal synapses, but not long enough to reach distal synap­ses. In support of this, line scans of GLR-1::GFP fluorescence in unc-116(RNAi) and unc-116(rh24) mutants revealed dramati­cally reduced fluorescence in the distal processes when compared to fluorescence in more proximal regions, a pattern consistent with a diffusion-driven process (Figure S6A). We considered whether other motors might contribute to GLR-1 transport earlier in development, thereby confounding the inter­pretation of our line-scan analysis. However, in larval L2 stage unc-116 mutants (Figures S6B and S6C), we found defects in GLR-1 puncta and transport similar to those of adult unc-116 mutants (Figures 4 and 5). To directly test the contribution of motor-mediated delivery of GLR-1 to synapses, we photobleached the entire distal half of the AVA processes and monitored the return of GFP fluores­cence in three regionswithin the photobleached area (Figure 5G). Four hours after photobleaching, we observed significant fluorescence recovery in all distal regions in wild-type worms (67.5% ± 8.2%, n = 4; Figures 5H and 5I). In contrast, essentially no recovery was observed in unc-116(RNAi) mutants (3.5% ± 1.4%, n = 4, p < 0.01). Thus, in unc-116 mutants, receptors diffused out of the cell body to proximal synapses where they accumulated secondary to defective removal. Blocking Protein Synthesis Does Not Appreciably Alter Delivery of GLR-1 to Synapses While our data indicate that UNC-116/KIF5 has a critical role in receptor removal and delivery, other mechanisms, such as local synthesis of GLR-1, might also contribute to the number of syn­aptic receptors. For example, UNC-116/KIF5 might transport mRNA encoding GLR-1 to distal synapses, thus complicating the interpretation of our photobleaching studies. To evaluate the role of local GLR-1 synthesis, we blocked protein synthesis by acutely treating worms with the drug cycloheximide (CHX) for 6 hr (Jensen et al., 2012; Kourtis and Tavernarakis, 2009). We reasoned that if local synthesis of GLR-1 contributed to new synaptic receptors, treatmentwith CHXshould significantly slow fluorescence recovery following photobleaching of GLR- 1::GFP. Although CHX blocked new protein synthesis (Fig­ure S7A), it did not disrupt existing GLR-1::GFP puncta or motor-mediated transport of GLR-1 (Figures S7B and S7C). Importantly, we did not observe an appreciable difference in the recovery of CHX-treated and untreated wild-type worms (Figure S7C). These data indicate that the repopulation of synap­tic GLR-1 during the 4 hr following photobleaching is primarily dependent on motor-driven transport. The Intensity of GLR-1::GFP Puncta Is Decreased in klp-4 Mutants Although we did not observe any transport in unc-116(RNAi) worms, it is possible that additional kinesin motors might contribute to GLR-1 transport. In contrast to the accumulation of receptors in unc-116 mutants, we observed a decrease in synaptic GLR-1::GFP fluorescence in mutants that lacked the Kinesin-3 motor KLP-4 (Figures S8A and S8B), which is consis­tent with an earlier report on klp-4 mutants (Monteiro et al., 2012). Further analysis revealed that GLR-1::GFP puncta inten­sities were similarly diminished in klp-4 mutants and unc-116; klp-4 double mutants, indicating that klp-4 is epistatic to unc- 116 and suggesting that KLP-4 functions upstream of UNC- 116/KIF5-mediated transport of GLR-1. Although GLR-1::GFP synaptic puncta were smaller in klp-4 mutants, analysis of the kymographs revealed apparently normal transport of vesicles in the AVA processes compared to the severely disrupted trans­port in unc-116 mutants and unc-116; klp-4 double mutants (Figures S8C and S8D). Additionally, we did not detect any apparent difference in the intensity of GLR-1::GFP transport vesicles in klp-4 mutants (data not shown). Our data suggest that KLP-4 motors likely act in the cell body to regulate the num­ber of exported GLR-1, but they apparently do not have a direct role in the long-range transport of GLR-1 vesicles in neuronal processes. GLR-1 Surface Expression Is Increased in unc-116 Mutants In unc-116 mutants, the intensity of synaptic GLR-1::GFP fluo­rescence in the proximal processes was increased compared to wild-type. However, two populations of GLR-1 contribute to this fluorescent signal, i.e., receptors at the surface and receptors localized to subcellular compartments. To determine whetherthe number ofsurface receptors was modified by motor transport, we examined the relative levels of GLR-1 tagged with SEP (SEP::GLR-1) in wild-type and unc-116 mutants. Interest­ingly, we found that surface SEP::GLR-1 fluorescence was considerably increased following RNAi knockdown of UNC- 116 in AVA compared to that observed in wild-type (Figures 6A and 6B). Since both the total pool of GLR-1 (GLR-1::GFP) and surface-expressed GLR-1 (SEP::GLR-1) were increased in unc-116 mutants, we next asked whether the ratio of surface to total receptors was modified. We examined SEP::mCherry::GLR-1 at synapses and found that the ratio of surface to total receptors was considerably increased in unc-116 mutants compared to wild-type (Figures 6C and 6D). This increase was similar to that in transgenic worms that expressed SEP::mCherry::GLR- 1(4KR)-an ubiquitination-defectivevariantofGLR-1 thatis pre­dicted to increase the number of cell-surface synaptic receptors (Burbea et al., 2002; Grunwald et al., 2004). Together, our data suggest a model in which GLR-1 receptors in unc-116 mutants diffuse within the membrane to synaptic sites, where they preferentially remain at the surface. The altered ratio of surface to internal receptors might reflect a decreased rate of local receptor endocytosis, or an increased rate of receptor recycling to the cell surface. To distinguish between these possibilities, we photobleached SEP::mCherry:: GLR-1 in either transgenic wild-type worms or unc-116 mutants and quantified the rate of GLR-1 surface insertion by the appearance of SEP fluorescence. In transgenic wild-type worms that expressed either SEP::mCherry::GLR-1 or SEP:: mCherry::GLR-1(4KR), we were able to detect insertion events, with GLR-1(4KR) having a higher rate of insertion (Figures 6E and 6F). In contrast, we did not observe any insertion events in unc-116 mutants (Figures 6E and 6F). The observed defects in receptor removal and recycling in unc-116 mutants suggest that UNC-116/KIF5 might also be required for the delivery of endosomal machinery. Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 1429 Cell P R E S S 57 Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs Figure 6. Surface Expression of GLR-1 Is Increased in unc-116 Mutants (A and B) Images o f SEP::GLR-1 fluorescence (A) and quantification (B) of total fluorescence intensity normalized to WT. n = 10 worms. (C) Confocal images o f SEP::mCherry::GLR-1 and SEP::mCherry::GLR-1(4KR). (D) Ratio quantification o f total synaptic SEP and mCherry signals from SEP::mCherry::GLR-1 in WT and u n c-116 m utants, and SEP::mCherry::GLR-1(4KR). For all genotypes, n > 15 w orms. (E) Kymographs from simultaneous tw o -c o lo r streaming confocal movies of SEP::mCherry::GLR-1 in WT and unc-116(rh24), and SEP::mCherry::GLR-1(4KR). (F) Quantification o f the overall insertion events. ‘‘0 '' indicates no measurable insertion events. For all genotypes, n > 15 worms. Scale bars represent 5 mm. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate SEM. 1430 Neuron 80, 1421-1437, December 18, 2013 ©2013 Elsevier Inc. 58 Neuron UNC-116/KIF5 Mediates Transport of Synaptic AMPARs Cel Figure 7. Glutamate-Gated Currents Are Reduced in unc-116 Mutants (A) Representative traces of glutamate-gated currents in AVA of transgenic w orm s that expressed GLR-1::GFP or GLR-1(4KR)::GFP. (B) Quantification of glutamate-gated currents. For all genotypes, n > 6 worms. (C) Response to nose touch stimulation. n = 10 worms. (D) Glutamate-gated currents in AVA o f transgenic w orm s that overexpressed GLR-1::GFP e ither with or without overexpression of the GLR-1 signaling complex (complex = SOL-1 + SOL-2 + STG-2 + GLR-2). (E) Quantification of glutamate-gated currents. For all genotypes, n > 5 worms. *p < 0.05; **p < 0.01; ***p < 0.001. For all recordings, cells w ere held at - 6 0 mV. Bars indicate 3 mM glutamate application. Error bars indicate SEM. See also Figure S9. Glutamate-Gated Currents Are Decreased in unc-116 Mutants Based on our finding that loss of UNC-116/KIF5 function is asso­ciated with an increase in GLR-1::GFP surface expression, we predicted that voltage-clamp recordings from the proximal pro­cesses of AVA in these transgenic unc-116 mutant worms would reveal an increase in glutamate-gated currents. Instead, we found that glutamate-gated currents in AVA were significantly decreased. This defect was cell autonomous as we could rescue the current by specifically expressing UNC-116 in the AVA neurons (Figures 7A and 7B). This decrease in current was inde­pendent of the GLR-1::GFP transgene as we found similar decreases in current when recording from unc-116(RNAi) or unc-116(wy270) mutants that did not express the transgene (Fig­ure S9). In contrast, the increased surface expression of GLR- 1(4KR) resulted in larger glutamate-gated currents compared to wild-type GLR-1 (Figures 7A and 7B). However, current ampli­tudes in transgenic unc-116(RNAi) mutants that expressed either GLR-1::GFP or GLR-1(4KR)::GFP were both indistinguishable and dramatically reduced compared to wild-type transgenic worms (Figures 7A and 7B). Thus, although surface GLR-1 was increased in unc-116 mutants, glutamate-gated currents were paradoxically decreased. Because of the diminished GLR-1-mediat